Purification of Chloroplast Envelope, Thylakoids, and Stroma from Angiosperm Leaves.

Plant cell chloroplasts are bounded by a two-membrane envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are distinct from the chloroplast envelope, and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three compartments. A protocol is detailed for leaves of spinach, Arabidopsis or pea.

Isolation of the Inner and Outer Membranes of the Chloroplast Envelope from Angiosperms.

The outer and the inner membranes of the chloroplast envelope, also called OEM and IEM, have distinct lipid and protein compositions. They host molecular systems involved in the biogenesis of the organelle, its cellular function, and its communication with other compartments. Here we describe a method for the isolation of these two membranes starting from intact chloroplast preparations, with two alternative procedures based on the starting material. One was developed from spinach leaves, the other from pea leaves. The two procedures differ in the method used to isolate and rupture chloroplasts and separate each membrane.

Goat milk extracellular vesicles: Separation comparison of natural carriers for theragnostic application.

Goat milk is a complex biological fluid, which in addition to having a high nutritional value, it is an interesting source of extracellular vesicles (EVs). Despite the countless potential applications that they offer in many biological fields, is not easy to compare the different proposed systems, and this is a major limitation for the real translatability of these natural nanoplatforms for theragnostic purposes. Thus, it is useful to further investigate reproducible methods to separate goat milk EVs. The choice of methods but also the preprocessing of milk has an immense impact on the separation, quality, and yield of EVs. Here, we tested four protocols to separate EVs from unpasteurised goat milk: two based on differential ultracentrifugation (DUC) and two on size-exclusion chromatography (SEC). Moreover, we assessed two different approaches of pre-treatment (acidification and precipitation) to facilitate milk protein discharge. To the best of our knowledge, a similar comparison of all performed protocols on raw goat milk has never been published before. Therefore, enriched EV samples were successfully obtained from goat milk using both DUC and SEC. Taken together, our results may be helpful to obtain natural carriers for future theragnostic applications in personalised medicine.

Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles.

Extracellular vesicles (EVs), which are found in almost all cells and human body fluids, are currently being studied as a source of pathophysiological information. Previously, we demonstrated that at least two types of EVs can be isolated from human whole saliva (WS) using enzymatic activity of dipeptidyl peptidase IV (DPP IV) as a marker for differentiating the EV subsets. In the present study, EV fractions, termed EV-I 20 k-ppt and EV-II 100 k-ppt, were prepared by a combination of size-exclusion chromatography of improved condition and sequential centrifugation. The EV-I 20 k-ppt fraction contained medium/large EVs with a diameter of 100-1,000 nm, including aminopeptidase N (APN), mucin 1, ezrin, and Annexin A1. EV-II 100 k-ppt contained small EVs with a diameter of 20-70 nm, with DPP IV and CD9, programmed cell death 6-interacting protein, and tumor susceptibility gene 101 as characteristic proteins. Proteomic analyses also revealed distinctive repertoires of constituent proteins. Immunoprecipitation of several membrane proteins of the EVs with respective antibodies suggested their differential local membrane environment between the two types of salivary vesicles. Thus, we identified two distinctive types of EVs, one is APN/MUC1- rich EVs (EV-I, large/medium EVs) and the other is DPP IV/CD9-rich EVs (EV-II, small EVs). Furthermore, analysis of the binding of the EVs to coronavirus spike proteins showed that EV-II 100 k-ppt, but not EV-I 20 k-ppt, significantly bound to the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we developed a simple method to prepare two distinctive EVs from only 1 mL of human WS using sequential immunoprecipitation. Elucidating the features and functions of these two types of salivary EVs may help us understand their pathophysiological roles in the oral cavity and gastrointestinal tract.

Extracellular vesicles separated from goat milk by differential centrifugation coupled with sodium citrate pretreatments.

Satisfactory separation of milk-derived extracellular vesicles (MEVs) is important for the downstream analysis of the functions and properties of MEVs. However, the presence of abundant proteins in milk hindered the separation of MEVs. In this study, three pretreatment methods, including sodium citrate (SC), acetic acid (AA), and high-speed centrifugation, were adopted to separate MEVs from goat milk while minimizing the impact of protein. The MEVs were then characterized by nanoparticle tracking, transmission electron microscopy and western blotting experiments. The results indicated that pretreatments with AA and SC greatly decreased the impact of casein, but AA pretreatment damaged the surface structure of MEVs. Additionally, the differential centrifugation process resulted in a slight loss of MEVs. Overall, MEVs with small size and high purity can be obtained under 125 k × g centrifugation combined with SC pretreatment, which suggests a promising method for separation of MEVs from goat milk.

An Improved Method to Enrich Large Extracellular Vesicles Derived from Giardia intestinalis through Differential Centrifugation.

Giardia intestinalis is a flagellated unicellular protozoan that colonizes the small intestine, causing the diarrheal disease called giardiasis. The production of extracellular vesicles (EVs) by G. intestinalis and the role of these EVs in the parasite's interaction with the host have been described. According to biogenesis, EVs are grouped mainly into large (microvesicles-derived from the plasma membrane) and small (exosomes-derived from multivesicular bodies). Populations of EVs are heterogeneous, and improved methods to separate and study them are needed to understand their roles in cell physiology and pathologies. This work aimed to enrich the large extracellular vesicles (LEVs) of G. intestinalis in order to better understand the roles of these vesicles in the interaction of the parasite with the host. To achieve the enrichment of the LEVs, we have modified our previously described method and compared it by protein dosage and using Nano tracking analysis. Giardia intestinalis vesiculation was induced by incubation in a TYI-S-33 medium without serum, to which 1 mM of CaCl2 was added at 37 °C for 1 h. Then, the supernatant was centrifuged at 15,000× g for 1 h (15 K 1 h pellet), 15,000× g for 4 h (15 K 4 h pellet) and 100,000× g for 1.5 h (100 K 1h30 pellet). The pellet (containing EVs) was resuspended in 1× PBS and stored at 4 °C for later analysis. The EVs were quantified based on their protein concentrations using the Pierce BCA assay, and by nanoparticle tracking analysis (NTA), which reports the concentration and size distribution of the particles. The NTA showed that direct ultracentrifugation at 100,000× g for 1.5 h and centrifugation at 15,000× g for 4 h concentrated more EVs compared to centrifugation at 15,000× g for 1 h. Additionally, it revealed that centrifugation at 15,000× g 4 h was able to concentrate at the same particle concentration levels as a direct ultracentrifugation at 100,000× g for 1.5 h. As for the enrichment of LEVs, the NTA has shown a higher concentration of LEVs in direct ultracentrifugation at 100,000× g for 1.5 h, and in centrifugation at 15,000× g for 4 h, compared to centrifugation at 15,000× g for 1 h. Our results have shown that the most used method at 15,000× g for 1 h is not enough to obtain a representative population of large EVs, and we suggest that LEVs released by G. intestinalis can be better enriched by direct ultracentrifugation at 100,000× g for 1.5 h, or by centrifugation at 15,000× g for 4 h.

A new sight separation for collecting starch nanocrystals with small size and high crystallinity based on the hydrolysis mechanism.

To prevent starch nanocrystals (SNCs) that are generated at an early stage from being hydrolyzed excessively, this study proposed a new separation method, named "neutral dispersion and acidic precipitation." SNCs were prepared from waxy potato starch by sulfuric acid hydrolysis. Based on the results of kinetics and molecular weight, the hydrolysis was divided into three stages, e.g., rapid (initial 1 day), medium (subsequent 1 day) and slow stage (2-5 days). The rapid and medium stages were related to the degradation of amorphous region in starch, and the slow stage mainly referred to SNC release. Therefore, the method was developed to separate SNCs at the slow stage. After centrifugation at 6000 rpm, large particles were removed from the SNC suspension under pH 7. The SNCs with small average size and crystallite size, high relative crystallinity (RC), and high dispersion stability in the supernatant were retained and were then precipitated entirely under pH 5, because pH 5 led to the reduction of dispersion stability of SNCs. Meanwhile, the hydrothermal and dry-thermal stability of separated SNCs were significantly promoted. The separation method has the potential in SNC preparation for increasing the yield and collecting products with small size and high RC.

A Modified Differential Centrifugation Protocol for Isolation and Quantitation of Extracellular Heat Shock Protein 90 (eHsp90).

Studies of the past 15 years have revealed a critical role for extracellular heat shock protein 90alpha (eHsp90α) in the development of several human disorders, including wound healing, cachexia (muscle wasting), inflammatory diseases, and cancers. The two established functions of highly purified eHsp90α protein are to promote cell survival and to stimulate cell migration. However, the mechanism of secretion and the method of isolation of eHsp90α remained to be standardized. Among the half a dozen reported methodologies, differential centrifugation is considered the "gold standard" largely for its quantitative recovery of eHsp90α from a conditioned medium of cultured cells. Herein, we describe a revised protocol that isolates three fractions of extracellular vesicles with distinct ranges of diameters and the leftover vesicle-free supernatant for biochemical analyses, especially eHsp90α, from tumor cell-conditioned media. Quantitation of the relative amount of eHsp90α can be carried out with known amounts of recombinant Hsp90α protein on the same SDS-PAGE. We believe that this modified methodology will prove to be a useful tool for studying eHsp90α in cultured cells and beyond.

A practical guide to separate and concentrate ALNs and femtoplankton entities.

The development of new technologies of microscopy, flow cytometry and genomics has allowed a profound reconsideration of the diversity and ecological role of femtoplankton entities (i.e., viruses, vesicles, aster like nanoparticles -ALNs-). Among these, the discovery of ALNs, raise serious questions about their exact nature and their biological and environmental roles. The elaboration of a practical guide for the concentration and separation of femtoplankton entities, including ALNs, is necessary for a better understanding of their diversity, ontogeny, and ecology. Here, we propose a step-by-step procedure for the enrichment and isolation of femtoplankton entities and prokaryotes. The established protocol couples tangential flow filtration to differential centrifugation, leading to differentiate enriched samples (with different target entity contents), usable as a matrix for sorting by flow cytometry. All entities were identified, characterized and counted by transmission electron microscopy and flow cytometry. The procedure allows an efficient detection, concentration and separation of femtoplankton entities (up to purity rate of 92, 67, 81 and 85% for virus like particles, vesicles, prokaryotes and ALNs, respectively), and different morphotypes of ALNs into different fractions (up to 51, 72, 52, 40 and 79% of total ALNs for 20-, 11-, budding 11-, 5-10- and 4-armed ALNs, respectively).

A simple double differential centrifugation-wash procedure to rapidly obtain bacterial identification and direct antimicrobial susceptibility testing from positive blood cultures / Método sencillo basado en una doble centrifugación diferencial para obtener una identificación bacteriana y sensibilidad a antibióticos rápida a partir de hemocultivos positivos

Introduction: This study proposes a simple and rapid method for both bacterial identification and direct antimicrobial susceptibility testing (AST) by using MALDI-TOF and a double differential centrifugation-wash procedure from positive blood cultures. Methods: Fifty-two positive blood cultures (37 gramnegative bacilli and 15 grampositive cocci) were studied by two methods for identification and AST: a reference method, and the rapid MALDI-TOF method obtaining a purified pellet by using a double differential centrifugation procedure. Results : A total of 1101 MIC values (mg/l) were interpreted according to EUCAST clinical breakpoints and compared using the two methods simultaneously. Discrepancies in 81 MIC values (7.35%) were detected. By analyzing standard parameters, we obtained 98.28% essential agreement and 92.65% categorical agreement considering all isolates tested. Conclusion: This method provides rapid bacterial identification and AST, offering definitive results 24–48h earlier than the conventional method (p<0.001) and improving the turnaround time in blood culture diagnostics, especially in laboratories without 24-h on-call.(AU)

Introducción: Este trabajo propone un método sencillo, rápido y barato de identificación bacteriana y sensibilidad antibiótica utilizando MALDI-TOF y una doble centrifugación diferencial a partir de hemocultivo positivo. Métodos: Se estudiaron 52 hemocultivos positivos (37 bacilos gramnegativos y 15 cocos grampositivos). Se compararon 2 métodos: un método convencional de identificación y determinación de sensibilidad a antibióticos automatizada partiendo de colonia crecida, y un método rápido utilizando MALDI-TOF, caracterizado por la obtención de un pellet purificado procedente de un hemocultivo positivo, mediante un procedimiento basado en una doble centrifugación diferencial. Resultados: Se analizaron y compararon 1.101 valores de CMI (mg/l) de acuerdo con los criterios establecidos por EUCAST y obtenidos por ambos métodos. Se detectaron discrepancias en 81 valores de CMI (7,35%). Considerando todos los aislados, la concordancia esencial fue del 98,28% y la concordancia categórica del 92,65%. Conclusión: Este método proporciona resultados de identificación y sensibilidad a antibióticos definitivos 24-48h antes que un método convencional (p<0,001), mejorando el tiempo de respuesta en el diagnóstico microbiológico de bacteriemias, especialmente en laboratorios sin servicio de guardias de 24h.(AU)

Analysis of Yeast Peroxisomes via Spatial Proteomics.

Peroxisomes are ubiquitous organelles with essential functions in numerous cellular processes such as lipid metabolism, detoxification of reactive oxygen species, and signaling. Knowledge of the peroxisomal proteome including multi-localized proteins and, most importantly, changes of its composition induced by altering cellular conditions or impaired peroxisome biogenesis and function is of paramount importance for a holistic view on peroxisomes and their diverse functions in a cellular context. In this chapter, we provide a spatial proteomics protocol specifically tailored to the analysis of the peroxisomal proteome of baker's yeast that enables the definition of the peroxisomal proteome under distinct conditions and to monitor dynamic changes of the proteome including the relocation of individual proteins to a different cellular compartment. The protocol comprises subcellular fractionation by differential centrifugation followed by Nycodenz density gradient centrifugation of a crude peroxisomal fraction, quantitative mass spectrometric measurements of subcellular and density gradient fractions, and advanced computational data analysis, resulting in the establishment of organellar maps on a global scale.

Isolation and Quality Control of Yeast Mitochondria.

The isolation of organelles devoid of other cellular compartments is crucial for studying organellar proteomes and the localization of newly identified proteins, as well as for assessing specific organellar functions. Here, we describe a protocol for the isolation of crude and highly pure mitochondria from Saccharomyces cerevisiae and provide methods for testing the functional integrity of the isolated organelles.

A simple double differential centrifugation-wash procedure to rapidly obtain bacterial identification and direct antimicrobial susceptibility testing from positive blood cultures.

INTRODUCTION: This study proposes a simple and rapid method for both bacterial identification and direct antimicrobial susceptibility testing (AST) by using MALDI-TOF and a double differential centrifugation-wash procedure from positive blood cultures. METHODS: Fifty-two positive blood cultures (37 gramnegative bacilli and 15 grampositive cocci) were studied by two methods for identification and AST: a reference method, and the rapid MALDI-TOF method obtaining a purified pellet by using a double differential centrifugation procedure. RESULTS: A total of 1101 MIC values (mg/l) were interpreted according to EUCAST clinical breakpoints and compared using the two methods simultaneously. Discrepancies in 81 MIC values (7.35%) were detected. By analyzing standard parameters, we obtained 98.28% essential agreement and 92.65% categorical agreement considering all isolates tested. CONCLUSION: This method provides rapid bacterial identification and AST, offering definitive results 24-48h earlier than the conventional method (p<0.001) and improving the turnaround time in blood culture diagnostics, especially in laboratories without 24-h on-call.

Formation mechanism of starch nanocrystals from waxy rice starch and their separation by differential centrifugation.

Starch nanocrystals (SNCs) were prepared from waxy rice starch via sulfuric acid hydrolysis. The objective focused on the following: i) the hydrolysis kinetics and structural properties of SNCs; ii) the effects of differential centrifugation on the yield and size distribution of SNCs. The hydrolysis was divided into a rapid hydrolysis stage in the initial two days and a slow hydrolysis stage after two days. During the two-day hydrolysis, the average diameter of SNCs reached 244 nm. After two days of hydrolysis, the degree of crystallinity, crystallite size, and melting temperature and enthalpy increased. The proportion of A-branched chains decreased, whereas the proportion of B1-branched chains and molecular weight did not change considerably. Thus, the reaction in the slow hydrolysis stage could be considered as the surface modification and gradual release of SNCs. Furthermore, SNCs with a small size and high charge density could be used for differential centrifugation.

Micro-dynamic process of cadmium removal by microbial induced carbonate precipitation.

Microbially induced carbonate precipitation (MICP) is a technique used extensively to address heavy metal pollution but its micro-dynamic process remains rarely explored. In this study, A novel Cd-tolerant ureolytic bacterium DL-1 (Pseudochrobactrum sp.) was used to study the micro-dynamic process. With conditions optimized by response surface methodology, the removal efficiency of Cd2+ could achieve 99.89%. Three components were separated and characterized in the reaction mixture of Cd2+ removal by MICP. The quantitative-dynamic distribution of Cd2+ in different components was revealed. Five synergistic effects for Cd2+ removal were found, including co-precipitation, adsorption by precipitation, crystal precipitation on the cell surface, intracellular accumulation and extracellular chemisorption. Importantly, during Cd2+ removal by MICP, the phenomenon that crystalline nanoparticles adhere to the cell surface, but without any micrometer-sized precipitation encapsulated bacterial cells was observed. This indicated that the previously studied model of bacterial cells as nucleation sites for metal cation precipitation and crystal growth is oversimplified. Our findings provided valuable insights into the mechanism of heavy metals removal by MICP, and a more straightforward method for studying biomineralization-related dynamic process.

Infrared and Raman spectroscopy for purity assessment of extracellular vesicles.

Extracellular vesicles (EVs) are a complex and heterogeneous population of nanoparticles involved in cell-to-cell communication. Recently, numerous studies have indicated the potential of EVs as therapeutic agents, drug carriers and diagnostic tools. However, the results of these studies are often difficult to evaluate, since different characterization methods are used to assess the purity, physical and biochemical characteristics of the EV samples. In this study, we compared four methods for the EV sample characterization and purity assessment: i) the particle-to-protein ratio based on particle analyses with nanoparticle tracking and protein concentration by bicinchoninic acid assay, ii) Western Blot analysis for specific EV biomarkers, iii) two spectroscopic lipid-to-protein ratios by either the attenuated total reflection Fourier transform infrared (ATR-FTIR) or Raman spectroscopy. The results confirm the value of Raman and ATR-FTIR spectroscopy as robust, fast and operator independent tools that require only a few microliters of EV sample. We propose that the spectroscopic lipid-to-protein (Li/Pr) ratios are reliable parameters for the purity assessment of EV preparations. Moreover, apart from determining protein concentrations, we show that ATR-FTIR spectroscopy can also be used for indirect measurements of EV concentrations. Nevertheless, the Li/Pr ratios do not represent full characterization of the EV preparations. For a complete characterization of selected EV preparations, we recommend also additional use of particle size distribution and EV biomarker analysis.

Isolation of Mitochondria from Ustilago maydis Protoplasts.

Ustilago maydis, a basidiomycete that infects Zea mays, is one of the top ten fungal models for studying DNA repair, signal transduction pathways, and dimorphic transitions, among other processes. From a metabolic point of view, U. maydis lacks fermentative capacity, pointing to mitochondria as a key player in central metabolism. Oxidative phosphorylation, synthesis of heme groups, Krebs cycle, ß-oxidation of fatty acids, and synthesis of amino acids are some of the processes that take place in mitochondria. Given the importance of this organelle in eukaryotic cells in general, and in fungal cells in particular, we present a protocol for the isolation of U. maydis mitochondria based on the enzymatic disruption of U. maydis cell wall and differential centrifugation. The method can easily be extrapolated to other fungal species, by using appropriate lytic enzymes.

Comparative Analysis of T-Cell Spatial Proteomics and the Influence of HIV Expression.

As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses, we found that classifier performance in this system was organelle dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine learning for mitochondrial and endoplasmic reticulum proteins but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, Bayesian analysis of differential localization experiments, on row-normalized data. Comparative Bayesian analysis of differential localization experiment analysis of cells induced to express the WT viral genome versus cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as T-cell receptor signaling components and coatomer complex. Finally, we found that support vector machine classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.

Extracellular Vesicle Collection from Human Stem Cells Grown in Suspension Bioreactors.

Extracellular vesicles (EVs) are particles with 100-1000 nm sizes which are secreted by cells for intercellular communication. Meanwhile, studies have found that EVs secreted by human stem cells carry similar characteristics (microRNAs, proteins, metabolites, etc.) from their cell counterpart. Thus, EVs derived from stem cells, especially human induced pluripotent stem cells (hiPSCs) and human mesenchymal stromal/stem cells (hMSCs) are promising candidates for cell-free therapy. However, conventional planar culture is insufficient to produce a large amount of cells or EVs to satisfy clinical requirements. In this chapter, we described feasible approaches to harvest EVs secreted by lineage-specific hiPSCs and undifferentiated hMSCs in suspension bioreactors. Differentiation of hiPSCs to cortical organoids can be performed in suspension bioreactors and the corresponding EVs can be isolated and purified. This scale-up protocol can be applied to a majority of stem cell types with EV collection thus provides useful information for both experimental and biomanufacturing purposes.

Purification and Characterization of Extracellular Vesicles from Mycobacterium ulcerans Culture.

Extracellular vesicles (EVs) from both eukaryotic and prokaryotic cells have been characterized over decades and present many biological properties. Since it has been shown that mycobacterial extracellular vesicles (MEVs) of M. ulcerans contain the macrolide toxin mycolactone, MEVs are known to be associated with the pathogenesis of mycobacteria. This chapter describes a method for purifying and characterizing vesicles from in vitro cultures of M. ulcerans. We also describe how purified vesicles can be used in cellular tests, to determine their role in the pathophysiology of M. ulcerans infection.