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Fusarium graminearum not only causes Fusarium head blight (FHB) on wheat but also produces fungal toxins that pose a serious threat to food safety. Biological control is one of the safe and most effective alternative methods. In this study, cyclic lipopeptides (CLPs) produced from Bacillus mojavensis B1302 were extracted and identified by LC-MS/MS. After preparing mesoporous silica nanoparticles-NH2 (MSNsN) and encapsulating CLPs, the characterization analysis showed that the interaction between CLPs and MSNsN enhanced the crystal structure of CLPs-MSNsN. The antimicrobial activity and antioxidant capacity of CLPs-MSNsN stored at 20 °C and 45 °C were decreased more slowly than those of free CLPs with increasing storage time, indicating the enhancement of the antimicrobial and antioxidant stability of CLPs. Moreover, the field control efficacy of long-term stored CLPs-MSNsN only decreased from 78.66% to 63.2%, but the efficacy of free CLPs decreased significantly from 84.34% to 26.01%. The deoxynivalenol (DON) content of wheat grains in the CLPs-MSNsN treatment group was lower than that in the free CLPs treatment group, which showed that long-term stored CLPs-MSNsN reduced the DON content in wheat grains. Further analysis of the action mechanism of CLPs-MSNsN on F. graminearum showed that CLPs-MSNsN could disrupt mycelial morphology, cause cell apoptosis, lead to the leakage of proteins and nucleic acids, and destroy the cell permeability of mycelia. This work puts a novel insight into the antimicrobial and antioxidant stability enhancement of CLPs-MSNsN through encapsulation and provides a potential fungicide to control F. graminearum, reduce toxins and ensure food safety.
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Antioxidantes , Fusarium , Lipopéptidos , Péptidos Cíclicos , Enfermedades de las Plantas , Triticum , Fusarium/efectos de los fármacos , Antioxidantes/farmacología , Antioxidantes/química , Triticum/microbiología , Triticum/química , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/química , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Lipopéptidos/farmacología , Lipopéptidos/química , Nanopartículas/química , Composición de Medicamentos , Antiinfecciosos/farmacología , Antiinfecciosos/químicaRESUMEN
To investigate the alterations of yolk protein during embryonic development in Wanxi white goose, the egg yolk protein composition at days 0, 4, 7, 14, 18, and 25 of incubation (D0, D4, D7, D14, D18, and D25) was analyzed by two-dimensional gel electrophoresis combined with mass spectrometry. A total of 65 spots representing 11 proteins with significant abundance changes were detected. Apolipoprotein B-100, vitellogenin-1, vitellogenin-2-like, riboflavin-binding protein, and serotransferrin mainly participated in nutrient (lipid, riboflavin, and iron ion) transport, and vitellogenin-2-like showed a lower abundance after D14. Ovomucoid-like were involved in endopeptidase inhibitory activity and immunoglobulin binding and exhibited a higher expression after D18, suggesting a potential role in promoting the absorption of immunoglobulin and providing passive immune protection for goose embryos after D18. Furthermore, myosin-9 and actin (ACTB) were involved in the tight junction pathway, potentially contributing to barrier integrity. Serum albumin mainly participated in cytolysis and toxic substance binding. Therefore, the high expression of serum albumin, myosin-9, and ACTB throughout the incubation might protect the developing embryo. Apolipoprotein B-100, vitellogenin-1, vitellogenin-2-like, riboflavin-binding protein, and serotransferrin might play a crucial role in providing nutrition for embryonic development, and VTG-2-like was preferentially degraded/absorbed.
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Gansos , Vitelogeninas , Animales , Vitelogeninas/análisis , Gansos/metabolismo , Apolipoproteína B-100/análisis , Apolipoproteína B-100/metabolismo , Proteómica , Transferrina , Proteínas del Huevo/química , Desarrollo Embrionario , Albúmina Sérica/metabolismo , Inmunoglobulinas/análisis , Miosinas/análisis , Miosinas/metabolismo , Yema de Huevo/químicaRESUMEN
The development of in vitro models recapitulating nanoparticle transport under physiological flow conditions is of great importance for predicting the efficacy of nanoparticle drug carriers. Liposomes are extensively used for drug delivery owing to their biocompatibility and biodegradability and the ability to carry both hydrophilic and hydrophobic compounds. Here, we used a library of liposomes with various dimensions and a microfluidic platform comprising a large array of uniformly sized breast cancer spheroids to explore size-dependent liposome internalization and retention in the spheroids under close-to-physiological interstitial conditions. Such a platform showed promising applications in the preclinical screening of small molecule drugs; however, the capability to deliver nanoparticles in the spheroid interior under close-to-physiological flow conditions was not explored. For the liposomes with diameters in the range of 45-200 nm, we show experimentally and by simulations that in comparison with liposome delivery solely by diffusion, flow significantly enhances liposome internalization in the microgels and mitigates the size-dependent spheroid penetration by the liposomes. The utility of the microfluidic platform was validated by evaluating the efficacy of clinically approved doxorubicin-loaded liposomes (Doxil), which exhibited superior retention in the spheroids under flow conditions, in comparison with free doxorubicin. This MF platform can serve as an in vitro model for screening the efficacy of drugs encapsulated in liposomes and find applications for screening other types of nanoparticle carriers for vaccine delivery, diagnostics, and skincare.
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Doxorrubicina/análogos & derivados , Liposomas , Neoplasias , Humanos , Liposomas/química , Portadores de Fármacos/química , Microfluídica , Esferoides Celulares , Doxorrubicina/farmacología , PolietilenglicolesRESUMEN
The fibrous network of an extracellular matrix (ECM) possesses mechanical properties that convey critical biological functions in cell mechanotransduction. Engineered fibrous hydrogels show promise in emulating key aspects of ECM structure and functions. However, varying hydrogel mechanics without changing its architecture remains a challenge. A composite fibrous hydrogel is developed to vary gel stiffness without affecting its structure by controlling intrafibrillar crosslinking. The hydrogel is formed from aldehyde-modified cellulose nanocrystals and gelatin methacryloyl that provide the capability of intrafibrillar photocrosslinking. By varying the degree of gelatin functionalization with methacryloyl groups and/or photoirradiation time, the hydrogel's elastic modulus is changed by more than an order of magnitude, while preserving the same fiber diameter and pore size. The hydrogel is used to seed primary mouse lung fibroblasts and test the role of ECM stiffness on fibroblast contraction and activation. Increasing hydrogel stiffness by stronger intrafibrillar crosslinking results in enhanced fibroblast activation and increased fibroblast contraction force, yet at a reduced contraction speed. The developed approach enables the fabrication of biomimetic hydrogels with decoupled structural and mechanical properties, facilitating studies of ECM mechanics on tissue development and disease progression.
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Hidrogeles , Mecanotransducción Celular , Animales , Ratones , Hidrogeles/química , Matriz Extracelular , Fibroblastos , Módulo de ElasticidadRESUMEN
Fibrous networks formed by biological polymers such as collagen or fibrin exhibit nonlinear mechanical behavior. They undergo strong stiffening in response to weak shear and elongational strains, but soften under compressional strain, in striking difference with the response to the deformation of flexible-strand networks formed by molecules. The nonlinear properties of fibrous networks are attributed to the mechanical asymmetry of the constituent filaments, for which a stretching modulus is significantly larger than the bending modulus. Studies of the nonlinear mechanical behavior are generally performed on hydrogels formed by biological polymers, which offers limited control over network architecture. Here, we report an engineered covalently cross-linked nanofibrillar hydrogel derived from cellulose nanocrystals and gelatin. The variation in hydrogel composition provided a broad-range change in its shear modulus. The hydrogel exhibited both shear-stiffening and compression-induced softening, in agreement with the predictions of the affine model. The threshold nonlinear stress and strain were universal for the hydrogels with different compositions, which suggested that nonlinear mechanical properties are general for networks formed by rigid filaments. The experimental results were in agreement with an affine model describing deformation of the network formed by rigid filaments. Our results lend insight into the structural features that govern the nonlinear biomechanics of fibrous networks and provide a platform for future studies of the biological impact of nonlinear mechanical properties.
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Colágeno , Hidrogeles , Hidrogeles/química , Colágeno/química , Elasticidad , Polímeros , Citoesqueleto , Estrés MecánicoRESUMEN
Spheroid-on-a-chip platforms are emerging as promising in vitro models that enable screening of the efficacy of biologically active ingredients. Generally, the supply of liquids to spheroids occurs in the steady flow mode with the use of syringe pumps; however, the utilization of tubing and connections, especially for multiplexing and high-throughput screening applications, makes spheroid-on-a-chip platforms labor- and cost-intensive. Gravity-induced flow using rocker platforms overcomes these challenges. Here, a robust gravity-driven technique was developed to culture arrays of cancer cell spheroids and dermal fibroblast spheroids in a high-throughput manner using a rocker platform. The efficiency of the developed rocker-based platform was benchmarked to syringe pumps for generating multicellular spheroids and their use for screening biologically active ingredients. Cell viability, internal spheroid structure as well as the effect of vitamin C on spheroids' protein synthesis was studied. The rocker-based platform not only offers comparable or enhanced performance in terms of cell viability, spheroids formation, and protein production by dermal fibroblast spheroids but also, from a practical perspective, offers a smaller footprint, requires a lower cost, and offers an easier method for handling. These results support the application of rocker-based microfluidic spheroid-on-a-chip platforms for in vitro screening in a high-throughput manner with industrial scaling-up opportunities.
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Many fibrous biological tissues exhibit structural anisotropy due to the alignment of fibers in the extracellular matrix. To study the impact of such anisotropy on cell proliferation, orientation, and mobility, it is important to recapitulate and achieve control over the structure of man-made hydrogel scaffolds for cell culture. Here, we report a chemically crosslinked fibrous hydrogel due to the reaction between aldehyde-modified cellulose nanofibers and gelatin. We explored two ways to induce structural anisotropy in this gel by extruding the hydrogel precursor through two different printheads. The cellulose nanofibers in the hydrogel ink underwent shear-induced alignment during extrusion and retained it in the chemically crosslinked hydrogel. The degree of anisotropy was controlled by the ink composition and extrusion flow rate. The structural anisotropy of the hydrogel extruded through a nozzle affected the orientation of human dermal fibroblasts that were either seeded on the hydrogel surface or encapsulated in the extruded hydrogel. The reported straightforward approach to constructing fibrillar hydrogel scaffolds with structural anisotropy can be used in studies of the biological impact of tissue anisotropy.
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Autophagy is an important conserved homeostatic process related to nutrient and energy deficiency and organelle damage in diverse eukaryotic cells and has been reported to play an important role in cellular responses to pathogens and bacterial replication. The respiratory bacterium Mycoplasma hyopneumoniae has been identified to enter porcine alveolar macrophages, which are considered important immune cells. However, little is known about the role of autophagy in the pathogenesis of M. hyopneumoniae infection of porcine alveolar macrophages. Our experiments demonstrated that M. hyopneumoniae infection enhanced the formation of autophagosomes in porcine alveolar macrophages but prevented the fusion of autophagosomes with lysosomes, thereby blocking autophagic flux and preventing the acidification and destruction of M. hyopneumoniae in low-pH surroundings. In addition, using different autophagy regulators to intervene in the autophagy process, we found that incomplete autophagy promoted the intracellular proliferation of M. hyopneumoniae. We also found that blocking the phosphorylation of JNK and Akt downregulated the autophagy induced by M. hyopneumoniae, but pathways related to two mitogen-activated protein kinases (Erk1/2 and p38) did not affect the process. Collectively, M. hyopneumoniae induced incomplete autophagy in porcine alveolar macrophages through the JNK and Akt signalling pathways; conversely, incomplete autophagy prevented M. hyopneumoniae from entering and degrading lysosomes to realize the proliferation of M. hyopneumoniae in porcine alveolar macrophages. These findings raise the possibility that targeting the autophagic pathway may be effective for the prevention or treatment of M. hyopneumoniae infection.
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Mycoplasma hyopneumoniae , Neumonía Porcina por Mycoplasma , Enfermedades de los Porcinos , Animales , Autofagia , Proliferación Celular , Macrófagos Alveolares , Mycoplasma hyopneumoniae/fisiología , Neumonía Porcina por Mycoplasma/microbiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Porcinos , Enfermedades de los Porcinos/metabolismoRESUMEN
Over the past decade, droplet microfluidics has attracted growing interest in biology, medicine, and engineering. In this feature article, we review the advances in droplet microfluidics, primarily focusing on the research conducted by our group. Starting from the introduction to the mechanisms of microfluidic droplet formation and the strategies for cell encapsulation in droplets, we then focus on droplet transformation into microgels. Furthermore, we review three biomedical applications of droplet microfluidics, that is, 3D cell culture, single-cell analysis, and in vitro organ and disease modeling. We conclude with our perspective on future directions in the development of droplet microfluidics for biomedical applications.
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Microfluídica , Microgeles , Análisis de la Célula IndividualRESUMEN
Osteoarthritis (OA) is a prevalent debilitating age-related joint degenerative disease. It is a leading cause of pain and functional disability in older adults. Unfortunately, there is no cure for OA once the damage is established. Therefore, it promotes an urgent need for early detection and intervention of OA. Theranostics, combining therapy and diagnosis, emerges as a promising approach for OA management. However, OA theranostics is still in its infancy. Three fundamental needs have to be firstly fulfilled: i) a reliable OA model for disease pathogenesis investigation and drug screening, ii) an effective and precise diagnostic platform, and iii) an advanced fabrication approach for drug delivery and therapy. Meanwhile, microfluidics emerges as a versatile technology to address each of the needs and eventually boost the development of OA theranostics. Therefore, this review focuses on the applications of microfluidics, from benchtop to bedside, for OA modelling and drug screening, early diagnosis, and clinical therapy. We first introduce the basic pathophysiology of OA and point out the major unfilled research gaps in current OA management including lack of disease modelling and drug screening platforms, early diagnostic modalities and disease-modifying drugs and delivery approaches. Accordingly, we then summarize the state-of-the-art microfluidics technology for OA management from in vitro modelling and diagnosis to therapy. Given the existing promising results, we further discuss the future development of microfluidic platforms towards clinical translation at the crossroad of engineering and biomedicine.
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Microfluídica , Osteoartritis , Animales , Técnicas Biosensibles , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Microfluídica/tendencias , Osteoartritis/diagnóstico , Osteoartritis/tratamiento farmacológico , Osteoartritis/fisiopatología , Osteoartritis/terapia , Sistemas de Atención de Punto , Medicina de PrecisiónRESUMEN
Organotypic micrometre-size 3D aggregates of skin cells (multicellular spheroids) have emerged as a promising in vitro model that can be utilized as an alternative of animal models to test active ingredients (AIs) of skincare products; however, a reliable dermal spheroid-based microfluidic (MF) model with a goal of in vitro AI screening is yet to be developed. Here, we report a MF platform for the growth of massive arrays of dermal fibroblast spheroids (DFSs) in a biomimetic hydrogel under close-to-physiological flow conditions and with the capability of screening AIs for skincare products. The DFSs formed after two days of on-chip culture and, in a case study, were used in a time-efficient manner for screening the effect of vitamin C on the synthesis of collagen type I and fibronectin. The computational simulation showed that the uptake of vitamin C was dominated by the advection flux. The results of screening the benchmark AI, vitamin C, proved that DFSs can serve as a reliable in vitro dermal model. The proposed DFS-based MF platform offers a high screening capacity for AIs of skincare products, as well as drug discovery and development in dermatology.
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Técnicas de Cultivo de Célula , Microfluídica , Animales , Hidrogeles , Esferoides CelularesRESUMEN
Fibrosis is a pathological condition that leads to excessive deposition of collagen and increased tissue stiffness. Understanding the mechanobiology of fibrotic tissue necessitates the development of effective in vitro models that recapitulate its properties and structure; however, hydrogels that are currently used for this purpose fail to mimic the filamentous structure and mechanical properties of the fibrotic extracellular matrix (ECM). Here, we report a nanofibrillar hydrogel composed of cellulose nanocrystals and gelatin, which addresses this challenge. By altering the composition of the hydrogel, we mimicked the changes in structure, mechanical properties, and chemistry of fibrotic ECM. Furthermore, we decoupled the variations in hydrogel structure, properties, and ligand concentration. We demonstrate that this biocompatible hydrogel supports the three-dimensional culture of cells relevant to fibrotic diseases. This versatile hydrogel can be used for in vitro studies of fibrosis of different tissues, thus enabling the development of novel treatments for fibrotic diseases.
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Matriz Extracelular , Hidrogeles , Celulosa , Colágeno , Fibrosis , HumanosRESUMEN
Interactions between tumor cells and the extracellular matrix (ECM) are an important factor contributing to therapy failure in cancer patients. Current in vitro breast cancer spheroid models examining the role of mechanical properties on spheroid response to chemotherapy are limited by the use of two-dimensional cell culture, as well as simultaneous variation in hydrogel matrix stiffness and other properties, e.g., hydrogel composition, pore size, and cell adhesion ligand density. In addition, currently used hydrogel matrices do not replicate the filamentous ECM architecture in a breast tumor microenvironment. Here, we report a collagen-alginate hydrogel with a filamentous architecture and a 20-fold variation in stiffness, achieved independently of other properties, used for the evaluation of estrogen receptor-positive breast cancer spheroid response to doxorubicin. The variation in hydrogel mechanical properties was achieved by altering the degree of cross-linking of alginate molecules. We show that soft hydrogels promote the growth of larger MCF-7 tumor spheroids with a lower fraction of proliferating cells and enhance spheroid resistance to doxorubicin. Notably, the stiffness-dependent chemotherapeutic response of the spheroids was temporally mediated: it became apparent at sufficiently long cell culture times, when the matrix stiffness has influenced the spheroid growth. These findings highlight the significance of decoupling matrix stiffness from other characteristics in studies of chemotherapeutic resistance of tumor spheroids and in development of drug screening platforms.
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Neoplasias de la Mama , Esferoides Celulares , Neoplasias de la Mama/tratamiento farmacológico , Técnicas de Cultivo de Célula , Matriz Extracelular , Femenino , Humanos , Hidrogeles , Microambiente TumoralRESUMEN
Under internal or external insults such as aging and oxidative stresses, cells are induced into a senescent state and stop cellular division permanently. As senescent cells (SnCs) accumulate, the regeneration capacity of biological tissue would be compromised, which has been found to be associated with a plethora of age-related disorders. Therefore, isolating SnCs becomes necessary. To address the lack of effective surface markers for SnCs isolation, a label-free microfluidic device was proposed in this paper, in which a spiral microchannel was deployed to isolate SnCs based on their size differences. We adopted a well-received cellular senescence model by exerting excessive oxidative stress to murine mesenchymal stem cells. This model was then validated through a series of SnCs characterizations including size measurement, p16INK4a expression level, senescence-associated beta-galactosidase, and doubling time. The senescence chip demonstrated an efficiency of 75% and viability over 85% at a flow rate of 5 ml/min. The average cell size from the inner outlet was 5 µm larger than that from the outer outlet. The isolated cells had a sixfold higher p16INK4a expression level. Overall, the chip had an area under curve of 0.719 in the receiver operating characteristic analysis, showing decent performance in sorting SnCs. By having the ability to perform size-based sorting at a high flow rate, such a microfluidic device can provide high-throughput and label-free isolation of SnCs. To further improve the isolation performance, the device can be modified to introduce additional physical biomarkers of SnCs such as stiffness. This device poses a good potential in purification for cytotherapy or estimation of biological age.
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Isolating active mesenchymal stem cells from a heterogeneous population is an essential step that determines the efficacy of stem cell therapy such as for osteoarthritis. Nowadays, the gold standard of cell sorting, fluorescence-activated cell sorting, relies on labelling surface markers via antibody-antigen reaction. However, sorting stem cells with high stemness usually requires the labelling of multiple biomarkers. Moreover, the labelling process is costly, and the high operating pressure is harmful to cell functionality and viability. Although label-free cell sorting, based on physical characteristics, has gained increasing interest in the past decades, it has not shown the ability to eliminate stem cells with low stemness. Cell motility, as a novel sorting marker, is hence proposed for label-free sorting active stem cells. Accumulating evidence has demonstrated the feasibility in manipulating directional cell migration through patterning the biophysical, biochemical or both gradients of the extracellular matrix. However, applying those findings to label-free cell sorting has not been well discussed and studied. This review thus first provides a brief overview about the effect of biophysical and biochemical gradients of the extracellular matrix on cell migration. State-of-the-art fabrication techniques for generating such gradients of hydrogels are then introduced. Among current research, the authors suggest that hydrogels with dual-gradients of biochemistry and biophysics are potential tools for accurate label-free cell sorting with satisfactory selectivity and efficiency. TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The reviewed label-free cell sorting approaches enable us to isolate active cell for cytotherapy. The proposed system can be further modified for single-cell analysis and drug screening.
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One new diphenyl ether, diorcinol K (1), along with three known compounds, diorcinols D (2), F (3) and I (4) were isolated from the fermentation media of a marine-derived fungus Aspergillus sp. CUGB-F046 which was isolated from a sediment sample collected from the Bohai Sea, China. Their structures were elucidated by detailed spectroscopic methods. Compounds 1, 2 and 4 displayed significant antibacterial activities against Staphylococcus aureus and methicillin-resistant S. aureus with MIC values of 3.125, 6.25 and 6.25 µg/mL, respectively.
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Antibacterianos/farmacología , Aspergillus/química , Éteres Fenílicos/química , Antibacterianos/química , Organismos Acuáticos , Aspergillus/aislamiento & purificación , Aspergillus/metabolismo , China , Evaluación Preclínica de Medicamentos/métodos , Sedimentos Geológicos/microbiología , Espectroscopía de Resonancia Magnética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Fenoles/química , Fenoles/farmacología , Éteres Fenílicos/farmacología , Metabolismo Secundario , Staphylococcus aureus/efectos de los fármacosRESUMEN
Trichopeptides A (1) and B (2), new linear tetrapeptide and tripeptide, respectively, and three new diketopiperazines trichocyclodipeptides A-C (3-5) were isolated from the fermentation of the ascomycete fungus Stagonospora trichophoricola, a fungus isolated from the soil sample surrounding the fruiting body of Ophiocordyceps sinensis in Maqin Country, Qinghai Province, People's Republic of China. Their structures were primarily elucidated by interpretation of NMR and MS experiments. The absolute configurations of 1-5 were assigned through Marfey's method on their acid hydrolyzates. Compound 3 showed antifungal activity against Candida albicans with the IC50 and MIC values of 22 and 90 µg ml-1, respectively.
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Antibacterianos/aislamiento & purificación , Antibióticos Antineoplásicos/aislamiento & purificación , Antifúngicos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Ascomicetos/metabolismo , Descubrimiento de Drogas , Células A549 , Adenocarcinoma Bronquioloalveolar/tratamiento farmacológico , Secuencia de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacología , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Antifúngicos/química , Antifúngicos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/crecimiento & desarrollo , Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Supervivencia Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Fermentación , Humanos , Concentración 50 Inhibidora , Células K562 , Leucemia Eritroblástica Aguda/tratamiento farmacológico , Pruebas de Sensibilidad Microbiana , Oligopéptidos/química , Oligopéptidos/aislamiento & purificación , Oligopéptidos/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , EstereoisomerismoRESUMEN
Chemical investigation of a Chinese collection of a marine-derived fungus, Fusarium sp., led to the characterization of beauvericin.K, a new analogue of beauvericin. The structure of the new compound was elucidated by detailed spectroscopic analysis, including ID and 2D NMR methods, and HRMS. Beauvericin K showed significant activity against the yeast Candida albicans with an IC50 value of 6.25 üg/mL..