Combination Of Ultracentrifugation Research Articles

Investigating Neuroplasticity Changes Reflected by BDNF Levels in Astrocyte-Derived Extracellular Vesicles in Patients with Depression.

To investigate the neuroplasticity hypothesis of depression by measuring brain-derived neurotrophic factor (BDNF) levels in plasma astrocyte-derived extracellular vesicles (ADEVs) and to evaluate their potential as biomarkers for depression compared with plasma BDNF levels. Thirty-five patients with major depressive disorder (MDD) and 35 matched healthy controls (HCs) were enrolled. Plasma ADEVs were isolated using a combination of ultracentrifugation and immunoaffinity capture. Isolated ADEVs were validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. BDNF levels were quantified in both ADEVs and plasma. ALG-2-interacting protein X (Alix) and cluster of differentiation 81 (CD81) levels, two established extracellular vesicle markers, were measured in ADEVs. After false discovery rate correction, patients with MDD exhibited higher CD81 levels (P FDR = 0.040) and lower BDNF levels (P FDR = 0.043) in ADEVs than HCs at baseline. BDNF levels in ADEVs normalized to CD81 (P FDR = 0.002) and Alix (P FDR = 0.040) remained consistent with this finding. Following four weeks of selective serotonin reuptake inhibitor treatment (n=10), CD81 levels in ADEVs decreased (P FDR = 0.046), while BDNF levels normalized to CD81 increased (P FDR = 0.022). BDNF levels in ADEVs were more stable than in plasma. Exploratory analysis revealed no correlation between BDNF levels in ADEVs and plasma (ρ=0.117, P = 0.334). This study provides human in vivo evidence supporting the neuroplasticity hypothesis of depression by demonstrating altered BDNF levels in ADEVs. ADEVs may be more suitable for developing biomarkers of depression than plasma-derived biomarkers.

Exosomes isolation from bovine serum: qualitative and quantitative comparison between ultracentrifugation, combination ultracentrifugation and size exclusion chromatography, and exoEasy methods.

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.

Proteomic Analysis of Human Chylomicron Remnants Isolated by Apolipoprotein B-48 Immunoprecipitation.

Postprandial hypertriglyceridemia (PHTG) is an independent risk factor for coronary heart diseases. PHTG exhibits accumulation of apoB-48 containing chylomicron remnants (CM-Rs) and apoB-100 containing VLDL remnants (VLDL-Rs), which are both known to be atherogenic. However, unlike VLDL-Rs, structural and functional characterization of CM-Rs remains to be elucidated due to challenges in separating CM-Rs from VLDL-Rs. Recently, we successfully isolated CM-Rs and VLDL-Rs utilizing anti-apoB-48 or apoB-100 specific antibodies. This study aimed to characterize the proteome of CM-Rs along with that of VLDL-Rs. Eight healthy subjects were enrolled. Venous blood was drawn 3 hours after high-fat-containing meals. We isolated CM-Rs and VLDL-Rs from sera through combination of ultracentrifugation and immunoprecipitation using apoB-48 or apoB-100 specific antibodies, followed by shotgun proteomic analysis. We identified 42 CM-Rs or VLDL-Rs-associated proteins, including 11 potential newly identified proteins such as platelet basic protein (PPBP) and platelet factor 4, which are chemokines secreted from platelets. ApoA-I, apoA-IV, and clusterin, which are also known as HDL-associated proteins, were significantly more abundant in CM-Rs. Interestingly, apoC-I, which reduces the activity of lipoprotein lipase and eventually inhibits catabolism of remnant proteins, was also more abundant in CM-Rs. Moreover, we identified proteins involved in complement regulation such as complement C3 and vitronectin, and those involved in acute-phase response such as PPBP, serum amyloid A protein 2, and protein S100-A8, in both CM-Rs and VLDL-Rs. We have firstly characterized the proteome of CM-Rs. These findings may provide an explanation for the atherogenic properties of CM-Rs.

Therapeutic potential of plant-derived extracellular vesicles as nanocarriers for exogenous miRNAs

Cell-to-cell communication strategies include extracellular vesicles (EVs) in plants and animals. The bioactive molecules in a diet rich in vegetables and fruits are associated with disease-preventive effects. Plant-derived EVs (PDEVs) are biogenetically and morphologically comparable to mammalian EVs and transport bioactive molecules, including miRNAs. However, the biological functions of PDEVs are not fully understood, and standard isolation protocols are lacking. Here, PDEVs were isolated from four foods with a combination of ultracentrifugation and size exclusion chromatography, and evaluated as vehicles for enhanced transport of synthetic miRNAs. In addition, the role of food-derived EVs as carriers of dietary (poly)phenols and other secondary metabolites was investigated. EVs from broccoli, pomegranate, apple, and orange were efficiently isolated and characterized. In all four sources, 4 miRNA families were present in tissues and EVs. miRNAs present in broccoli and fruit-derived EVs showed a reduced RNase degradation and were ferried inside exposed cells.EVs transfected with a combination of ath-miR159a, ath-miR162a-3p, ath-miR166b-3p, and ath-miR396b-5p showed toxic effects to human cells, as did natural broccoli EVs alone. PDEVs transport trace amounts of phytochemicals, including flavonoids, anthocyanidins, phenolic acids, or glucosinolates. Thus, PDEVs can act as nanocarriers for functional miRNAs that could be used in RNA-based therapy.

Optimal isolation of extracellular vesicles from pleural fluid and profiling of their microRNA cargo

AbstractPleural effusion occurs in both benign and malignant pleural disease. In malignant pleural effusions, the diagnostic accuracy and sensitivity of pleural fluid cytology is less than perfect, particularly for the diagnosis of malignant pleural mesothelioma, but also in some cases for the diagnosis of metastatic pleural malignancy with primary cancer in the lung, breast or other sites. Extracellular vesicles (EVs) carry an enriched cargo of microRNAs (miRNAs) which are selectively packaged and differentially expressed in pleural disease states. To investigate the diagnostic potential of miRNA cargo in pleural fluid extracellular vesicles (PFEVs), we evaluated methods for isolating the extracellular vesicle (EV) fraction including combinations of ultracentrifugation, size‐exclusion chromatography (SEC) and ultrafiltration (10 kDa filter unit). PFEVs were characterized by total and EV–associated protein, nanoparticle tracking analysis and visualisation by transmission electron microscopy. miRNA expression was analyzed by Nanostring nCounter® in separate EV fractions isolated from pleural fluid with or without additional RNA purification by ultrafiltration (3 kDa filter unit). Optimal PFEV yield, purity and miRNA expression were observed when PFEV were isolated from a larger volume of pleural fluid processed through combined ultracentrifugation and SEC techniques. Purification of total RNA by ultrafiltration further enhanced the detectability of PFEV miRNAs. This study demonstrates the feasibility of isolating PFEVs, and the potential to examine PFEV miRNA cargo using Nanostring technology to discover disease biomarkers.

Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography.

In recent years, the study of extracellular vesicles (EVs) in the context of various diseases has dramatically increased due to their diagnostic and therapeutic potential. Typically, EVs are isolated in vitro from the cell culture of primary cells or cell lines or from bodily fluids. However, these cell culture methods do not represent the whole complexity of an in vivo microenvironment, and bodily fluids contain a high heterogeneous population of vesicles since they originate from different tissues. This highlights the need to develop new methods to isolate EVs directly from tissue samples. In the present study, we established a protocol for isolating EVs from hepatic and adipose tissue of mice, using a combination of ultracentrifugation and iodixanol-sucrose density gradient separation. EV isolation was confirmed with EV protein marker enrichment in Western blot assays, total protein quantification, and transmission electron microscopy. Regarding the liver tissue, we additionally implemented size exclusion chromatography (SEC) to further increase the purity grade of the EVs. The successful isolation of EVs from tissue samples will allow us to uncover a more precise molecular composition and functions, as well as their role in intercellular communication in an in vivo microenvironment.

P161 Systemic Sclerosis Dermal Fibroblast-derived Exosomes trigger a Type I Interferon Response in Keratinocytes, through Tank Binding Kinase 1

Abstract Background/Aims Activation of Type I IFN response has been shown to be present in blood and tissue in systemic sclerosis (SSc) and correlates with disease pathogenesis and therapy response. It is currently unknown whether the tissue-specific Type I IFN activation is a consequence of the response observed in blood or rather its source. SSc fibroblasts share many cellular and molecular features with cancer-associated fibroblasts (CAF), and there is a wealth of evidence supporting the role of CAF in inducing Type I IFN activation in epithelial cancer cells. Here, we aimed to determine whether keratinocytes in SSc show evidence of Type I IFN activation and the potential role of SSc in the process. Methods Skin biopsies were obtained from healthy and SSc patients’ forearms and processed for immunohistochemistry and subculture of primary fibroblasts and keratinocytes. The human skin keratinocyte cell line (HaCats) were stimulated with conditioned media from healthy (n = 3) and SSc (n = 3) fibroblasts. Exosomes were isolated from healthy and SSc patient fibroblast cultured supernatant by a combination of ultracentrifugation and polymer-based precipitation and used to stimulate HaCats. Cellular exosomes were investigated by immunoblotting and electron microscopy. TANK-binding kinase (TBK) was inhibited using a small molecule inhibitor (GSK8612). RT-PCR based IFN array and western blot for pSTAT1 (Tyr701) were employed to assess Type I IFN activation in these cells. Results Consistent with previous studies we found high levels of pSTAT1 in keratinocytes from SSc patient biopsies. Contrary to observations in dermal fibroblasts, primary keratinocytes isolated from SSc skin biopsies lost IFN activation once cultured in-vitro. Conversely, conditioned media from SSc patient fibroblasts triggered ISG induction; MX1 (2.7-fold, p < 0.05), CXCL10 (2.8-fold, p < 0.01) and CXCL11 (1.9-fold, p < 0.05). Media fractionation by ultracentrifugation indicated that the effect on keratinocytes was driven mainly by pelleted micro-vesicles, suggesting a potential role for cellular exosomes. Fibroblasts from SSc patients shed cellular exosomes of similar quantities and biophysical qualities, compared to healthy. SSc exosomes induced a 2.2-fold increase in phosphorylation of STAT1 (p < 0.05), as well as an induction in ISGs; MX1 (1.45-fold, p < 0.05), CXCL10 (1.84-fold, p < 0.01), CXCL11 (2.11-fold, p < 0.01) and OAS (1.24-fold, p < 0.05). Suppression of TBK activity by 10μM of GSK8612 suppressed the induction of pSTAT1. Consistent with these findings IFN array of HaCats stimulated with SSc exosomes showed significant (p < 0.05) upregulation of 16 ISGs by at least 1.2-fold compared to the effect of healthy control exosomes. Conclusion We have shown for the first time that scleroderma keratinocytes ex-vivo IFN activation is lost in-vivo and can be regained following exposure to fibroblast-derived exosomes though a TBK-dependent mechanism. Our data indicates that exosomes from SSc fibroblasts may carry the “signal zero” of local Type I IFN activation, potentially though activation of toll-like receptors and/or pattern recognition receptors. Disclosure J.A. Bryon: None. C. Wasson: None. R.L. Ross: None. E. Zeqiraj: None. F. Del Galdo: Consultancies; FDG received consultancies and research support from Abbvie, AstraZeneca, Boheringer-Ingelheim, Capella, Chemomab, Ergomed, Janssen, Kymab, Mitsubishi-Tanabe.. Grants/research support; FDG received consultancies and research support from Abbvie, AstraZeneca, Boheringer-Ingelheim, Capella, Chemomab, Ergomed, Janssen, Kymab, Mitsubishi-Tanabe.

Comparison of proteomic landscape of extracellular vesicles in pleural effusions isolated by three strategies.

Extracellular vesicles (EVs) derived from pleural effusion (PE) is emerging as disease biomarkers. However, the methods for isolation of EVs from PE (pEVs) were rarely studied. In our study, three methods for isolating pEVs of lung cancer patients were compared, including ultracentrifugation (UC), a combination of UC and size exclusion chromatography (UC-SEC) and a combination of UC and density gradient ultracentrifugation (UC-DGU). The subpopulation of pEVs was identified by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Western blotting (WB) and nano-flow cytometry (nFCM). Additionally, the proteomic landscape of pEVs was analyzed by Label-free proteomics. The results showed that, compared with UC and UC-DGU, the UC-SEC method separated pEVs with the highest purity. In the proteomic analysis, on average, 1595 proteins were identified in the pEVs isolated by UC-SEC, much more than pEVs isolated by UC (1222) or UC-DGU (807). Furthermore, approximately 90% of identified proteins in each method were found in the EVs public database ExoCarta. Consistent with this, GO annotation indicated that the core proteins identified in each method were mainly enriched in "extracellular exosome." Many of the top 100 proteins with high expression in each method were suggested as protein markers to validate the presence of EVs in the MISEV2018 guidelines. In addition, combined with lung tissue-specific proteins and vesicular membrane proteins, we screened out and validated several novel protein markers (CD11C, HLA DPA1 and HLA DRB1), which were enriched in pEVs rather than in plasma EVs. In conclusion, our study shows that the method of UC-SEC could significantly improve the purity of EVs and the performance of mass spectrometry-based proteomic profiling in analyzing pEVs. The exosomal proteins CD11C, HLA DPA1 and HLA DRB1 may act as potential markers of pEVs. The proteomic analysis of pEVs provides important information and new ideas for studying diseases complicated with PE.

TMT-Based Quantitative Proteomics Analysis of Synovial Fluid-Derived Exosomes in Inflammatory Arthritis.

ObjectivesTo compare the proteomics of synovial fluid (SF)-derived exosomes in rheumatoid arthritis (RA), axial spondyloarthritis (axSpA), gout, and osteoarthritis (OA) patients.MethodsExosomes were separated from SF by the Exoquick kit combined ultracentrifugation method. Tandem mass tags (TMT)-labeled liquid chromatography mass spectrometry (LC-MS/MS) technology was used to analyze the proteomics of SF-derived exosomes. Volcano plot, hierarchical cluster, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted.ResultsA total of 1,678 credible proteins were detected. Sixty-nine differentially expressed proteins were found in gout, compared with OA, axSpA, and RA simultaneously. Twenty-five proteins were found highly expressed in gout uniquely, lysozyme C and protein S100-A9 included, whose bioinformatic analysis was significantly involved in “neutrophil degranulation” and “prion diseases”. Eighty-four differentially expressed proteins were found in axSpA, compared with OA, gout, and RA simultaneously. Thirty-nine proteins were found highly expressed in axSpA uniquely, RNA-binding protein 8A and protein transport protein Sec24C included, whose bioinformatic analysis was significantly involved in “acute-phase response” and “citrate cycle”. One hundred and eighty-four differentially expressed proteins were found in RA, compared with OA, gout, and axSpA simultaneously. Twenty-eight proteins were found highly expressed in RA uniquely, pregnancy zone protein (PZP) and stromelysin-1 included, whose bioinformatic analysis was significantly involved in “serine-type endopeptidase inhibitor activity” and “complement and coagulation cascades”. Enzyme-linked immunosorbent assay (ELISA) result showed that the exosome-derived PZP level of SF in RA was higher than that in OA (p < 0.05).ConclusionOur study for the first time described the protein profiles of SF-derived exosomes in RA, axSpA, gout, and OA patients. Some potential biomarkers and hypothetical molecular mechanisms were proposed, which may provide helpful diagnostic and therapeutic insights for inflammatory arthritis (IA).

Prognostic significance of exosomal programmed death-ligand 1 in advanced gastric cancer patients treated with first-line chemotherapy.

Retraction The abstract by Shin et al entitled, “Prognostic significance of exosomal programmed death-ligand 1 in advanced gastric cancer patients treated with first-line chemotherapy,” ( Journal of Clinical Oncology 40, no. 4, suppl 665) published on February 1, 2022, has been retracted due to statistical errors that significantly changed the results and conclusion of the abstract. This abstract was retracted on November 4, 2022. 665 Background: Recent studies have found that exosomal PD-L1 could be associated with prognosis in several malignancies by its potential immunosuppressive role. Prognostic role of exosomal PD-L1 in advanced gastric cancer patients treated with systemic chemotherapy has not been well explored. The aim of the present study was to explore prognostic and predictive significance of exosomal PD-L1 in advanced gastric cancer patients. Methods: We prospectively collected plasma samples of patients with advanced gastric cancer receiving first-line chemotherapy at pre and post treatment. Combined ultracentrifugation with polymer-based precipitation was applied to isolate exosome and enzyme-linked immunosorbent assay(ELISA) was applied to measure exosomal PD-L1. The correlation between exosomal PD-L1 and circulating immune cells in the plasma was evaluated. Patients were divided into two groups according to pretreatment exosomal PD-L1 level. Survival analysis was performed using Kaplan-Meier method and multivariate analyses based on Cox-proportional hazards regression model. Results: A total of 99 patients were enrolled in the study, and serum samples collected at pre-treatment and post-treatment were pooled. Median value of the pre-treatment exosomal PD-L1 was 0.42 pg/ml (range 0.01 ̃3.75). Patients with pre-treatment exoPD-L1 &lt; 0.42 pg/mL (exoPD-L1low) showed significantly better overall survival than those with exoPD-L1 ≥ 0.42 pg/mL(exoPD-L1high) (p = 0.033). Progression-free survival was not different between the groups (p = 0.754). exoPD-L1lowgroup showed significantly higher percentages of CD8+T cells than exoPD-L1high group. (p = 0.024). In multivariate analysis, pretreatment exosomal PD-L1, poorly differentiated tumors and No. of metastatic sites(≥2) were independent prognostic factors for overall survival. Conclusions: In summary, the level of pre-treatment exosomal PD-L1 could be used as a prognostic marker for patients with advanced gastric cancer receiving systemic chemotherapy.

Bovine Milk-Derived Exosomes as a Drug Delivery Vehicle for miRNA-Based Therapy

MicroRNAs (miRNAs) are small non-coding RNAs with a known role as mediators of gene expression in crucial biological processes, which converts them into high potential contenders in the ongoing search for effective therapeutic strategies. However, extracellular RNAs are unstable and rapidly degraded, reducing the possibility of successfully exerting a biological function in distant target cells. Strategies aimed at enhancing the therapeutic potential of miRNAs include the development of efficient, tissue-specific and nonimmunogenic delivery methods. Since miRNAs were discovered to be naturally transported within exosomes, a type of extracellular vesicle that confers protection against RNase degradation and increases miRNA stability have been proposed as ideal delivery vehicles for miRNA-based therapy. Although research in this field has grown rapidly in the last few years, a standard, reproducible and cost-effective protocol for exosome isolation and extracellular RNA delivery is lacking. We aimed to evaluate the use of milk-derived extracellular vesicles as vehicles for extracellular RNA drug delivery. With this purpose, exosomes were isolated from raw bovine milk, combining ultracentrifugation and size exclusion chromatography (SEC) methodology. Isolated exosomes were then loaded with exogenous hsa-miR148a-3p, a highly expressed miRNA in milk exosomes. The suitability of exosomes as delivery vehicles for extracellular RNAs was tested by evaluating the absorption of miR-148a-3p in hepatic (HepG2) and intestinal (Caco-2) cell lines. The potential exertion of a biological effect by miR-148a-3p was assessed by gene expression analysis, using microarrays. Results support that bovine milk is a cost-effective source of exosomes which can be used as nanocarriers of functional miRNAs with a potential use in RNA-based therapy. In addition, we show here that a combination of ultracentrifugation and SEC technics improve exosome enrichment, purity, and integrity for subsequent use.

Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues.

Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches—(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols.

Proteomic Analysis Reveals Proteins and Pathways Associated with Lactation in Bovine Mammary Epithelial Cell-Derived Exosomes.

Milk-derived exosomes have been reported, which are involved in many biological processes. The exosomes derived from mammary glands are not known yet, and their relationship with mammary gland lactation and the origin of milk-derived exosomes are largely unclear. The present study aimed to investigate the proteome of exosomes derived from bovine mammary epithelial cells (BMECs) and compare them with milk-derived exosomes in the database. BMEC-derived exosomes were successfully separated from the culture supernatant of BMECs by a combined ultracentrifugation approach, and the purity of exosomes was identified by western blot analysis. Liquid chromatography with tandem mass spectrometry identified 638 proteins in BMEC-derived exosomes. The MS data were deposited into the PUBLIC repository ProteomeXchange, dataset identifier(s): https://www.iprox.org/page/PSV023.html;?url=1590961453176tKpa. Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these proteins were associated with specific biological processes and molecular functions of metabolism. Cross comparison of these proteins with the protein database of milk exosomes showed that 77 common expressed proteins (CEPs) were in both BMEC- and milk-derived exosomes. The KEGG pathway analysis for these CEPs showed that they were mainly involved in signaling pathways associated with milk biosynthesis in BMECs. Among these CEPs, six proteins have been previously reported to be associated with the lactation function. The western blot analysis detected that expression of these six proteins in BMEC-derived exosomes was increased after the stimulation of methionine and β-estradiol on BMECs. In summary, the proteome of BMEC-derived exosomes reveals that they are associated with milk biosynthesis in BMECs and might be a source of milk-derived exosomes.

Accurate quantification of TiO2 nanoparticles in commercial sunscreens using standard materials and orthogonal particle sizing methods for verification

The implementation and enforcement of product labeling obligation as required, for example, by the cosmetic product regulation, needs simple and precise validated analytical methods. This also applies to the analysis of nanoparticles in products such as cosmetics. However, the provision of such methods is often hampered by inaccurate sizing due to unwanted nanoparticle changes, interference of matrix components with sizing and interactions between nanoparticles and analytical instrumentation. It is, therefore, necessary to develop appropriate sample preparation methods that preserve NP properties and reduce or remove matrix compounds that interfere with sizing. Further, accurate particle size analysis of samples containing unknown and possibly multiple nanoparticulate constituents is needed. In this study, we evaluated three sample preparation methods to identify and quantify TiO2 nanoparticles in sunscreens. Specifically, we used a combination of ultracentrifugation and hexane washing, thermal destruction of the matrix, and surfactant assisted particle extraction. The method accuracy was assessed by two internal reference samples: pristine TiO2 nanoparticles (NM104) and similar TiO2 nanoparticles dispersed in a sunscreen matrix. The PSDs were determined using an asymmetrical flow field-flow fractionation hyphenated with multi-angle light scattering and inductively coupled plasma-mass spectroscopy. Particle sizing was based on size calibration of the particle retention time in the AF4. Computation of radius of gyration from MALS data was used as an orthogonal particle sizing approach to verify ideal elution and particle size data from the AF4 calibration. Among the three tested sample preparation methods surfactant assisted particle extraction revealed TiO2 nanoparticle recoveries of above 90% and no increase in particle size due to sample preparation was observed. Finally, the sample preparation methods were applied to two commercial sunscreen samples revealing the existence of TiO2-NP < 100 nm. Conclusively, the surfactant assisted particle extraction method can provide valid data for TiO2-NPs in sunscreen and possibly for cosmetic samples of similar matrix.

Isolation of Small Extracellular Vesicles From Human Serum Using a Combination of Ultracentrifugation With Polymer-Based Precipitation.

Methods for reproducibly isolating and enriching small extracellular vesicles (EVs) from blood are essential for clinical utilization of small EVs in cancer patients. We combined ultracentrifugation (UC) with polymer-based precipitation (ExoQuick [EQ] or Total Exosome Isolation [TEI] kit) to isolate small EVs (diameter, 30–150 nm) from the serum of breast cancer patients. We compared the performance of four cycles of UC (UC4x) with that of two cycles of UC followed by enrichment using the EQ (UC2x→EQ) or TEI (UC2x→TEI) kits. The mean concentration of small EVs isolated from 1 mL of serum using UC2x→EQ (139.0±29.1 µg) and UC2x→TEI (140.4±5.0 µg) did not differ from that obtained using UC4x (141.8±26.9 µg). The mean number of EV particles obtained using UC4x was 29.2±9.9×109 per mL of serum, whereas UC2x→EQ and UC2x→TEI yielded higher numbers of EVs (50.7±17.0×109 and 59.3±20.6×109, respectively). Concentrations of EV microRNAs, including miR-21 and miR-155, did not differ between the three methods. In conclusion, performing UC prior to the use of polymer-based precipitation kits could be feasible for isolating small EVs from human serum in large sample-based translational researches.

An Evaluation of Palladin Overexpression in Pancreatic Cancer Cells

Pancreatic cancer (PC) is the fourth leading cause of cancer in United States with overall survival rates of less than 5% (ACS, 2018). Its aggressive nature and late onset of physical symptoms lead to poor prognoses. Recognition of biomarkers is clearly an imperative undertaking for the disease. To tackle this problem, we evaluated the secreted protein profile of pancreatic cancer cell lines derived from different stages. In total, nine human pancreatic cancer cell lines were successfully adapted into serum‐free environments from which conditioned media was collected by a combination of ultracentrifugation, molecular weight cutoff, protein precipitation, gel‐based separation, and tryptic digestion followed by mass spectrometry protein identification. A small subset of proteins has been identified and validated biochemically including Palladin (PALLD), a pancreatic cancer susceptibility protein shown to be up‐regulated in previous studies. PALLD is a scaffolding protein that supports remodeling of the actin cytoskeleton in association with integrins at focal adhesion sites to coordinate cell migration. We demonstrate a possible interaction between PALLD and the beta5 integrin (ITGB5) indicative of the increased motile phenotype in PC. Taken together, our results suggest that PALLD overexpression is associated with enhanced motility in pancreatic cancer cells and could represent a potential biomarker or therapeutic strategy against pancreatic cancer progression.Support or Funding InformationThis work was funded in part through intramural programs at Fresno State to KV, RCB, JAB, and through NCI P20CA138025 to JAB.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proteome profiling of exosomes derived from plasma of heifers with divergent genetic merit for fertility

The current study evaluated exosomes isolated from plasma of heifers bred to have high or low fertility through developing extreme diversity in fertility breeding values, however, key animal traits (e.g., body weight, milk production, and percentage of North American genetics) remained similar between the 2 groups. The exosomes were isolated by a combined ultracentrifugation and size exclusion chromatography approach and characterized by their size distribution (nanoparticle tracking analysis), morphology (transmission electron microscopy), and presence of exosomal markers (immunoblotting). In addition, a targeted mass spectrometry approach was used to confirm the presence of 2 exosomal markers, tumor susceptibility gene 101 and flotillin 1. The number of exosomes from plasma of high fertility heifers was greater compared with low fertility heifers. Interestingly, the exosomal proteomic profile, evaluated using mass spectrometry, identified 89 and 116 proteins in the high and low fertility heifers respectively, of which 4 and 31 were unique, respectively. These include proteins associated with specific biological processes and molecular functions of fertility. Most notably, the tetratricopeptide repeat protein 41-related, glycodelin, and kelch-like protein 8 were identified in plasma exosomes unique to the low fertility heifers. These proteins are suggested to play a role in reproduction; however, the role of these proteins in dairy cow reproduction remains to be elucidated. Their identification underscores the potential for proteins within exosomes to provide information on the fertility status and physiological condition of the cow. This may potentially lead to the development of prognostic tools and interventions to improving dairy cow fertility.

Effect of carrageenan addition on the rennet-induced gelation of skim milk.

Carrageenan (CG) (κ-CG, ι-CG and λ-CG) was added to skim milk and the rennet-induced aggregation was studied. Caseinomacropeptide (CMP) release, diffusing wave spectroscopy (DWS) and rheology were used to follow the structural dynamics of casein micelles during gelation. The influence of carrageenan on the nature of protein interactions in the gels was investigated using a combination of ultracentrifugation and specific dissociating agents. For the recombined samples containing κ-CG and low concentrations of ι-CG and λ-CG, the CMP release was slowed down; however, the development of DWS and rheological parameters was similar to that of the control sample, and the increase in the incorporation of proteins through calcium bridges and hydrophobic interactions may be the most likely contributors. For the recombined samples containing high concentrations of ι-CG and λ-CG, other factors may impede the gel formation process. High concentrations of ι-CG and λ-CG strongly interfered with the rennet-induced aggregation, interrupted the interaction of caseins and therefore may contribute to good quality of low-fat cheese. © 2016 Society of Chemical Industry.

Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array

Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen–antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios.

Combination of ultracentrifugation and solid-phase extraction with subsequent chromatographic analysis of α-tocopherol in erythrocyte membranes.

A novel and rapid sample pretreatment technique based on a combination of ultracentrifugation and solid-phase extraction for the determination of α-tocopherol in human erythrocyte membranes by high-performance liquid chromatography with ultraviolet detection is presented in this work. Red blood cell samples were ultracentrifuged (288 000 × g, 3 min, 4°C) in the presence of d-mannitol, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and calcium chloride. The α-tocopherol was then extracted from the erythrocyte membranes by solid-phase extraction with n-hexane in the presence of ascorbic acid. Tocopherol acetate was used as the internal standard. The extract was dissolved in methanol and separated on the monolithic column Chromolith Performance RP-18e (100 × 4.6 mm) using 100% methanol as the mobile phase. The absorbance of α-tocopherol was measured at a wavelength of 295 nm. The method was validated and showed sufficient accuracy and precision, ranging from 96.4 to 100.8% and from 4.5 to 6.3%, respectively. Moreover, the developed method was applied to the determination of erythrocyte α-tocopherol in real samples from patients. The combined ultracentrifugation and solid-phase extraction technique substantially decreased the time for the sample pretreatment step compared to liquid-liquid extraction and could be applicable for the quantitation of other analytes in erythrocyte membranes.