RESUMO
Small extracellular vesicles (sEVs) are proven to hold great promise for diverse therapeutic and diagnostic applications. However, batch preparation of sEVs with high purity and bioactivity is a prerequisite for their clinical translations. Herein, we present an electric field assisted tangential flow filtration system (E-TFF), which integrates size-based filtration with electrophoretic migration-based separation to synergistically achieve the isolation of high-quality sEVs from cell culture medium. Compared with the gold-standard ultracentrifugation (UC) method, E-TFF not only improved the purity of sEVs by 1.4 times but also increased the yield of sEVs by 15.8 times. Additionally, the entire isolation process of E-TFF was completed within 1 h, about one-fourth of the time taken by UC. Furthermore, the biological activity of sEVs isolated by E-TFF was verified by co-incubation of sEVs derived from human umbilical cord mesenchymal stem cells (hUCMSCs) with HT22 mouse hippocampal neuronal cells exposed to amyloid-ß (Aß). The results demonstrated that the sEVs isolated by E-TFF exhibited a significant neuroprotective effect. Overall, the E-TFF platform provides a promising and robust strategy for batch preparation of high-quality sEVs, opening up a broad range of opportunities for cell-free therapy and precision medicine.
Assuntos
Vesículas Extracelulares , Filtração , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Animais , Camundongos , Humanos , Filtração/instrumentação , Filtração/métodos , Células-Tronco Mesenquimais/citologia , Meios de Cultura/química , Peptídeos beta-Amiloides/metabolismo , Linhagem CelularRESUMO
A Gram-stain-positive, aerobic, non-mobile and spherical strain, designated ZS9-10T, belonging to the genus Deinococcus was isolated from soil sampled at the Chinese Zhong Shan Station, Antarctica. Growth was observed in the presence of 0-4â% (w/v) NaCl, at pH 7.0-8.0 and at 4-25â°C. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZS9-10T formed a lineage in the genus Deinococcus. It exhibited highest sequence similarity (97.4â%) to Deinococcus marmoris DSM 12784T. The major phospholipids of ZS9-10T were unidentified phosphoglycolipid, unidentified glycolipids and unidentified lipids. The major fatty acids were summed feature 3 (C16â:â1 ω7c and/or C16â:â1 ω6c), C16â:â0 and C16â:â1 ω7c. MK-8 was the predominant respiratory quinone. The digital DNA-DNA hybridization and average nucleotide identity values between strain ZS9-10T and its close relative D. marmoris DSM 12784T were 27.4 and 83.9â%, respectively. Based on phenotypic, phylogenetic and genotypic data, a novel species, named Deinococcus arenicola sp. nov., is proposed. The type strain iis ZS9-10T (=CCTCC AB 2019392T=KCTC43192T).
Assuntos
Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano , Deinococcus , Ácidos Graxos , Hibridização de Ácido Nucleico , Fosfolipídeos , Filogenia , RNA Ribossômico 16S , Análise de Sequência de DNA , Microbiologia do Solo , Regiões Antárticas , RNA Ribossômico 16S/genética , Deinococcus/genética , Deinococcus/classificação , Deinococcus/isolamento & purificação , Ácidos Graxos/análise , Ácidos Graxos/química , DNA Bacteriano/genética , Fosfolipídeos/análise , Fosfolipídeos/química , Vitamina K 2/análogos & derivados , Vitamina K 2/análise , Vitamina K 2/química , Areia/microbiologiaRESUMO
Polymer micelles now have promising applications in the treatment of cancer, increasing the water solubility and bioavailability of drugs. Previous studies have found that micelles of niclosamide have good anti-liver cancer effect. In view of the poor water solubility of niclosamide (NIC), we decided to prepare niclosamide micelles. However, its therapeutic mechanism is not clear, so this paper conducted a preliminary study on its vitro anti-tumour mechanism and metabonomics to find out its impact. It was found that the drug-loaded micelles (PEG2K -FIbu/NIC) had an inhibitory effect on HepG2 cells. Moreover, it can promote apoptosis of HepG2 cells and block S and G2/M phase of cell cycle. The plasma and liver metabolomics of mice in normal group, model group and administration group were studied by UPLC-MS and 1 H-NMR. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to process the data and find the relevant metabolites. metaboanalyst 5.0 was used to integrate the relevant metabolites to find the main related metabolic pathways. Thus, the anti-tumour mechanism of PEG2K -FIbu/NIC was analysed. Fifty-one biomarkers were detected in plasma, and 43 biomarkers were detected in liver. After comprehensive biomarker and metabolic pathway analysis, it was found that PEG2K -FIbu/NIC micelles could affect the changes of many metabolites, mainly affecting amino acid metabolism. This article is an in-depth study based on the published Preparation and pharmacodynamics of niclosamide micelles (DOI: 10.1016/j.jddst.2021.103088).
Assuntos
Micelas , Niclosamida , Animais , Biomarcadores , Cromatografia Líquida , Metabolômica , Camundongos , Niclosamida/farmacologia , Espectrometria de Massas em Tandem , ÁguaRESUMO
Exosomes are extracellular vesicles with unique size distribution derived from the parent cells. They are involved in intercellular communication and transport, and are also biomarkers for early diagnosis and prognosis of disease. However, the isolation and characterization of exosomes face the challenges of large sample requirements and low enrichment efficiency of traditional methods. Herein, a simple method is proposed for the preparation of an artificial antibody that has a synergistic effect by featuring nanocavities obtained by dull template imprinting and molecular recognition conferred by electrostatic interaction. With this artificial antibody, highly efficient capture and proteome analysis of exosomes from urine and cell culture media are achieved: for urine, the abundance of Top100 exosomal proteins enriched by this artificial antibody increased from 1.85% to 9.66%. For the cell culture medium, the abundance of the Top100 proteins enriched by this artificial antibody was 28.4%. Moreover, this artificial antibody has a comparable effect to the commercial precipitation-based method in capturing exosomes and has advantages in removing contaminants such as prothymosin alpha (PTMA), demonstrating the superior selectivity of the artificial antibody. Overall, this artificial antibody holds promise to capture exosomes from biofluids and is compatible with subsequent proteome analysis.
Assuntos
Exossomos , Anticorpos , Biomarcadores/metabolismo , Exossomos/metabolismo , Proteoma/metabolismo , TecnologiaRESUMO
Protein N-phosphorylation plays a critical role in central metabolism and two/multicomponent signaling of prokaryotes. However, the current enrichment methods for O-phosphopeptides are not preferred for N-phosphopeptides due to the intrinsic lability of P-N bond under acidic conditions. Therefore, the effective N-phosphoproteome analysis remains challenging. Herein, bis(zinc(II)-dipicolylamine)-functionalized sub-2 µm core-shell silica microspheres (SiO2@DpaZn) are tailored for rapid and effective N-phosphopeptides enrichment. Due to the coordination of phosphate groups to Zn(II), N-phosphopeptides can be effectively captured under neutral conditions. Moreover, the method is successfully applied to an E.coli and HeLa N-phosphoproteome study. These results further broaden the range of methods for the discovery of N-phosphoproteins with significant biological functions.
Assuntos
Microesferas , Compostos Organometálicos/química , Fosfoproteínas/metabolismo , Picolinas/química , Proteoma/análise , Proteômica/métodos , Dióxido de Silício/química , Proteínas de Escherichia coli/análise , Células HeLa , Células Hep G2 , Humanos , Proteínas de Neoplasias/análise , Tamanho da Partícula , Fosfopeptídeos/análise , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The selective and highly efficient capture of circulating tumor cells (CTCs) from blood and their subsequent release without damage are very important for the early diagnosis of tumors and for understanding the mechanism of metastasis. Herein, a universal strategy is proposed for the fabrication of an antibody-free hydrogel that has a synergistic effect by featuring microinterfaces obtained by cell imprinting and molecular recognition conferred by boronate affinity. With this artificial antibody, highly efficient capture of human hepatocarcinoma SMMC-7721 cells is achieved: as many as 90.3 ± 1.4% (n = 3) cells are captured when 1 × 105 SMMC-7721 cells are incubated on a 4.5 cm2 hydrogel, and 99% of these captured cells are subsequently released without any loss of proliferation ability. In the presence of 1000 times as many nontarget cells, namely, leukaemia Jurkat cells, the SMMC-7721 cells can be captured with an enrichment factor as high as 13.5 ± 3.2 (n = 3), demonstrating the superior selectivity of the artificial antibody for the capture of the targeted CTCs. Most importantly, the SMMC-7721 cells can be successfully captured even when spiked into whole blood, indicating the great promise of this approach for the further molecular characterization of CTCs.
Assuntos
Separação Celular , Técnicas e Procedimentos Diagnósticos , Hidrogéis , Neoplasias , Células Neoplásicas Circulantes , Contagem de Células , Linhagem Celular Tumoral , Separação Celular/métodos , Humanos , Hidrogéis/química , Neoplasias/diagnósticoRESUMO
Epitope imprinting is a promising tool to generate antibody-like specific recognition sites. Recently, because of the ease of obtaining templates, the flexibility in selecting monomers, their resistance to harsh environments, and the high specificity toward targets, epitope-imprinted materials have attracted much attention in various fields, such as bioanalysis, clinical therapy, and pharmacy. Here, the discussion is focused on the current representative epitope imprinting technologies, including epitope bulk imprinting and epitope surface imprinting. Moreover, the application of epitope-imprinted materials to the recognition of peptides, proteins, and cells is reviewed. Finally, the remaining challenges arising from the intrinsic properties of epitope imprinting are discussed, and future development in the field is prospected.
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Anticorpos/imunologia , Materiais Biomiméticos , Epitopos/imunologia , Especificidade de Anticorpos , HumanosRESUMO
Since circulating tumor cells (CTCs) in the blood carry the genetic and phenotypic information of tumor tissues, they are regarded as important markers for liquid biopsy, especially for the early diagnosis of cancer, determination of treatment options and evaluation of prognosis. However, the capture and detection of CTCs is challenging because of their extremely low concentration in blood. In this review, recent advances in capture methods of CTCs based on their biophysical properties, affinity, and artificial antibodies are summarized. The advantages and drawbacks of these methods are discussed from the aspects of capture efficiency, capture purity and maintenance of activity after release. Future trends in CTCs capture are also discussed.
Assuntos
Separação Celular/métodos , Células Neoplásicas Circulantes , HumanosRESUMO
Isolation of circulating tumor cells (CTCs) from blood holds great potential to diagnose cancers and discover therapeutic targets. Herein, we reported a novel kind of artificial antibody, the cell-imprinted hydrogel with site-directed modification of aptamers (APT-CIH) to achieve the specific capture of CTCs. Cell-imprinted sites not only could be used to recognize target cells but also could be used as efficient scaffolds for assembling aptamers to enhance the capture efficiency and selectivity. Due to the synergistic effect of conformation recognition and multivalent interaction between the aptamers and target cells, APT-CIH showed high capture efficiency and selectivity to SMMC-7721 cells. In the coexistence of the 1000 times leukemia Jurkat cells, the enrichment factor of APT-CIH could reach as high as 21.6 ± 3.1 toward target cells, while that relied only on cell imprinting or aptamer affinity was 8.1 ± 5.0 or 10.1 ± 1.3, respectively. Furthermore, the capture efficiency could reach 58.2% ± 10.9% with 1000 SMMC-7721 cells spiked in 1 mL of blood. Moreover, 92% of the captured cells could be released, beneficial to carry out further biological and clinical study of CTCs. These results demonstrated that APT-CIH might have great potential in CTCs analysis.
Assuntos
Aptâmeros de Nucleotídeos/química , Separação Celular/métodos , Hidrogéis/química , Células Neoplásicas Circulantes , Resinas Acrílicas/síntese química , Resinas Acrílicas/química , Sequência de Bases , Contagem de Células/métodos , Linhagem Celular Tumoral , Humanos , Hidrogéis/síntese química , Células Neoplásicas Circulantes/químicaRESUMO
Tailor-made materials for the purification of proteins with His-tag was designed through synergizing the selectivity of surface sieving and metal ion affinity. By excluding impurity proteins out of the surface polymer network, such materials could purify His-tagged proteins from the crude cell lysis with purity up to 90%, improved by 14% compared to that obtained by the commercial metal chelating affinity materials. This study might promote the His-tagged protein purification to a new level.
Assuntos
Cromatografia de Afinidade/métodos , Histidina/química , Nanopartículas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Quelantes/química , Proteínas de Fluorescência Verde/química , Íons/química , Cinética , Imãs/química , Peso Molecular , Ligação Proteica , Dióxido de Silício/químicaRESUMO
Circulating tumor cells hold the key to predicting the prognosis and discovering the therapeutic targets. Herein, we proposed a strategy to develop an aptamer-immobilized open tubular capillary column by which SMMC-7721 human hepatoma cells (SMMC-7721 cells) could be captured with an over 70% of capture efficiency and a 3.0 ± 0.2 of enrichment factor. Owing to the compatibility of the column, the captured cells by the column could be analyzed by LC-MS from protein level and 5 unique proteins of SMMC-7721 cells were identified which could be used as markers to identify SMMC-7721 cells when Jurkat T-leukemia cells (Jurkat cells) were employed as interfering cells. As the key component, the aptamer-immobilized column had the potential to be integrated into the platform for separating, enriching and characterizing rare cells simultaneously.
Assuntos
Aptâmeros de Nucleotídeos/química , Separação Celular/métodos , Células Neoplásicas Circulantes/química , Proteoma/análise , Linhagem Celular Tumoral , Separação Celular/instrumentação , Cromatografia Líquida/métodos , Desenho de Equipamento , Humanos , Células Neoplásicas Circulantes/patologia , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
The complications of hemodialysis accompanied the hemodialysis and threaten the patients' life. Besides the loss of nutrient substance, such as amino acid and vitamin, we found new clues that the adsorbed proteins on common-used polysulfone-based dialysis membrane might be the reason according to the qualitative proteomic study by ionic liquid assisted sample preparation method. Our results indicated that the adsorbed proteins on the membrane were related with complement activation, blood coagulation, and leukocyte-related biological process. The quantitative proteome further demonstrated some significant changes of signal proteins in the post-dialysis plasma after the hemodialysis, such as beta-2-microglobulin and platelet factor-4, which would further verify these new clues.
RESUMO
To achieve the simultaneous capture of various target proteins, the multiepitope templates imprinted particles were developed by phase inversion-based poly(ether sulfone) (PES) self-assembly. Herein, with the top three high-abundance proteins in the human plasma, serum albumin, immunoglobulin G, and transferrin, as the target proteins, their N-terminal peptides were synthesized as the epitope templates. After the preorganization of three epitopes and PES in dimethylacetamide, the multiepitope templates imprinted particles were formed in water through self-assembly, by which the simultaneous recognition of three target proteins in human plasma was achieved with high selectivity. Furthermore, the binding kinetics study proved that the adsorption mechanism in this imprinting system toward three epitope templates was the same as that on the single-epitope imprinting polymer. These results demonstrate that our proposed multiepitope templates imprinting strategy might open a new era of artificial antibodies to achieve the recognition of various targets simultaneously.
Assuntos
Imunoglobulina G/sangue , Impressão Molecular , Albumina Sérica Humana/análise , Transferrina/análise , Epitopos , Humanos , Cinética , Tamanho da Partícula , Polímeros/síntese química , Polímeros/química , Sulfonas/síntese química , Sulfonas/químicaRESUMO
Among various artificial antibodies, epitope imprinted polymer has been paid increasingly attention. To modulate the "adsorption and release" behavior by environment stimuli, N-isopropylacrylamide, was adopted to fabricate the thermoresponsive epitope imprinted sites. The prepared imprinted materials could adsorb 46.6 mg/g of target protein with the imprinting factor of 4.0. The template utilization efficiency could reach as high as 8.21%. More importantly, in the real sample, the materials could controllably capture the target protein from the human plasma at 45 °C and release it at 4 °C, which demonstrated the "on-demand" application potentials of such materials in the biomolecule recognition field.
Assuntos
Proteínas Sanguíneas/metabolismo , Epitopos/metabolismo , Impressão Molecular , Nanopartículas/química , Resinas Acrílicas/química , Sequência de Aminoácidos , Proteínas Sanguíneas/química , Eletroforese em Gel de Poliacrilamida , Epitopos/química , Humanos , Ligação de Hidrogênio , Tamanho da Partícula , Albumina Sérica/química , Dióxido de Silício/química , TemperaturaRESUMO
A molecular simulation method was introduced to compute the phenol-monomer pre-assembled system of a molecularly imprinted polymer. The interaction type and intensity between phenol and monomer were evaluated by combining binding energy and charge transfer with complex conformation. The simulation results indicate that interaction energies are simultaneously affected by the type of monomer and the ratio between phenol and monomers. At the same time, we considered that by increasing the amount of functional monomer is not always better for preparing molecularly imprinter polymers. In this study, three kinds of novel magnetic phenol-imprinted polymers with favorable specific adsorption effects were prepared by the surface imprinting technique combined with atom transfer radical polymerization. Various measures were selected to characterize the structure and morphology to obtain the optimal polymer. The characterization results show that the optimal polymer has suitable features for further adsorption process. A series of static adsorption experiments were conducted to analyze its adsorption performance, which follows the Elovich model from the kinetic analysis and the Sips equation from the isothermal analysis. To further verify the reliability and accuracy of the simulation results, the effects of different monomers on the adsorption selectivity were also determined. They display higher selectivity towards phenol than 4-nitrophenol.The results from the simulation of the pre-assembled complexes are in reasonable agreement with those from the experiment.
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Recombinant protein technology occupies an important position in fields including biopharmaceutics, proteomics, structural and functional biology. However, the purification of His-tagged protein, the majority portion of recombinant protein, is seriously hindered by impurities. These impurities, including host proteins with inherent cysteine and histidine-rich regions or metal centers, are usually beyond the purification ability of commonly used IMAC materials. To remove this barrier, a novel purification material was developed through enhancing the selectivity of IMAC by means of surface epitope imprinting using His-tag, the common terminal of His-tagged protein, as the template. Characterizations including TEM, thermogravimetric analysis, X-ray photoelectron spectroscopy, measurement of DLS size and zeta potential were carried out to prove the fabrication of the imprinted shell. Results exhibited a high imprinting factor of 7.1. Besides, the adsorption kinetics were not affected by the surface imprinted shell and could reach adsorption equilibrium within 15 min. Compared with the substrate IMAC, the novel epitope imprinting enhanced IMAC (EI-IMAC) showed an obvious improvement (5% increase of purity) in the selectivity of His-tagged recombinant protein from crude cell lysis.
RESUMO
Correction for 'Epitope imprinting enhanced IMAC (EI-IMAC) for highly selective purification of His-tagged protein' by Senwu Li et al., J. Mater. Chem. B, 2016, 4, 1960-1967.