Bioprospecting of Selected Species of Polypore Fungi from the Western Balkans.

Growing mushrooms means meeting challenges while aiming for sustainability and circularity. Wherever the producer is located, commercial strains are the same originating from several producers. Customized strains adapted to local conditions are urgently needed. Before introducing new species to the strain development pipeline, the chemical characterization and biological activity of wild ones need to be assessed. Accordingly, the mycoceutical potential of five polypore mushroom species from Serbia was evaluated including: secondary metabolite composition, oxidative damage prevention, anti-tyrosinase, and anti-angiotensin converting enzyme (ACE). The phenolic pattern was comparable in all samples, but the amounts of specific chemicals varied. Hydroxybenzoic acids were the primary components. All samples had varying quantities of ascorbic acid, carotene, and lycopene, and showed a pronounced inhibition of lipid peroxidation (LPx) and ability to scavenge HO•. Extracts were more potent tyrosinase inhibitors but unsuccessful when faced with ACE. Fomitopsis pinicola had the strongest anti-tumor efficacy while Ganoderma lucidum demonstrated strong selectivity in anti-tumor effect in comparison to normal cells. The evaluated species provided a solid foundation for commercial development while keeping local ecology in mind.

Self-Heating Flower-like Nanoconstructs with Limited Incorporation of Yttrium in Maghemite: Effect of Chemical Composition on Heating Efficiency, Cytotoxicity and Genotoxicity.

Partial cation substitution can significantly change the physical properties of parent compounds. By controlling the chemical composition and knowing the mutual relationship between composition and physical properties, it is possible to tailor the properties of materials to those that are superior for desired technological application. Using the polyol synthesis procedure, a series of yttrium-substituted iron oxide nanoconstructs, γ-Fe2-xYxO3 (YIONs), was prepared. It was found that Y3+ could substitute Fe3+ in the crystal structures of maghemite (γ-Fe2O3) up to a limited concentration of ~1.5% (γ-Fe1.969Y0.031O3). Analysis of TEM micrographs showed that crystallites or particles were aggregated in flower-like structures with diameters from 53.7 ± 6.2 nm to 97.3 ± 37.0 nm, depending on yttrium concentration. To be investigated for potential applications as magnetic hyperthermia agents, YIONs were tested twice: their heating efficiency was tested and their toxicity was investigated. The Specific Absorption Rate (SAR) values were in the range of 32.6 W/g to 513 W/g and significantly decreased with increased yttrium concentration in the samples. Intrinsic loss power (ILP) for γ-Fe2O3 and γ-Fe1.995Y0.005O3 were ~8-9 nH·m2/Kg, which pointed to their excellent heating efficiency. IC50 values of investigated samples against cancer (HeLa) and normal (MRC-5) cells decreased with increased yttrium concentration and were higher than ~300 µg/mL. The samples of γ-Fe2-xYxO3 did not show a genotoxic effect. The results of toxicity studies show that YIONs are suitable for further in vitro/in vivo studies toward to their potential medical applications, while results of heat generation point to their potential use in magnetic hyperthermia cancer treatment or use as self-heating systems for other technological applications such as catalysis.

Iron salt-promoted oxidation of steroidal phenols by m-chloroperbenzoic acid: a route to possible antitumor agents.

A new oxidant, containing m-chloroperoxybenzoic acid (MCPBA) and an iron salt, was developed and used for oxidation of steroidal phenols to a quinol/epoxyquinol mixture. Reaction was optimized for estrone, by varying initiators (Fe-salts), reaction temperature, time and mode of MCPBA application. A series of five more substrates (17ß-estradiol and its hydrophobized derivatives) was subjected to the optimized oxidation, providing corresponding p-quinols and 4ß,5ß-epoxyquinols in good to moderate yields. The obtained epoxyquinols were additionally transformed by oxidation, as well as the acid-catalyzed oxirane opening. In a preliminary study of the antiproliferative activity against human cancer cell lines, all newly synthesized compounds expressed moderate to high activity.

Fragment-type 4-azolylcoumarin derivatives with anticancer properties.

Several coumarin derivatives with a directly attached azole substituent at C-4 were synthesized and biologically studied for their anticancer properties. The cell lines used for this investigation (HeLa, K-562, MDA-MB-53, and MCF-7) demonstrated different sensitivities. The best response in the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay was shown by K-562 cells, with compounds displaying activity (3c, IC50 3.06 µM; 4a, IC50 5.24 µM; 4c, IC50 4.7 µM) similar to that of cisplatin (IC50 ~6 µM), which was used as the standard. The studied azole-substituted coumarins demonstrated weaker activity toward other cell lines, except for compound 4c, which was equally potent in the case of MCF-7 cells. Additional biological evaluations supported interference with the cell cycle as a potential mechanism of action and confirmed the absence of toxicity in zebrafish embryos. On the basis of these initial results, 4-azole coumarins should be explored further. Although their activity would need additional optimization, the fact that these compounds are fragment-like structures with MW <300 and clog P <3 offers enough flexibility to fine-tune their drug-like properties.

Evaluation of cytokine expression and circulating immune cell subsets as potential parameters of acute radiation toxicity in prostate cancer patients.

One of the challenges of radiation oncology in the era of personalized medicine is identification of biomarkers associated with individual radiosensitivity. The aim of research was to evaluate the possible clinical value of the associations between clinical, physical, and biological factors, and risk for development of acute radiotoxicity in patients with prostate cancer. The study involved forty four patients treated with three-dimensional conformal radiotherapy. The concentrations of IL-1ß, IL-2, IL-6, IFN-γ and TGF-ß1 were assessed before radiotherapy, after 5th, 15th and 25th radiotherapy fractions, at the end, and 1 month after the end of radiotherapy. Cytokine gene expression was determined in peripheral blood mononuclear cells. The univariate analysis of circulating cytokine levels during radiotherapy showed that increased serum concentrations of IL-6 were significantly associated with higher grade of acute genitourinary toxicity. The multivariate analysis demonstrated that increased level of IL-6 during the radiotherapy was significantly associated with higher grade of acute genitourinary toxicity across treatment. TGF-ß expression levels significantly decreased during course of radiotherapy. Research indicates that changes in circulating cytokine levels might be important parameter of radiotoxicity in patients with prostate cancer. These findings suggest that future studies based on multi-parameter examination are necessary for prediction of individual radiosensitivity.

Chitosan nanobeads loaded with Biginelli hybrids as cell-selective toxicity systems with a homogeneous distribution of the cell cycle in cancer treatment.

Tetrahydropyrimidines are a class of azaheterocycles, also called Biginelli hybrids (obtained from the Biginelli reaction), that have attracted an enormous interest in the medicinal chemistry community in recent years, due to a broad biological activity, such as anticancer, antiviral, anti-inflammatory, antidiabetic, antituberculosis activities, etc. According to SciFinder®, more than 70 000 different Biginelli-like compounds have been covered in publications. However, although the Biginelli reaction can yield a large number of compounds with a broad range of activities, none of them have been captured in a carrier. In this study, chitosan-based (Ch) nanoparticles (NPs) containing three different molecules (Biginelli hybrids) were developed and tested for the first time as simple and promising vehicles for anticancer Biginelli-based drugs. The key features of NPs, such as size, surface morphology, drug encapsulation efficiency, and in vitro release were systematically investigated. Rather weak cell selectivity of pure Biginelli hybrids (A-C) to selected cancer cell lines has improved and this has been accompanied with two-to-four times stronger cytotoxic effect of A-C loaded Ch NPs, with a triple reduction in toxicity to healthy cells (MRC-5). It has been observed that the examined NPs induce apoptosis. The cell cycle analysis has confirmed the influence of A-loaded Ch (A-Ch), B-loaded Ch (B-Ch), and C-loaded Ch (C-Ch) on the cell cycle distribution, which was homogenously affected. This is the difference with regard to the effect of A, B, and C on the cell cycle. It has been established that the increased selectivity and antitumor activity of NPs are related to the presence of the carrier.

Subcritical Water for Recovery of Polyphenols from Comfrey Root and Biological Activities of Extracts.

In the present study, subcritical water was used for extraction of bioactive compounds of Symphytum officinale root. Temperature (120-200 °C), extraction time (10-30 min) and HCl concentration in extraction solvent (0-1.5%) were investigated as independent variables in order to obtain the optimal conditions for extraction and to maximize the yield of total phenols, flavonoids and antioxidant activity of obtained extracts. The application of optimal conditions (200 °C, 25.6 min and 0.0075%) provided extracts rich in total phenols and flavonoids and high antioxidant activity. Results also demonstrated that subcritical water extraction showed significant advantages for recovery of comfrey root bioactive compounds comparing to maceration and ultrasound-assisted extraction techniques. In addition, subcritical water extracts of S. officinale root are the promising sources of compounds with antioxidant, ACE inhibition, and antiproliferative properties and could potentially be used for production of new pharmacologically-active formulations.

Antiproliferative and antibacterial activity of some glutarimide derivatives.

Antiproliferative and antibacterial activities of nine glutarimide derivatives (1-9) were reported. Cytotoxicity of compounds was tested toward three human cancer cell lines, HeLa, K562 and MDA-MB-453 by MTT assay. Compound 7 (2-benzyl-2-azaspiro[5.11]heptadecane-1,3,7-trione), containing 12-membered ketone ring, was found to be the most potent toward all tested cell lines (IC50 = 9-27 µM). Preliminary screening of antibacterial activity by a disk diffusion method showed that Gram-positive bacteria were more susceptible to the tested compounds than Gram-negative bacteria. Minimum inhibitory concentration (MIC) determined by a broth microdilution method confirmed that compounds 1, 2, 4, 6-8 and 9 inhibited the growth of all tested Gram-positive and some of the Gram-negative bacteria. The best antibacterial potential was achieved with compound 9 (ethyl 4-(1-benzyl-2,6-dioxopiperidin-3-yl)butanoate) against Bacillus cereus (MIC 0.625 mg/mL; 1.97 × 10(-3 )mol/L). Distinction between more and less active/inactive compounds was assessed from the pharmacophoric patterns obtained by molecular interaction fields.

Nutraceutical properties of the methanolic extract of edible mushroom Cantharellus cibarius (Fries): primary mechanisms.

The methanolic extract of the wild edible mushroom Cantharellus cibarius Fr. (chanterelle) was analyzed for in vitro antioxidative, cytotoxic, antihypertensive and antibacterial activities. Various primary and secondary metabolites were found. Phenols were the major antioxidant components found in the extract (49.8 mg g(-1)), followed by flavonoids, whose content was approximately 86% of the total phenol content. Antioxidant activity, measured by four different methods, was high for inhibition of lipid peroxidation (EC50 = 1.21 mg mL(-1)) and chelating ability (EC50 = 0.64 mg mL(-1)). The antioxidant activity of the C. cibarius methanol extract was achieved through chelating iron compared to hydrogen atom and/or electron transfer. The extract showed good selectivity in cytotoxicity on human cervix adenocarcinoma HeLa, breast carcinoma MDA-MB-453 and human myelogenous leukemia K562, compared to normal control human fetal lung fibroblasts MRC-5 and human lung bronchial epithelial cells BEAS-2B. The extract had inhibitory activity against angiotensin converting I enzyme (ACE) (IC50 = 0.063 mg mL(-1)). The extract revealed selective antimicrobial activity against Gram-positive bacteria with the highest potential against E. faecalis. The medicinal and health benefits, observed in wild C. cibarius mushroom, seem an additional reason for its traditional use as a popular delicacy food.

Did the Iceman Know Better? Screening of the Medicinal Properties of the Birch Polypore Medicinal Mushroom, Piptoporus betulinus (Higher Basidiomycetes).

The birch polypore Piptoporus betulinus was among two mushrooms that were found in the Iceman's bag. Recent studies indicated that P. betulinus was probably used as a religious and medicinal item. In order to examine the medicinal potential of P. betulinus, hot water (HW), partially purified (PP), and alkali extract (HA) were prepared and tested for antioxidant, antimicrobial, cytotoxic, and angiotensin I-converting enzyme (ACE) inhibitory activity. All tested samples exhibited moderate cytotoxic activity, and HW appeared as the most effective (IC50 = 0.8 ± 0.1 mg/ml for HeLa cells). HA proved to be a good 1,1-diphenyl-2-picrylhydrazyl radical scavenger and exhibited the strongest ferric-reducing power (EC50 = 0.07 ± 0.3 mg/ml). The same extract (HA) also expressed the strongest ferric-reducing power (EC50 = 0.99 ± 0.1 mg/ml). Hot alkali extraction contributed significantly to ACE inhibitory activity (EC50 = 0.06 ± 0.00 mg/ml) and to antimicrobial activity, especially against highly resistant Enterococcus faecalis (minimum inhibitory concentration: 0.156 ± 0.000 mg/ml; and minimum bactericidal concentration: 1.25 ± 0.00 mg/ml).

Correction: Synthesis, cytotoxic and hydrolytic studies of titanium complexes anchored by a tripodal diamine bis(phenolate) ligand.

Correction for 'Synthesis, cytotoxic and hydrolytic studies of titanium complexes anchored by a tripodal diamine bis(phenolate) ligand' by Sónia Barroso et al., Dalton Trans., 2014, 43, 17422-17433.

Synthesis, cytotoxic and hydrolytic studies of titanium complexes anchored by a tripodal diamine bis(phenolate) ligand.

The reactivity, cytotoxic studies and hydrolytic behaviour of diamine bis(phenolate) titanium complexes are reported. The reactions of [Ti((tBu2)O2NN')Cl]2(µ-O) (1) with LiO(I)Pr or HO(I)Pr in the presence of NEt3, aiming at the synthesis of the alkoxido derivative of 1 led to no reaction or to the synthesis of the monomeric complex [Ti((tBu2)O2NN')(O(i)Pr)2] (3), respectively. A small amount of the alkoxidotitanium dimer [Ti((tBu2)O2NN')(O(i)Pr)]2(µ-O) (2) crystallized out of a solution of 3 and DFT calculations showed that the transformation of 1 into 3 is a thermodynamically favorable process in the presence of a base (NEt3) (ΔG = -14.7 kcal mol(-1)). 2 was quantitatively obtained through the direct reaction of the ligand precursor H2((tBu2)O2NN') with titanium tetra(isopropoxido). Further reaction of 2 with an excess of TMSCl was revealed to be the most suitable method for the preparation of [Ti((tBu2)O2NN')Cl2] (4). 1 and 3 disclosed cytotoxic activity towards HeLa, Fem-x, MDA-MB-361 and K562 cells and 1 exhibited moderate binding affinity to FS-DNA. (1)H NMR hydrolysis studies attested the fast decomposition of 4 in the presence of D2O. The hydrolysis of 3 is slower and proceeds through the formation of [Ti((tBu2)O2NN')(OH)]2(µ-O) (5) that was crystallographically characterized. Upon D2O addition 1 immediately forms complex new species, stable in solution for long periods (weeks).

Phenylpropiophenone derivatives as potential anticancer agents: synthesis, biological evaluation and quantitative structure-activity relationship study.

Series of twelve chalcone and propafenone derivatives has been synthesized and evaluated for anticancer activities against HeLa, Fem-X, PC-3, MCF-7, LS174 and K562 cell lines. The 2D-QSAR and 3D-QSAR studies were performed for all compounds with cytotoxic activities against each cancer cell line. Partial least squares (PLS) regression has been applied for selection of the most relevant molecular descriptors and QSAR models building. Predictive potentials of the created 2D-QSAR and 3D-QSAR models for each cell line were compared, by use of leave-one-out cross-validation and external validation, and optimal QSAR models for each cancer cell line were selected. The QSAR studies have selected the most significant molecular descriptors and pharmacophores of the chalcone and propafenone derivatives and proposed structures of novel chalcone and propafenone derivatives with enhanced anticancer activity on the HeLa, Fem-X, PC-3, MCF-7, LS174 and K562 cells.

In vitro antitumor actions of extracts from endemic plant Helichrysum zivojinii.

BACKGROUND: The aim of this research was to determine the intensity and mechanisms of the cytotoxic actions of five extracts isolated from the endemic plant species Helichrysum zivojinii Cernjavski & Soska (family Asteraceae) against specific cancer cell lines. In order to evaluate the sensitivity of normal immunocompetent cells implicated in the antitumor immune response, the cytotoxicity of extracts was also tested against healthy peripheral blood mononuclear cells (PBMC). METHODS: The aerial parts of the plants were air-dried, powdered, and successively extracted with solvents of increasing polarity to obtain hexane, dichloromethane, ethyl-acetate, n-butanol and methanol extracts. The cytotoxic activities of the extracts against human cervix adenocarcinoma HeLa, human melanoma Fem-x, human myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 cells and PBMC were evaluated by the MTT test. The mode of HeLa cell death was investigated by morphological analysis. Changes in the cell cycle of HeLa cells treated with the extracts were analyzed by flow cytometry. The apoptotic mechanisms induced by the tested extracts were determined using specific caspase inhibitors. RESULTS: The investigated Helichrysum zivojinii extracts exerted selective dose-dependent cytotoxic actions against selected cancer cell lines and healthy immunocompetent PBMC stimulated to proliferate, while the cytotoxic actions exerted on unstimulated PBMC were less pronounced. The tested extracts exhibited considerably stronger cytotoxic activities towards HeLa, Fem-x and K562 cells in comparison to resting and stimulated PBMC. It is worth noting that the cytotoxicity of the extracts was weaker against unstimulated PBMC in comparison to stimulated PBMC. Furthermore, each of the five extracts induced apoptosis in HeLa cells, through the activation of both intrinsic and extrinsic signaling pathways. CONCLUSION: Extracts obtained from the endemic plant Helichrysum zivojinii may represent an important source of novel potential antitumor agents due to their pronounced and selective cytotoxic actions towards malignant cells.

Cinnamic acid derivatives induce cell cycle arrest in carcinoma cell lines.

Cinnamic acid derivatives can be found in plant material, and they possess a remarkable variety of biological effects. In the present study, we have investigated the cytotoxic effects of representative cinnamic acid esters and amides. The cytotoxicity was determined by MTT test on human cervix adenocarcinoma (HeLa), myelogenous leukemia (K562), malignant melanoma (Fem-x), and estrogen-receptor-positive breast cancer (MCF-7) cells, versus peripheral blood mononuclear cells (PBMCs) without or with the addition of the plant lectin phytohemaglutinin (PHA). The compounds tested showed significant cytotoxicity (IC50s between 42 and 166 µM) and furthermore selectivity of these cytotoxic effects on the malignant cell lines versus the PBMCs was also seen, especially when electron-withdrawing groups, such as a cyano group (compound 5), were present on the aromatic rings of the alcohol or amine parts of the cinnamic acid derivatives. The additional study on cell cycle phase distribution indicated that novel cinnamic acid derivatives inhibit cell growth by induction of cell death. Thus, cinnamic acids derivatives represent important lead compounds for further development of antineoplastic agents.

Antiproliferative activity of aroylacrylic acids. Structure-activity study based on molecular interaction fields.

Antiproliferative activity of 27 phenyl-substituted 4-aryl-4-oxo-2-butenoic acids (aroylacrylic acids) toward Human cervix carcinoma (HeLa), Human chronic myelogenous leukemia (K562) and Human colon tumor (LS174) cell lines in vitro are reported. Compounds are active toward all examined cell lines. The most active compounds bear two or three branched alkyl or cycloalkyl substituents on phenyl moiety having potencies in low micromolar ranges. One of most potent derivatives arrests the cell cycle at S phase in HeLa cells. The 3D QSAR study, using molecular interaction fields (MIF) and derived alignment independent descriptors (GRIND-2), rationalize the structural characteristics correlated with potency of compounds. Covalent chemistry, most possibly involved in the mode of action of reported compounds, was quantitatively accounted using frontier molecular orbitals. Pharmacophoric pattern of most potent compounds are used as a template for virtual screening, to find similar ones in database of compounds screened against DTP-NCI 60 tumor cell lines. Potency of obtained hits is well predicted.

An alignment independent 3D QSAR study of the antiproliferative activity of 1,2,4,5-tetraoxanes.

An alignment-free 3D QSAR study on antiproliferative activity of the thirty-three 1,2,4,5-tetraoxane derivatives toward two human dedifferentiated cell lines was reported. GRIND methodology, where descriptors are derived from GRID molecular interaction fields (MIF), were used. It was found that pharmacophoric pattern attributed to the most potent derivatives include amido NH of the primary or secondary amide, and the acetoxy fragments at positions 7 and 12 of steroid core which are, along with the tetraoxane ring, common for all studied compounds. Independently, simple multiple regression model obtained by using the whole-molecular properties, confirmed that the hydrophobicity and the H-bond donor properties are the main parameters influencing potency of compounds toward human cervix carcinoma (HeLa) and human malignant melanoma (FemX) cell lines. Corollary, similar structural motifs are found to be important for the potency toward both examined cell lines.

Cytotoxic effect of wine polyphenolic extracts and resveratrol against human carcinoma cells and normal peripheral blood mononuclear cells.

Red and white wine polyphenols have been reported to provide substantial health benefits. In this study, the cytotoxic activity of red and white wine polyphenolic extracts and of resveratrol was evaluated against different cancer cell lines--human cervix adenocarcinoma HeLa, human breast adenocarcinoma MDA-MB-361, and human breast carcinoma MDA-MB-453--and normal human peripheral blood mononuclear cells (PBMCs). Qualitative and quantitative compositions of wine polyphenolic extracts obtained by fractional vacuum distillation of corresponding wines were determined using spectrophotometric methods and high-performance liquid chromatography with diode array detection and liquid chromatography with electrospray ionization-time of flight mass spectrometry analysis. It was demonstrated that wine polyphenolic extracts and resveratrol exerted higher cytotoxic activity against HeLa and MDA-MB-453 cells in comparison to MDA-MB-361 cells and unstimulated and stimulated PBMCs. Furthermore, white wine polyphenolic extract exhibited a significantly higher antiproliferative action on cancer cell lines than red wine extract. The presence of condensed or fragmented nuclei in HeLa cells, pretreated with extract of white wine and stained with a mixture of acridine orange and ethidium bromide, pointed to the morphological signs of apoptosis. In addition, HeLa cells in late stages of apoptosis or secondary necrosis were also observed. Results from our study suggest that polyphenolic extracts from red and white wine may have anticarcinogenic potential.

Improvement of cytotoxicity of titanocene-functionalized mesoporous materials by the increase of the titanium content.

The reaction of [Ti(eta(5)-C(5)H(5))(2)Cl(2)] (1), with 3-mercaptopropyltrimethoxysilane or 3-mercaptopropyltriethoxysilane in the presence of triethylamine leads to the formation of the thiolate complexes [Ti(eta(5)-C(5)H(5))(2){SCH(2)CH(2)CH(2)Si(OMe)(3)}(2)] (2) and [Ti(eta(5)-C(5)H(5))(2){SCH(2)CH(2)CH(2)Si(OEt)(3)}(2)] (3), respectively. Complexes 2 and 3 have been characterized by traditional methods, in addition, structural studies based on DFT calculations are reported. 1-3 have been grafted onto dehydroxylated MCM-41 to give the novel materials MCM-41/[Ti(eta(5)-C(5)H(5))(2)Cl(2)] (S1), MCM-41/[Ti(eta(5)-C(5)H(5))(2){SCH(2)CH(2)CH(2)Si(OMe)(3)}(2)] (S2) and MCM-41/[Ti(eta(5)-C(5)H(5))(2){SCH(2)CH(2)CH(2)Si(OEt)(3)}(2)] (S3) which have been characterized by powder X-ray diffraction, X-ray fluorescence, nitrogen gas sorption, multinuclear MAS NMR spectroscopy, thermogravimetry, UV spectroscopy, SEM and TEM. Materials S2 and S3 present much higher values of Ti wt% (ca. 3%) than S1 (ca. 1%), indicating the higher functionalization rate induced by the substitution of the chloro ligands by the thiolato ligands in the starting titanocene derivatives. The cytotoxicity of the non-functionalized MCM-41 and S1-S3 toward human cancer cell lines such as adenocarcinoma HeLa, human myelogenous leukemia K562 and human malignant melanoma Fem-x has been studied. In addition the cytotoxicity of these materials on normal immunocompetent cells such as stimulated (PBMC+PHA) and non-stimulated (PBMC-PHA) peripheral blood mononuclear cells have been also studied. M(50) values (quantity of material needed to inhibit normal cell survival by 50%) of the studied surfaces show that non-functionalized MCM-41 was not active against any of the studied cells, while the functionalized surfaces S1-S3 were active against all the tested human cancer cells. The cytotoxic activity of surfaces S2 and S3 were very similar, however, S1 showed lower cytotoxic activity. This phenomenon indicates that the cytotoxicity of the titanocene-functionalized materials strongly depends on the titanium content.