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1.
Sci Rep ; 13(1): 1165, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36670195

RESUMO

Cell-free systems have become valuable investigating tools for metabolic engineering research due to their easy access to metabolism without the interference of the membrane. Therefore, we applied Zymomonas mobilis cell-free system to investigate whether ethanol production is controlled by the genes of the metabolic pathway or is limited by cofactors. Initially, different glucose concentrations were added to the extract to determine the crude extract's capability to produce ethanol. Then, we investigated the genes of the metabolic pathway to find the limiting step in the ethanol production pathway. Next, to identify the bottleneck gene, a systemic approach was applied based on the integration of gene expression data on a cell-free metabolic model. ZMO1696 was determined as the bottleneck gene and an activator for its enzyme was added to the extract to experimentally assess its effect on ethanol production. Then the effect of NAD+ addition at the high concentration of glucose (1 M) was evaluated, which indicates no improvement in efficiency. Finally, the imbalance ratio of ADP/ATP was found as the controlling factor by measuring ATP levels in the extract. Furthermore, sodium gluconate as a carbon source was utilized to investigate the expansion of substrate consumption by the extract. 100% of the maximum theoretical yield was obtained at 0.01 M of sodium gluconate while it cannot be consumed by Z. mobilis. This research demonstrated the challenges and advantages of using Z. mobilis crude extract for overproduction.


Assuntos
Etanol , Zymomonas , Etanol/metabolismo , Fermentação , Zymomonas/genética , Zymomonas/metabolismo , Misturas Complexas/metabolismo , Glucose/metabolismo , Trifosfato de Adenosina/metabolismo
2.
Chemosphere ; 315: 137705, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36592838

RESUMO

Humans are exposed to increasingly complex mixtures of hormone-disrupting chemicals from a variety of sources, yet, traditional research methods only evaluate a small number of chemicals at a time. We aimed to advance novel methods to investigate exposures to complex chemical mixtures. Silicone wristbands were worn by 243 office workers in the USA, UK, China, and India during four work shifts. We analyzed extracts of the wristbands for: 1) 99 known (targeted) chemicals; 2) 1000+ unknown chemical features, tentatively identified through suspect screening; and 3) total hormonal activities towards estrogen (ER), androgen (AR), and thyroid hormone (TR) receptors in human cell assays. We evaluated associations of chemicals with hormonal activities using Bayesian kernel machine regression models, separately for targeted versus suspect chemicals (with detection ≥50%). Every wristband exhibited hormonal activity towards at least one receptor: 99% antagonized TR, 96% antagonized AR, and 58% agonized ER. Compared to men, women were exposed to mixtures that were more estrogenic (180% higher, adjusted for country, age, and skin oil abundance in wristband), anti-androgenic (110% higher), and complex (median 836 detected chemical features versus 780). Adjusted models showed strong associations of jointly increasing chemical concentrations with higher hormonal activities. Several targeted and suspect chemicals were important co-drivers of overall mixture effects, including chemicals used as plasticizers, fragrance, sunscreen, pesticides, and from other or unknown sources. This study highlights the role of personal care products and building microenvironments in hormone-disrupting exposures, and the substantial contribution of chemicals not often identifiable or well-understood to those exposures.


Assuntos
Disruptores Endócrinos , Praguicidas , Masculino , Humanos , Feminino , Silicones , Teorema de Bayes , Estrogênios , Praguicidas/análise , Misturas Complexas , Androgênios , Disruptores Endócrinos/análise
3.
Microb Cell Fact ; 22(1): 5, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36609255

RESUMO

BACKGROUND: New antibiotics are urgently needed in clinical treatment of superdrug-resistant bacteria. Nonribosomal peptides (NRPs) are a major source of antibiotics because they exhibit structural diversity, and unique antibacterial mechanisms and resistance. Analysis of gene clusters of S. agglomeratus 5-1-3 showed that Clusters 3, 6, 12, 21, and 28 were used to synthesize NRPs. Here, we examined secondary metabolites of S. agglomeratus 5-1-3 isolated from soils in the Qinghai-Tibet Plateau, China, for NRPs with antibacterial activity. RESULTS: We isolated a total of 36 Streptomyces strains with distinct colony morphological characteristics from 7 soil samples. We screened 8 Streptomyces strains resistant to methicillin-resistant Staphylococcus aureus (MRSA). We then selected S. agglomeratus 5-1-3 for further study based on results of an antibacterial activity test. Here, we isolated three compounds from S. agglomeratus 5-1-3 and characterized their properties. The crude extract was extracted with ethyl acetate and purified with column chromatography and semipreparative high-performance liquid chromatography (HPLC). We characterized the three compounds using NMR analyses as echinomycin (1), 5,7,4'-trihydroxy-3.3',5'-trimethoxy flavone (2), and 2,6,2', 6'-tetramethoxy-4,4-bis(2,3-epoxy-1-hydroxypropyl)-biphenyl (3). We tested the antibacterial activity of pure compounds from strain 5-1-3 with the Oxford cup method. NRP echinomycin (1) showed excellent anti-MRSA activity with a minimum inhibitory concentration (MIC) of 2.0 µg/mL. Meanwhile, MIC of compound 2 and 3 was 128.0 µg/mL for both. In addition, 203 mg of echinomycin was isolated from 10 L of the crude extract broth of strain 5-1-3. CONCLUSION: In this study, S. agglomeratus 5-1-3 with strong resistance to MRSA was isolated from the soils in the Qinghai-Tibet Plateau. Strain 5-1-3 had a high yield of echinomycin (1) an NRP with a MIC of 2 µg/mL against MRSA. We propose that echinomycin derived from S. agglomeratus 5-1-3 may be a potent antibacterial agent for pharmaceutical use.


Assuntos
Equinomicina , Staphylococcus aureus Resistente à Meticilina , Streptomyces , Tibet , Antibacterianos/química , Streptomyces/química , Testes de Sensibilidade Microbiana , Misturas Complexas , Solo
4.
Environ Int ; 171: 107680, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36502700

RESUMO

Modern society continues to pollute the environment with larger quantities of chemicals that have also become more structurally and functionally diverse. Risk assessment of chemicals can hardly keep up with the sheer numbers that lead to complex mixtures of increasing chemical diversity including new chemicals, substitution products on top of still abundant legacy compounds. Fortunately, over the last years computational tools have helped us to identify and prioritize chemicals of concern. These include toxicokinetic models to predict exposure to chemicals as well as new approach methodologies such as in-vitro bioassays to address toxicodynamic effects. Combined, they allow for a prediction of mixtures and their respective effects and help overcome the lack of data we face for many chemicals. In this study we propose a high-throughput approach using experimental and predicted exposure, toxicokinetic and toxicodynamic data to simulate mixtures, to which a virtual population is exposed to and predict their mixture effects. The general workflow is adaptable for any type of toxicity, but we demonstrated its applicability with a case study on neurotoxicity. If no experimental data for neurotoxicity were available, we used baseline toxicity predictions as a surrogate. Baseline toxicity is the minimal toxicity any chemical has and might underestimate the true contribution to the mixture effect but many neurotoxicants are not by orders of magnitude more potent than baseline toxicity. Therefore, including baseline-toxic effects in mixture simulations yields a more realistic picture than excluding them in mixture simulations. This workflow did not only correctly identify and prioritize known chemicals of concern like benzothiazoles, organochlorine pesticides and plasticizers but we were also able to identify new potential neurotoxicants that we recommend to include in future biomonitoring studies and if found in humans, to also include in neurotoxicity screening.


Assuntos
Monitoramento Biológico , Hidrocarbonetos Clorados , Humanos , Toxicocinética , Medição de Risco , Misturas Complexas
5.
Anal Chem ; 95(2): 1002-1007, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36579454

RESUMO

Diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY) plays a vital role in mixture studies. However, its applications to complex mixture samples are generally limited by spectral congestion along the chemical shift domain caused by extensive J coupling networks and abundant compounds. Herein, we develop the in-phase multidimensional DOSY strategy for complex mixture analyses by simultaneously revealing molecular self-diffusion behaviors and multiplet structures with optimal spectral resolution. As a proof of concept, two pure shift-based three-dimensional (3D) DOSY protocols are proposed to record high-resolution 3D spectroscopic view with separated mixture components and their resolved multiplet coupling structures, thus suitable for analyzing complex mixtures that contain abundant compounds and complicated molecular structures, even under adverse magnetic field conditions. Therefore, this study shows a promising tool for component analyses and multiplet structure studies on practical mixture samples.


Assuntos
Misturas Complexas , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodos , Difusão , Estrutura Molecular
6.
Talanta ; 252: 123824, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36027618

RESUMO

Mpro represents one of the most promising drug targets for SARS-Cov-2, as it plays a crucial role in the maturation of viral polyproteins into functional proteins. HTS methods are currently used to screen Mpro inhibitors, and rely on searching chemical databases and compound libraries, meaning that they only consider previously structurally clarified and isolated molecules. A great advancement in the hit identification strategy would be to set-up an approach aimed at exploring un-deconvoluted mixtures of compounds such as plant extracts. Hence, the aim of the present study is to set-up an analytical platform able to fish-out bioactive molecules from complex natural matrices even where there is no knowledge on the constituents. The proposed approach begins with a metabolomic step aimed at annotating the MW of the matrix constituents. A further metabolomic step is based on identifying those natural electrophilic compounds able to form a Michael adduct with thiols, a peculiar chemical feature of many Mpro inhibitors that covalently bind the catalytic Cys145 in the active site, thus stabilizing the complex. A final step consists of incubating recombinant Mpro with natural extracts and identifying compounds adducted to the residues within the Mpro active site by bottom-up proteomic analysis (nano-LC-HRMS). Data analysis is based on two complementary strategies: (i) a targeted search applied by setting the adducted moieties identified as Michael acceptors of Cys as variable modifications; (ii) an untargeted approach aimed at identifying the whole range of adducted peptides containing Cys145 on the basis of the characteristic b and y fragment ions independent of the adduct. The method was set-up and then successfully tested to fish-out bioactive compounds from the crude extract of Scutellaria baicalensis, a Chinese plant containing the catechol-like flavonoid baicalin and its corresponding aglycone baicalein which are well-established inhibitors of Mpro. Molecular dynamics (MD) simulations were carried out in order to explore the binding mode of baicalin and baicalein, within the SARS-CoV-2 Mpro active site, allowing a better understanding of the role of the nucleophilic residues (i.e. His41, Cys145, His163 and His164) in the protein-ligand recognition process.


Assuntos
SARS-CoV-2 , Animais , Proteases 3C de Coronavírus , Peptídeo Hidrolases , Proteômica , Inibidores de Proteases/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Simulação de Acoplamento Molecular , Misturas Complexas , Antivirais/farmacologia , Antivirais/química
7.
Molecules ; 27(24)2022 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-36558021

RESUMO

The conversion of lignocellulosic biomass by pyrolysis or hydrothermal liquefaction gives access to a wide variety of molecules that can be used as fuel or as building blocks in the chemical industry. For such purposes, it is necessary to obtain their detailed chemical composition to adapt the conversion process, including the upgrading steps. Petroleomics has emerged as an integral approach to cover a missing link in the investigation bio-oils and linked products. It relies on ultra-high-resolution mass spectrometry to attempt to unravel the contribution of many compounds in complex samples by a non-targeted approach. The most recent developments in petroleomics partially alter the discriminating nature of the non-targeted analyses. However, a peak referring to one chemical formula possibly hides a forest of isomeric compounds, which may present a large chemical diversity concerning the nature of the chemical functions. This identification of chemical functions is essential in the context of the upgrading of bio-oils. The latest developments dedicated to this analytical challenge will be reviewed and discussed, particularly by integrating ion source features and incorporating new steps in the analytical workflow. The representativeness of the data obtained by the petroleomic approach is still an important issue.


Assuntos
Misturas Complexas , Óleos , Espectrometria de Massas/métodos , Energia Renovável , Biomassa , Biocombustíveis/análise
8.
Proc Natl Acad Sci U S A ; 119(52): e2211406119, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36534806

RESUMO

Surface-enhanced Raman spectroscopy (SERS) holds exceptional promise as a streamlined chemical detection strategy for biological and environmental contaminants compared with current laboratory methods. Priority pollutants such as polycyclic aromatic hydrocarbons (PAHs), detectable in water and soil worldwide and known to induce multiple adverse health effects upon human exposure, are typically found in multicomponent mixtures. By combining the molecular fingerprinting capabilities of SERS with the signal separation and detection capabilities of machine learning (ML), we examine whether individual PAHs can be identified through an analysis of the SERS spectra of multicomponent PAH mixtures. We have developed an unsupervised ML method we call Characteristic Peak Extraction, a dimensionality reduction algorithm that extracts characteristic SERS peaks based on counts of detected peaks of the mixture. By analyzing the SERS spectra of two-component and four-component PAH mixtures where the concentration ratios of the various components vary, this algorithm is able to extract the spectra of each unknown component in the mixture of unknowns, which is then subsequently identified against a SERS spectral library of PAHs. Combining the molecular fingerprinting capabilities of SERS with the signal separation and detection capabilities of ML, this effort is a step toward the computational demixing of unknown chemical components occurring in complex multicomponent mixtures.


Assuntos
Poluentes Ambientais , Hidrocarbonetos Policíclicos Aromáticos , Humanos , Hidrocarbonetos Policíclicos Aromáticos/análise , Análise Espectral Raman/métodos , Água , Poluentes Ambientais/análise , Misturas Complexas , Aprendizado de Máquina
9.
Drug Des Devel Ther ; 16: 4179-4204, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36514526

RESUMO

Aim: Depression is a chronic recurrent neuropsychiatric disorder associated with inflammation. This study explored the pharmacological activities of Aerva javanica leaves crude extract (Aj.Cr) on lipopolysaccharide (LPS)-induced depressive-like behavior in experimental mice. Methods: Aj.Cr was evaluated for its phenolic and flavonoid contents, bioactive potential, amino acid profiling and enzyme inhibition assays using different analytical techniques followed by in-silico molecular docking was performed. In addition, three ligands identified in HPLC analysis and standard galantamine were docked to acetyl cholinesterase (AchE) enzyme to assess the ligand interaction along with their binding affinities. In in-vivo analysis, mice were given normal saline (10 mL/kg), imipramine (10 mg/kg) and Aj.Cr (100, 300, and 500 mg/kg) orally for 14-consecutive days. On the 14th day, respective treatment was given 30-minutes before intra-peritoneal administration of (0.83 mg/kg) LPS. Open field, forced swim and tail suspension tests were performed 24-hours after LPS injection, followed by a sucrose preference test 48-hours later. Serum corticosterone levels, as well as levels of nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), tumor necrosis factor-alpha (TNF-), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), brain-derived neurotrophic factor (BDNF) and catecholamines were determined in brain tissues. Results: In-vitro results revealed that crude extract of Aj.Cr possesses anti-depressant agents with solid antioxidant potential. In-vivo analysis showed that LPS significantly increased depressive-like behavior followed by alteration in serum and tissue biomarkers as compared to normal control (p < 0.001). While imipramine and Aj.Cr (100, 300, and 500 mg/kg) treated groups significantly (p<0.05) improved the depressive-like behavior and biomarkers when compared to the LPS group. Conclusion: The mitigation of LPS-induced depressive-like behavior by Aj.Cr may be linked to the modulation of oxidative stress, neuro-inflammation and catecholamines due to the presence of potent bioactive compounds exerting anti-depressant effects.


Assuntos
Amaranthaceae , Lipopolissacarídeos , Animais , Camundongos , Antidepressivos/metabolismo , Comportamento Animal , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Catecolaminas/metabolismo , Catecolaminas/farmacologia , Misturas Complexas/farmacologia , Depressão/induzido quimicamente , Depressão/tratamento farmacológico , Glutationa/metabolismo , Imipramina/metabolismo , Imipramina/farmacologia , Inflamação/tratamento farmacológico , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Metanol/farmacologia , Simulação de Acoplamento Molecular , Fator de Necrose Tumoral alfa/metabolismo
10.
Sci Rep ; 12(1): 21153, 2022 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-36477418

RESUMO

An accelerated solvent extraction method was used to recover polyphenol-rich crude extract from rambutan (Nephelium lappaceum L.) peel, a waste product from the canning industry. The influence of extraction parameters including temperature, extraction time and ethanol concentration on extraction yield, total phenolic content, total anthocyanin content, and ABTS antioxidant activity was investigated. A Box-Behnken design and response surface methodology were used to optimize the extraction process. Optimal conditions were obtained at temperature, extraction time, and ethanol concentration of 60 °C, 34 min, and 54 vol%, respectively. These optimum conditions gave 333.01 ± 5.84 mg gallic acid/g, 318.28 ± 5.56 mg cyanidin-3-O-glucoside/g, and 3.05 ± 0.04 mmol Trolox/mg for total phenolic content, total anthocyanins content, and ABTS activity, respectively with extraction yield of 28.68 ± 1.48 wt%. Important active compounds found in the extract were geraniin, ellagic acid, shikimic acid and corilagin. Crude extract concentrations of 50-500 mg/kg retarded linoleic acid oxidation but efficacy was lower than synthetic antioxidants at 200 mg/kg. The current findings indicated that accelerated aqueous ethanol extraction was an effective method for the recovery of a crude extract rich in polyphenols from rambutan peel with the potential to be used as a natural antioxidant.


Assuntos
Polifenóis , Sapindaceae , Antioxidantes , Etanol , Antocianinas , Misturas Complexas
11.
Braz Dent J ; 33(6): 56-64, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36477965

RESUMO

Neem has been used as a medicine due to its beneficial properties such as anti-microbial effects. Neem products for oral application are on the rise. Before recommendation for therapeutic use in human, its effects on cellular activities need to be examined. Therefore, the aim of this study was to test the effects of the ethanolic neem crude extract on dental pulp cells and osteoblasts in terms of cell viability, mineralization, and gene expressions. The ethanolic neem extract derived from dry neem leaves was subjected to chemical identification using GC-MS. Human dental pulp stem cells (hDPSCs) and pre-osteoblasts (MC3T3) were treated with various concentrations of the neem crude extract. Cell viability, mineralization, and gene expressions were investigated by MTT assay, real-time PCR, and alizarin red S assay, respectively. Statistical analysis was performed by one-way ANOVA followed by Dunnett test. GC-MS detected several substance groups such as sesquiterpene. Low to moderate doses of the neem crude extract (4 - 16 µg/ml) did not affect hDPSC and MC3T3 viability, while 62.5 µg/ml of the neem extract decreased MC3T3 viability. High doses of the neem crude extract (250 - 1,000 µg/ml) significantly reduced viability of both cells. The neem crude extract at 1,000 µg/ml also decreased viability of differentiated hDPSC and MC3T3 and their mineralization. Furthermore, 4 µg/ml of neem inhibited viability of differentiated hDPSC. There is no statistical difference in gene expressions related to cell differentiation. In conclusion, the neem crude extract affected cell viability and mineralization. Cell viability altered differently depending on the doses, cell types, and cell stages. The neem crude extract did not affect cell differentiation. Screening of its effect in various aspects should be examined before the application for human use.


Assuntos
Misturas Complexas , Polpa Dentária , Humanos , Camundongos , Animais
12.
Pak J Biol Sci ; 25(10): 922-928, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36404746

RESUMO

<b>Background and Objective:</b> The AL22 strain was isolated from the rhizosphere soil of <i>Alpinia galanga</i> (L.) Willd (Zingiberaceae) and identified as <i>Microbispora</i> sp., by analysing its morphology, chemotaxonomy and 16S rDNA sequence. Previous studies demonstrated the bactericidal effects of its crude extract against <i>Bacillus cereus</i>, <i>Bacillus subtilis</i>, <i>Staphylococcus aureus</i> and methicillin-resistant <i>Staphylococcus aureus</i>. The present study aimed to isolate the major compounds and evaluate their biological properties. <b>Materials and Methods:</b> Silica gel column chromatography and thin-layer chromatography were used for the purification and identification of 3,4-dihydro-lactucin (compound <b>1</b>) and umbelliferone (compound <b>2</b>) by NMR and mass spectrometry, respectively. Antibacterial and anticancer activities were carried out. <b>Results:</b> The bioassay studies illustrated that compound <b>1</b> had antibacterial activity against gram-positive bacteria, with its minimum inhibitory concentration and minimum bactericidal concentration of 16-32 and 64-128 µg mL<sup></sup><sup>1</sup>, respectively. The crude extract and purified compounds showed weak cytotoxic activity on the L929 and Vero cells with IC<sub>50</sub> values >512.00 µg mL<sup></sup><sup>1</sup>. The cytotoxicity of compound <b>1</b> was observed in the MDA-MB-231 and HeLa cells with IC<sub>50</sub> values of 37.62 and 75.34 µg mL<sup></sup><sup>1</sup>, respectively, while its IC<sub>50</sub> value against the HepG2 cells was 456.67 µg mL<sup></sup><sup>1</sup>. <b>Conclusion:</b> These findings showed that compound <b>1</b> of <i>Microbispora</i> sp., AL22 exhibited antibacterial and anticancer activities. Extensive studies on 3,4-dihydro-lactucin could lead to the development of beneficial approaches for managing bacterial infections and cancer.


Assuntos
Alpinia , Staphylococcus aureus Resistente à Meticilina , Humanos , Animais , Chlorocebus aethiops , Endófitos , Células HeLa , Células Vero , Antibacterianos , Misturas Complexas/farmacologia
14.
Oecologia ; 200(3-4): 515-528, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36342526

RESUMO

Climate change is increasing water temperature and intensifying the incidence of cyanobacterial blooms worldwide. However, the combined effects of increased temperature and microcystin concentrations as co-stressors on survival and ecological processes in freshwater species are unclear. Here, using purified MC-LR and crude extract of toxigenic Microcystis aeruginosa, we tested the individual and combined effects of three water temperatures (15, 20, 25 °C) and a range of environmentally relevant concentrations of dissolved microcystin and crude extract (0.01-10 µg·L-1) on survival, growth inhibition, grazing and predation rates in three freshwater species: phytoplankton (Scenedesmus quadricauda), zooplankton (Daphnia pulex), and an invertebrate predator (Ischnura elegans). Purified MC-LR exerted a higher growth inhibitory effect on S. quadricauda compared to crude extract with the same concentration of MC-LR, while neither treatment affected its chlorophyll-a content or survival of D. pulex. Crude extract reduced grazing and survival of D. pulex and I. elegans, respectively. The combined effect of higher temperature and crude extract reduced I. elegans survival by 50%. Increased temperature reduced prey handing time in I. elegans by 49%, suggesting a higher predation rate. However, warming together with higher concentrations of crude extract jointly increased zooplankton grazing and reduced damselfly predation. Taken together, these results suggest crude extract, and not necessarily microcystin, can affect survival and productivity in freshwater species, although these effects may vary unevenly across trophic levels. Our findings highlight the importance of complex ecological mechanisms by which warming can exacerbate toxic effects of cyanobacterial bloom extracts on survival and functions among species in eutrophic freshwaters.


Assuntos
Cianobactérias , Sifonápteros , Animais , Água , Microcistinas/toxicidade , Temperatura , Água Doce , Zooplâncton , Misturas Complexas
15.
Sci Rep ; 12(1): 18614, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36329158

RESUMO

In nature, bacteria can form biofilms, multi-layered structures that adhere microbial populations to solid surfaces by exopolysaccharides, proteins, and nucleic acids. In addition to causing foodborne infections, biofilms can be a major problem in aquaculture. Actinomycetes extracts have previously demonstrated antibiofilm activity against multiple foodborne and fish pathogens, and further characterization of these extracts is needed. In this study, we identified the chemical structures and antibiofilm properties of four extracts and determined the genetic similarity of the isolates to known Streptomyces isolates. We found that several extracts contained multiple antibiofilm compounds, and the antibiofilm activities of all extracts were most stable at pH 6. Furthermore, the antibiofilm inhibition and destruction activities of the isolates were stable at different temperatures. All of crude extracts demonstrated activity against biofilms formed by foodborne and fish pathogens on the surface of stainless-steel coupons as well as polystyrene that commonly used in industrial equipment. Using PCR 16S-rRNA gene and DNA sequencing analysis, the four Actinomycetes isolates were found to be 99% (1 AC), 97% (20 PM), 95% (16 PM), and 85% (18 PM) similar to Streptomyces. Biofilm structure were analyzed using Scanning Electron Microscopy coupled with Energy-Dispersive Spectrometry analysis. Coniine/(S)-2-propylpiperidine was the most active fraction of the crude extracts of the 1 AC, 20 PM, and 16 PM isolates, and piperidine, 2-(tetrahydro-2-furanyl) was most active in the 18 PM isolate.


Assuntos
Actinobacteria , Streptomyces , Animais , Actinomyces , Biofilmes , Misturas Complexas , Antibacterianos/farmacologia
16.
J Chromatogr A ; 1685: 463583, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36323100

RESUMO

Online comprehensive two-dimensional liquid chromatography (LC×LC) is the preferred method currently for the separation of highly complex mixtures of non-volatile compounds. With fully automated commercial instrumentation and software making the method appealing to both researchers and industry, the demand for systems with improved separation capabilities for highly complex mixtures such as those found in natural products or proteomics has increased. In this study we report an approach that enables variable second dimension analysis times based on the use of multiple heart-cutting valves and stop-flow operation to circumvent the requirement for very fast second dimension (2D) analyses in online LC×LC. As application, the HILIC×RP-LC analysis of condensed tannins (proanthocyanidins) in cocoa, grape seed and quebracho extracts is used to demonstrate the performance on the proposed methodology. The method offers increased flexibility compared to conventional online LC×LC separations in that longer 2D gradients can be used to accommodate more complex portions of the chromatogram, while shorter 2D gradients can be used in sections containing fewer peaks, while largely maintaining the benefits of comprehensive separation. We present an evaluation of the performance of the variable gradient time stop-flow HILIC×RP-LC method compared to a comparable, conventional online HILIC×RP-LC system in terms of practical peak capacities using established 2D-LC theory. The improved separation of especially low-level intermediate molecular weight proanthocyanidin oligomers by the former method demonstrates the benefits of the developed approach.


Assuntos
Cacau , Proteômica , Cromatografia Líquida/métodos , Cromatografia Líquida de Alta Pressão/métodos , Misturas Complexas
17.
Exp Oncol ; 44(3): 213-216, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36325705

RESUMO

BACKGROUND: The anticancer effects of phytohormones of cytokinin nature are similar to those of medicinal mushrooms, which are able to synthesize cytokinins in large amounts. AIM: To determine the antiproliferative effect of crude extracts and cytokinin fractions from the mycelial biomass of seven fungi species on colon cancer cells in vitro. MATERIALS AND METHODS: Cytokinin content in mycelial biomass of Ganoderma lucidum, Lentinula edodes, Trametes versicolor, Pleurotus ostreatus, Morchella esculenta, Hericium coralloides, and Fomitopsis officinalis was determined by high performance liquid chromatography mass spectrometry. The antiproliferative effect of the mushroom extracts on the human colon adenocarcinoma Colo 205 cells was assessed by MTT-test. RESULTS: The content of cytokinins (trans-zeatin, zeatin riboside, isopentenyladenosine, isopentenyladenine and zeatin-O-glucoside) was determined in the mycelial biomass of the medicinal macromycetes. Zeatin-type hormones prevailed in all species, though trans-zeatin was the most abundant in H. coralloides and M. esculenta. In P. ostreatus, only zeatin-O-glucoside was detected. The lowest IC50 was found for both the cytokinin fraction (0.21 µg/ml) and the crude extract (0.17 µg/ml) from mycelial biomass of H. coralloides. F. officinalis also demonstrated high antiproliferative effect against Colo 205 cells: IC50 was 0.9 µg/ml for the crude extract and almost twice lower for the cytokinin fraction. In the studied concentration range (0.016-2 µg/ml), the crude extracts from G. lucidum and M. esculenta and the cytokinin fraction from L. edodes did not reach IC50 values. CONCLUSIONS: The present study showed that crude extracts and/or cytokinin fractions of several medicinal Basidiomycetes species are capable to inhibit proliferation of colon cancer cells in vitro. Crude extract cytotoxicity of H. coralloides, P. ostreatus and T. versicolor was higher than that of cytokinin fraction while antiproliferative effect of cytokinin fraction from F. officinalis was higher than that in its crude extract.


Assuntos
Adenocarcinoma , Neoplasias do Colo , Humanos , Zeatina , Biomassa , Trametes , Neoplasias do Colo/tratamento farmacológico , Citocininas/química , Misturas Complexas
18.
Toxicol Ind Health ; 38(11): 733-744, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36271631

RESUMO

Contamination of human habitats with complex mixtures of heavy metals and polycyclic aromatic hydrocarbons (PAHs) is an important environmental and industrial health problem. Hexavalent chromium (Cr(VI)) and benzo(a)pyrene (B[a] P) are typical of the two, respectively. In recent decades, a great deal of research has focused on their carcinogenicity and mechanisms of action. However, few studies have been conducted to evaluate their combined effects on humans and cells, which has important implications for overall understanding of their toxicity and interaction. In the current study, the combined toxic effects of B[a] P and Cr(VI) were studied in human bronchial epithelial cells (16 HBE). We measured the genotoxic activity and epigenetic changes of these two toxicants alone and in combination on these cells and analyzed the difference between their single and combined toxicity. The results showed that B[a]P caused DNA damage in 16HBE cells in a concentration-dependent manner, while the presence of Cr(VI) showed a sharp decrease in DNA damage, and it inhibited the expression of genes related to base excision repair induced by B[a]P. In addition, Cr(VI) also reduced B[a]P-triggered epigenetic changes in 16HBE cells. In conclusion, the combined effect of B[a]P and Cr(VI) on 16HBE cells was less toxic than single B[a]P exposure, indicating that the combined toxicity of the two toxicants is partially antagonistic. Further research is required to explore the mechanism of this antagonism.


Assuntos
Benzo(a)pireno , Cromo , Humanos , Benzo(a)pireno/toxicidade , Cromo/toxicidade , Reparo do DNA , Misturas Complexas
19.
Nutrients ; 14(20)2022 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-36297055

RESUMO

Barley (Hordeum vulgare L.) proteins are taxonomically homologous to wheat proteins and react with sera from patients with baker's asthma. In the current work, the crude extract of barley proteins was divided into six fractions on DEAE-Sepharose. Their immunoreactivity in reacting with sera from patients with a confirmed food allergy varied, and the 15-kDa fraction (B-FrVI) showed the strongest response. In silico analysis confirmed that 15-kDa B-FrVI protein belongs to the trypsin/amylase inhibitor family and to a group of MHC type II allergens. In the next step, the immunogenicity of the B-FrVI was examined in a mouse model. It was shown that, compared to the PBS group, administration of B-FrVI to mice induced almost 2× higher amounts of specific IgG, ~217, and IgA ~29, as early as day 28 after immunization, regardless of the route (intraperitoneal or oral) of antigen administration (p < 0.0001). An ELISpot for B-cell responses confirmed it. Stimulation of mesenteric lymphocytes with pure B-FrVI significantly increased (p < 0.001) the proliferation of lymphocytes from all groups compared to cells growing in media only and stimulated with lyophilized beer. The experiments prove the strong immunogenicity of the 15-kDa B-FrVI protein and provide a basis for future studies of the allergenic nature of this protein.


Assuntos
Hordeum , Camundongos , Animais , Tripsina , Sefarose , Alérgenos , Inibidores da Tripsina , Sistema Imunitário , Amilases , Misturas Complexas , Imunoglobulina A , Imunoglobulina G
20.
Nutrients ; 14(20)2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-36297084

RESUMO

Bioactive peptides are found in foods and dietary supplements and are responsible for health benefits with applications in human and animal medicine. The health benefits include antihypertensive, antimicrobial, antithrombotic, immunomodulatory, opioid, antioxidant, anti-allergic and anti-inflammatory functions. Bioactive peptides can be obtained by microbial action, mainly by the gastrointestinal microbiota from proteins present in food, originating from either vegetable or animal matter or by the action of different gastrointestinal proteases. Proteomics can play an important role in the identification of bioactive peptides. High-resolution mass spectrometry is the principal technique used to detect and identify different types of analytes present in complex mixtures, even when available at low concentrations. Moreover, proteomics may provide the characterization of epitopes to develop new food allergy vaccines and the use of immunomodulating peptides to induce oral tolerance toward offending food allergens or even to prevent allergic sensitization. In addition, food-derived bioactive peptides have been investigated for their anti-inflammatory properties to provide safer alternatives to nonsteroidal anti-inflammatory drugs (NSAIDs). All these bioactive peptides can be a potential source of novel drugs and ingredients in food and pharmaceuticals. The following review is focused on food-derived bioactive peptides with antiallergic and anti-inflammatory properties and summarizes the new insights into the use of proteomics for their identification and quantification.


Assuntos
Antialérgicos , Anti-Infecciosos , Peptídeos , Analgésicos Opioides , Antialérgicos/farmacologia , Anti-Infecciosos/farmacologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Hipertensivos , Antioxidantes/farmacologia , Misturas Complexas , Suplementos Nutricionais , Epitopos , Fibrinolíticos , Hipersensibilidade Alimentar/prevenção & controle , Peptídeo Hidrolases , Peptídeos/farmacologia , Peptídeos/química , Proteômica
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