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.

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.

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.

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.

Targeted analysis of eicosanoids derived from cytochrome P450 pathway by high-resolution multiple-reaction monitoring mass spectrometry.

Cytochrome P450 (CYP450) pathway is one of the critical enzymatic via eicosanoid biosynthesis. Nevertheless, their metabolites are far less explored. This pathway plays a crucial role in converting arachidonic acid to hydroxyeicosatetraenoic (HETEs), epoxyeicosatrienoic (EETs), dihydroxyeicosatetraenoic acids (DiHETEs), and dihydroxyeicosatrienoic acids (DiHETrEs), which mediate several physiological and pathological functions. However, CYP450-derived eicosanoids are structurally complex, making those analyses a challenge in lipidomics studies. Herein, a high-resolution multiple-reaction monitoring (MRMHR ) method has been proposed as a powerful tool for the simultaneous analysis of CYP450-eicosanoids on different biological samples. The developed liquid chromatography (LC)-MRMHR method was partially validated according to the Food and Drug Administration (FDA) criteria, demonstrating adequate specificity, linearity, precision, and accuracy. Besides, several biological samples were analyzed to guarantee the feasibility of the method. The proposed strategy may improve the understanding of CYP450-derived eicosanoids in biological systems, which could be fundamental to reveal new aspects of those in physiologic and pathologic conditions.

Actinobacteria from Antarctica as a source for anticancer discovery.

Although many advances have been achieved to treat aggressive tumours, cancer remains a leading cause of death and a public health problem worldwide. Among the main approaches for the discovery of new bioactive agents, the prospect of microbial secondary metabolites represents an effective source for the development of drug leads. In this study, we investigated the actinobacterial diversity associated with an endemic Antarctic species, Deschampsia antarctica, by integrated culture-dependent and culture-independent methods and acknowledged this niche as a reservoir of bioactive strains for the production of antitumour compounds. The 16S rRNA-based analysis showed the predominance of the Actinomycetales order, a well-known group of bioactive metabolite producers belonging to the Actinobacteria phylum. Cultivation techniques were applied, and 72 psychrotolerant Actinobacteria strains belonging to the genera Actinoplanes, Arthrobacter, Kribbella, Mycobacterium, Nocardia, Pilimelia, Pseudarthrobacter, Rhodococcus, Streptacidiphilus, Streptomyces and Tsukamurella were identified. The secondary metabolites were screened, and 17 isolates were identified as promising antitumour compound producers. However, the bio-guided assay showed a pronounced antiproliferative activity for the crude extracts of Streptomyces sp. CMAA 1527 and Streptomyces sp. CMAA 1653. The TGI and LC50 values revealed the potential of these natural products to control the proliferation of breast (MCF-7), glioblastoma (U251), lung/non-small (NCI-H460) and kidney (786-0) human cancer cell lines. Cinerubin B and actinomycin V were the predominant compounds identified in Streptomyces sp. CMAA 1527 and Streptomyces sp. CMAA 1653, respectively. Our results suggest that the rhizosphere of D. antarctica represents a prominent reservoir of bioactive actinobacteria strains and reveals it as an important environment for potential antitumour agents.

Extraction of carotenoid-rich palm pressed fiber oil using mixtures of hydrocarbons and short chain alcohols.

Solvent extraction is the most efficient method for recovering residual oil from palm pressed fiber (PPFO), which may contain up to eight times the carotenoid content of that found in crude palm oil. The objective of the present study is the use of binary mixtures of hydrocarbons (HC), hexane (Hex), cyclohexane (CHex) or heptane (Hep), and alcohols (ALC), ethanol (Eth) or isopropanol (IPA), in order to promote the highest recovery of a carotenoid-rich PPFO, in which the compositions of the mixtures are defined based on the calculation of solute-solvent distance (Ra) considering ß-carotene as the solute. The extraction experiments were conducted in batch, at 60 ± 2 °C, or in a fixed-bed packed column, at 55 ± 3 °C. Hex and Hep:IPA provided 80% of batch PPFO extraction yield, while in column, the highest yields were obtained with Eth and Hex:IPA (66%). The total carotenoid content obtained was the same independent of the solvent and extraction configuration (from 1790 ± 230 up to 2539 ± 78 mg ß-carotene/kg PPFO). In terms of the carotenoid profile, ß-carotene was mostly extracted by Hex, Hex:Eth stood out in the extraction of α-carotene, and Eth extracted the highest content of lycopene. It is possible to infer that mixtures of HC and ALC with compositions defined based on Hansen Solubility Parameters (HSPs) demonstrated good ability to extract carotenoid-rich PPFO, maintaining their relatively stable fatty acids composition and free acidity, showing that partial substitution of HC by ALC is technically possible.

Tandem mass spectrometry methods to accelerate the identification of phytotoxic metabolites produced by Streptomyces sp. 39 PL.

Natural products isolated by microorganisms are interesting in the search for new compounds with several biological activities. However, low concentration and structural diversity make the isolation a time-consuming step. Tandem mass spectrometry is a well-established technology for the identification and characterization of target microbial natural products due to high sensitivity and selectivity of these experiments. We developed a method employing neutral loss experiments (LC-ESI-MS/MS) to identify luminacins in microbial crude extracts. The luminacins class exhibited conserved fragmentation pattern with loss at 172 Da relative to glycosides fragment and this loss was used in searching for compounds belonging to this class. Therefore, the crude extract produced by Streptomyces sp. 39 PL was analysed and five luminacins were isolated - one is a novel luminacin I at 466 Da.

Plasma Eicosanoid Profile in Plasmodium vivax Malaria: Clinical Analysis and Impacts of Self-Medication.

The participation of cytokines and chemokines in Plasmodium vivax malaria (Pv-malaria) activates the immune response and thus causes the production of several inflammatory mediators. This process is already well-established, but little is known about eicosanoids in malaria physiopathology, especially in regards to inflammation and immunity. Malaria is an acute febrile syndrome similar to any other less important infectious disease and people may self-medicate with any anti-inflammatory drugs in order to cease the recurrent symptoms of the disease. Based on this information, the study describes the eicosanoid profile and its possible influence on the production of cytokines and chemokines in P. vivax infections. In addition, we investigated the influence of self-medication with anti-inflammatory drugs in this immune profile. Twenty-three patients were included in the study, with or without self-medication by anti-inflammatory drugs prior to diagnosis. A total 12 individuals were selected for the control group. Eicosanoid profiles were quantified by HPLC-MS/MS, and cytokines and chemokines by flow cytometry and ELISA. The Pv-malaria infection significantly reduces the production of several lipid mediators, and its action is increased by self-medication. We observed that the eicosanoids we found derive from the lipoxygenase and cyclooxygenase pathways, and present positive and negative correlations with chemokines and cytokines in the follow-up of patients. Our data suggest that self-medication may interfere in the immunological characteristics in P. vivax infection and may modify the follow-up of the disease.

Solid phase microextraction as a powerful alternative for screening of secondary metabolites in actinomycetes.

Actinobacteria are one of the most promising producers of medically and industrially relevant secondary metabolites. However, screening of such compounds in actinobacteria growth demands simple, fast, and efficient extraction procedures that enable detection and precise quantification of biologically active compounds. In this regard, solid phase microextraction (SPME) emerges as an ideal extraction technique for screening of secondary metabolites in bacteria culture due to its non-exhaustive, minimally invasive, and non-destructive nature: its integrated sample preparation workflow; balanced coverage feature; metabolism quenching capabilities; and superior cleanup, as well as its versatility in configuration, which enables automation and high throughput applications. The current work provides a comparison of micro-scale and direct immersion SPME (DI-SPME) for screening of secondary metabolites, describes the optimization of the developed DI-SPME method, and introduces the developed technique for mapping of target secondary metabolites as well as its direct coupling to mass spectrometry for such applications. The optimized DI-SPME method provided higher amounts of extracted ions and intensity signals, yielding superior extraction and desorption efficiency as compared with micro-scale extraction. Studied compounds presented stability on the coating for 24 h at room temperature. The DI-SPME mapping approach revealed that lysolipin I and the lienomycin analog are distributed along the center and edges of the colony, respectively. Direct coupling of SPME to MS provided a similar ions profile as SPME-LC-MS while enabling a significant decrease in analysis time, demonstrating its suitability for such applications. DI-SPME is herein presented as an alternative to micro-scale extraction for screening of secondary metabolites in actinobacteria solid medium, as well as a feasible alternative to DESI-IMS for mapping of biologic radial distribution of secondary metabolites and cell life cycle studies. Lastly, the direct coupling of DI-SPME to MS is presented as a fast, powerful technique for high throughput analysis of secondary metabolites in this medium.

Pradimicin-IRD exhibits antineoplastic effects by inducing DNA damage in colon cancer cells.

DNA-damaging agents are widely used in cancer therapy; however, their use is limited by dose-related toxicities, as well as the development of drug resistance. Drug discovery is essential to overcome these limitations and offer novel therapeutic options. In a previous study by our research group, pradimicin-IRD-a new polycyclic antibiotic produced by the actinobacteria Amycolatopsis sp.-displayed antimicrobial and potential anticancer activities. In the present study, cytotoxic activity was further confirmed in a panel of five colon cancer, including those with mutation in TP53 and KRAS, the most common ones observed in cancer colon patients. While all tested colon cancer cells were sensitive to pradimicin-IRD treatment with IC50 in micromolar range, non-tumor fibroblasts were significantly less sensitive (p < 0.05). The cellular and molecular mechanism of action of pradimicin-IRD was then investigated in the colorectal cancer cell line HCT 116. Pradimicin-IRD presented antitumor effects occurring after at least 6 h of exposure. Pradimicin-IRD induced statistically significant DNA damage (γH2AX and p21), apoptosis (PARP1 and caspase 3 cleavage) and cell cycle arrest (reduced Rb phosphorylation, cyclin A and cyclin B expression) markers. In accordance with these results, pradimicin-IRD increased cell populations in the subG1 and G0/G1 phases of the cell cycle. Additionally, mass spectrometry analysis indicated that pradimicin-IRD interacted with the DNA double strand. In summary, pradimicin-IRD exhibits multiple antineoplastic activities-including DNA damage, cell cycle arrest, reduction of clonal growth and apoptosis-in the HCT 116 cell line. Furthermore, pradimicin-IRD displays a TP53-independent regulation of p21 expression in HCT 116 TP53-/-, HT-29, SW480, and Caco-2 cells. This exploratory study identified novel targets for pradimicin-IRD and provided insights for its potential anticancer activity as a DNA-damaging agent.

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.

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.

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.

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.

Dose-response effect of crude extracts produced by actinobacteria on in vitro rumen fermentation / Padrão dose-resposta de extratos brutos produzidos por actinobactérias sobre a fermentação ruminal in vitro

Actinobacteria have been researched as a source that produces crude extracts, which contain bioactive compounds able to act as antimicrobial agents. The present investigation evaluated the dose-response effect of two crude extracts, obtained at Caatinga rhizosphere (Caat) and Rhizophora mangle (AMC), on in vitro ruminal fermentation by:cumulative gas production, digestibility of dry (IVDMD) and organic matter (IVOMD), and short-chain fatty acids concentration (SCFA). Three multiparous Holstein dairy cows with ruminal fistula were used as the inoculum donors and fed a basal diet consisting of corn silage, soybean meal, urea, ground corn and mineral supplement. Ruminal fluid samples were incubated in glass bottles with 1 g of the dried and milled diet, a buffer solution, and the crude extracts evaluated in four doses (0.3, 0.6, 0.9 and 1.20 mg/10 mL inoculum) in a randomized block design, and the donators were considered as blocks with random effects. Additionally, negative controls were used. The results were expressed as average values based on triplicate analyses. Decreased cumulative gas production was observed according to linear dose response at 24, 48 and 72 h of incubation with the addition of Caat extract. The IVOMD showed a linear decrease at 72 h of incubation with dose Caat inclusion. Furthermore, the inclusion of Caat extract linearly reduced butyric and isovaleric acid concentrations, as well as acetate:propionate ratio. Finally, the Caat inclusion increased the propionic acid concentration in comparison to AMC extract. However, the inclusion of AMC extract did not affect any of the analyzed variables at the used doses. The Caat extract could be used as a modulator of in vitro ruminal fermentation, since it reduced acetate:propionate ratio and cumulative gas production.(AU)

As actinobactérias têm sido pesquisadas como fonte produtoras de extratos brutos que contêm compostos bioativos capazes de atuar como agentes antimicrobianos. O presente trabalho investigou o efeito dose-resposta de dois extratos brutos, AMC e Caat, na fermentação ruminal in vitro por: produção cumulativa de gás, digestibilidade in vitro da matéria seca (IVDMD) e matéria orgânica (IVOMD) e concentração de ácidos graxos de cadeia curta (SCFA). Três vacas leiteiras da raça Holandesa, multíparas e portadoras de fístula ruminal foram utilizadas como doadoras de inóculo ruminal e foram alimentadas com uma dieta basal composta por silagem de milho, farelo de soja, ureia, milho moído e suplemento mineral. As amostras de inóculo ruminal foram incubadas em garrafas de vidro com 1 g da dieta seca e moída, solução tampão e os extratos brutos avaliados em quatro doses (0,3, 0,6, 0,9 e 1,20 mg/10 mL de inóculo) em delineamento em blocos casualizados, sendo as doadoras consideradas os blocos como efeito aleatório. Além disso, foram utilizados controles negativos para a correção da produção de gás. Os resultados foram expressos como valores médios com base em análises triplicadas. A diminuição da produção cumulativa de gás foi observada de acordo com a dose em resposta linear às 24, 48 e 72 h de incubação com a adição de extrato de Caat. A IVOMD mostrou uma diminuição linear com 72 h de incubação com inclusão de Caat. Além disso, a inclusão do Caat reduziu linearmente as concentrações de ácido butírico e isovalérico, bem como a proporção de acetato/propionato. Diferentemente, a inclusão do extrato de AMC não afetou nenhuma das variáveis analisadas nas doses utilizadas. O extrato de Caat pode ser usado como um modulador da fermentação ruminal in vitro, uma vez que reduziu a proporção de acetato/propionato e a produção de gás acumulada. (AU)

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.

Comprehensive high-resolution multiple-reaction monitoring mass spectrometry for targeted eicosanoid assays.

Eicosanoids comprise a class of bioactive lipids derived from a unique group of essential fatty acids that mediate a variety of important physiological functions. Owing to the structural diversity of these lipids, their analysis in biological samples is often a major challenge. Advancements in mass spectrometric have been helpful for the characterization and quantification of these molecular lipid species in complex matrices. However, there are technical limitations to this approach, including low-abundant and/or poorly ionizable lipids. Using high-resolution multiple-reaction monitoring (MRMHR), we were able to develop a targeted bioanalytical method for eicosanoid quantification. For this, we optimized the LC-MS/MS conditions and evaluated several parameters, including linearity, limits of quantification, matrix effects and recovery yields. For validation purposes, we looked at the method's precision and accuracy. A library of high-resolution fragmentation spectra for eicosanoids was developed. Our comprehensive dataset meets benchmark standards for targeted analysis, having been derived using best-practice workflows and rigorous quality assessments. As such, our method has applications for determining complex eicosanoid profiles in the biomedical field.

High-resolution multiple reaction monitoring method for quantification of steroidal hormones in plasma.

Multiple reaction monitoring (MRM) is one of the most powerful modes of analysis in liquid chromatographic tandem mass spectrometry for quantification of low-concentration metabolites in biological samples. The advances in mass spectrometry enabled the development of high-resolution multiple reaction monitoring (MRMHR ) and became suitable for the more specific analysis of target analytes. This is important for lipidomic studies and contributes in the medical and pharmaceutical fields, primarily in investigating alterations in cells or fluids relevant to various diseases. Therefore, this work proposes the development of the MRMHR method for quantification of circulating steroids. We focused on the determination of corticosterone, 11-dehydrocorticosterone (11-DHC), cortisol, cortisone, aldosterone, and progesterone concentration in serum, by using 129sv male mice exposed to chronic unpredictable stress to validate the quantification. The method was conducted according to the ANVISA normative, adopting a coefficient of variation, as well as relative standard deviation and relative error lower than 15% in linearity, intraday and interday precision, and accuracy. For cortisol, corticosterone, and their inert metabolites (cortisone and 11-DHC), the lower limit of quantification was 3.9 ng· mL-1 , while that for progesterone and aldosterone was 7.8 and 15.6 ng· mL-1 , respectively. MRMHR analysis showed that animals submitted to stressors have 4.5 times more corticosterone in their serum than nonstressed mice. However, 11-DHC concentration does not vary significantly in response to stress for these animals. The results indicate that the method can be applied for quantification of steroids in several biological samples, such as human plasma.