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Protein is one of the most important biological macromolecules in life, which plays a vital role in cell growth, development, movement, heredity, reproduction and other life activities. High quality isolation and purification is an essential step in the study of the structure and function of target proteins. Therefore, the development of protein purification technologies has great theoretical and practical significance in exploring the laws of life activities and guiding production practice. Up to now, there is no forthcoming method to extract any proteins from a complex system, and the field of protein purification still faces significant opportunities and challenges. Conventional protein purification generally includes three steps: pretreatment, rough fractionation, and fine fractionation. Each of the steps will significantly affect the purity, yield and the activity of target proteins. The present review focuses on the principle and process of protein purification, recent advances, and the applications of these technologies in the life and health industry as well as their far-reaching impact, so as to promote the research of protein structure and function, drug development and precision medicine, and bring new insights to researchers in related fields.

This study presents the results of HPLC, a gentler and rapid separation method in comparison with the conventional ultracentrifugation, for 55 human serum samples. The elution patterns were analysed parametrically, and the attribute of each class was confirmed biochemically. Human samples contained 12 classes of lipoproteins, each of which may consist primarily of proteins. There are three classes of VLDLs. The level of each class was distributed lognormally, and the standard amount and the 95% range were estimated. Some lipoprotein classes with a narrow range could become ideal indicators of specific diseases. This lognormal character suggests that the levels are controlled by the synergy of multiple factors; multiple undesirable lifestyle habits may drastically increase the levels of specific lipoprotein classes. Lipoproteins in medical samples have been measured by enzymatic methods that coincide with conventional ultracentrifugation; however, the high gravity and time required for ultracentrifugation can cause sample degradation. Actually, the enzymatic methods measured the levels of several mixed classes. The targets of enzymatic methods have to be revised. [ABSTRACT FROM AUTHOR]

The Oseberg Viking ship burial is one of the most extensive collections of Viking wooden artefacts ever excavated in Norway. In the early twentieth century, many of these artefacts were treated with alum in order to preserve them, inadvertently leading to their current degraded state. It is therefore crucial to develop new bioinspired polymers which could be used to conserve these artefacts and prevent further disintegration. Two hydroxylated polymers were synthesised (TPA6 and TPA7), using α-pinene- and oleic acid-derived monomers functionalised with an acrylate moiety. Characterisation using biomolecular hydrodynamics (analytical ultracentrifugation and high precision viscometry) has shown that these polymers have properties which would potentially make them good wood consolidants. Conformation analyses with the viscosity increment (ν) universal hydrodynamic parameter and ELLIPS1 software showed that both polymers had extended conformations, facilitating in situ networking when applied to wood. SEDFIT-MSTAR analyses of sedimentation equilibrium data indicates a weight average molar mass Mw of (3.9 ± 0.8) kDa and (4.2 ± 0.2) kDa for TPA6 and TPA7 respectively. Analyses with SEDFIT (sedimentation velocity) and MultiSig however revealed that TPA7 had a much greater homogeneity and a lower proportion of aggregation. These studies suggest that both these polymers—particularly TPA7—have characteristics suitable for wood consolidation, such as an optimal molar mass, conformation and a hydroxylated nature, making them interesting leads for further research. [ABSTRACT FROM AUTHOR]

• The adenosine deaminase assay showed acceptable performance in various body fluids. • Significant icteric interference was noted using several sources of bilirubin. • One of two types of hyaluronidase evaluated showed significant interference. Adenosine deaminase (ADA) can be increased in various body fluids during infectious and inflammatory states. The objective of this study was to evaluate the performance characteristics of the Diazyme ADA assay for serum, pleural, pericardial, peritoneal, and cerebrospinal fluids using the Roche cobas c501 analyzer. Accuracy, linearity, recovery, precision, sensitivity, specificity, reference interval, and stability studies were conducted. Potential interference of hyaluronidase and ultracentrifugation pre-treatment for viscosity on ADA concentrations were further evaluated. Assay method comparison to two separate external laboratories showed the following results (slope, intercept, %bias): serum (1.053, −0.478, 4.4 %); pleural (1.046, −1.41, 2.6 %). Accuracy (109.6 % recovery) was further demonstrated using a commercially available ADA reference material (BCR647). Linearity and spiked recovery studies showed percent recoveries ranging 94.3–109.3 %. Precision across all specimen types was ≤4.7 %CV. Interference was observed with increasing concentrations of various sources of conjugated and unconjugated bilirubin. Reference intervals were established for serum and pleural fluids, and previously published reference intervals were verified for pericardial, peritoneal, and cerebrospinal fluids. All specimen types were stable for 24 h ambient (8–25 °C), 1 week refrigerated (2–8 °C), and 1 month frozen (−20 °C). Of the two types of hyaluronidase evaluated, one showed positive interference for ADA (Sigma-Aldrich, H3506; 4.59 to 17.90 average % difference from baseline). Ultracentrifugation did not interfere with results (−2.32 to 0.87 average % difference from baseline). The Diazyme ADA assay was validated for use in our laboratory for all fluid types evaluated. Interference was observed with increasing concentrations of bilirubin and one source of hyaluronidase. [ABSTRACT FROM AUTHOR]

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Nesfatin-1 and -2 are produced from a reaction in which the N-terminus of human Nucleobindin-2 undergoes proteolytical processing. To date, Nucleobindin-2 and/or nesfatin-1 have only been shown to act as peptide hormones. On the other hand, the purpose of nesfatin-2 remains unknown. Since Nucleobindin-2/nesfatin-1 is thought impact the control of a wide range of physiological processes, including energy homeostasis, neurodegenerative processes and carcinogenesis, its ligands/interactions deserve special studies and attention. However, there are no reports about the molecular properties of the proteolytical products of human Nucleobindin-2 in the literature. Hence, this study aimed to analyze the effect of Zn(II) and Ca(II) on human nesfatin-1, -2, and -1/2 structures. Herein, we report that human nesfatin-1 is a member of the intrinsically disordered protein family, as indicated by circular dichroism and analytical ultracentrifugation experiments. In contrast, we found that the human nesfatin-2 and nesfatin-1/2 structures were globular with intrinsically disordered regions. Under Zn(II) treatment, we observed concentration-dependent structurization and compaction of intrinsically disordered nesfatin-1 and its propensity for oligomerization, as well as destabilization of both nesfatin-2 and nesfatin-1/2. Furthermore, dissociation constants for Zn(II) binding by nesfatin-1, nesfatin-2, and nesfatin-1/2 were also reported. Moreover, structurally distinct nesfatin-1 and -2 seem to be interdependent when linked together, as indicated by the observed molecular properties of nesfatin-1/2, which in turn are not a simple sum of the properties exhibited by the former peptides. Thus, herein, we shed new light on the molecular behavior of human nesfatins, which might help to elucidate the complex function of those peptides. 4pJh3LW78TdZ__LiE1dLpQ Video abstract [ABSTRACT FROM AUTHOR]

Exosomes are membrane-bound nanovesicles released by cells into their extracellular environment. They carry different types of RNA including mRNA which may be useful in the diagnosis of various diseases. Exosome isolation has been a challenge because of their small size; therefore, two exosome isolation methods were compared in this study. The Exoquick-TC PLUS™ exosome isolation kit (kit) was compared with the classic ultracentrifugation (UC) method for exosome isolation. In samples obtained using both methods, cryo-electron microscopy showed round or slightly elongated vesicles with diameters ranging from 50 to 150 nm and delimited by a bilayered membrane. Dynamic light scattering resulted in multiple peaks for kit exosomes, whereas a single peak was observed for UC exosomes. Significantly, more total RNA was present in UC exosomes in contrast to kit exosomes (P < 0.0001). This was reflected in subsequent mRNA analysis using qPCR, where UC exosomes had lower Ct values compared to kit exosomes. In conclusion, exosome characterization revealed the presence of exosomes in both UC and the kit samples. The kit samples presented additional peaks from DLS which might be due to impurities. Overall, due to a higher total RNA and mRNA content, UC is a better option for subsequent mRNA analysis; nevertheless, the kit can still be used if an ultracentrifuge is not available as four out of the five genes selected were detected and quantified using the kit. [ABSTRACT FROM AUTHOR]

Urine features an ideal source of non-invasive diagnostic markers. Some intrinsic and methodological issues still pose barriers to its full potential as liquid biopsy substrate. Unlike blood, urine concentration varies with nutrition, hydration and environmental factors. Urine is enriched with EVs from urinary-genital tract, while its conservation, purification and normalization can introduce bias in analysis of EV subsets in inter-and intra-individual comparisons. The present study evaluated the methods that decrease such biases such as appropriate and feasible urine storage, optimal single-step EV purification method for recovery of proteins and RNAs from small urine volumes and a normalization method for quantitative analysis of urine EV RNAs. Ultracentrifugation, chemical precipitation and immuno-affinity were used to isolate EVs from healthy donors' urine that was stored frozen or at room temperature for up to 6 months. Multiple urine biochemical and EV parameters, including particle count and protein content, were compared across urine samples. To this purpose nanoparticle tracking analysis (NTA) and protein assessment by BCA, ELISA and WB assays were performed. These measurements were correlated with relative abundances of selected EV mRNAs and miRNAs assessed by RT-PCR and ranked for the ability to reflect and correct for EV content variations in longitudinal urine samples. All purification methods enabled recovery and downstream analysis of EVs from as few as 1 ml of urine. Our findings highlight long term stability of EV RNAs upon urine storage at RT as well as excellent correlation of EV content in urine with some routinely measured biochemical features, such as total urine protein and albumin, but not creatinine most conventionally used for urine normalization. Comparative evaluation of mRNA and miRNAs in EV isolates revealed specific RNAs, in particular RNY4 and small miRNA panel, levels of which well reflected the inter-sample EV variation and therefore useful as possible post-analytical normalizers of EV RNA content. We describe some realistic urine processing and normalization solutions for unbiased readout of EV biomarker studies and routine clinical sampling and diagnostics providing the input for design of larger validation studies employing urine EVs as biomarkers for particular conditions and diseases. [ABSTRACT FROM AUTHOR]

Activating mutations in the receptor KIT promote the dysregulated proliferation of human mast cells (huMCs). The resulting neoplastic huMCs secrete extracellular vesicles (EVs) that can transfer oncogenic KIT among other cargo into recipient cells. Despite potential contributions to diseases, KIT‐containing EVs have not been thoroughly investigated. Here, we isolated and characterized KIT‐EV subpopulations released by neoplastic huMCs using an immunocapture approach that selectively isolates EVs containing KIT in its proper topology. Immunocapture of EVs on KIT antibody‐coated electron microscopy (EM) affinity grids allowed to assess the morphology and size of KIT‐EVs. Immunoblot analysis demonstrated KIT‐EVs have a distinct protein profile from KIT‐depleted EVs, contain exosome and microvesicle markers, and are separated into these subtypes by ultracentrifugation. Cell treatment with sphingomyelinase inhibitors shifted the protein content among KIT‐EV subtypes, suggesting different biogenesis routes. Proteomic analysis revealed huMC KIT‐EVs are enriched in proteins involved in signalling, immune responses, and cell migration, suggesting diverse biological functions, and indicated neoplastic huMCs disseminate KIT via shuttling in heterogeneous microvesicle‐ and exosome‐like EVs. Further, selective KIT‐immunocapture will enable the enrichment of specific huMC‐derived EVs from complex human biosamples and facilitate an understanding of their in vivo functions and potential to serve as biomarkers of specific biological pathologies. [ABSTRACT FROM AUTHOR]

CONTEXT: Low-density lipoprotein cholesterol (LDL-C) is the classical target in cardiovascular (CV) disease management and is usually estimated by Friedewald's formula. However, this formula may over- or underestimate LDL-C levels. AIMS: Our aim is to compare eight LDL-C-estimating formulas to the direct LDL-C measurement and validate their use in the Saudi population. SETTINGS AND DESIGN: This was a retrospective study. SUBJECTS AND METHODS: A blood sample of fasting 669 Saudi subjects was tested for lipid profiles in King Abdulaziz University Hospital Laboratory, from which directly measured LDL-C was obtained. LDL-C was then estimated from eight different formulas: Friedewald's, Cordova's, Hata's, Puavilai's, Chen's, Ahmadi's, Hattori's, and Vujovic's, which were all compared to direct LDL-C. STATISTICAL ANALYSIS USED: Mean and standard deviation, paired t-test, and Pearson's correlation were used for statistical analysis. RESULTS: The mean differences between the direct LDL-C and Hattori and Chen's LDL-C were 0.03 and 0.08 mmol/L, respectively; P < 0.001, while the mean difference observed with Hata, Friedewald, Puavilai, and Vujovic's formulas were higher in comparison: 0.15, 0.24, 0.29, and 0.33 mmol/L, respectively, P < 0.001. Ahmadi and Cordova's LDL-C were estimated to be 0.60 and 0.64 mmol/L more than direct LDL-C levels, respectively, which showed the highest discordance with direct LDL-C, P < 0.001. At a triglyceride (TG) level of <4.5 mmol/L, Hattori also had the best agreement with direct LDL-C, with a mean difference of 0.04 mmol/L, and with TG >4.5 mmol/L, their mean difference was 0.21 mmol/L. All estimated LDL-C strongly correlated with direct LDL-C, except for Ahmadi's. CONCLUSIONS: Hattori's LDL-C had the best agreement with the direct LDL-C, and across all TG levels. However, we recommend directly measuring LDL-C in those with high CV risk. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) have been identified as cell-cell communication agents, and EVs derived from mesenchymal stem cells (MSCs) exhibit therapeutic effects similar to those of the cells of origin. Precipitation methods have been used extensively for EV harvests, such as UC- (ultracentrifugation-) or PEG- (polyethylene glycol-) based methods, and the difference in EVs derived from MSCs by UC and PEG is not fully understood. We harvested EVs from amniotic fluid MSCs (AF-MSCs) by UC- or PEG-based precipitation methods and conducted a comparison study of those EVs derived by the two methods: output, RNA, and protein expression of EVs and EV biological reaction in a THP-1-cell model of LPS induction, which was considered an infection model. There was no difference in morphology, size, or specific marker-positive ratio of PEG-EVs and UC-EVs, but PEG obtained more EV particles, protein, and RNA than the UC method. In our THP-1 model of LPS induction, MSC-EVs did not lead to a change in protein expression but inhibited the LPS-induced increase in cytokine secretion. UC-EVs were more effective for TNF-α inhibition, and PEG-EVs were more effective for IL10 inhibition. Thus, our findings provide evidence that PEG-based precipitation is a more efficient mesenchymal stem cell-extracellular vesicle-derived method than UC. [ABSTRACT FROM AUTHOR]

To investigate the proliferation cycle of a virus, virus-host interaction, and pathogenesis of a virus, virion particles must be concentrated from the media of virus cell culture or the sera of virus-infected patients. Ultracentrifugation of the culture media is a standard method for concentrating virion particles. However, this method is time-consuming and requires special equipment (ultracentrifuge). Moreover, a large number of infectious viruses are lost during enrichment. We developed a new method of hepatitis C virus (HCV) concentration to overcome the issues associated with traditional methods of virus concentration. We used an aqueous two-phase system (ATPS) to concentrate the virus. HCV, which causes various liver diseases, such as liver fibrosis, cirrhosis, and hepatocellular carcinoma, was used as a model virus to test the efficacy and reliability of the ATPS. The efficiency of HCV concentration by the ATPS was approximately three times higher than that by ultracentrifugation. Moreover, the infectivity of the concentrated HCV, which is a labile virus, remained the same after concentration of the virus by the ATPS. Considering the simplicity and effectiveness of the ATPS, it is the method of choice for concentrating viruses. [ABSTRACT FROM AUTHOR]

The photosystem II (PSII) reaction centre is the critical supramolecular pigment–protein complex in the chloroplast which catalyses the light-induced transfer of electrons from water to plastoquinone. Structural studies have demonstrated the existence of an oligomeric PSII. We carried out radiation inactivation target analysis (RTA), together with sucrose gradient ultracentrifugation (SGU) of PSII, to study the functional size of PSII in diverse plant species under physiological and stress conditions. Two PSII populations, made of dimeric and monomeric core particles, were revealed in Pisum sativum, Spinacea oleracea, Phaseulus vulgaris, Medicago sativa, Zea mais and Triticum durum. However, this core pattern was not ubiquitous in the higher plants since we found one monomeric core population in Vicia faba and a dimeric core in the Triticum durum yellow-green strain, respectively. The PSII functional sizes measured in the plant seedlings in vivo, as a decay of the maximum quantum yield of PSII for primary photochemistry, were in the range of 75–101 ± 18 kDa, 2 to 3 times lower than those determined in vitro. Two abiotic stresses, heat and drought, imposed individually on Pisum sativum, increased the content of the dimeric core in SGU and the minimum functional size determined by RTA in vivo. These data suggest that PSII can also function as a monomer in vivo, while under heat and drought stress conditions, the dimeric PSII structure is predominant. [ABSTRACT FROM AUTHOR]

• Uterine lavage sampling two days before FET did not affect the implantation rate. • Uterine lavage sampling before FET did not affect the clinical pregnancy rate. • EVs can be extracted by ultracentrifugation, sucrose cushion, and qEV column. • qEV column isolates EVs from small amounts of UFL gently, quickly and specifically. Does pre-implantation uterine fluid lavage (UFL) of patients undergoing IVF and frozen embryo transfer (FET) affect implantation and clinical pregnancy rates? Which methods among ultracentrifugation, sucrose cushion and qEV column are suitable for isolating UFL extracellular vesicles? First, UFL was collected from 20 patients undergoing IVF and FET 2 days before embryo transfer as the case group. The control group consisted of 20 patients undergoing IVF and FET patients without lavage. All patients were monitored for 6 weeks. In the next step, the UFLs (n = 30) were collected and pooled. The UFL-derived extracellular vesicles were extracted by ultracentrifugation, sucrose cushion and qEV column methods and characterized. Preimplantation uterine lavage sampling did not affect implantation and clinical pregnancy rates. Extracellular vesicles were successfully isolated from UFL by all three methods. Scanning electron microscopy and dynamic light scattering analysis showed that the isolated vesicles were morphologically spherical. The qEV technique showed that they were smaller and homogenized in size. SDS-PAGE of extracellular vesicles showed a weaker albumin band in the qEV column. Western blot analysis indicated that the isolated extracellular vesicles by the qEV column were more immunoreactive for all the common extracellular vesicle markers (CD81, CD9, CD63, and TSG101). Six reference genes were compared by real-time polymerase chain reaction in the isolated extracellular vesicle subpopulations, and lowest cycle threshold value was observed for the 18SrRNA gene. The isolation of endometrial secretome extracellular vesicles is a minimally invasive procedure for individual assessment of endometrial receptivity and can be carried out during conception cycles along with transvaginal ultrasonography. Molecular analysis of UFL-derived extracellular vesicle components could suggest biomarkers to determine precise extracellular vesicle timing. [ABSTRACT FROM AUTHOR]

Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum‐containing media. The majority of researchers use serum‐containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non‐sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA‐MB‐231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano‐flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large‐scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations. [ABSTRACT FROM AUTHOR]

The understanding of the role played by extracellular vesicles (EVs) in different tissues has improved significantly in the last years, but remains limited concerning the conjunctiva, a complex eye tissue whose role is pivotal for corneal protection. Here, we conducted a comparative study to isolate and characterize EVs from human conjunctival epithelial (IM-HConEpiC) and human conjunctival mesenchymal stromal cell (Conj-MSCs) secretomes using different isolation methods: differential ultracentrifugation (UC), and a combination of ultrafiltration (UF) with precipitation or size exclusion chromatography (SEC). EVs were characterized by total protein content, size, morphology, and expression of protein markers. EV functional effect was tested in an in vitro oxidative stress model. We successfully recovered EVs with the three methods, although significantly higher yields were obtained with UF-precipitation. Dynamic light scattering analysis confirmed the presence of nano-sized particles, being UC-isolated EVs larger than those isolated by UF-precipitation and UF-SEC. Atomic Force Microscopy showed EVs with a slightly ellipsoidal morphology. EVs enriched with UF-precipitation method were further analyzed, confirming the expression of Alix, CD63, TSG101, and Syntenin-1 by Western blotting and showing that Conj-MSC-derived EVs significantly reduced oxidative stress on IM-HConEpiC. Therefore, we conclude that UF-precipitation is the most efficient method for conjunctival EV enrichment., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Extracellular vesicles (EVs) are involved in intercellular communication and various biological processes. They hold clinical promise for the diagnosis and management of a wide range of pathologies, including cancer, cardiovascular diseases and degenerative diseases, and are of interest as regenerative therapies. Understanding the complex structure of these EVs is essential to perceive the current challenges associated with their analysis and characterization. Today, challenges remain in terms of access to high-yield, high-purity isolation methods, as well as analytical methods for characterizing and controlling the quality of these products for clinical use., Results: We isolated EVs from the same immortalized human cell culture supernatant using two commonly used approaches, namely differential ultracentrifugation and membrane affinity. Then we evaluated EV morphology, size, zeta potential, particle and protein content, as well as protein identity using cryogenic electron microscopy, nanoparticle tracking analysis, asymmetric field flow fractionation (AF4) and size exclusion chromatography (SEC) coupled to multi angle light scattering, bicinchoninic acid assay, electrophoretic light scattering, western blotting and high-resolution mass spectrometry. Compared to membrane affinity isolation, dUC is a more efficient isolation process for obtaining particles with the characteristics expected for EVs and more specifically for exosomes. To validate an isolation process, cryogenic electron microscopy is essential to confirm vesicles with membranes. High resolution mass spectrometry is powerful for understanding the mechanism of action of vesicles. Separative methods, such as AF4 and SEC, are interesting for separating vesicle subpopulations and contaminants., Significance: This study provides a critical assessment of eight different techniques for analyzing EVs, some of which are mandatory for in-depth characterization and deciphering, while others are more appropriate for routine analysis, once the production and isolation process has been validated. The strengths and limitations of the different approaches used are highlighted., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The cerebrospinal fluid (CSF) is a key matrix for discovery of biomarkers relevant for prognosis and the development of therapeutic targets in pediatric central nervous system malignancies. However, the wide range of protein concentrations and age-related differences in children makes such discoveries challenging. In addition, pediatric CSF samples are often sparse and first prioritized for clinical purposes. The present work focused on optimizing each step of the proteome analysis workflow to extract the most detailed proteome information possible from the limited CSF resources available for research purposes. The strategy included applying sequential ultracentrifugation to enrich for extracellular vesicles (EV) in addition to analysis of a small volume of raw CSF, which allowed quantification of 1351 proteins (+55% relative to raw CSF) from 400 μL CSF. When including a spectral library, a total of 2103 proteins (+240%) could be quantified. The workflow was optimized for CSF input volume, tryptic digestion method, gradient length, mass spectrometry data acquisition method and database search strategy to quantify as many proteins a possible. The fully optimized workflow included protein aggregation capture (PAC) digestion, paired with data-independent acquisition (DIA, 21 min gradient) and allowed 2989 unique proteins to be quantified from only 400 μL CSF, which is a 340% increase in proteins compared to analysis of a tryptic digest of raw CSF.

Increasing studies have highlighted the significance of milk-derived extracellular vesicles (MEV) in mother-newborn integration, as well as their application as novel drug delivery systems and diagnostic biomarkers. However, conventional ultracentrifugation (UC) often results in the co-precipitation of casein micelles in MEV pellets. In this study, we compared methods with different principles to screen the optimal pretreatment in caseins removal, and found that isoelectric precipitation by hydrochloric acid (HA) could most effectively remove caseins in porcine milk. We further characterized MEV populations isolated by UC and HA/UC from diverse aspects, including particle methodology via nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), RNA and protein contents, and purity analysis. Importantly, the proliferative and anti-inflammatory effects of MEV were evaluated in vitro, showing the superiority of MEV via HA/UC in functionality compared with UC. Our results suggest that HA pretreatment before ultracentrifugation could effectively remove caseins and other protein complexes, making MEV with higher purity and more significant effects in vitro. This study provides valuable insights for the advancement of MEV isolation techniques across different species and accurate function analysis of MEV., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Introduction: The clinical potential of extracellular vesicles (EVs) is widely acknowledged, yet the standardization and reproducibility of its separation remain challenging. This study compares three protocols: ultracentrifugation (UC), UC with purification step (UC + PS), and a combined protocol using polymer-based precipitation and UC (PBP + UC)., Methods: Salivary samples were collected from healthy donors. EVs were separated (UC, UC + PS, and PBP + UC) and characterized using transmission electron microscopy, nanoparticle tracking analysis, EV purity, RNA concentration, and Western blotting. miRNA expression was evaluated by quantitative RT-PCR. Statistical analyses comparing groups were performed using ANOVA., Results: All methods successfully separated CD9+ and CD63+ EVs from saliva. The UC + PS and PBP + UC protocols yielded the highest concentrations of EVs, enriched in < 200 nm vesicles. EV purity and RNA recovery were comparable among all methods. Expression of miR-16, miR-27a, and miR-99a was successfully detected using all methods., Conclusions: The UC + PS and PBP + UC protocols demonstrate comparable efficiency in separating salivary EVs. However, the combined PBP + UC protocol, with its simplified processing capability, offers a significant advantage, particularly in the initial phase of EV separation. This finding suggests its potential application in clinical settings where time-sensitive simple processing is critical. Further validation is needed to confirm its effectiveness for transcriptomic and proteomic analyses., (© 2024 The Author(s). Oral Diseases published by John Wiley & Sons Ltd.)

Introduction: The market offers a wide range of extracellular vesicles (EVs) isolation products, but their lack of standardization is a concern. Therefore, it is important to carefully assess the quality of the EVs obtained using these products. In this study, we compared the EXOCIB kit with the ultracentrifuge method, which is considered the gold standard for small EV isolation., Methods: After overnight fasting, small plasma EVs were extracted from four individuals using both the ultracentrifuge and the EXOCIB kit methods. The pooled EVs were then compared for the presence of the cluster of differentiation 63 (CD63) protein using the western blot analysis, and their size and zeta potential were performed by Dynamic Light Scattering (DLS). In addition, the size and morphology of small EVs were determined by using the Transmission Electron Microscopy (TEM) technique., Results: An average hydrodynamic size of 135.7 nm and a zeta potential of -6.33 Mv at 25°C was found for small EVs isolated by the ultracentrifuge, whereas the kit method resulted in small EVs with a hydrodynamic size of 102.8 nm and a zeta potential of -0.907. Notably, the size of the particles in the kit samples was smaller compared to those obtained through the ultracentrifuge (P < 0.001). The western blot method confirmed the expression of CD63 in both methods, so the ultracentrifuge yielded small EVs with a higher level of purity compared to the kit-based approach (P = 0.036)., Conclusion: The DLS findings revealed the existence of vesicles within the appropriate size range for small EVs like exosomes in both isolation techniques. The results of the western blot analysis, in conjunction with DLS, displayed that the ultracentrifuge method extracted small EVs with a greater degree of purity than the kit-based approach., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Lymphatic filariasis and onchocerciasis are neglected tropical diseases and cause significant public health problems in endemic countries, especially in sub-Saharan Africa. Since the human parasites are not viable in immune-competent mice, animal models have been developed, among them Litomosoides sigmodontis which permits a complete life cycle in BALB/c mice, including the development of patent infections with circulating microfilariae (Mf, the worm's offspring). To investigate the immunomodulatory properties of helminths in vitro, antigenic extracts can be prepared from different life cycle stages of the L. sigmodontis model, including adult worms, but the methods to prepare these antigens differ between research groups. This study analyzed whether different centrifugation methods during the preparation of an antigenic extract, the gender of used worms, or the different fractions (soluble or pellet) altered filarial-specific CD4 + T cell responses. These cells were isolated from pre-patent or patent/chronic infected mice, hence those without and those with Mf, respectively. Ex vivo immune responses were compared at these two different time points of the infection as well as the parasitic parameters. Worm burden and cell infiltration were elevated in the thoracic cavity (TC) and draining mediastinal lymph nodes at the pre-patent stage. Within the TC, eosinophils were significantly up-regulated at the earlier time point of infection which was further reflected by the eosinophil-related eotaxin-1 levels. Regarding the production of cytokines by re-stimulated CD4 + T cells in the presence of different antigen preparations, cytokine levels were comparable for all used extracts. Our data show that immune responses differ between pre-patent and patent filarial infection, but not in response to the different antigenic extracts themselves., (© 2024. The Author(s).)

Bringing effective cancer therapy in the form of chimeric antigen receptor technology to untapped markets faces numerous challenges, including a global shortage of therapeutic lentiviral or retroviral vectors on which all current clinical therapies using genetically modified T cells are based. Production of these lentiviral vectors in academic settings in principle opens the way to local production of therapeutic cells, which is the only economically viable approach to make this therapy available to patients in developing countries. The conditions for obtaining and concentrating lentiviral vectors have been optimized and described. The calcium phosphate precipitation method was found to be suitable for transfecting high cell-density cultures, a prerequisite for high titers. We describe protocols for gradually increasing production from 6-well plates to P100 plates, T-175 flasks, and 5-layer stacks while maintaining high titers, >10 8 transducing units. Concentration experiments using ultracentrifugation revealed the advantage of lower centrifugation speeds compared to competing protocols. The resulting batches of lentiviral vectors had a titer of 10 10 infectious particles and were used to transduce primary human T lymphocytes generating chimeric antigen receptor T cells, the quality of which was checked and found potential applicability for treatment., (© 2024 Wiley Periodicals LLC.)

Exosomes have been extensively studied as disease biomarker in humans, given their role in transporting bioactive molecules. However, despite the great potential of exosomes as noninvasive diagnostic markers and therapeutic nanocarriers for bovine diseases, few studies have been conducted on bovine exosome. Thus, this study aimed to quantitatively and qualitatively compare three isolation methods to identify a suitable method for bovine serum. Exosomes were isolated using ultracentrifugation alone (UC), a combination of ultracentrifugation and size exclusion chromatography (US), or membrane affinity-based exoEasy kit (EE). Isolated particles were evaluated using a range of complementary techniques. Transmission electron microscopy showed that all three isolation methods resulted in particles with a cup-shaped morphology. The particle concentration measured by nanoparticle trafficking analyzer of US was lower compared to those of UC and EE method. As a result of immunoblotting, exosome markers including TSG101, CD81, and HSP70 were detected in US particles, while in UC and EE, only TSG101 expression was confirmed. Particles isolated from UC and EE showed a contamination with the blood protein albumin, whereas particles from US did not show albumin contamination. In addition, to evaluate the possibility of using exosomes as biomarkers, the profiles of the small RNA in the exosomes were compared using the bioanalyzer 2100. As a result, in the EE method, the band of small RNA (25-200 nt) was most prominent, and in the US methods, a distinct band was observed in the small RNA range. Collectively, the purity of exosomes without non-exosomal contamination was highest in the US method. However, for the detection of small RNA, the EE method was found to be the most suitable. Therefore, the results suggest that the optimal isolation method varies depending on the specific purpose of exosome isolation., Competing Interests: No potential conflict of interest relevant to this article was reported., (© Copyright 2024 Korean Society of Animal Science and Technology.)

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric β-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1., (© 2024. The Author(s).)

Abstract Background Because of their specific and biologically relevant cargo, urine extracellular vesicles (EVs) constitute a valuable source of potential non-invasive biomarkers that could support the clinical decision-making to improve the management of prostate cancer (PCa) patients. Different EV isolation methods differ in terms of complexity and yield, conditioning, as consequence, the analytical result. Methods The aim of this study was to compare three different isolation methods for urine EVs: ultracentrifugation (UC), size exclusion chromatography (SEC), and a commercial kit (Exolute® Urine Kit). Urine samples were collected from 6 PCa patients and 4 healthy donors. After filtered through 0.22 µm filters, urine was divided in 3 equal volumes to perform EVs isolation with each of the three approaches. Isolated EVs were characterized by spectrophotometric protein quantification, nanoparticle tracking analysis, transmission electron microscopy, AlphaScreen Technology, and whole miRNA Transcriptome. Results Our results showed that UC and SEC provided better results in terms of EVs yield and purity than Exolute®, non-significant differences were observed in terms of EV-size. Interestingly, luminescent AlphaScreen assay demonstrated a significant enrichment of CD9 and CD63 positive microvesicles in SEC and UC methods compared with Exolute®. This heterogeneity was also demonstrated in terms of miRNA content indicating that the best correlation was observed between UC and SEC. Conclusions Our study highlights the importance of standardizing the urine EV isolation methods to guaranty the analytical reproducibility necessary for their implementation in a clinical setting.

Jian Yang,1,2 Xin Gao,1,2 Xiaotao Xing,1,2 Haisen Huang,1,2 Qi Tang,1,3 Shixing Ma,1,2 Xun Xu,1,2 Cheng Liang,1,2 Maojiao Li,1,2 Li Liao,1,2 Weidong Tian1,2 1State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Engineering Research Center of Oral Translational Medicine, Ministry of Education & National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, People’s Republic of China; 2Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, People’s Republic of China; 3West China School of Public Health & West China Fourth Hospital, Sichuan University, Chengdu, 610041, People’s Republic of ChinaCorrespondence: Li Liao; Weidong TianState Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Engineering Research Center of Oral Translational Medicine, Ministry of Education & National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, People’s Republic of ChinaTel/ Fax +86-28-85503499Email lliao@scu.edu.cn; drtwd@sina.comPurpose: 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.Keywords: extracellular vesicles, size-exclusion chromatography, ultracentrifugation, total exosomes isolation, combination methods

Wastewater-based epidemiology (WBE) may be successfully used to comprehensively monitor and determine the scale and dynamics of some infections in the community. We monitored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in raw wastewater samples from Porto Alegre, Southern Brazil. The samples were collected and analyzed every week between May 2020 to May 2021. Meanwhile, different social restrictions were applied according to the number of hospitalized patients in the region. Weekly samples were obtained from two wastewater treatment plants (WWTP), named Navegantes and Serraria. To determine the SARS-CoV-2 RNA titers in wastewater, we performed RT-qPCR analysis targeting the N gene (N1). The highest titer of SARS-CoV-2 RNA was observed between epidemiological weeks (EWs) 33–37 (August), 42–43 (October), 45–46 (November), 49–51 (December) in 2020, and 1–3 (January), 7–13 (February to March) in 2021, with viral loads ranging from 1 × 106–3 × 106 genomic copies/Liter. An increase in positive confirmed cases followed such high viral loads. Depending on the sampling method used, positive cases increased in 6–7 days and 15 days after the rise of viral RNA titers in wastewater, with composite sampling methods showing a lower time lag and a higher resolution on the analyses. The results showed a direct relation between strict social restrictions and the loads of detected RNA reduction in wastewater, corroborating the number of confirmed cases. Differences in viral loads between different sampling points and methods were observed, as composite samples showed more stable results during the analyzed period. Besides, viral loads obtained from samples collected at Serraria WWTP were consistently higher than the ones obtained at Navegantes WWTP, indicating differences in local dynamics of SARS-CoV-2 spread in different regions of Porto Alegre. In conclusion, wastewater sampling to monitor SARS-CoV-2 is a robust tool to evaluate the viral loads contributing to hospitalized patients’ data and confirmed cases. In addition, SARS-CoV-2 detection in sewage may inform and alert the government when there are asymptomatic or non-tested patients.

Blastocystis is a genus of single-celled protist belonging to the stramenopile group. Prior studies have shown that isolates of Blastocystis subtype 7 (ST7) induced higher levels of intestinal epithelial cell damage and gut microbiota dysbiosis in comparison to other subtypes in in vivo and in vitro settings. Prior research has shown a link between gut dysbiosis and exposure to extracellular vesicles (EVs) produced by pathogenic microorganisms. This study demonstrates a protocol for the isolation of EVs from Blastocystis ST7 via ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy were used to assess EV size and morphology. The protein content of isolated EVs was assessed by mass spectrophotometry and the presence of EV markers were evaluated by Western blotting. Finally, the EVs were cocultured with prominent human gut microbiome species to observe their effect on prokaryote growth. Our data shows that Blastocystis ST7 secretes EVs that are similar in morphology to previously characterized EVs from other organisms and that these EVs contain a limited yet unique protein cargo with functions in host-parasite intercellular communication and cell viability. This cargo may be involved in mediating the effects of Blastocystis on its surrounding environment.

The study addresses Enterotoxigenic Escherichia coli (ETEC), a significant concern in low-income countries. Despite its prevalence, there is no licensed vaccine against ETEC. Bacterial vesicle-based vaccines are promising due to their safety and diverse virulence factors. However, cost-effective production requires enhancing vesicle yield while considering altered properties due to isolation methods. The proposed method involves heat treatment and ultrafiltration to recover vesicles from bacterial cultures. Two vesicle types, collected from heat-treated (HT-OMV) or untreated (NT-OMV) cultures, were compared. Vesicles were isolated via ultrafiltration alone (“complete”) or with ultracentrifugation (“sediment”). Preliminary findings suggest complete HT-OMV vesicles are suitable for an ETEC vaccine. They express important proteins (OmpA, OmpX, OmpW) and virulence factors (adhesin TibA). Sized optimally (50–200 nm) for mucosal vaccination, they activate macrophages, inducing marker expression (CD40, MHCII, CD80, CD86) and Th1/Th2 cytokine release (IL-6, MCP-1, TNF-α, IL12p70, IL-10). This study confirms non-toxicity in RAW 264.7 cells and the in vivo ability of complete HT-OMV to generate significant IgG2a/IgG1 serum antibodies. Results suggest promise for a cost-effective ETEC vaccine, requiring further research on in vivo toxicity, pathogen-specific antibody detection, and protective efficacy.

Background: Alternatives to the Friedewald low-density lipoprotein cholesterol (LDL-C) equation have been proposed. Objective: To compare the accuracy of available LDL-C equations with ultracentrifugation measurement. Methods: We used the second harvest of the Very Large Database of Lipids (VLDbL), which is a population-representative convenience sample of adult and pediatric patients (N = 5,051,467) with clinical lipid measurements obtained via the vertical auto profile (VAP) ultracentrifugation method between October 1, 2015 and June 30, 2019. We performed a systematic literature review to identify available LDL-C equations and compared their accuracy according to guideline-based classification. We also compared the equations by their median error versus ultracentrifugation. We evaluated LDL-C equations overall and stratified by age, sex, fasting status, and triglyceride levels, as well as in patients with atherosclerotic cardiovascular disease, hypertension, diabetes, kidney disease, inflammation, and thyroid dysfunction. Results: Analyzing 23 identified LDL-C equations in 5,051,467 patients (mean±SD age, 56±16 years; 53.3% women), the Martin/Hopkins equation most accurately classified LDL-C to the correct category (89.6%), followed by the Sampson (86.3%), Chen (84.4%), Puavilai (84.1%), Delong (83.3%), and Friedewald (83.2%) equations. The other 17 equations were less accurate than Friedewald, with accuracy as low as 35.1%. The median error of equations ranged from –10.8 to 18.7 mg/dL, and was best optimized using the Martin/Hopkins equation (0.3, IQR–1.6 to 2.4 mg/dL). The Martin/Hopkins equation had the highest accuracy after stratifying by age, sex, fasting status, triglyceride levels, and clinical subgroups. In addition, one in five patients who had Friedewald LDL-C 70 mg/dL by the Martin/Hopkins equation. Conclusions: Most proposed alternatives to the Friedewald equation worsen LDL-C accuracy, and their use could introduce unintended disparities in clinical care. The Martin/Hopkins equation demonstrated the highest LDL-C accuracy overall and across subgroups.

Both Gram-negative and Gram-positive bacteria can release vesicle-like structures referred to as bacterial extracellular vesicles (BEVs), which contain various bioactive compounds. BEVs play important roles in the microbial community interactions and host-microbe interactions. Markedly, BEVs can be delivered to host cells, thus modulating the development and function of the innate immune system. To clarify the compositions and biological functions of BEVs, we need to collect these vesicles with high purity and bioactivity. Here we propose an isolation strategy based on a broad-spectrum antimicrobial epsilon-poly-L-lysine (ϵ-PL) to precipitate BEVs at a relatively low centrifugal speed (10,000 × g). Compared to the standard ultracentrifugation strategy, our method can enrich BEVs from large volumes of media inexpensively and rapidly. The precipitated BEVs can be recovered by adjusting the pH and ionic strength of the media, followed by an ultrafiltration step to remove ϵ-PL and achieve buffer exchange. The morphology, size, and protein composition of the ϵ-PL-precipitated BEVs are comparable to those purified by ultracentrifugation. Moreover, ϵ-PL-precipitated BEVs retained the biological activity as observed by confocal microscopy studies. And THP-1 cells stimulated with these BEVs undergo marked reprogramming of their transcriptome. KEGG analysis of the differentially expressed genes showed that the signal pathways of cellular inflammatory response were significantly activated. Taken together, we provide a new method to rapidly enrich BEVs with high purity and bioactivity, which has the potential to be applied to BEVsrelated immune response studies. [ABSTRACT FROM AUTHOR]

Dysbetalipoproteinemia (hyperlipoproteinemia type III, HLP3) is a genetic disorder that results in the accumulation of cholesterol on highly atherogenic remnant particles. Traditionally, the diagnosis of HLP3 depended upon lipoprotein gel electrophoresis or density gradient ultracentrifugation. Because these two methods are not performed by most clinical laboratories, we describe here two new equations for estimating the cholesterol content of VLDL (VLDL-C), which can then be used for the diagnosis of HLP3. Using results from the beta-quantification (BQ) reference method on a large cohort of dyslipidemic patients (N = 24,713), we identified 115 patients with HLP3 based on having a VLDL-C to plasma TG ratio greater than 0.3 and plasma TG between 150 and 1,000 mg/dl. Next, we developed two new methods for identifying HLP3 and compared them to BQ and a previously described dual lipid apoB ratio method. The first method uses results from the standard lipid panel and the Sampson-NIH equation 1 for estimating VLDL-C (S-VLDL-C), which is then divided by plasma TG to calculate the VLDL-C/TG ratio. The second method is similar, but the Sampson-NIH equation 1 is modified or enhanced (eS-VLDL-C) by including apoB as an independent variable for predicting VLDL-C. At a cut-point of 0.194, the first method showed a modest ability for identifying HLP3 (sensitivity = 73.9%; specificity = 82.6%; and area under the curve (AUC) = 0.8685) but was comparable to the existing dual lipid apoB ratio method. The second method based on eS-VLDL-C showed much better sensitivity (96.5%) and specificity (94.5%) at a cutpoint of 0.209. It also had an excellent AUC score of 0.9912 and was superior to the two other methods in test classification. In summary, we describe two new methods for the diagnosis of HLP3. The first one just utilizes the results of the standard lipid panel and the Sampson-NIH equation 1 for estimating (VLDL-C) (S-VLDL-C) and can potentially be used as a screening test. The second method (eS-VLDL-C), in which the Sampson-NIH equation 1 is modified to include apoB, is nearly as accurate as the BQ reference method. Because apoB is widely available at most clinical laboratories, the second method should improve both the accessibility and the accuracy of the HLP3 diagnosis. [ABSTRACT FROM AUTHOR]

Dispersion of objects in a fluid phase can be classified as solutions (Gibbs free energy of mixing, ΔGmix < 0) or suspensions (ΔGmix > 0) depending on their thermodynamic stability. Small objects tend to form solutions, larger ones suspensions, e.g., molecules versus micrometer‐sized colloids. Proteins and nanomaterials fall between these two size regimes. The long‐standing issue of whether proteins and nanoparticles are dissolved or suspended remains an important research question. Here, a simple, versatile, and experimentally robust method, based on sedimentation equilibrium analytical ultracentrifugation (SE‐AUC), which can determine whether proteins, nanoparticles, or polymers form solutions or suspensions, is presented. SE‐AUC determines the osmotic pressure profile for a dispersion. Such a profile for solutions (equilibrium one‐phase systems) is independent of the initial and the operating conditions. The opposite is true for suspensions that are nonequilibrium two‐phase systems. This study proves that bovine serum albumin and lysozyme form solutions while ferritin and apoferritin form suspensions. [ABSTRACT FROM AUTHOR]

Analytical ultracentrifugation (AUC) is a critical analytical tool supporting the development and manufacture of protein therapeutics. AUC is routinely used as an assay orthogonal to size exclusion chromatography for aggregate quantitation. This article distills the experimental and analysis procedures used by the authors for sedimentation velocity AUC into a series of best-practices considerations. The goal of this distillation is to help harmonize aggregate quantitation approaches across the biopharmaceutical industry. We review key considerations for sample and instrument suitability, experimental design, and data analysis best practices and conversely, highlight potential pitfalls to accurate aggregate analysis. Our goal is to provide experienced users benchmarks against which they can standardize their analyses and to provide guidance for new AUC analysts that will aid them to become proficient in this fundamental technique. [ABSTRACT FROM AUTHOR]

[Display omitted] • Methyglyoxal (MG) is a key pathogenic molecule in chronic metabolic disease, difficult to measure. • Its end metabolite D-lactate can be used as a surrogate marker of MG fluxes. • We present a modified, optimized and validated method to measure serum D-lactate using a commercial kit. • We increased the specificity and the sensitivity of the method to 2 orders of magnitude. • It is a cost-efficient resource for researchers to explore the D-lactate pathway in metabolic disorders. Plasma D-lactate levels can be considered a surrogate indicator of MG flux. There are commercial methods specifically designed to cover pathological ranges (mmol/l in sepsis or leaky gut) that are not suitable to encompass the metabolically relevant ranges. We modified and optimized the D-lactate Colorimetric Assay kit MAK058 from Sigma adding an ultracentrifugation step, kinetic reading and increasing reaction temperature. We present a modified, optimized and validated method to measure serum D-lactate using a commercial kit to increase the sensitivity of the method to 2 orders of magnitude lower as well as minimizing interferences so that it may be used by researchers to explore the D-lactate pathway in metabolic disorders at a low cost (colorimetric method and a plate reader) and with a much higher specificity. [ABSTRACT FROM AUTHOR]

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Background/Introduction: Plasma protein binding (PPB) continues to be a key aspect of antibiotic development and clinical use. PPB is essential to understand several properties of drug candidates, including antimicrobial activity, drug-drug interaction, drug clearance, volume of distribution, and therapeutic index. Focus areas of the review: In this review, we discuss the basics of PPB, including the main drug binding proteins i.e., Albumin and α-1-acid glycoprotein (AAG). Furthermore, we present the effects of PPB on the antimicrobial activity of antibiotics and the current role of PPB in in vitro pharmacodynamic (PD) models of antibiotics. Moreover, the effect of PPB on the PK/PD of antibiotics has been discussed in this review. A key aspect of this paper is a concise evaluation of PPB between animal species (dog, rat, mouse, rabbit and monkey) and humans. Our statistical analysis of the data available in the literature suggests a significant difference between antibiotic binding in humans and that of dogs or mice, with the majority of measurements from the pre-clinical species falling within five-fold of the human plasma value. Conversely, no significant difference in binding was found between humans and rats, rabbits, or monkeys. This information may be helpful for drug researchers to select the most relevant animal species in which the metabolism of a compound can be studied for extrapolating the results to humans. Furthermore, state-of-the-art methods for determining PPB such as equilibrium dialysis, ultracentrifugation, microdialysis, gel filtration, chromatographic methods and fluorescence spectroscopy are highlighted with their advantages and disadvantages. [ABSTRACT FROM AUTHOR]

Background: Measuring lipoprotein particle concentrations may help to improve cardiovascular risk stratification. Both the lipofit (Numares) and lipoprofile (LabCorp) NMR methods are widely used for the quantification of lipoprotein particle concentrations. Objective: The aim of the present work was to perform a method comparison between the lipofit and lipoprofile NMR methods. In addition, there was the objective to compare lipofit and lipoprofile measurements of standard lipids with clinical chemistry-based results. Methods: Total, LDL, and HDL cholesterol and triglycerides were measured with ß-quantification in serum samples from 150 individuals. NMR measurements of standard lipids and lipoprotein particle concentrations were performed by Numares and LabCorp. Results: For both NMR methods, differences of mean concentrations compared to ß-quantification-derived measurements were ≤5.5% for all standard lipids. There was a strong correlation between ß-quantification- and NMR-derived measurements of total and LDL cholesterol and triglycerides (all r > 0.93). For both, the lipofit (r = 0.81) and lipoprofile (r = 0.84) methods, correlation coefficients were lower for HDL cholesterol. There was a reasonable correlation between LDL and HDL lipoprotein particle concentrations measured with both NMR methods (both r > 0.9). However, mean concentrations of major and subclass lipoprotein particle concentrations were not as strong. Conclusions: The present analysis suggests that reliable measurement of standard lipids is possible with these two NMR methods. Harmonization efforts would be needed for better comparability of particle concentration data. [ABSTRACT FROM AUTHOR]

Exosomes are extracellular endosomal nanoparticles, which are formed under complex processes during the formation of multivesicular bodies. They are also achieved from conditioned media of a variety of cell types, especially mesenchymal stem cells (MSCs). Exosomes can modulate intracellular physiological actions via signaling molecules on the surface or secretion of components to the extracellular spaces. Furthermore, they are potentially used as crucial agents for cell-free therapy; however, their isolation and characterization can be challenging. In the current study, two methods of exosome isolation have been characterized and compared using a culture media of adipose-derived mesenchymal stem cells, namely ultracentrifugation and a commercial kit; moreover, the efficiency of these two methods was highlighted in this study. Two different isolation methods of exosomes from MSCs were used to compare the efficiency of exosomes. For both isolation methods, transmission electron microscopy, dynamic light scattering (DLS), and bicinchoninic acid (BCA) assay have been performed. The electron microscopy and DLS indicated the presence of exosomes. Moreover, the kit and ultracentrifugation isolates contained approximately comparable amounts of protein measured by the BCA. Overall, the two isolation methods had similar performances. Although ultracentrifugation is used as a gold standard for exosome isolation, the commercial kit has some advantages and can be applied alternatively according to its cost-effectiveness and time-saving properties. [ABSTRACT FROM AUTHOR]

Individual single–walled carbon nanotubes (SWNTs) with distinct electronic types are crucial for the fabrication of SWNTs–based electronic and magnetic devices. Herein, the water–soluble polymethyl(1–butyric acidyl)silane (BA–PMS) was synthesized via the hydrosilylation reaction between 3–butenoic acid and polymethylsilane catalyzed by 2,2′–azodibutyronitrile. As a new dispersant, BA–PMS displayed a quite good dispersing capacity to arc–discharged SWNTs and moderate selectivity for metallic species. The application of sucrose–DGU, the density gradient ultracentrifugation with sucrose as the gradient medium, to the co–surfactants (BA–PMS and sodium dodecyl sulfonate) individually dispersed SWNTs yielded metallic SWNTs of 85.6% purity and semiconducting SWNTs of 99% purity, respectively. This work paves a path to the DGU separation of the SWNTs dispersed by polymer–based dispersants with hydrophobic alkyl chains. [ABSTRACT FROM AUTHOR]

Single nuclei RNA sequencing (snRNA-seq) has evolved as a powerful tool to study complex human diseases. Single cell resolution enables the study of novel cell types, biological processes, cell trajectories, and cell–cell signaling pathways. snRNA-seq largely relies on the dissociation of intact nuclei from human tissues. However, the study of complex tissues using small core biopsies presents many technical challenges. Here, an optimized protocol for single nuclei isolation is presented for frozen and RNAlater preserved human kidney biopsies. The described protocol is fast, low cost, and time effective due to the elimination of cell sorting and ultra-centrifugation. Samples can be processed in 90 min or less. This method is effective for obtaining normal nuclei morphology without signs of structural damage. Using snRNA-seq, 16 distinct kidney cell clusters were recovered from normal and peri-transplant acute kidney injury allograft samples, including immune cell clusters. Quality control measurements demonstrated that these optimizations eliminated cellular debris and allowed for a high yield of high-quality nuclei and RNA for library preparation and sequencing. Cellular disassociation did not induce cellular stress responses, which recapitulated transcriptional patterns associated with standardized methods of nuclei isolation. Future applications of this protocol will allow for thorough investigations of small biobank biopsies, identifying cell-specific injury pathways and driving the discovery of novel diagnostics and therapeutic targets. [ABSTRACT FROM AUTHOR]

Fuculose phosphate aldolases play an important role in glycolysis and gluconeogenesis pathways. L-fuculose 1-phosphate aldolase catalyzes the reversible cleavage of L-fuculose 1-phosphate to DHAP and L-lactaldehyde. Class II aldolases found in bacteria are linked to pathogenesis of human pathogens, and have potential applications in the biosynthesis of carbohydrates and other chiral compounds. Here we report the structure of a putative L-fuculose 1-phosphate aldolase (Kp FucA) from the nosocomial pathogen Klebsiella pneumoniae to 1.85 Å resolution. The enzyme crystallizes in space group P422 with one monomer per asymmetric unit. Analytical ultracentrifugation analysis confirms that Kp FucA is a tetramer in solution. A magnesium ion cofactor and sulfate ion were identified in the active pocket. Enzyme activity assays confirmed that Kp FcuA has a strong preference for L-fuculose 1-phosphate as a substrate, but can also catalyze the cleavage of fructose-1,6-bisphosphate and glucose-6-phosphate. This work should provide a starting point for further investigation of the role of Kp FucA in K. pneumoniae pathogenesis or in industrial applications. • The crystal structure of the putative Klebsiella pneumoniae L-fuculose 1-phosphate aldolase (Kp FucA) was determined. • Kp FucA forms a stable tetramer in solution. • The structure reveals a class II aldolase, including a metal cofactor and a conserved His-His-His catalytic triad. • Kp FcuA has a strong preference for L-fuculose 1-phosphate as a substrate. • This Kp FcuA structure provides a starting point for further investigation of its role in K. pneumoniae pathogenesis. [ABSTRACT FROM AUTHOR]

Edible plant-derived nanovesicles have been explored as effective materials for preventing colorectal cancer (CRC) incidence, dependent on gene status, as a K-Ras-activating mutation via the macropinocytosis pathway. Approximately 70% of CRC harbors the p53 mutation, which is strongly associated with a poor prognosis for CRC. However, it has not been revealed whether p53 inactivation activates the macropinocytosis pathway or not. In this study, we investigated parental cells, wild-type or null for p53 treated with Citrus limon L.-derived nanovesicles, as potential materials for CRC prevention. Using ultracentrifugation, we obtained C. limon L.-derived nanovesicles, the diameters of which were approximately 100 nm, similar to that of the exosomes derived from mammalian cells. C. limon L.-derived nanovesicles showed inhibitory effects on cell growth in not p53-wild, but also in p53-inactivated CRC cells. Furthermore, we revealed that the macropinocytosis pathway is activated by p53 inactivation and C. limon L.-derived nanovesicles were up taken via the macropinocytosis pathway. Notably, although C. limon L.-derived nanovesicles contained citrate, the inhibitory effects of citrate were not dependent on the p53 status. We thus provide a novel mechanism for the growth inhibition of C. limon L.-derived nanovesicles via macropinocytosis and expect to develop a functional food product containing them for preventing p53-inactivation CRC incidence. [ABSTRACT FROM AUTHOR]

Exosomes are secreted by different types of cells and known as biological packages. Exosomes have significant role in intercellular communications and involved in the development and progression of various diseases such as cancer. Inhibin B and anti-mullerian hormone (AMH) are markers of granulosa cell tumors (GCT) and due to the role of exosomes in the progression of cancer, in this experimental study, the effect of exosomes derived from human ovarian cancer cells on the secretion of Inhibin B and antimullerian hormone (AMH) by granulosa cells was investigated. First, A2780 human ovarian cancer cells were cultured, then the supernatant was collected to extract the exosomes by ultracentrifugation and subsequently, the extracted exosomes were checked out using dynamic light scattering (DLS) and Scanning electron microscopy (SEM). In addition, granulosa cells were isolated and cultured from the ovaries immature female Balb / C mice and treated with 25 μg/ml of exosomes derived from the ovarian cancer cell-line. Inhibin B and AMH hormones levels were then measured. The results showed the significant (P

In this study, Shiitake mushroom-derived extracellular nanovesicles (SMDENVs) were isolated from fresh Shiitake mushrooms by ultracentrifugation and sucrose gradient ultracentrifugation. The morphological characteristics of SMDENVs were investigated via Transmission Electron Microscopy and Laser Scanning Confocal Microscopy. SMDENVs were spherical, hollow, and uniform in size, with an average diameter of 177.6 ± 51.4 nm. Based on the analysis of lipidomics and proteomics, 383 lipids species and 1290 proteins were identified in SMDENVs. Compared with the conventional liposomes, SMDENVs demonstrated higher stability in different environmental conditions. Furthermore, we observed that SMDENVs were cytocompatible and inhibited the proliferation of Caco-2 cells. SMDENVs could be phagocytized by Caco-2 cells in a time-dependent manner. Further, SMDENVs also inhibited the proliferation of Caco-2 cells in a dose-dependent manner, and the half-maximal inhibitory concentration (IC 50) was 236.2 ± 3.2 μg/mL. Additionally, SMDENVs induced cellular apoptosis by increasing the levels of reactive oxygen species and decreasing the mitochondrial membrane potential. [Display omitted] • The nanovesicles were isolated from shiitake mushrooms (SMDENVs). • SMDENVs were purified by sucrose gradient ultracentrifugation. • The morphology, structure, and composition of SMDENVs were characterized. • SMDENVs showed higher stability compared with conventional liposomes. • SMDENVs could be phagocytosed by Caco-2 cells and induce cellular apoptosis. [ABSTRACT FROM AUTHOR]

In order to more fully elucidate the biogenesis of exosomes, a type of extracellular vesicles (EVs) and understand the role of EVs in disease processes, it is necessary to develop methods to capture EVs and induce the unloading of viable cargo. Traditionally, ultracentrifugation followed by chemical based EV lysis techniques is used to isolate these extracellular vesicles and release their internal content. Here, we describe a novel technique for capturing and releasing exosomal content through magnetic bead-based EV extraction coupled with electric field induced release and measurement (EFIRM). The usage of low-voltage electric fields allows the EV to be lysed without chemical treatment, and surface immobilized probes can allow for the rapid capture of the content of lysed EVs. EFIRM as an integrated EV lysing and analysis system offers great potential for the investigation of EVs in clinical and basic science contexts.

Exosomes, one type of extracellular vesicle (EV) secreted by cells, participate in intercellular communication and other biological processes as carriers of lipids, functional proteins, mRNAs, miRNAs, lncRNAs, and DNA fragments. Their presence in biofluids makes them attractive candidates as innovative clinical diagnostic tools. However, the conventional isolation and analysis of high-purity exosomes in clinical application is challenging, with traditional methods facing a number of shortcomings, including low yield or purity, long periods of processing, high cost, and difficulties in standardization. In this study, we provide an overview of commonly used exosome isolation approaches with a focus on magnetic bead-based capture, an ideal methodology with high purity and integrality of exosomes. The current challenges on exosome isolation methods are also described to highlight areas for future research and development.

Simulated SV-AUC data for an adeno-associated virus (AAV) sample consisting of four components having closely spaced sedimentation coefficients were used to develop a high-speed protocol that optimized the size distribution analysis resolution. The resulting high speed (45K rpm) SV-AUC (hs-SV-AUC) protocol poses several experimental challenges: 1) the need for rapid data acquisition, 2) increased potential for optical artifacts from steep and fast moving boundaries and 3) the increased potential for convection. To overcome these challenges the protocol uses interference detection at low temperatures and data that are confined to a limited radial-time window. In addition to providing higher resolution AAV SV-AUC data and very short run times (<20 min after temperature equilibration), the need to match the sample and reference solvent composition and meniscus positions is relaxed making interference detection as simple to employ as absorbance detection. Finally, experimental data comparing hs-SV-AUC (at 45K rpm) with standard low-speed (15K rpm) SV-AUC on the same AAV sample demonstrate the size distribution resolution improvement. These experiments also validate the use of a radial-time window and show how quickly data can be acquired using the hs-SV-AUC protocol. [Display omitted] • High-resolution SV-AUC protocol for AAV and other supramolecular structures. • High speed analytical ultracentrifugation improves AAV size distribution resolution. • High speed AAV SV-AUC leads to short run times of less than 20 minutes. • Simplifying operational procedure when doing AAV SV-AUC with interference. [ABSTRACT FROM AUTHOR]

Exosomes which are tiny extracellular vesicles (30–150 nm), transport vital proteins and gene materials such as miRNA, mRNA, or DNA, whose role in cell communication and epithelia regulation is critical. Many techniques have been developed as a result of studying exosomes' biochemical and physicochemical properties, although there is still no standard method to isolate exosomes simply with high yield. Commercial kits have gained popularity for exosome extraction despite concerns about their effectiveness in scientific research. On the other hand, ultracentrifugation remains the gold standard isolation method. This study compares these two common exosome isolation methods to determine their impact on the quality and quantity of exosomes isolated from bone marrow (BM) and Wharton's jelly (WJ)-derived mesenchymal stem cells. Isolated exosomes from the two sources of the cell's conditioned medium by two methods (polymer kit and ultracentrifuge) were characterized using western blotting, scanning electron microscopy (SEM), dynamic light scattering (DLS), and the Bradford assay. Western blot analysis confirmed separation efficiency based on CD81 and CD63 markers, with the absence of calnexin serving as a negative control. The Morphology of exosomes studied by SEM image analysis revealed a similar round shape appearance and their sizes (30–150 nm) were the same in both isolation techniques. The DLS analysis of the sample results was consistent with the SEM ones, showing a similar size range and very low disparity. The exosome protein content concentration analysis revealed that exosomes isolated by the polymer-based kits contained higher protein concentration density and purity (p <0.001). In general, though the protein yield was higher when the polymer-based kits were used, there were no significant differences in morphology, or size between WJ-derived and BM-derived exosomes, regardless of the isolation method employed. [Display omitted] • Polymer-based kit-isolated exosomes provided significantly higher protein yield compared with ultracentrifugation isolation. • Polymer-based kit-isolated and ultracentrifugation isolation techniques produced exosome with the same sizes. • Kit-based and ultracentrifugation techniques produced exosome with high homogeneous size and the lowest polydispersity index. • The SEM image analysis revealed the same morphology in exosomes produced by both methods. • The cell sources had little effect on the characteristics of exosome isolated by both methods. [ABSTRACT FROM AUTHOR]