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.

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.

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)

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.

Degradation of textile dyes by cyanobacteria.

Dyes are recalcitrant compounds that resist conventional biological treatments. The degradation of three textile dyes (Indigo, RBBR and Sulphur Black), and the dye-containing liquid effluent and solid waste from the Municipal Treatment Station, Americana, São Paulo, Brazil, by the cyanobacteria Anabaena flos-aquae UTCC64, Phormidium autumnale UTEX1580 and Synechococcus sp. PCC7942 was evaluated. The dye degradation efficiency of the cyanobacteria was compared with anaerobic and anaerobic-aerobic systems in terms of discolouration and toxicity evaluations. The discoloration was evaluated by absorption spectroscopy. Toxicity was measured using the organisms Hydra attenuata, the alga Selenastrum capricornutum and lettuce seeds. The three cyanobacteria showed the potential to remediate textile effluent by removing the colour and reducing the toxicity. However, the growth of cyanobacteria on sludge was slow and discoloration was not efficient. The cyanobacteria P. autumnale UTEX1580 was the only strain that completely degraded the indigo dye. An evaluation of the mutagenicity potential was performed by use of the micronucleus assay using Allium sp. No mutagenicity was observed after the treatment. Two metabolites were produced during the degradation, anthranilic acid and isatin, but toxicity did not increase after the treatment. The cyanobacteria showed the ability to degrade the dyes present in a textile effluent; therefore, they can be used in a tertiary treatment of effluents with recalcitrant compounds.

The gut microbiota of insecticide-resistant insects houses insecticide-degrading bacteria: A potential source for biotechnological exploitation.

The exploration of new niches for microorganisms capable of degrading recalcitrant molecules is still required. We hypothesized the gut microbiota associated with insect-resistant lines carry pesticide degrading bacteria, and predicted they carry bacteria selected to degrade pesticides they were resistant to. We isolated and accessed the pesticide-degrading capacity of gut bacteria from the gut of fifth instars of Spodoptera frugiperda strains resistant to lambda-cyhalothrin, deltamethrin, chlorpyrifos ethyl, spinosad and lufenuron, using insecticide-selective media. Sixteen isolates belonging to 10 phylotypes were obtained, from which four were also associated with the susceptible strain. However, growth of gut bacteria associated with larvae from the susceptible strain was not obtained in any of the insecticide-based selective media tested. Growth of isolates was affected by the concentration of insecticides in the media, and all grew well up to 40 µg/ml. The insecticide-degrading capacity of selected isolates was assessed by GC or LC-MS/MS analyses. In conclusion, resistant strains of S. frugiperda are an excellent reservoir of insecticide-degrading bacteria with bioremediation potential. Moreover, gut-associated bacteria are subjected to the selection pressure imposed by insecticides on their hosts and may influence the metabolization of pesticides in insects.

Degradation of textile dyes by cyanobacteria

Abstract Dyes are recalcitrant compounds that resist conventional biological treatments. The degradation of three textile dyes (Indigo, RBBR and Sulphur Black), and the dye-containing liquid effluent and solid waste from the Municipal Treatment Station, Americana, São Paulo, Brazil, by the cyanobacteria Anabaena flos-aquae UTCC64, Phormidium autumnale UTEX1580 and Synechococcus sp. PCC7942 was evaluated. The dye degradation efficiency of the cyanobacteria was compared with anaerobic and anaerobic-aerobic systems in terms of discolouration and toxicity evaluations. The discoloration was evaluated by absorption spectroscopy. Toxicity was measured using the organisms Hydra attenuata, the alga Selenastrum capricornutum and lettuce seeds. The three cyanobacteria showed the potential to remediate textile effluent by removing the colour and reducing the toxicity. However, the growth of cyanobacteria on sludge was slow and discoloration was not efficient. The cyanobacteria P. autumnale UTEX1580 was the only strain that completely degraded the indigo dye. An evaluation of the mutagenicity potential was performed by use of the micronucleus assay using Allium sp. No mutagenicity was observed after the treatment. Two metabolites were produced during the degradation, anthranilic acid and isatin, but toxicity did not increase after the treatment. The cyanobacteria showed the ability to degrade the dyes present in a textile effluent; therefore, they can be used in a tertiary treatment of effluents with recalcitrant compounds.