Magnetic Mesoporous Silica for Targeted Drug Delivery of Chloroquine: Synthesis, Characterization, and In Vitro Evaluation.

Malaria is a dangerous tropical disease, with high morbidity in developing countries. The responsible parasite has developed resistance to the existing drugs; therefore, new drug delivery systems are being studied to increase efficacy by targeting hemozoin, a parasite paramagnetic metabolite. Herein, magnetic mesoporous silica (magMCM) was synthesized using iron oxide particles dispersed in the silica structure for magnetically driven behavior. The X-ray diffractogram (XRD) and Mössbauer spectra show patterns corresponding to magnetite and maghemite. Furthermore, Mössbauer spectroscopy revealed superparamagnetic behavior, attributed to single magnetic domains in particles smaller than 10 nm. Even in the presence of iron oxide particles, the hexagonal structure of MCM is clearly identified in XRD (low-angle region) and the channels are visible in TEM images. The drug chloroquine (CQ) was encapsulated by incipient wetness impregnation (magMCM-CQ). The N2 adsorption-desorption isotherms show that CQ molecules were encapsulated in the pores, without completely filling the mesopores. BET surface area values were 630 m2 g-1 (magMCM) and 467 m2 g-1 (magMCM-CQ). Encapsulated CQ exhibited rapid delivery (99% in 3 h) in buffer medium and improved solubility compared to the non-encapsulated drug, attributed to CQ encapsulation in amorphous form. The biocompatibility assessment of magMCM, magMCM-CQ, and CQ against MRC5 non-tumoral lung fibroblasts using the MTT assay after 24 h revealed no toxicity associated with magMCM. On the other hand, the non-encapsulated CQ and magMCM-CQ exhibited comparable dose-response activity, indicating a similar cytotoxic effect.

Mushroom ß-glucans: application and innovation for food industry and immunotherapy.

Among the most important sources of ß-glucans are edible and medicinal mushrooms. These molecules are components of the cellular wall of basidiomycete fungi (mushrooms) and can be extracted even from the basidiocarp as the mycelium and its cultivation extracts or biomasses. Mushroom ß-glucans are recognized by their potential effects as immunostimulants and immunosuppressants. They are highlighted as anticholesterolemic, anti-inflammatory, adjuvant in diabetes mellitus, mycotherapy for cancer treatment, as well as adjuvants for COVID-19 vaccines. Due to their relevance, several techniques of ß-glucans extraction, purification, and analysis have already been described. Despite the previous knowledge of ß-glucans' benefits for human nutrition and health, the main information about this topic refers to the molecular identification, properties, and benefits, as well as their synthesis and action on cells. Studies on biotechnology industry applications (product development) and the registered products of ß-glucans from mushrooms are still limited and more common for feed and healthcare. In this context, this paper reviews the biotechnological production of food products containing ß-glucans from basidiomycete fungi, focusing on food enrichment, and presents a new perspective on fungi ß-glucans' use as potential immunotherapy agents. KEY POINTS: • Mushrooms' ß-glucans for product development in the biotechnology industry • Biotechnological production of food products containing mushrooms' ß-glucans • Basidiomycete fungi ß-glucans are used as potential immunotherapy agents.

Antiprotozoal Activity of Mycelial Extracts of Several Medicinal Agaricomycetes Mushrooms against Giardia duodenalis.

Despite knowledge on the therapeutic properties of fungal bio-compounds, few studies have been reported on their anti-parasitic activities. The anti-parasitic activity (APA) of mycelial extracts from seven medicinal agaricomycetous mushrooms (Polyporus lipsiensis, Ganoderma applanatum, Pleurotus ostreatus, P. flabellatus, Oudemansiella canarii, Lentinula edodes, and Pycnoporus sanguineus) against Giardia duodenalis, and identification of chemical compounds produced by mycelium P. lipsiensis mycelium, have been reported. The extracts of mycelia and fermented culture broths of tested mushroom species were evaluated against G. duodenalis by biological assays. P. lipsiensis showed the highest APA. The chemical analysis of mycelial extract of P. lipsiensis by gas chromatography-mass spectrometry (GC-MS) identified 73 molecules, including steroids, terpenes, and lipids. According to literature data, among these molecules, 11 possess APA. The present study revealed the diversity of compounds with anti-protozoal potential produced by mycelia of Agaricomycetes mushrooms, particularly P. lipsiensis against G. duodenalis.

Bioadhesive 3D-Printed Skin Drug Delivery Polymeric Films: From the Drug Loading in Mesoporous Silica to the Manufacturing Process.

The alliance between 3D printing and nanomaterials brings versatile properties to pharmaceuticals, but few studies have explored this approach in the development of skin delivery formulations. In this study, clobetasol propionate (CP) was loaded (about 25% w/w) in mesoporous silica nanomaterial (MSN) to formulate novel bioadhesive and hydrophilic skin delivery films composed of pectin (5% w/v) and carboxymethylcellulose (5% w/v) by 3D printing. As a hydrophobic model drug, CP was encapsulated in MSN at a 3:1 (w/w) ratio, resulting in a decrease of CP crystallinity and an increase of its dissolution efficiency after 72 h (65.70 ± 6.52%) as compared to CP dispersion (40.79 ± 4.75%), explained by its partial change to an amorphous form. The CP-loaded MSN was incorporated in an innovative hydrophilic 3D-printable ink composed of carboxymethylcellulose and pectin (1:1, w/w), which showed high tensile strength (3.613 ± 0.38 N, a homogenous drug dose (0.48 ± 0.032 mg/g per film) and complete CP release after 10 h. Moreover, the presence of pectin in the ink increased the skin adhesion of the films (work of adhesion of 782 ± 105 mN·mm). Therefore, the alliance between MSN and the novel printable ink composed of carboxymethylcellulose and pectin represents a new platform for the production of 3D-printed bioadhesive films, opening a new era in the development of skin delivery systems.

Synthesis of magnetic nanoparticles functionalized with histidine and nickel to immobilize His-tagged enzymes using ß-galactosidase as a model.

The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using ß-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant ß-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized ß-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant ß-galactosidase, showing its potential application in other high-molecular-weight enzymes.

High performance biocatalyst based on ß-d-galactosidase immobilized on mesoporous silica/titania/chitosan material.

A new support for the immobilization of ß-d-galactosidase from Kluyveromyces lactis was developed, consisting of mesoporous silica/titania with a chitosan coating. This support presents a high available surface area and adequate pore size for optimizing the immobilization efficiency of the enzyme and, furthermore, maintaining its activity. The obtained supported biocatalyst was applied in enzyme hydrolytic activity tests with o-NPG, showing high activity 1223 Ug-1, excellent efficiency (74%), and activity recovery (54%). Tests of lactose hydrolysis in a continuous flow reactor showed that during 14 days operation, the biocatalyst maintained full enzymatic activity. In a batch system, after 15 cycles, it retained approximately 90% of its initial catalytic activity and attained full conversion of the lactose 100% (±12%). Additionally, with the use of the mesoporous silica/titania support, the biocatalyst presented no deformation and fragmentation, in both systems, demonstrating high operational stability and appropriate properties for applications in food manufacturing.

A novel electrochemical platform based on mesoporous silica/titania and gold nanoparticles for simultaneous determination of norepinephrine and dopamine.

An amorphous and mesoporous silica/titania (SiTi) material was synthesized by sol-gel method and its surface was modified with gold nanoparticles (AuNP) previously stabilized in a chitosan solution. The presence of small AuNP, with diameter lower than 10 nm was confirmed by transmission electron microscopy (TEM) and UV-Vis spectroscopy. Carbon paste electrodes were prepared to test the electrochemical properties by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in [Fe(CN)6]3-/4- solution probe whereby the material silica-titania/gold nanoparticles (SiTi/AuNP) showed a huge improvement in the redox peak current and low charge transfer resistance. This electrode presented a good response for both norepinephrine and dopamine by means of square wave voltammetry (SWV) measurements; great sensitivity for both analytes, in an extensive linear range, was obtained. The limits of detection were 0.35 µmol L-1 and 0.57 µmol L-1 for norepinephrine and dopamine, respectively. Additionally, this electrode showed high selectivity for both analytes and it was applied in the simultaneous determination of norepinephrine and dopamine. The sensor was also tested in simulated biological fluids presenting a good recovery. The SWV electrochemical response of norepinephrine was also investigated in the presence of possible interferers such as uric acid, ascorbic acid and glucose and there was no significant interference. The prepared electrode also exhibits good reproducibility for norepinephrine detection, with relative standard deviation of 5.19%.

Identification and antigiardial activity of biocompounds produced in the Ganoderma lipsiense mycelium in submerged fermentation.

Natural antiparasitic agents are used as a strategy for advances in the treatment of neglected tropical diseases (NTDs), such as giardiasis. The present paper identified the compounds produced by Ganoderma lipsiense mycelium grown in submerged fermentation and investigated the antigiardial activity potential of extracts in vitro. G. lipsiense mycelium crude extract (CE) was obtained by the maceration process and after purification in column, thirty compounds were identified by gas chromatography - mass spectrometry (GC-MS) in hexane sub-fraction (HSF). To determine the antigiardial potential, different parts of the purified CE have been evaluated, where hexane (HEXf) and dichloromethane (DCMf) fractions showed in vitro reduction levels higher than 90% of the Giardia duodenalis trophozoites at 100 µg mL-1. This antigiardial activity was investigated aiming to explore the possibility of G. lipsiense mycelium extract as a novel antigiardial compound to the treatment of human giardiasis.

Designing a Support for Lipase Immobilization Based On Magnetic, Hydrophobic, and Mesoporous Silica.

A mesoporous, magnetic, and hydrophobic material was designed step by step to act as a support for lipase immobilization. Its pore size (8.0 nm) is compatible with the size of lipase from Thermomyces lanuginosus (TLL), and its hydrophobic surface (contact angle of a water drop = 125°) was planned to interact with lipase on its interfacially activated form (open conformation). The presence of magnetite particles provides magnetic retrieval of the material and enables recyclability of the biocatalysts. Regarding immobilization parameters, the hydrophobic support was tested in comparison to the unmodified hydrophilic support in phosphate buffer solution (50 mmol L-1, pH 7.5) at 25 °C. Hydrophobicity was found to be critical for the amount of immobilized TLL (immobilization yield of 97% versus 36% for the hydrophilic support), whereas the hydrophilic support favors the native conformational state and substrate access to the enzyme's catalytic site (specific activity of 5.7 versus 4.7 U g-1 for the hydrophobic support, even when it has higher TLL content). Therefore, the hydrophobic support immobilizes higher amounts of TLL and the hydrophilic support keeps the enzyme hyperactivated. Last, due to the stronger interactions of TLL with hydrophobic surfaces, the hydrophobic support offers better preservation of enzyme activity in repeated cycles (76% of activity retained after three cycles versus 50% for the hydrophilic support).

Kinetics Analysis of the Inhibitory Effects of Alpha-Glucosidase and Identification of Compounds from Ganoderma lipsiense Mycelium.

The studies on natural compounds to diabetes mellitus treatment have been increasing in recent years. Research suggests that natural components can inhibit alpha-glucosidase activities, an important strategy in the management of blood glucose levels. In this work, for the first time in the literature, the compounds produced by Ganoderma lipsiense extracts were identified and evaluated on the inhibitory effect of these on alpha-glucosidase activity. Four phenolic compounds were identified by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to crude extract from G. lipsiense grown in red rice medium (RCE) and synthetic medium (SCE), being syringic acid identified in both extracts. Gas chromatography-mass spectrometry (GC-MS) analysis showed fatty acids and their derivatives, terpene, steroid, niacin, and nitrogen compounds to SCE, while RCE was rich in fatty acids and their derivatives. Both extracts demonstrated alpha-glucosidase inhibition (RCE IC50 = 0.269 ± 8.25 mg mL-1; SCE IC50 = 0.218 ± 9.67 mg mL-1), and the purified hexane fraction of RCE (RHEX) demonstrated the highest inhibition of enzyme (81.1%). Studies on kinetic inhibition showed competitive inhibition mode to RCE, while SCE showed uncompetitive inhibition mode. Although the inhibitory effects of RCE and SCE were satisfactory, the present findings identified some unpublished compounds to G. lipsiense in the literature with important therapeutic properties.

Liposoluble compounds from Ganoderma lipsiense grown on solid red rice medium with antiparasitic and antibacterial properties.

Liposoluble molecules are a group of compounds that display potent biological and therapeutic properties. The present study aimed to identify liposoluble molecules produced by Ganoderma lipsiense grown in red rice medium using solid-state fermentation (SSF) techniques, and to investigate the antigiardial and antibacterial activities potential of extracts in vitro. Eighteen fatty acids and derivatives were identified by gas chromatograph-mass spectrometry (GC-MS) analysis in G. lipsense extract. Qualitative (Fourier transform infrared spectroscopy and nuclear magnetic resonance) characterizations identified the steroid ergosta-6,22-diene-3ß,5α,8α-triol in purified hexane subfraction (HEXsf) F19 isolated from hexane fraction (HEXf) of crude extract (CE). Ergosta-6,22-diene-3ß,5α,8α-triol exhibited significant inhibitory activity against Giardia duodenalis throphozoites (93.6%) in in vitro assays. CE and HEXf inhibited 95.38% and 92.74% of the G. duodenalis throphozoites in 100 µg mL-1 , whereas CE and their fractions dichloromethane (DCMf) and ethyl acetate (EAf) showed antibacterial activities against Pseudomonas aeruginosa and Staphylococcus aureus at 500 µg mL-1 . Importantly, some liposoluble compounds produced and identified in G. lipsiense are unpublished for this species. This is first report for the production of ergosta-6,22-diene-3ß,5α,8α-triol by G. lipsiense and its antiparasitic activity.

Incorporation of zeaxanthin nanoparticles in yogurt: Influence on physicochemical properties, carotenoid stability and sensory analysis.

Zeaxanthin nanoparticles (Zea-NP) and zeaxanthin nanoemulsion (Zea-NE) were incorporated in yogurt. Control yogurt (CY), yogurt added of nanoparticles (Y-NP) and yogurt added of nanoemulsion (Y-NE) were evaluated weekly regarding pH, titratable acidity, color, textural parameters, viscosity and syneresis during 28 days. Zeaxanthin retention in Y-NP and Y-NE was also determined over storage. Sensory attributes and morphology were evaluated in all yogurt samples, and zeaxanthin bioaccessibility after in vitro digestion was analyzed in Y-NP and Y-NE after preparation. At the end of storage time, zeaxanthin retention was higher in Y-NP (22.31 ±â€¯2.53%) than in Y-NE (16.84 ±â€¯0.53%). Despite the lower firmness and viscosity observed in Y-NP, these changes were not sensory perceived. The bioaccessibility after in vitro digestion suggested that nanoencapsulation provided a controlled release of the carotenoid. Zea-NP can be incorporated in yogurt, allowing the dispersion of a hydrophobic compound in a hydrophilic matrix, providing stability.

Kinetic identification of phenolic compounds and potential production of caffeic acid by Ganoderma lipsiense in solid-state fermentation.

Basidiomycetes fungi have been utilized for the production of several compounds with bioactive properties, such as phenolic compounds. The present work quantified and identified the phenolic compounds produced in a kinetic study (63 days) and evaluated the antimicrobial activity from the extract obtained by Ganoderma lipsiense cultivation in solid-state fermentation using red rice. Phenolic compounds were identified by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) and caffeic acid content was measured by high-performance liquid chromatography with diode-array detection (HPLC-DAD). Caffeic and syringic acids were produced by G. lipsiense. In the control medium (red rice), the following compounds were identified: p-coumaric acid, salicylic acid, ferulic acid and vanillin. High concentrations of caffeic acid (0.977 µg g-1) were measured in 49 days. Antimicrobial activity was investigated against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus using a minimum inhibitory concentration (MIC) technique. Ganoderma lipsiense extract was only effective against P. aeruginosa. These data have proved to be satisfactory in the study of biosynthesis of caffeic acid and antibacterial compounds by G. lipsiense in solid-state fermentation with red rice.

Nanoencapsulation of linseed oil with chia mucilage as structuring material: Characterization, stability and enrichment of orange juice.

Linseed oil was nanoencapsulated with chia seed mucilage (CSM) as structuring material. Linseed oil nanoparticles (LO-NP) were evaluated regarding particle size distribution, zeta potential, pH, viscosity, encapsulation efficiency, loading capacity, morphology, FT-IR and thermal properties. Furthermore, the nanoparticles were spray-dried, and oxidative stability was evaluated during 28 days under storage at accelerated conditions (40 °C). The bioaccessibility of spray dried nanoparticles (SP LO-NP) was also evaluated after in vitro digestion. Thereafter, SP LO-NP were utilized in the enrichment of orange juice, and physicochemical and sensory evaluation of pure orange juice and orange juice with SP LO-NP were evaluated. Nanoparticles in suspension presented a mean diameter of 356 ±â€¯2.83 nm, zeta potential of -22.75 ±â€¯3.89 mV and encapsulation efficiency of 52%. No significant differences regarding consumer acceptance were observed between pure orange juice and orange juice with SP LO-NP. The results suggest that CSM can be used as structuring material to nanoencapsulate hydrophobic compounds, allowing its solubility in foods with high water content. Furthermore, the SP LO-NP provided a good bioaccessibility to linseed oil after in vitro digestion, which represents an advantage to incorporate the nanoparticles in food.

Medicinal fungi: a source of antiparasitic secondary metabolites.

Regions with a tropical climate are frequently affected by endemic diseases caused by pathogenic parasites. More than one billion people worldwide are exposed directly to tropical parasites. The literature cites several antiparasitic metabolites obtained from medicinal plants or via synthetic pathways. However, fungi produce a diversity of metabolites that play important biological roles in human well-being. Thus, they are considered a potential source of novel natural agents for exploitation in the pharmaceutical industry. In this brief review article, we will provide an overview of the current situation regarding antiparasitic molecules derived from filamentous fungi, in particular, those which are effective against protozoan parasites, such as Plasmodium, Trypanosoma, and Leishmania, vectors of some neglected tropical diseases. Diseases and parasitic agents are described and classified, and the antiparasitic properties of natural compounds produced by the fungi of the phyla Basidiomycota and Ascomycota are reviewed herein, in order to explore a topic only sparsely addressed in the scientific literature.

Highly stable novel silica/chitosan support for ß-galactosidase immobilization for application in dairy technology.

ß-d-Galactosidase is an important enzyme in the dairy industry, and the enzyme from the yeast Kluyveromyces lactis is most widely used. Here, we report immobilization of the enzyme on a silica/chitosan composite support, devised to have 10% and 20% chitosan (SiQT10 and SiQT20, respectively). Morphological and textural characterizations showed that chitosan is dispersed in micrometric regions in silica. For comparison, a silica organofunctionalized with 3-aminopropyltrimethoxysilane (SiO2aptms) was prepared. Performance of the biocatalysts was tested for lactose hydrolysis, and the enzyme immobilized in SiQT10 and SiQT20 composites showed higher efficiency (62% and 47%, respectively) compared with the enzyme in SiO2aptms. Operational stability in this system was evaluated for the first time. After 200 h of continuous use in a fixed-bed reactor, SiQT10 remained with approximately 90% activity. Thus, in addition to demonstrating compatibility for food processing, these results align the enzyme stabilization properties of chitosan with the mechanical resistance of silica.

Effects of immobilization, pH and reaction time in the modulation of α-, ß- or γ-cyclodextrins production by cyclodextrin glycosyltransferase: Batch and continuous process.

This study reports the immobilization of a ß-CGTase on glutaraldehyde pre-activated silica and its use to production of cyclodextrins in batch and continuous reactions. We were able to modulate the cyclodextrin production (α-, ß- and γ-CD) by immobilization and changing the reaction conditions. In batch reactions, the immobilized enzyme reached to maximum productions of 4.9mgmL-1 of α-CD, 3.6mgmL-1 of ß-CD and 3.5mgmL-1 of γ-CD at different conditions of temperature, pH and reaction time. In continuous reactor, varying the residence time and pH it was possible to produce at pH 4.0 and 141min of residence time preferentially γ-CD (0.75 and 3.36mgmL-1 of α- and γ-CD, respectively), or at pH 8.0 and 4.81min α- and ß-CDs (3.44 and 3.51mgmL-1).

Nanoencapsulation of chia seed oil with chia mucilage (Salvia hispanica L.) as wall material: Characterization and stability evaluation.

In this study, chia seed oil was nanoencapsulated utilizing chia seed mucilage (CSM) as wall material. The viscosity, encapsulation efficiency, loading capacity, transmission electron microscopy, FT-IR spectroscopy and thermal properties of chia seed oil nanoparticles (CSO-NP) were performed after preparation. Particle size, zeta potential, span value, and pH of CSO-NP and oxidation stability of nanoencapsulated and unencapsulated oil were evaluated during 28days of storage at accelerated conditions (40°C). The CSO-NP showed spherical shape, an average size of 205±4.24nm and zeta potential of -11.58±1.87mV. The encapsulation efficiency (82.8%), loading capacity (35.38%) and FT-IR spectroscopy demonstrated the interaction between oil and mucilage. Furthermore, CSO-NP were thermally stable at temperatures up 300°C and nanoencapsulated oil showed higher stability against oxidation than unencapsulated oil. The results suggest that chia seed mucilage represents a promising alternative to substitute synthetic polymers in nanoencapsulation.

Trends in mortality from ill-defined causes among the elderly in Brazil, 1979-2013: ecological study.

CONTEXT AND OBJECTIVE:: Mortality measurements are traditionally used as health indicators and are useful in describing a population's health situation through reporting injuries that lead to death. The aim here was to analyze the temporal trend of proportional mortality from ill-defined causes (IDCs) among the elderly in Brazil from 1979 to 2013. DESIGN AND SETTING:: Ecological study using data from the Mortality Information System of the Brazilian Ministry of Health. METHODS:: The proportional mortality from IDCs among the elderly was calculated for each year of the study series (1979 to 2013) in Brazil, and the data were disaggregated according to sex and to the five geographical regions and states. To analyze time trends, simple linear regression coefficients were calculated. RESULTS:: During the study period, there were 2,646,194 deaths from IDCs among the elderly, with a decreasing trend (ß -0.545; confidence interval, CI: -0.616 to -0.475; P < 0.000) for both males and females. This reduction was also observed in the macroregions and states, except for Amapá. The states in the northeastern region reported an average reduction of 80%. CONCLUSIONS:: Mortality from IDCs among the elderly has decreased continuously since 1985, but at different rates among the different regions and states. Actions aimed at improving data records on death certificates need to be strengthened in order to continue the trend observed.

Synthesis of biodegradable films with antioxidant properties based on cassava starch containing bixin nanocapsules.

Biodegradable and active packaging based on cassava starch incorporated bixin nanocapsules with different concentrations were developed. The physical, mechanical, barrier properties and antioxidant activity of the active packaging were studieds. The films incorporated with bixin nanocapsules were found to be homogeneous and thermally stable. Films with higher concentrations of bixin nanocapsules exhibited a significant decrease in tensile strength, water solubility and increase in elongation at break and water vapour permeability, well as, significant improvement in protection against UV and visible light. The films were used to pack sunflower oil under accelerated oxidation conditions (65 % RH/35 °C). Sunflower oil packaged in films with bixin exhibited lower oxidation rates, thus maintaining its freshness according to Codex Alimentarius guidelines (<10 mEq kg-1). Films containing bixin nanocapsules are very promising materials for use as packaging with antioxidant properties for maintaining food safety and extending the shelf life.