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1.
J Colloid Interface Sci ; 607(Pt 1): 698-710, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34530190

RESUMO

Anisotropic nanoparticles offer considerable promise for applications but also present significant challenges in terms of their characterization. Recent developments in the electroless deposition of silver patches directly onto colloidal silica particles have opened up a simple and scalable synthesis method for patchy particles with tunable optical properties. Due to the reliance on patch nucleation and growth, however, the resulting coatings are distributed in coverage and thickness and some core particles remain uncoated. To support process optimization, new methods are required to rapidly determine patch yield, thickness and coverage. Here we present a novel approach based on multiwavelength analytical ultracentrifugation (MWL-AUC) which permits simultaneous hydrodynamic and spectroscopic characterization. The patchy particle colloids are produced in a continuous flow mixing process that makes use of a KM-type micromixer. By varying the process flow rate or metal precursor concentration we show how the silver to silica mass ratio distribution derived from the AUC-measured sedimentation coefficient distribution can be influenced. Moreover, through reasoned assumptions we arrive at an estimation of the patch yield that is close to that determined by arduous analysis of scanning electron microscopy (SEM) images. Finally, combining MWL-AUC, electrodynamic simulations and SEM image analysis we establish a procedure to estimate the patch thickness and coverage.


Assuntos
Nanopartículas , Prata , Coloides , Dióxido de Silício , Ultracentrifugação
2.
Int J Nanomedicine ; 16: 6681-6692, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34616151

RESUMO

Purpose: Extracellular vesicles (EVs) are membrane-encapsulated nanoparticles that function as carriers and play a role in intercellular communication. There are a large number of EVs in the blood and serve as an indicator of pathophysiological conditions. Studies on the basics and application of EVs are hampered by the limitations of current protocols to isolate EVs from blood. However, current isolation methods are difficult to achieve a balance between yield and purity. Methods: Firstly, we use Sepharose-4B to build a self-made size exclusion chromatography (SEC) column and perform separation and characteristics. Then, we use the SEC column to systematically compare the efficiency with the most common EV isolation methods: Ultracentrifugation (UC) and total exosomes isolation commercial kit (TEI). The EVs isolated through different methods were characterized the yield and size of EVs, analyzed their protein profiles, the morphology and purity were observed under the transmission electron microscope. To further improve the quality and purity, we combined SEC and UC methods and established a two-steps method to isolated EVs from serum. Results: Self-made SEC column can well separate EVs from complex serum protein, and EVs enriched in the 8-13 fractions with good morphology and yield. By systematically compare SEC with the commonly used UC and TEI kit, SEC is outstanding in all aspects and balances both isolation purity and yield. However, using the SEC method alone still has certain limitations and residual impurities. The SEC+UC combined method can cleverly solve the shortcomings of SEC and optimize the quality and purity of EVs from serum, which is much better than using one method alone. Conclusion: Our study presents the combination of size-exclusion chromatography and ultracentrifugation as a feasible and time-saving method to isolate high-quality and purity extracellular vesicles from serum.


Assuntos
Exossomos , Vesículas Extracelulares , Proteínas Sanguíneas , Cromatografia em Gel , Ultracentrifugação
3.
Molecules ; 26(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34641622

RESUMO

Analytical ultracentrifugation is a versatile approach for analysing the molecular mass, molecular integrity (degradation/aggregation), oligomeric state and association/dissociation constants for self-association, and assay of ligand binding of kinase related membrane proteins and glycans. It has the great property of being matrix free-providing separation and analysis of macromolecular species without the need of a separation matrix or membrane or immobilisation onto a surface. This short review-designed for the non-hydrodynamic expert-examines the potential of modern sedimentation velocity and sedimentation equilibrium and the challenges posed for these molecules particularly those which have significant cytoplasmic or extracellular domains in addition to the transmembrane region. These different regions can generate different optimal requirements in terms of choice of the appropriate solvent (aqueous/detergent). We compare how analytical ultracentrifugation has contributed to our understanding of two kinase related cellular or bacterial protein/glycan systems (i) the membrane erythrocyte band 3 protein system-studied in aqueous and detergent based solvent systems-and (ii) what it has contributed so far to our understanding of the enterococcal VanS, the glycan ligand vancomycin and interactions of vancomycin with mucins from the gastrointestinal tract.


Assuntos
Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Bactérias/metabolismo , Ligantes , Peso Molecular , Ultracentrifugação
4.
PLoS One ; 16(9): e0257633, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34591894

RESUMO

Milk is a highly complex, heterogeneous biological fluid that contains non-nutritive, bioactive extracellular vesicles called exosomes. Characterization of milk-derived exosomes (MDEs) is challenging due to the lack of standardized methods that are currently being used for milk pre-processing, storage, and exosome isolation. In this study, we tested: 1) three pre-processing methods to remove cream, fat, cellular debris, and casein proteins from bovine milk to determine whether pre-processing of whole milk prior to long-term storage improves MDE isolations, 2) the suitability of two standard exosome isolation methods for MDE fractionation, and 3) four extraction protocols for obtaining high quality RNA from bovine and human MDEs. MDEs were characterized via Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western immunoblotting for CD9, CD63, and Calnexin protein markers. We also present an optimized method of TEM sample preparation for MDEs. Our results indicate that: 1) Removal of cream and fat globules from unpasteurized bovine milk, prior to long-term storage, improves the MDE yield but not purity, 2) Differential ultracentrifugation (DUC) combined with serial filtration is better suited for bovine MDE isolation compared to ExoQuick (EQ) combined with serial filtration, however both methods were comparable for human milk, and 3) TRIzol LS is better suited for RNA extraction from bovine MDEs isolated by EQ and DUC methods. 4) TRIzol LS, TRIzol+RNA Clean and Concentrator, and TRIzol LS+RNA Clean and Concentrator methods can be used for RNA extractions from human MDEs isolated by EQ, yet the TRIzol LS method is better suited for human MDEs isolated by DUC. The QIAzol + miRNeasy Mini Kit produced the lowest RNA yield for bovine and human MDEs.


Assuntos
Exossomos/química , Precipitação Fracionada , Leite Humano/metabolismo , Leite/metabolismo , RNA/isolamento & purificação , Ultracentrifugação , Animais , Bovinos , Exossomos/metabolismo , Feminino , Filtração , Humanos , Microscopia Eletrônica de Transmissão , RNA/metabolismo
5.
Se Pu ; 39(9): 968-980, 2021 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-34486836

RESUMO

Exosomes are membrane-bound nanovesicles that are secreted by most types of cells and contain a range of biologically important molecules, including lipids, proteins, ribonucleic acids, etc. Emerging evidences show that exosomes can affect cells' physiological status by transmitting molecular messages among cells. As such, exosomes are involved in various pathological processes. Studying exosomes is of great importance for understanding their biological functions and relevance to disease diagnosis. However, it is difficult to separate and analyze exosomes due to their small size, and because their density is similar to that of bodily fluids. Traditional methods, including ultracentrifugation and ultrafiltration are time-consuming and require expensive equipment. Other methods for exosome separation, including immunoaffinity-based methods, are expensive and rely heavily on specific antibodies. Precipitation-based methods do not yield acceptable purity for downstream analysis, due to polymer contamination. Thus, urgent demand exists for a portable, simple, affordable method for exosome separation. Microfluidic chip technology offers a potential platform for separation and detection of exosomes, with several remarkable characteristics, including low sample consumption, high throughput, and easy integration. This paper provides an overview of current microfluidic strategies for separation and analysis of circulating exosomes. In our introduction to exosome separation, we divide existing separation methods into two categories. Category one is based on exosome physical properties, and includes membrane filtration, nano-column array sorting, and physical isolation. The other is immune capture, which is based on biochemical characteristics of exosomes, and includes fixed base immune capture and unfixed base immune capture. In our introduction to exosome analyses, some commonly used methods, including western blotting, scanning electron microscopy, and flow cytometry are briefly described. Some new systems, which combine microfluidic technology with fluorescence, electrochemical sensing, surface plasmon resonance, or other multimodal analysis methods for integrated detection of exosomes are then described in detail. Finally, the challenges faced by microfluidic technology in improving exosome purity and making systems more portable are analyzed. Prospects for application of microfluidic chips in this area are also discussed. With the rapid development of micro/nano-manufacturing, new materials, and information technology, microfluidic exosome separation and analysis systems will become smaller, more integrated, and more automated. Microfluidic chip technology will play important roles in exosome separation, biochemical detection, and mechanism analysis.


Assuntos
Exossomos , Microfluídica , Proteínas , Ultracentrifugação
6.
Nanoscale ; 13(35): 14760-14776, 2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34473170

RESUMO

Given the emerging diagnostic utility of extracellular vesicles (EVs), it is important to account for non-EV contaminants. Lipoprotein present in EV-enriched isolates may inflate particle counts and decrease sensitivity to biomarkers of interest, skewing chemical analyses and perpetuating downstream issues in labeling or functional analysis. Using label free surface enhanced Raman scattering (SERS), we confirm that three common EV isolation methods (differential ultracentrifugation, density gradient ultracentrifugation, and size exclusion chromatography) yield variable lipoprotein content. We demonstrate that a dual-isolation method is necessary to isolate EVs from the major classes of lipoprotein. However, combining SERS analysis with machine learning assisted classification, we show that the disease state is the main driver of distinction between EV samples, and largely unaffected by choice of isolation. Ultimately, this study describes a convenient SERS assay to retain accurate diagnostic information from clinical samples by overcoming differences in lipoprotein contamination according to isolation method.


Assuntos
Vesículas Extracelulares , Neoplasias , Cromatografia em Gel , Humanos , Lipoproteínas , Neoplasias/diagnóstico , Análise Espectral Raman , Ultracentrifugação
7.
Water Res ; 203: 117509, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34388497

RESUMO

Nanoplastics are an emerging contaminant in aquatic environments. However, analytical methods for the separation, concentration, and identification of nanoplastics, which are essential to assess nanoplastic presence in the environment, are lacking. Here, we developed a new and easy-to-use method to separate and enrich nanoplastics in field water samples with ultracentrifugation. River water was spiked with polystyrene fragments (< 1000 nm) at an environmentally relevant concentration (108-109 particles/L). The polystyrene fragments were successfully separated and enriched by a factor of nearly 50 times with a high recovery rate (87.1%) after undergoing our process. Particles were then characterized using UV-vis spectroscopy, scanning electron microscopy (SEM), and enhanced darkfield microscopy with a hyperspectral imaging (HSI) spectrometer. These techniques are non-destructive and allow the assessment of plastic concentration, morphology, and polymer type. Our method can potentially be applied to other water samples to supply clean, enriched nanoplastic samples that can facilitate their identification in environmental samples.


Assuntos
Microplásticos , Plásticos , Poliestirenos , Ultracentrifugação , Água
8.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445427

RESUMO

Carbonic anhydrases (CAs) are a family of ubiquitous enzymes that catalyze the interconversion of CO2 and HCO3-. The "iota" class (ι-CA) was first found in the marine diatom Thalassiosira pseudonana (tpι-CA) and is widespread among photosynthetic microalgae and prokaryotes. The ι-CA has a domain COG4875 (or COG4337) that can be repeated from one to several times and resembles a calcium-calmodulin protein kinase II association domain (CaMKII-AD). The crystal structure of this domain in the ι-CA from a cyanobacterium and a chlorarachniophyte has been recently determined. However, the three-dimensional organization of the four domain-containing tpι-CA is unknown. Using biophysical techniques and 3-D modeling, we show that the homotetrameric tpι-CA in solution has a flat "drone-like" shape with a core formed by the association of the first two domains of each monomer, and four protruding arms formed by domains 3 and 4. We also observe that the short linker between domains 3 and 4 in each monomer confers high flexibility, allowing for different conformations to be adopted. We propose the possible 3-D structure of a truncated tpι-CA containing fewer domain repeats using experimental data and discuss the implications of this atypical shape on the activity and metal coordination of the ι-CA.


Assuntos
Anidrases Carbônicas/química , Diatomáceas/enzimologia , Cristalografia por Raios X , Diatomáceas/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fotossíntese , Domínios Proteicos , Espectrometria de Massas por Ionização por Electrospray , Ultracentrifugação
9.
Sci Rep ; 11(1): 16088, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373477

RESUMO

Two-cycle cesium chloride (2 × CsCl) gradient ultracentrifugation is a conventional approach for purifying recombinant adenoviruses (rAds) for research purposes (gene therapy, vaccines, and oncolytic vectors). However, rAds containing the RGD-4C peptide in the HI loop of the fiber knob domain tend to aggregate during 2 × CsCl gradient ultracentrifugation resulting in a low infectious titer yield or even purification failure. An iodixanol-based purification method preventing aggregation of the RGD4C-modified rAds has been proposed. However, the reason explaining aggregation of the RGD4C-modified rAds during 2 × CsCl but not iodixanol gradient ultracentrifugation has not been revealed. In the present study, we showed that rAds with the RGD-4C peptide in the HI loop but not at the C-terminus of the fiber knob domain were prone to aggregate during 2 × CsCl but not iodixanol gradient ultracentrifugation. The cysteine residues with free thiol groups after the RGD motif within the inserted RGD-4C peptide were responsible for formation of the interparticle disulfide bonds under atmospheric oxygen and aggregation of Ad5-delta-24-RGD4C-based rAds during 2 × CsCl gradient ultracentrifugation, which could be prevented using iodixanol gradient ultracentrifugation, most likely due to antioxidant properties of iodixanol. A cysteine-to-glycine substitution of the cysteine residues with free thiol groups (RGD-2C2G) prevented aggregation during 2 × CsCl gradient purification but in coxsackie and adenovirus receptor (CAR)-low/negative cancer cell lines of human and rodent origin, this reduced cytolytic efficacy to the levels observed for a fiber non-modified control vector. However, both Ad5-delta-24-RGD4C and Ad5-delta-24-RGD2C2G were equally effective in the murine immunocompetent CT-2A glioma model due to a primary role of antitumor immune responses in the therapeutic efficacy of oncolytic virotherapy.


Assuntos
Adenoviridae/isolamento & purificação , Césio/química , Cloretos/química , Vetores Genéticos/genética , Células A549 , Infecções por Adenoviridae/terapia , Animais , Antioxidantes/química , Linhagem Celular , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Glioma/terapia , Glioma/virologia , Células HEK293 , Humanos , Camundongos , Oligopeptídeos/genética , Terapia Viral Oncolítica/métodos , Ratos , Ácidos Tri-Iodobenzoicos/química , Ultracentrifugação/métodos
10.
Sci Rep ; 11(1): 16086, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373542

RESUMO

High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.


Assuntos
Lipoproteínas HDL/sangue , Lipoproteínas HDL/isolamento & purificação , Cromatografia em Gel/métodos , Cromatografia Líquida/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Tamanho da Partícula , Proteínas/isolamento & purificação , Ultracentrifugação/métodos
11.
J Biol Chem ; 297(3): 100995, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34302810

RESUMO

Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved Fc glycosylation sites are unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s020,w of 5.82 to 6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier RG values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) showed a maximum length of 25 to 28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modeling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures, respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each form of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively restricted Fab and Fc conformations joined by an extended semirigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2, and IgG4.


Assuntos
Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Mieloma Múltiplo/imunologia , Proteínas do Mieloma/química , Receptores Fc/química , Sequência de Aminoácidos , Cromatografia Líquida/métodos , Glicosilação , Humanos , Espectrometria de Massas/métodos , Simulação de Dinâmica Molecular , Nêutrons , Conformação Proteica , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Ultracentrifugação/métodos , Difração de Raios X
12.
J Dairy Sci ; 104(9): 9478-9493, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34218910

RESUMO

Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, ß-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV.


Assuntos
Queijo , Exossomos , Vesículas Extracelulares , Nanopartículas , Animais , Filtração , Ultracentrifugação
13.
Biomolecules ; 11(6)2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204944

RESUMO

Proteomics can map extracellular vesicles (EVs), including exosomes, across disease states between organisms and cell types. Due to the diverse origin and cargo of EVs, tailoring methodological and analytical techniques can support the reproducibility of results. Proteomics scans are sensitive to in-sample contaminants, which can be retained during EV isolation procedures. Contaminants can also arise from the biological origin of exosomes, such as the lipid-rich environment in human milk. Human milk (HM) EVs and exosomes are emerging as a research interest in health and disease, though the experimental characterization and functional assays remain varied. Past studies of HM EV proteomes have used data-dependent acquisition methods for protein detection, however, improvements in data independent acquisition could allow for previously undetected EV proteins to be identified by mass spectrometry. Depending on the research question, only a specific population of proteins can be compared and measured using isotope and other labelling techniques. In this review, we summarize published HM EV proteomics protocols and suggest a methodological workflow with the end-goal of effective and reproducible analysis of human milk EV proteomes.


Assuntos
Vesículas Extracelulares/química , Proteínas do Leite/análise , Leite Humano/química , Proteômica/métodos , Biologia Computacional/métodos , Biologia Computacional/normas , Exossomos/química , Humanos , Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Proteômica/normas , Reprodutibilidade dos Testes , Ultracentrifugação/métodos , Ultracentrifugação/normas
14.
J Vis Exp ; (172)2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34279508

RESUMO

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma membrane and then transported to the early endosomes. The internalized proteins could be transited to the lysosome for degradation or recycled back to the plasma membrane. A robust endocytic recycling pathway is required to balance the removal of membrane materials from endocytosis. Various proteins are reported to regulate the pathway, including ADP-ribosylation factor 6 (ARF6). Density gradient ultracentrifugation is a classical method for cell fractionation. After the centrifugation, organelles are sedimented at their isopycnic surface. The fractions are collected and used for other downstream applications. Described here is a protocol to obtain a recycling endosome-containing fraction from transfected mammalian cells using density gradient ultracentrifugation. The isolated fractions were subjected to standard Western blotting for analyzing their protein contents. By employing this method, we identified that the plasma membrane targeting of engulfment and cell motility 1 (ELMO1), a Ras-related C3 botulinum toxin substrate 1 (Rac1) guanine nucleotide exchange factor, is through ARF6-mediated endocytic recycling.


Assuntos
Endocitose , Endossomos , Animais , Membrana Celular/metabolismo , Endossomos/metabolismo , Transporte Proteico , Ultracentrifugação
15.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063036

RESUMO

Urinary extracellular vesicles (EVs) and their RNA cargo are a novel source of biomarkers for various diseases. We aimed to identify the optimal method for isolating small (<200 nm) EVs from human urine prior to small RNA analysis. EVs from filtered healthy volunteer urine were concentrated using three methods: ultracentrifugation (UC); a precipitation-based kit (PR); and ultrafiltration (UF). EVs were further purified by size-exclusion chromatography (SEC). EV preparations were analysed with transmission electron microscopy (TEM), Western blotting, nanoparticle tracking analysis (NTA) and an Agilent Bioanalyzer Small RNA kit. UF yielded the highest number of particles both before and after SEC. Small RNA analysis from UF-concentrated urine identified two major peaks at 10-40 nucleotides (nt) and 40-80 nt. In contrast, EV preparations obtained after UC, PR or SEC combined with any concentrating method, contained predominantly 40-80 nt sized small RNA. Protein fractions from UF+SEC contained small RNA of 10-40 nt in size (consistent with miRNAs). These data indicate that most of the microRNA-sized RNAs in filtered urine are not associated with small-sized EVs, and highlights the importance of removing non-vesicular proteins and RNA from urine EV preparations prior to small RNA analysis.


Assuntos
Cromatografia em Gel , Vesículas Extracelulares/genética , MicroRNAs/urina , Sistema Livre de Células , Vesículas Extracelulares/ultraestrutura , Humanos , Ultracentrifugação , Ultrafiltração
16.
Clin Chim Acta ; 520: 172-178, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34118239

RESUMO

BACKGROUND: The causal relationship between low-density lipoprotein (LDL) and atherosclerotic cardiovascular disease (CVD) has been firmly substantiated. LDL consists of a heterogeneous group of particles with different physicochemical and metabolic properties. Among them, small dense LDL (sdLDL) particles are considered an emerging CVD risk factor and a promising CVD risk biomarker. This paper reviews published analytical and calculation-based methods for sdLDL determination in plasma, present their principles, strengths, and weaknesses, and examine the challenges arising from method comparison. METHODS: A literature survey was conducted using the PubMed database. Subject headings and keywords facilitated the search strategy. Titles and abstracts were initially assessed, and the full-text article of the pre-selected ones was reviewed. RESULTS: A range of methods is currently available for the analysis of LDL subfractions and the measurement of sdLDL particle size, number, and cholesterol concentration. Ultracentrifugation (UC), vertical auto profile, gradient gel electrophoresis (GGE), nuclear magnetic resonance (NMR) spectroscopy, high-performance liquid chromatography, ion mobility analysis, and a homogeneous assay are the most prevalent. To date, there is no "gold standard". UC and GGE are the most established techniques, albeit significantly sophisticated. NMR and the homogeneous assay are options with potential clinical use as they yield results rapidly and can be high-throughput. None of the proposed equations for the calculated sdLDL determination has been sufficiently validated to serve as a clinical tool. CONCLUSIONS: Many analytical procedures have been developed for the study of sdLDL particles. Their use remains largely restricted to research laboratories since their analytical and clinical performance, along with the clinical- and cost-effectiveness of sdLDL determination have not been fully established.


Assuntos
Aterosclerose , Lipoproteínas LDL , Biomarcadores , Eletroforese , Humanos , Ultracentrifugação
17.
Methods Mol Biol ; 2310: 1-15, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34095994

RESUMO

Several studies have indicated the presence of microRNAs (miRNAs) within mitochondria although the origin, as well as the biological function, of these mitochondrially located miRNAs is largely unknown. The identification and significance of this subcellular localization is gaining increasing relevance to the pathogenesis of certain disease states. Here, we describe the isolation of highly purified mitochondria from rat liver by differential centrifugation, followed by RNAse A treatment to eliminate contaminating RNA. The coupled extraction of total RNA and protein is a more efficient design for allowing the downstream evaluation of miRNA and protein expression in mitochondria.


Assuntos
Fracionamento Celular , MicroRNAs/isolamento & purificação , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Animais , Ratos , Ribonuclease Pancreático/metabolismo , Ultracentrifugação
18.
J Food Sci ; 86(7): 2838-2850, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34151426

RESUMO

Plant-derived exosome-like nanoparticles (PDENs) are small vesicles released by multivesicular bodies mainly to communicate between cells and regulate immunity against pathogen attack. Current studies have reported that PDENs could modulate gene expression in a cross-kingdom fashion. Therefore, PDENs could be a potential future functional food ingredient as their cross-kingdom communication abilities were reported to exert multiple health benefits. Macrophage and other cells have been reported to absorb PDENs in a manner regulated by the membrane lipid and protein profile and the intactness of the PDENs lipid bilayer. PDENs could be extracted from plant materials by various techniques such as ultracentrifugation, immunoaffinity, size-based isolation, and precipitation, though each method has its pros and cons. PDENs mainly contain lipid, protein, and genetic materials, mainly micro RNAs, which could exert multiple health benefits and functionalities when consumed in sufficient amounts. However, most studies on the health functionalities of PDENs were conducted through in-vitro and in-vivo studies, and its potency to be used as a functional ingredient remains a question as PDENs are sensitive to storage and processing condition and requires costly extraction method. This concise review features various exosome extraction methods, contents of PDENs and their roles, the health functionalities of PDENs, and its potency as a functional food ingredient.


Assuntos
Exossomos/metabolismo , Ingredientes de Alimentos , Macrófagos/imunologia , Nanopartículas/química , Plantas/química , Plantas/metabolismo , Exossomos/imunologia , Humanos , Ultracentrifugação/métodos
19.
FEBS Lett ; 595(15): 2034-2046, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34115884

RESUMO

Bacterial α-2 macroglobulins (A2Ms) structurally resemble the large spectrum protease inhibitors of the eukaryotic immune system. In Pseudomonas aeruginosa, MagD acts as an A2M and is expressed within a six-gene operon encoding the MagA-F proteins. In this work, we employ isothermal calorimetry (ITC), analytical ultracentrifugation (AUC), and X-ray crystallography to investigate the function of MagC and show that MagC associates with the macroglobulin complex and with the peptidoglycan (PG). However, the catalytic residues of MagC display an inactive conformation that could suggest that it binds to PG but does not degrade it. We hypothesize that MagC could serve as an anchor between the MagD macroglobulin and the PG and could provide stabilization and/or regulation for the entire complex.


Assuntos
Proteínas de Bactérias/metabolismo , Peptidoglicano/metabolismo , alfa 2-Macroglobulinas Associadas à Gravidez/metabolismo , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Calorimetria/métodos , Cristalografia por Raios X , Ligação Proteica , Homologia de Sequência de Aminoácidos , Ultracentrifugação
20.
Sci Rep ; 11(1): 11585, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34079007

RESUMO

Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples.


Assuntos
Vesículas Extracelulares/metabolismo , Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Cromatografia em Gel/métodos , Glutamato Carboxipeptidase II/metabolismo , Humanos , Separação Imunomagnética/métodos , Masculino , Estudo de Prova de Conceito , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ultracentrifugação/métodos , Ultrafiltração/métodos
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