Characterizing lipid constituents of B. moojeni snake venom: a comparative approach for chemical and biological investigations.

Snake venoms are complex mixtures majorly composed of proteins with well-studied biological effects. However, the exploration of non-protein components, especially lipids, remains limited despite their potential for discovering bioactive molecules. This study compares three liquid-liquid lipid extraction methods for both chemical and biological analyses of Bothrops moojeni snake venom. The methods evaluated include the Bligh and Dyer method (methanol, chloroform, water), considered standard; the Acunha method, a modification of the Bligh and Dyer protocol; and the Matyash method (MTBE/methanol/water), featuring an organic phase less dense than the aqueous phase. Lipidomic analysis using liquid chromatography with high-resolution mass spectrometry (LC-HRMS) system revealed comparable values of lipid constituents' peak intensity across different extraction methods. Our results show that all methods effectively extracted a similar quantity of lipid species, yielding approximately 17-18 subclasses per method. However, the Matyash and Acunha methods exhibited notably higher proportions of biologically active lipids compared to the Bligh and Dyer method, particularly in extracting lipid species crucial for cellular structure and function, such as sphingomyelins and phosphatidylinositol-phosphate. In conclusion, when selecting a lipid extraction method, it is essential to consider the study's objectives. For a biological approach, it is crucial to evaluate not only the total quantity of extracted lipids but also their quality and biological activity. The Matyash and Acunha methods show promise in this regard, potentially offering a superior option for extracting biologically active lipids compared to the Bligh and Dyer method.

Microbial Metabolites Annotation by Mass Spectrometry-Based Metabolomics.

Since the discovery of penicillin, microbial metabolites have been extensively investigated for drug discovery purposes. In the last decades, microbial derived compounds have gained increasing attention in different fields from pharmacognosy to industry and agriculture. Microbial metabolites in microbiomes present specific functions and can be associated with the maintenance of the natural ecosystems. These metabolites may exhibit a broad range of biological activities of great interest to human purposes. Samples from either microbial isolated cultures or microbiomes consist of complex mixtures of metabolites and their analysis are not a simple process. Mass spectrometry-based metabolomics encompass a set of analytical methods that have brought several improvements to the microbial natural products field. This analytical tool allows the comprehensively detection of metabolites, and therefore, the access of the chemical profile from those biological samples. These analyses generate thousands of mass spectra which is challenging to analyse. In this context, bioinformatic metabolomics tools have been successfully employed to accelerate and facilitate the investigation of specialized microbial metabolites. Herein, we describe metabolomics tools used to provide chemical information for the metabolites, and furthermore, we discuss how they can improve investigation of microbial cultures and interactions.

Enzymatic Pretreatment with Laccases from Lentinus sajor-caju Induces Structural Modification in Lignin and Enhances the Digestibility of Tropical Forage Grass (Panicum maximum) Grown under Future Climate Conditions.

Since laccase acts specifically in lignin, the major contributor to biomass recalcitrance, this biocatalyst represents an important alternative to the pretreatment of lignocellulosic biomass. Therefore, this study investigates the laccase pretreatment and climate change effects on the hydrolytic performance of Panicum maximum. Through a Trop-T-FACE system, P. maximum grew under current (Control (C)) and future climate conditions: elevated temperature (2 °C more than the ambient canopy temperature) combined with elevated atmospheric CO2 concentration(600 µmol mol-1), name as eT+eC. Pretreatment using a laccase-rich crude extract from Lentinus sajor caju was optimized through statistical strategies, resulting in an increase in the sugar yield of P. maximum biomass (up to 57%) comparing to non-treated biomass and enabling hydrolysis at higher solid loading, achieving up to 26 g L-1. These increments are related to lignin removal (up to 46%) and lignin hydrophilization catalyzed by laccase. Results from SEM, CLSM, FTIR, and GC-MS supported the laccase-catalyzed lignin removal. Moreover, laccase mitigates climate effects, and no significant differences in hydrolytic potential were found between C and eT+eC groups. This study shows that crude laccase pretreatment is a potential and sustainable method for biorefinery solutions and helped establish P. maximum as a promising energy crop.

Williamsia aurantiacus sp. nov. a novel actinobacterium producer of antimicrobial compounds isolated from the marine sponge.

An antibiotic-producing actinobacterium, designated isolate B375T, was isolated from marine sponge Glodia corticostylifera collected from Praia Guaecá, São Paulo, Brazil (23°49S; 45°25W), and its taxonomic position established using data from a polyphasic study. The organism showed a combination of morphological, physiological, biochemical and chemotaxonomic characteristics consistent with its classification in the genus Williamsia. Comparative 16S rRNA gene sequence analysis indicated that the strain B375T was most closely related to Williamsia serinedens DSM 45037T and Williamsia spongiae DSM 46676T and having 99.43% and 98.65% similarities, respectively, but was distinguished from these strains by a low level of DNA-DNA relatedness (53.2-63.2%) and discriminatory phenotypic properties. Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and N-glycolated muramic acid residues present in the wall cells. The cells contained C16:0 (23.3%), C18:0 10-methyl (23.2%) and C18:1 ω9c (21.6%) as the major cellular fatty acids. The strain B375T inhibited growing of Staphylococcus aureus and Colletotrichum gloeosporioides strains and was considered a producer of antimicrobial compounds. Based on the data obtained, the isolate B375T (= CBMAI 1090T = DSM 46677T) should, therefore, be classified as the type strain of a novel species of the genus Williamsia, for which the name Williamsia aurantiacus sp. nov. is proposed.

Microbial Diversity and Chemical Multiplicity of Culturable, Taxonomically Similar Bacterial Symbionts of the Leaf-Cutting Ant Acromyrmex coronatus.

Insects are a highly diverse group, exploit a wide range of habitats, and harbor bacterial symbionts of largely unknown diversity. Insect-associated bacterial symbionts are underexplored but promising sources of bioactive compounds. The community of culturable bacteria associated with the leaf-cutting ant Acromyrmex coronatus (Fabricius) and the diversity of their metabolites produced were investigated. Forty-six phylotypes belonging to Actinobacteria, Firmicutes, and Proteobacteria were identified. The chemical profiles of 65 isolates were further analyzed by LC-MS/MS, and principal components analysis (PCA) was used to group the isolates according to their chemical profiles. Historically, selection of bacterial strains for drug discovery has been based on phenotypic and/or genotypic traits. Use of such traits may well impede the discovery of new compounds; in this study, several indistinguishable phylotypes cultured in identical nutritional and environmental conditions produced completely different chemical profiles. Our data also demonstrated the wide chemical diversity to be explored in insect-associated symbionts.

Characterization of Casearin X Metabolism by Rat and Human Liver Microsomes.

Casearin X (CAS X) is the major clerodane diterpene isolated from the leaves of Casearia sylvestris and has been extensively studied due to its powerful cytotoxic activity at low concentrations. Promising results for in vivo antitumor action have also been described when CAS X was administered intraperitoneally in mice. Conversely, loss of activity was observed when orally administered. Since the advancement of natural products as drug candidates requires satisfactory bioavailability for their pharmacological effect, this work aimed to characterize the CAS X metabolism by employing an in vitro microsomal model for the prediction of preclinical pharmacokinetic data. Rat and human liver microsomes were used to assess species differences. A high-performance liquid chromatography with diode-array detection (HPLC-DAD) method for the quantification of CAS X in microsomes was developed and validated according to European Medicines Agency guidelines. CAS X was demonstrated to be a substrate for carboxylesterases via hydrolysis reaction, with a Michaelis-Menten kinetic profile. The enzyme kinetic parameters were determined, and the intrinsic clearance was 1.7-fold higher in humans than in rats. The hepatic clearance was estimated by in vitro-in vivo extrapolation, resulting in more than 90% of the hepatic blood flow for both species. A qualitative study was also carried out for the metabolite identification by mass spectrometry and indicated the formation of the inactive metabolite CAS X dialdehyde. These findings demonstrate that CAS X is susceptible to first-pass metabolism and is a substrate for specific carboxylesterases expressed in liver, which may contribute to a reduction in antitumor activity when administered by the oral route.

Characterization and mapping of secondary metabolites of Streptomyces sp. from caatinga by desorption electrospray ionization mass spectrometry (DESI-MS).

The discovery of new secondary metabolites is a challenge to biotechnologists due to the emergence of superbugs and drug resistance. Knowledge about biodiversity and the discovery of new microorganisms have become major objectives; thus, new habitats like extreme ecosystems have become places of interest to research. In this context, caatinga is an unexplored biome. The ecosystem caatinga is a rich habitat for thermophilic microbes. Its high temperature and dry climate cause selective microbes to flourish and become established. Actinobacteria (Caat 1-54 genus Streptomyces sp.) isolated from the soil of caatinga was investigated to characterize and map its secondary metabolites by desorption electrospray ionization mass spectrometry imaging (DESI-MSI). With this technique, the production of bioactive metabolites was detected and associated with the different morphological differentiation stages within a typical Streptomyces sp. life cycle. High-resolution mass spectrometry, tandem mass spectrometry, UV-Vis profiling and NMR analysis were also performed to characterize the metabolite ions detected by DESI-MS. A novel compound, which is presumed to be an analogue of the antifungal agent lienomycin, along with the antimicrobial compound lysolipin I were identified in this study to be produced by the bacterium. The potency of these bioactive compounds was further studied by disc diffusion assays and their minimum inhibitory concentrations (MIC) against Bacillus and Penicillium were determined. These bioactive metabolites could be useful to the pharmaceutical industry as candidate compounds, especially given growing concern about increasing resistance to available drugs with the emergence of superbugs. Consequently, the unexplored habitat caatinga affords new possibilities for novel bioactive compound discovery. Graphical Abstract ᅟ.

Bioguided isolation, characterization and media optimization for production of Lysolipins by actinomycete as antimicrobial compound against Xanthomonas citri subsp. citri.

Citrus Canker disease is one of the most important disease in citrus production worldwide caused by gram-negative bacterial pathogen Xanthomonas citri subsp. citri, leading to great economic losses. Currently, a spray of copper-based bactericides is the primary measure for citrus canker management. However, these measures can lead to the contamination of soil by metal contamination, but also the development of copper-resistant Xanthomonas populations. Considering the need to discovery new alternatives to control the citrus canker disease, actinomycetes isolated from the Brazilian Caatinga biome and their crude extracts were tested against different strains of Xanthomonas citri subsp. citri. Streptomyces sp. Caat 1-54 crude extract showed the highest antibiotic activity against Xcc. The crude extract dereplication was performed by LC-MS/MS. Through bioassay-guided fractionation strategy, the antimicrobial activity was assigned to Lysolipins, showing a MIC around 0.4-0.8 µg/mL. Growth media optimization using statistical experimental design increased the Lysolipins production in three-fold production. The preventive and curative effects of the optimized crude extract obtained by experimental design of Caat-1-54 against citrus canker were evaluated in potted 'Pera' sweet orange nursery trees. Caat 1-54 extract was effective in preventing new infections by Xcc on leaves but was not able to reduce Xcc population in pre-established citrus canker lesions. Streptomyces sp. Caat 1-54 extract is a promising, environmentally-friendly source of antimicrobial compound to protect citrus trees against citrus canker.

Tapping the biotechnological potential of insect microbial symbionts: new insecticidal porphyrins.

BACKGROUND: The demand for sustainable agricultural practices and the limited progress toward newer and safer chemicals for use in pest control maintain the impetus for research and identification of new natural molecules. Natural molecules are preferable to synthetic organic molecules because they are biodegradable, have low toxicity, are often selective and can be applied at low concentrations. Microbes are one source of natural insecticides, and microbial insect symbionts have attracted attention as a source of new bioactive molecules because these microbes are exposed to various selection pressures in their association with insects. Analytical techniques must be used to isolate and characterize new compounds, and sensitive analytical tools such as mass spectrometry and high-resolution chromatography are required to identify the least-abundant molecules. RESULTS: We used classical fermentation techniques combined with tandem mass spectrometry to prospect for insecticidal substances produced by the ant symbiont Streptomyces caniferus. Crude extracts from this bacterium showed low biological activity (less than 10% mortality) against the larval stage of the fall armyworm Spodoptera frugiperda. Because of the complexity of the crude extract, we used fractionation-guided bioassays to investigate if the low toxicity was related to the relative abundance of the active molecule, leading to the isolation of porphyrins as active molecules. Porphyrins are a class of photoactive molecules with a broad range of bioactivity, including insecticidal. The active fraction, containing a mixture of porphyrins, induced up to 100% larval mortality (LD50 = 37.7 µg.cm-2). Tandem mass-spectrometry analyses provided structural information for two new porphyrin structures. Data on the availability of porphyrins in 67 other crude extracts of ant ectosymbionts were also obtained with ion-monitoring experiments. CONCLUSIONS: Insect-associated bacterial symbionts are a rich source of bioactive compounds. Exploring microbial diversity through mass-spectrometry analyses is a useful approach for isolating and identifying new compounds. Our results showed high insecticidal activity of porphyrin compounds. Applications of different experiments in mass spectrometry allowed the characterization of two new porphyrins.

Saccharopolyspora spongiae sp. nov., a novel actinomycete isolated from the marine sponge Scopalina ruetzleri (Wiedenmayer, 1977).

A novel marine actinomycete, designated strain CMAA 1452T, was isolated from the sponge Scopalina ruetzleri collected from Saint Peter and Saint Paul Archipelago, in Brazil, and subjected to a polyphasic taxonomic investigation. The organism formed a distinct phyletic line in the Saccharopolyspora 16S rRNA gene tree and had chemotaxonomic and morphological properties consistent with its classification in this genus. It was found to be closely related to Saccharopolyspora dendranthemae KLBMP 1305T (99.5% 16S rRNA gene sequence similarity) and shared similarities of 99.3, 99.2 and 99.0 % with 'Saccharopolyspora endophytica' YIM 61095, Saccharopolyspora tripterygii YIM 65359T and 'Saccharopolyspora pathumthaniensis' S582, respectively. DNA-DNA relatedness values between the isolate and its closest phylogenetic neighbours, namely S. dendranthemae KLBMP 1305T, 'S. endophytica' YIM 61095 and S. tripterygii YIM 65359T, were 53.5, 25.8 and 53.2 %, respectively. Strain CMAA 1452T was also distinguished from the type strains of these species using a range of phenotypic features. On the basis of these results, it is proposed that strain CMAA 1452T (=DSM 103218T=NRRL B-65384T) merits recognition as the type strain of a novel Saccharopolyspora species, Saccharopolyspora spongiae sp. nov.

Plasma eicosanoid profiles determined by high-performance liquid chromatography coupled with tandem mass spectrometry in stimulated peripheral blood from healthy individuals and sickle cell anemia patients in treatment.

Eicosanoids play an important role in homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca(2+) ionophores and Ca(2+)-ATPase inhibitors, as well as natural agonists such as formylmethionine-leucyl-phenylalanine (fMLP), can stimulate eicosanoid biosynthesis. The aims of this work were to develop a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was partially validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient ≥0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of ≤15%, except for the lower limit of quantification, where these values were ≤20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were tested. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli for the production or liberation of eicosanoids. We next compared the eicosanoid profiles of stimulated whole blood samples of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients and those of healthy subjects, mainly for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method can detect significant changes in eicosanoid profiles in stimulated whole blood, which will contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.

Streptomyces atlanticus sp. nov., a novel actinomycete isolated from marine sponge Aplysina fulva (Pallas, 1766).

The taxonomic position of a novel marine actinomycete isolated from a marine sponge, Aplysina fulva, which had been collected in the Archipelago of Saint Peter and Saint Paul (Equatorial Atlantic Ocean), was determined by using a polyphasic approach. The organism showed a combination of morphological and chemotaxonomic characteristics consistent with its classification in the genus Streptomyces and forms a distinct branch within the Streptomyces somaliensis 16S rRNA gene tree subclade. It is closely related to Streptomyces violascens ISP 5183T (97.27 % 16S rRNA gene sequence similarity) and Streptomyces hydrogenans NBRC 13475T (97.15 % 16S rRNA gene sequence similarity). The 16S rRNA gene similarities between the isolate and the remaining members of the subclade are lower than 96.77 %. The organism can be distinguished readily from other members of the S. violacens subclade using a combination of phenotypic properties. On the basis of these results, it is proposed that isolate 103T (=NRRL B-65309T = CMAA 1378T) merits recognition as the type strain of a new Streptomyces species, namely Streptomyces atlanticus sp. nov.

Amycolatopsis rhabdoformis sp. nov., an actinomycete isolated from a tropical forest soil.

Strain SB026T was isolated from Brazilian rainforest soil and its taxonomic position established using data from a polyphasic study. The organism showed a combination of chemotaxonomic and morphological features consistent with its classification in the genus Amycolatopsis and formed a branch in the Amycolatopsis 16S rRNA gene tree together with Amycolatopsis bullii NRRL B-24847T, Amycolatopsis plumensis NRRL B-24324T, Amycolatopsis tolypomycina DSM 44544T and Amycolatopsis vancoresmycina NRRL B-24208T. It was related most closely to A. bullii NRRL B-24847T (99.0 % 16S rRNA gene sequence similarity), but was distinguished from this strain by a low level of DNA-DNA relatedness (~46 %) and discriminatory phenotypic properties. Based on the combined genotypic and phenotypic data, it is proposed that the isolate should be classified in the genus Amycolatopsis as representing a novel species, Amycolatopsis rhabdoformis sp. nov. The type strain is SB026T ( = CBMAI 1694T = CMAA 1285T = NCIMB 14900T).

Simultaneous determination of amino acids and neurotransmitters in plasma samples from schizophrenic patients by hydrophilic interaction liquid chromatography with tandem mass spectrometry.

A sensitive, reproducible, and rapid method was developed for the simultaneous determination of underivatized amino acids (aspartate, serine, glycine, alanine, methionine, leucine, tyrosine, and tryptophan) and neurotransmitters (glutamate and γ-aminobutyric acid) in plasma samples using hydrophilic interaction liquid chromatography coupled to triple quadrupole tandem mass spectrometry. The plasma concentrations of amino acids and neurotransmitters obtained from 35 schizophrenic patients in treatment with clozapine (27 patients) and olanzapine (eight patients) were compared with those obtained from 38 healthy volunteers to monitor the effectiveness of treatment. The chromatographic conditions separated ten target compounds within 3 min. This method presented linear ranges that varied from (lower limit of quantification: 9.7-13.3 nmol/mL) to (upper limit of quantification: 19.4-800 nmol/mL), intra- and interassay precision with coefficients of variation lower than 10%, and relative standard error values of the accuracy ranged from -2.1 to 9.9%. The proposed method appropriately determines amino acids and neurotransmitters in plasma from schizophrenic patients. Compared with the control group (healthy volunteers), the plasma levels of methionine in schizophrenic patients treated with olanzapine are statistically significantly higher. Moreover, schizophrenic patients treated with clozapine tend to have increased plasma levels of glutamate.

Erythropoietin Exacerbates Inflammation and Increases the Mortality of Histoplasma capsulatum-Infected Mice.

Erythropoietin (EPO) is a key hormone involved in red blood cell formation, but its effects on nonerythroid cells, such as macrophages, have not been described. Macrophages are key cells in controlling histoplasmosis, a fungal infection caused by Histoplasma capsulatum (Hc). Considering that little is known about EPO's role during fungal infections and its capacity to activate macrophages, in this study we investigated the impact of EPO pretreatment on the alveolar immune response during Hc infection. The consequence of EPO pretreatment on fungal infection was determined by evaluating animal survival, fungal burden, activation of bronchoalveolar macrophages, inflammatory mediator release, and lung inflammation. Pretreatment with EPO diminished mononuclear cell numbers, increased the recruitment of F4/80(+)/CD80(+) and F4/80(+)/CD86(+) cells to the bronchoalveolar space, induced higher production of IFN-γ, IL-6, MIP-1α, MCP-1, and LTB4, reduced PGE2 concentration, and did not affect fungal burden. As a consequence, we observed an increase in lung inflammation with extensive tissue damage that might account for augmented mouse mortality after infection. Our results demonstrate for the first time that EPO treatment has a deleterious impact on lung immune responses during fungal infection.

Streptomyces araujoniae Produces a Multiantibiotic Complex with Ionophoric Properties to Control Botrytis cinerea.

A recently described actinomycete species (Streptomyces araujoniae ASBV-1(T)) is effective against many phytopathogenic fungi. In this study, we evaluated the capacity of this species to inhibit Botrytis cinerea development in strawberry pseudofruit, and we identified the chemical structures of its bioactive compounds. An ethyl acetate crude extract (0.1 mg ml(-1)) of ASBV-1(T) fermentation broth completely inhibited fungus growth in strawberry pseudofruit under storage conditions. The crude extract was fractionated by preparative high-performance liquid chromatography; the active fraction was further evaluated by tandem mass spectrometry. ASBV-1(T) produced a multiantibiotic complex with ionophoric properties. This complex contained members of the macrotetralides class (including monactin, dinactin, trinactin, and tetranactin) and the cyclodepsipeptide valinomycin, all of which were active against B. cinerea. Furthermore, the addition of 2 mM MgSO4 and 1 mM ZnSO4 enhanced macrotetralide and valinomycin production, respectively, in the culture broth. These compounds are considered to be the main active molecules that S. araujoniae produces to control B. cinerea. Their low to moderate toxicity to humans and the environment justifies the application of ASBV-1(T) in biological control programs that aim to mitigate the damage caused by this phytopathogen.

Glycomic profiling identifies key-structural differences in three arabinoxylan fractions from sugarcane culms.

Sugarcane is an important food and bioenergy crop, and although the residual biomass is potentially available for biorefinery and biofuels production the complex plant cell wall matrix requires pretreatment prior to enzymatic hydrolysis. Arabinoxylans require multiple enzymes for xylose backbone and saccharide side-branch hydrolysis to release xylooligosaccharides and pentoses. The effect of arabinoxylan structure on xylooligosaccharide release by combinations of up to five xylanolytic enzymes was studied using three arabinoxylan fractions extracted from sugarcane culms by sodium chlorite, DMSO and alkaline treatments. Reducing sugar release and LC-MS detection with chemometric analysis identified different xylooligosaccharide profiles between extracts following enzyme treatments. The position and degree of side-branch decorations are determinants of enzyme activity and xylooligosaccharide diversity with the alkaline and post­sodium chlorite extracts as the most accessible and most recalcitrant, respectively, indicating acetyl substituents as a major recalcitrance factor. The complex xylooligosaccharide profile with the DMSO extract suggests regions with different levels of branching. Chemometric analysis identified GH10 xylanase hydrolysis products that act as substrates for other enzymes, such as α-glucuronidase. The strategy reported here can identify specific enzyme combinations to overcome barriers for biomass processing such as pretreatment selection, recalcitrance to enzyme digestion and optimization of reducing sugar release.

Exploring pradimicin-IRD antineoplastic mechanisms and related DNA repair pathways.

DNA-targeting agents have a significant clinical use, although toxicity remains an issue that plays against their widespread application. Understanding the mechanism of action and DNA damage response elicited by such compounds might contribute to the improvement of their use in anticancer chemotherapy. In a previous study, our research group characterized a new DNA-targeting agent - pradimicin-IRD. Since DNA-targeting agents and DNA repair are close-related subjects, the present study used in silico-modelling and a transcriptomic approach seeking to characterize the DNA repair pathways activated in HCT 116 cells following pradimicin-IRD treatment. Molecular docking analysis showed pradimicin-IRD as a DNA intercalating agent and a potential inhibitor of DNA-binding proteins. Furthermore, the transcriptomic study highlighted DNA repair functions related to genes modulated by pradimicin-IRD, such as nucleotide excision repair, telomeres maintenance and double-strand break repair. When validating these functions, PCNA protein levels decreased after exposure to pradimicin. Furthermore, molecular docking analysis suggested DNA-pradimicin-PCNA interaction. In addition, hTERT and POLH showed reduced mRNA levels after 6 h of treatment with pradimicin-IRD. Moreover, POLH-deficient cells displayed higher resistance to pradimicin-IRD than POLH-proficient cells and the compound prevented formation of the POLH/DNA complex (molecular docking). Since the modulation of DNA repair genes by pradimicin-IRD is TP53-independent, unlike doxorubicin, dissimilarities between the mechanism of action and the DNA damage response of pradimicin-IRD and doxorubicin open new insights for further studies of pradimicin-IRD as a new antineoplastic compound.

Effects of the antimycobacterial compound 2-phenoxy-1-phenylethanone on rat hepatocytes and formation of metabolites.

CONTEXT: Neolignans are usually dimers formed by oxidative coupling of allyl and propenyl phenols, and the neolignan analogue, 2-phenoxy-1-phenylethanone (LS-2) is a promising antimycobacterial compound showing very weak cytotoxicity in mammalian cells and lack of acute toxicity in murine models. OBJECTIVES: To investigate the mechanism of action of LS-2 in rat hepatocytes by evaluating the activity levels of enzymes related to oxidation status and drug-metabolizing activity. MATERIALS AND METHODS: Hepatocytes were treated with LS-2 from 0.05 up to 1 mM, for 24 and 48 h, and reduced glutathione (GSH), lipid peroxidation and cytochrome P450 enzyme (CYP450) activity were assayed. A homologous series of phenoxazone ethers were used as substrates to measure the enzymatic profile. The biotransformation of LS-2 was studied in hepatocytes by gas chromatography-mass spectrometry (GC-MS) for detection and analysis of possible metabolites. RESULTS: Hepatocytes treated with LS-2 up to 1 mM for 24 or 48 h did not induce the formation of GSH and lipid peroxidation. O-Dealkylation activities of the isoenzymes CYP4501A1, CYP4501A2, CYP4502B1 and CYP4502B2 were also not detected in the hepatocytes treated with LS-2 for 24 or 48 h. DISCUSSION AND CONCLUSION: The results indicate that LS-2 or its two detected metabolites, 2-phenoxy-1-phenylethanol and 2,4-(2-hydroxy-2-phenylethoxy)phenol, are not cytotoxic to rat hepatocytes. These compounds maintain a balance between the production of pro-oxidant agents and their respective antioxidant systems. The data show that enzymes related to oxidation status and drug-metabolizing activities are not involved in the mechanism of action of LS-2.

Biochemical characterization and biological properties of mycelium extracts from Lepista sordida GMA-05 and Trametes hirsuta GMA-01: new mushroom strains isolated in Brazil.

The objective of this study was to evaluate the antioxidant activity, determine and quantify the phenolic compounds and other compounds, and evaluate the cellular cytotoxicity of mycelium extracts of two new Basidiomycete mushrooms strains isolated in Brazil and identified as Lepista sordida GMA-05 and Trametes hirsuta GMA-01. Higher amounts of proteins, free amino acids, total and reducing carbohydrates, and phenolic compounds as chlorogenic, ferulic, caffeic, and gallic acids were found in extracts of T. hirsuta and L. sordida. Protocatechuic acid was found only in aqueous extracts of L. sordida. The TLC of the extracts showed the predominance of glucose and smaller amounts of xylose. It was observed through UPLC-MS higher amounts of phenolic compounds. The aqueous extract from T. hirsuta had the most noteworthy results in the antioxidant assays, especially the ABTS test. The cytotoxic activity was evaluated using two different cell lineages and showed higher toxicity for L. sordida in macrophages J774-A1. However, in Vero cells, it was 12.6-fold less toxic when compared to T. hirsuta. Thus, both mushrooms show potential as functional foods or additives, presenting phenolic content, antioxidant activity, and low cytotoxic activity in the tested cells.