Size Exclusion Chromatography Techniques Research Articles

Propagation rate coefficients of 2-(dimethylamino)ethyl methacrylate radical polymerization in bulk, alcohols and alcohol/water solutions determined by PLP-SEC

The pulsed laser polymerization – size exclusion chromatography technique is used to investigate the radical polymerization propagation kinetics of 2-(dimethylamino)ethyl methacrylate in n-butanol and ethanol (EtOH), as well as EtOH/water mixtures.

The Proteomic Signature of Extracellular Vesicles from Sickle Cell Anemia Patients Provides Insights into Their Possible Role in the Pathophysiology of the Disease

Sickle cell anemia (SCA) is marked by hemolysis and vaso-occlusion, which contribute to various other complications observed in the disease. Extracellular vesicles (EVs), released by activated cells, serve as carriers of various markers, proteins, and RNA derived from their parent cells. These structures have the ability to transfer their cargo to the recipient cells, modulating their activity. Despite EVs involvement in physiological processes, there is growing evidence of their role in certain blood diseases, making them a subject of extensive research in recent years. In sickle cell disease (SCD), EVs have been previously associated with hemolysis markers, leg ulcers, and endothelial cell activation and several other authors have emphasized the significant role of EVs in its pathophysiology. However, despite these advances, the precise protein content of these EVs, which may hold the key to unraveling their involvement in disease complications, remains largely unexplored. Here, we aimed to characterize the proteomic profile of EVs from patients with SCA. We conducted a comparative analysis, comparing the differentially abundant proteins in the SCA group to those in the control group. Additionally, we focused on analyzing the distinct protein profiles that were uniquely present in the SCA group studied. Nine healthy volunteers (HC) and nineteen patients with SCA were included in the study, with three receiving no treatment, twelve undergoing hydroxyurea (HU) therapy, and four on HU+Crizanlizumab (CRIZ) combination treatment. EVs were isolated from plasma using a combination of differential centrifugation and size exclusion chromatography techniques, and their quantification was performed using nano-tracking analysis. The protein profile of EVs was assessed through mass spectrometry after sample digestion and desalination in a developed protocol for EVs research. The obtained data were analyzed using MaxQuant and Perseus software. To determine differentially abundant proteins between the HC and SCA groups, statistical tests such as ANOVA followed by Benjamini-Hochberg correction were applied, considering a significance level of p<0.05. Additionally, gene ontology analysis was conducted to identify the relevant biological processes (FDR <0,05) associated with the differentially abundant proteins. Firstly, we identified 41 unique proteins in the SCA group. The relevant biological processes associated with these proteins are primarily related to erythroid differentiation, inflammation, cellular response to oxidative stress, and the upregulation of signaling pathways mediated by TNF and other cytokines. These findings align with the characteristic pathophysiology of SCA. The proteins found in higher concentrations in EVs of SCA patients compared to HC group (p<0,05) were mainly related to endocytosis and exocytosis, inflammation, immune response and coagulation process. Moreover, we observed higher levels of hemoglobin chains in SCA EVs. The delta chain was increased in the SCA group with no treatment in relation to the HC group and the SCA group with HU. Beta and alpha chains were also found in increased levels in the SCA group without HU compared to the HC group, SCA with HU and SCA with HU+CRIZ. Additionally, both transferrin (TF) and the transferrin receptor (TFRC-CD71) were increased in all three subgroups of SCA patients compared to the HC group. The identification of various types of hemoglobin in these EVs holds significant importance. This observation aligns with the potential role of these structures in promoting the generation of reactive oxygen species (ROS), activating endothelial cells, and reducing nitric oxide levels in individuals with SCA. Such findings may provide valuable insights into the underlying mechanisms contributing to the pathophysiology of SCA and its associated complications. Moreover, our data shows higher levels of Tenascin-C in the EVs of SCA patients (all subgroups) (p<0,05), a protein related to tissue damage and inflammation that has not yet been described in the pathophysiology of the disease. In summary, we access, for the first time, the protein content of EVs from patients with SCA. The data described here are novel and could help to elucidate the putative functions of EVs in the context of SCA providing valuable insights for future investigations and potential therapeutic strategies.

An iota-carrageenan isolated from marine alga Agardhiella ramosissima negatively modulates the inflammatory response in arthritis conditions

The present study involves the structural characterization of an iota-carrageenan from Agardhiella ramosissima (IC-Ar) and its ability to suppress the inflammatory response in experimental arthritis. The IC-Ar was structurally characterized by Size Exclusion Chromatography (SEC) and Nuclear Magnetic Resonance (NMR) techniques. Experimentally, the mice were treated with IC-Ar (3, 10, or 30 mg/kg) e submitted to arthritis with zymosan and Complete Freund's Adjuvant (CFA). IC-Ar has a molecular mass of 730 KDa and contains 3,6 anhydro-α-d-galactose-2 sulfate (DA2S), β-d-galactose-4 sulfate (G4S) and glucose, structural characteristic typical of an iota carrageenan. IC-Ar treatment reduced zymosan-induced hypernociception, in a dose-dependent manner, accompanied by reduced edema, vascular permeability, and histopathological scores. Likewise, IC-Ar also significantly decreases MPO activity, leukocytes, neutrophils, IL-1β, and nitrite levels in the synovial fluid. Therapeutically, IC-Ar inhibited the hypernociception, edema, and arthritic index in both acute, sub-chronic, and chronic phases of CFA-induced arthritis. In summary, we demonstrated the protective efficacy of an iota-carrageenan extracted from A. ramosissima in arthritis models by negatively modulating the inflammatory response and by reducing polymorphonuclear cell migration, IL-1β, and nitrite levels, culminating in the inhibition of hypernociception and edema. Thus, IC-Ar could be a promising molecule to treat arthritis conditions.

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.

Dual roles of fucoidan-GPIbα interaction in thrombosis and hemostasis: implications for drug development targeting GPIbα

BackgroundPlatelet GPIbα–von Willebrand factor (VWF) interaction initiates platelet adhesion, activation, and thrombus growth, especially under high shear conditions. Therefore, the GPIb–VWF axis has been suggested as a promising target against arterial thrombosis. The polysaccharide fucoidan has been reported to have opposing prothrombotic and antithrombotic effects; however, its binding mechanism with platelets has not been adequately studied. ObjectiveThe objective of this study was to explore the mechanism of fucoidan and its hydrolyzed products in thrombosis and hemostasis. MethodsNatural fucoidan was hydrolyzed by using hydrochloric acid and was characterized by using size-exclusion chromatography, UV-visible spectroscopy, and fluorometry techniques. The effects of natural and hydrolyzed fucoidan on platelet aggregation were examined by using platelets from wild-type, VWF and fibrinogen-deficient, GPIbα-deficient, and IL4Rα/GPIbα-transgenic and αIIb-deficient mice and from human beings. Platelet activation markers (P-selectin expression, PAC-1, and fibrinogen binding) and platelet-VWF A1 interaction were measured by using flow cytometry. GPIbα–VWF A1 interaction was evaluated by using enzyme-linked immunosorbent assay. GPIb-IX–induced signal transduction was detected by using western blot. Heparinized whole blood from healthy donors was used to test thrombus formation and growth in a perfusion chamber. ResultsWe found that GPIbα is critical for fucoidan-induced platelet activation. Fucoidan interacted with the extracellular domain of GPIbα and blocked its interaction with VWF but itself could lead to GPIbα-mediated signal transduction and, subsequently, αIIbβ3 activation and platelet aggregation. Conversely, low-molecular weight fucoidan inhibited GPIb-VWF–mediated platelet aggregation, spreading, and thrombus growth at high shear. ConclusionFucoidan-GPIbα interaction may have unique therapeutic potential against bleeding disorders in its high-molecular weight state and protection against arterial thrombosis by blocking GPIb–VWF interaction after fucoidan is hydrolyzed.

Balancing ring and stopper group size to control the stability of doubly threaded [3]rotaxanes.

Synthesizing doubly threaded [3]rotaxanes requires the use of larger rings than more traditional singly threaded [2]rotaxanes. A key challenge in accessing stable doubly threaded [3]rotaxanes with large rings is finding the right combination of ring to stopper size. In this study, a series of doubly threaded [3]rotaxanes derived from five different sized macrocycles in the size range of 40-48 atoms and two different stopper groups, which contain 1 or 2 tris(p-t-butylbiphenyl)methyl moieties, were prepared and their kinetic stability examined. These interlocked compounds were synthesized using a metal-templated approach and fully characterized utilizing a combination of mass spectrometry, NMR spectroscopy, and size-exclusion chromatography techniques. The effect of ring size on the stability of the doubly threaded [3]rotaxane was investigated via kinetic stability tests monitored using 1H-NMR spectroscopy. By tightening the macrocycle systematically every 2 atoms from 48 to 40 atoms, a wide range of doubly threaded interlocked molecules could be accessed in which the rate of room temperature slippage of the macrocycle from the dumbbells could be tuned. Using the larger stopper group with a 48-atom ring results in no observable rotaxane, 46-44 atom macrocycles result in metastable rotaxane species with a slippage half-life of ∼5 weeks and ∼9 weeks, respectively, while macrocycles of 42 atoms or smaller yield a stable rotaxane. The smaller sized stopper is not able to fully stabilize any of the [3]rotaxane structures but metastable [3]rotaxanes are obtained with slippage half-lives of 25 ± 2 hours and 13 ± 1 days using macrocycles with 42 or 40 atoms, respectively. These results highlight the dramatic effect that relatively small ring size changes can have on the structure of doubly threaded [3]rotaxanes and lay the synthetic groundwork for a range of higher order doubly threaded interlocked architectures.

Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell

The spliceosome, responsible for all mature protein-coding transcripts of eukaryotic intron-containing genes, consists of small uridine-rich nuclear ribonucleoproteins (UsnRNPs). The assembly of UsnRNPs depends, on one hand, on the arginine methylation of Sm proteins catalyzed by the PRMT5 complex. On the other hand, it depends on the phosphorylation of the PRMT5 subunit pICln by the Uncoordinated Like Kinase 1 (ULK1). In consequence, phosphorylation of pICln affects the stability of the UsnRNP assembly intermediate, the so-called 6 S complex. The detailed mechanisms of phosphorylation-dependent integrity and subsequent UsnRNP assembly of the 6 S complex in vivo have not yet been analyzed.By using a phospho-specific antibody against ULK1-dependent phosphorylation sites of pICln, we visualize the intracellular distribution of phosphorylated pICln. Furthermore, we detect the colocaliphosphor-pICln1 with phospho-pICln by size-exclusion chromatography and immunofluorescence techniques. We also show that phosphorylated pICln is predominantly present in the 6 S complex. The addition of ULK1 to in vitro produced 6 S complex, as well as the reconstitution of ULK1 in ULK1-deficient cells, increases the efficiency of snRNP biogenesis. Accordingly, inhibition of ULK1 and the associated decreased pICln phosphorylation lead to accumulation of the 6 S complex and reduction in the spliceosomal activity of the cell.

Optimizing Concentration of Polyethelene Glycol for Exosome Isolation from Plasma for Downstream Application.

Exosomes are ubiquitous extracellular nanovesicles secreted from almost all living cells that are thought to be involved in several important cellular processes, including cell-cell communication and signaling. Exosomes serve as a liquid biopsy tool for clinical and translational research. Although many techniques have been used to isolate exosomes, including ultracentrigation, size-exclusion chromatography, and immunocapturing-based techniques, these techniques are not convenient, they require expensive instrumentation, and they are unhandy for clinical samples. Precipitation techniques from available commercial kits that contain polyethelene glycol (PEG) are now widely used, but these kits are expensive, especially if a large number of biological samples are to be processed. the purpose of this study is to compare and optimize the efficacy of different concentrations of PEG with two commercial kits ExoQuick (SBI) and Total Exosome Isolation (TEI) from Invitrogen in human plasma. we determined exosome quantity, size distribution, marker expression, and downstream application. among the precipitation methods, we found the size of particles and concentrations with 10-20% PEG are similar to ExoQuick and better than TEI. Interestingly, we detected cfDNA with ExoQuick and 10-20% PEG but not TEI and 5% PEG. Moreover, 10% PEG detection of miR-122 and miR-16 expression was superior to ExoQuick and TEI. Furthermore, in proteomics results it also found the identified proteins better than commercial kits but there was a high level of contamination of other proteins in serum. together, these findings show that an optimal concentration of 10% PEG serves as a guide for use with clinical samples in exosome isolation for downstream applications.

On the Precipitation Copolymerization of 1‐Octene and Glycidyl Methacrylate in Supercritical Carbon Dioxide

The free radical precipitation copolymerization of the polar monomer glycidyl methacrylate (GMA) and the non-polar monomer 1-octene (Oct) in supercritical carbon dioxide (sc-CO2) is reported under different conditions of pressure and temperature. According to 1H NMR studies, in the obtained statistical copolymers (GMA-stat-Oct) the molar fraction of the olefin (FOct) increases when the pressure and temperature of the reaction increased from the reference values T = 125 °C and P = 34 bar. In particular, when the initial molar fraction of Oct (fOct) is 0.83, the molar fraction of this monomer in the final copolymer reaches a maximum value of 0.22 at 124 bar and T = 155 °C. On the other hand, for fOct = 0.89, it was obtained that FOct = 0.25 at 124 bar and T = 155 °C. Copolymers obtained were characterized by NMR, SEM and SEC techniques. Finally, two copolymers were selected to demonstrate the feasibility of modifying the GMA moiety in order to introduce OH polar groups giving rise to Oct-stat-glycerol methacrylate copolymers. This article is protected by copyright. All rights reserved

Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B-Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods.

Extracellular vesicles (EVs) are a versatile group of cell-secreted membranous nanoparticles present in body fluids. They have an exceptional diagnostic potential due to their molecular content matching the originating cells and accessibility from body fluids. However, methods for EV isolation are still in development, with size exclusion chromatography (SEC) emerging as a preferred method. Here we compared four types of SEC to isolate EVs from the CSF of patients with severe traumatic brain injury. A pool of nine CSF samples was separated by SEC columns packed with Sepharose CL-6B, Sephacryl S-400 or Superose 6PG and a ready-to-use qEV10/70 nm column. A total of 46 fractions were collected and analysed by slot-blot followed by Ponceau staining. Immunodetection was performed for albumin, EV markers CD9, CD81, and lipoprotein markers ApoE and ApoAI. The size and concentration of nanoparticles in fractions were determined by tunable resistive pulse sensing and EVs were visualised by transmission electron microscopy. We show that all four SEC techniques enabled separation of CSF into nanoparticle- and free protein-enriched fractions. Sepharose CL-6B resulted in a significantly higher number of separated EVs while lipoproteins were eluted together with free proteins. Our data indicate that Sepharose CL-6B is suitable for isolation of EVs from CSF and their separation from lipoproteins.

Temperature and pH-Dependent Behaviors of mAb Drugs: A Case Study for Trastuzumab

The distortions in the high-order structure of therapeutic monoclonal antibodies (mAbs) under different environmental conditions acutely affect mAb stability, resulting in altered safety, efficacy, and shelf-life profiles. The overall stability of mAbs depends on many factors, and it requires complementary techniques for an in-depth analysis. The stability of mAbs can be characterized by differential centrifugal sedimentation (DCS), differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), and size exclusion chromatography (SEC) techniques. In this report, temperature-ramped dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy were employed as complementary tools to show how temperature and pH affect the aggregation of a model mAb, trastuzumab, in solution. The results showed that the aggregation onset temperature of trastuzumab defined by DLS was 75 °C, which decreases the amount of β-sheets and causes a slight increase in helix structures. Moreover, the melting temperature of trastuzumab was determined to be between 80–83 °C by temperature-ramped CD spectrophotometry, which is in line with the Tm of trastuzumab’s Fab region tested with DSC. Thus, unfolding and aggregation of trastuzumab start simultaneously at 75 °C, and unfolding triggers the aggregation. The temperature-ramped CD and DLS methods are robust tools to determine the thermal behavior of biosimilars in various solution conditions. Their complementary usage provides solid scientific background for regulatory applications and a better understanding of mAb instability and its relationship with structural changes.

Purification and characterization of antibacterial surfactin isoforms produced by Bacillus velezensis SK

Bacillus velezensis SK having broad-spectrum antimicrobial activity has been isolated from soil. The efficient extraction of antimicrobial compounds produced in various mediums has been done using Diaion HP-20 resin. Further, characterization of an antimicrobial compound by TLC, FTIR, in-situ bioautography analysis revealed the presence of cyclic lipopeptides, which is then purified by the combination of silica gel, size exclusion, dual gradient, and RP-HPLC chromatography techniques. Growth kinetic studies showed that Bacillus velezensis SK produces a mixture of lipopeptides (1.33 gL−1). The lipopeptide exhibits good pH (2–10) and temperature stability up to 80 °C. LC–ESI–MS analysis of partially purified lipopeptide identified variant of surfactin, further analysis of purified chromatographic fractions revealed the occurrence of most abundant C15-surfactin homologues (m/z 1036.72 Da). The isolated surfactin exhibits good antimicrobial activity (1600 AU/ml) against drug-resistant food-born B. cereus and human pathogen Staphylococcus aureus. Hence, identified strain B. velezensis SK and its potent antibacterial surfactin lipopeptide could be used in various food and biomedical applications.

Green Eugenol Oligomers as Corrosion Inhibitors for Carbon Steel in 1M HCl

This study investigated the corrosion inhibition of polyeugenol on API P110 in 1 M HCl. Its monomer – eugenol – is obtained from clove and cinnamon, making the oligomer environmentally friendly. The influence of molecular weight and polymerization degree was evaluated by increasing the polymerization initiator/monomer ratio. This enabled the polymerization of three different oligomers (P10, P20 and P30), which were characterized by Fourier-transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H-NMR), thermogravimetric analysis (TGA/DTG), and size exclusion chromatography techniques. The inhibition performance of polyeugenol was determined by weight loss (WL) and electrochemical tests to assess the influence of structural differences. WL indicated that the oligomer with the lowest polymerization degree (P10) exhibited greater efficiency (82-84%) due to more uniform surface coverage. Electrochemical tests confirmed polyeugenol as a mixed inhibitor. Energy-dispersive X-ray spectroscopy (EDX) calculations indicated the presence of an organic layer covering the steel surface.

Biochemical characterization and insecticidal activity of isolated peptides from the venom of the scorpion Centruroides tecomanus

The venom of scorpions is a mixture of components that constitute a source of bioactive molecules. The venom of the scorpion Centruroides tecomanus contains peptides toxic to insects, however, to date no toxin responsible for this activity has yet been isolated and fully characterized. This communication describes two new peptides Ct-IT1 and Ct-IT2 purified from this scorpion. Both peptides contain 63 amino acids with molecular weight 6857.85 for Ct-IT1 and 6987.77 Da for Ct-IT2. The soluble venom was separated using chromatographic techniques of molecular size exclusion, cationic exchange, and reverse phase chromatography, allowing the identification of at least 99 components of which in 53 the insecticidal activity was evaluated. The LD50 determined for Ct-IT1 is 3.81 μg/100 mg of cricket weight, but low amounts of peptides (0.8 μg of peptide) already cause paralysis in crickets. The relative abundance of these two peptides in the venom is 2.1% for Ct-IT1 and 1% for Ct-IT2. The molecular masses and N-terminal sequences of both insecticidal toxins were determined by mass spectrometry and Edman degradation. The primary structure of both toxins was compared with other known peptides isolated from other scorpion venoms. The analysis of the sequence alignments revealed the position of a highly conserved amino acid residue, Gly39, exclusively present in anti-insect selective depressant β-toxins (DBTXs), which in Ct-IT1 and Ct-IT2 is at position Gly40. Similarly, a three-dimensional structure of this toxins was obtained by homology modeling and compared to the structure of known insect toxins of scorpions. An important similarity of the cavity formed by the trapping apparatus region of the depressant toxin LqhIT2, isolated from the scorpion Leiurus quinquestriatus hebraeus, was found in the toxins described here. These results indicate that Ct-IT1 and Ct-IT2 toxins have a high potential to be evaluated on pests that affect economically important crops to eventually consider them as a potential biological control method.

Glycoconjugation of Shigella flexneri type 2a O-polysaccharide with CRM197 as a potential vaccine candidate for shigellosis

Shigellosis, a diarrheal disorder caused by an entero-invasive bacterium Shigella, is a major concern among children often leading to mortality. As most of these strains have developed universal antibiotic resistance, the development of a vaccine is crucial in combating the infection. The O-specific polysaccharide (O-PSs) from S. flexneri type 2a is considered to be the major disease-causing antigen in shigellosis. Therefore, the O-PSs conjugated with carrier proteins, can serve as a potential high molecular weight vaccine candidate. Accordingly, in the present study, O-PS extracted from S. flexneri 2a is conjugated with Cross-Reactive Material (CRM197), a non-toxic mutant of diphtheria toxin. We derivatized CRM197 and O-PS separately with adipic acid dihydrazide (ADH) and reacted with their counterparts to probe the conjugation efficacy. Among the two strategies, the CRM197-ADH treated with O-PS has yielded a stable glycoconjugate of 311 kDa. The conjugation efficiency has been probed by estimating the free protein, free O-PS and O-PS:CRM197 ratio using slot-blot, size exclusion and high-performance anion exchange chromatography techniques. The conjugate exhibited enhanced shelf-life of three months. The cytotoxicity studies with Vero/MRC-5 cells have confirmed the non-toxicity of the conjugate, which makes the glycoconjugate a potential vaccine candidate for shigellosis.

Expression, purification and molecular dynamics simulation of extracellular domain of glucagon-like peptide-2 receptor linked to teduglutide

Teduglutide is the only drug approved for long-term treatment of short bowel syndrome (SBS). This drug exerts its pharmacological effects via binding to the GLP-2 receptors (ECD-GLP2R) located in intestinal tissue. The three dimensional (3D) structure of ECD-GLP2R hasn't been determined yet and hence its mode of interaction with agonists/antagonists is not clear. Therefore, it would be of great importance to develop a structural scaffold for investigation of ECD-GLP2R interactions with its binders. For this, the current study aimed to produce fusion protein of ECD-GLP2R-teduglutide. The ECD-GLP2R-teduglutide protein was expressed in bacterial expression system and purified using affinity and size exclusion chromatography techniques. Using circular dichroism the secondary structure content of purified protein was determined which was comparable to that of theoretical calculations. The low structural stability of purified protein (ΔG = 3.64 kJ.mol−1) was elucidated by monitoring its fluorescence emission at the presence of various concentrations of GdnHCl as a denaturant. Finally, a 3D model for ECD-GLP2R-teduglutide protein was generated and validated using molecular dynamics simulation whose information alongside the experimental studies can be useful for providing new insight into the mode of interaction of ECD-GLP2R with its specific ligands in order to design potent and specific GLP2R agonists.

Toward the enrichment of dietary proanthocyanidins: In vitro investigation of their concentration‐dependent complexation with β‐casein

Considering the proanthocyanidins’ (PA) potential health effects, nutritional, and organoleptic properties, their structure should be carefully preserved during food technological processing. Particularly, their interaction with proteins, leading to complex formation, alters their functional performance. In this context, proanthocyanidins-rich persimmon and pomegranate peels were used to investigate the effect of variable PA/β-casein relative concentrations on their in vitro interaction. High-Performance Liquid Chromatography coupled to Diode Array Detector (HPLC) analyses showed that, using 0.008, 0.017, 0.028, and 0.035 mg catechin equivalent (CE) per milliliter of PA in the reaction medium, the proanthocyanidin–β-casein interaction’s potencies varied, respectively, from 29.12% versus 27.70%, 37.64% versus 33.04%, 16.44% versus 44.60% to 15.96% versus 38.73% (p < .05) with regards to pomegranate versus persimmon peel starting material. Moreover, Size-Exclusion Chromatography (SEC) technique confirmed those findings. From the technological point of view, the relative concentration of PA-protein should be considered to manage their interaction, thus, saving the aliment nutritional values as well as sensorial traits, particularly, astringency and haze formation. Practical applications From the technological point of view, this work shows how proanthocyanidin-protein’ relative concentration should be considered to manage their interaction, thus, saving the aliment nutritional values as well as sensorial traits, particularly, astringency and haze formation. The pick of appropriate concentrations will be assumed to enhance the proanthocyanidin content of beverages and foods, during natural product handling by diminishing their inevitable adsorption to proteins, and so, avoiding haze formation. Additionally, in winemaking, targeting the fitting concentrations can be extremely useful since that winemakers can apply proteins as fining agents which can bind tannins molecules and precipitate them out as sediments to have a softer and less tannic taste.

Size exclusion chromatography: An effective tool for the estimation of Sirolimus levels

Sirolimus, a lipophilic macrolide, is a well-known immunosuppressant drug used for coating coronary stents and for preventing rejection of kidney transplants in humans. Since Sirolimus is a relatively large molecule with an average mass 914.172 g/mol, size exclusion chromatography (SEC) was employed for exploring its potential for the estimation of Sirolimus content from blood samples. When human blood samples were spiked with known concentrations of Sirolimus, it was observed that it could be estimated with an average recovery of >90% and relative standard deviation <5%. Results indicated that Sirolimus could be estimated with impressive accuracy and repeatability by SEC technique.

Macrophage M2 polarization induced by exosomes from adipose-derived stem cells contributes to the exosomal proangiogenic effect on mouse ischemic hindlimb

BackgroundM2 macrophages and exosomes from adipose-derived stem cells (ASCs) are both reported to promote angiogenesis. However, the possible synergistic effects between exogenous exosomes and endogenous M2 macrophages are poorly understood.MethodsExosomes were isolated from conditioned medium of normoxic and hypoxic ASCs using the combined techniques of ultrafiltration and size-exclusion chromatography and were identified with nanoparticle tracking analysis and immunoblotting for exosomal markers. Macrophages were collected from the mouse peritoneal cavity. M1 and M2 macrophages were detected by immunoblotting for the intracellular markers inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1) and by flow cytometry for the surface markers F4/80, CD86, and CD206. Murine models of Matrigel plug and hindlimb ischemia were employed as in vivo angiogenic assays.ResultsWhen M1 macrophages were treated with exosomes from normoxic ASCs (Nor/Exo), and particularly from hypoxic ASCs (Hyp/Exo), the expression of the M1 marker iNOS decreased, and the M2 marker Arg-1 increased in a time- and dose-dependent manner. Additionally, a decrease in the M1 surface marker CD86 and an increase in the M2 surface marker CD206 were observed, which suggested that M1 macrophages were polarized to an M2-like phenotype. Conditioned medium from these M2-like macrophages presented lower levels of proinflammatory cytokines and higher levels of proangiogenic factors and promoted endothelial cell proliferation, migration, and tube formation. Furthermore, M2 polarization and angiogenesis were induced upon the administration of exosomes in mouse Matrigel plug and hindlimb ischemia (HLI) models. Interestingly, these exosomal effects were attenuated by using a colony stimulating factor 1 receptor (CSF-1R) inhibitor, BLZ945, in vitro and in vivo. Downregulation of microRNA-21 (miR-21) in hypoxic ASCs reduced the exosomal effects on M2 polarization, Akt phosphorylation, and CSF-1 secretion. A similar reduction in exosomal activity was also observed when exosomes were administered along with BLZ945.ConclusionOur findings provide evidence that exosomes from ASCs polarize macrophages toward an M2-like phenotype, which further enhances the exosomal proangiogenic effects. Exosomal delivery of miR-21 and positive feedback of secreted CSF-1 may be involved in macrophage polarization.

Cytotoxic activity of proteins extracted from fennel (Foeniculum vulgare) seeds against human cancer cell lines

Fennel (Foeniculum vulgare) is an aromatic flowering medicinal plant classified into the family Umbelliferae (Apiaceae). It is native to southern Europe and the Mediterranean region with a long history of use by humans as a spice, fresh vegetable, and in traditional folk medicine. Despite its popularity for its medicinal value, the majority of data in scientific literature is based on the crude extracts and essential oils such as trans‐anethole and estragole, fenchone, limonene, and 1‐octen‐3‐ol. Besides, other phytochemicals found from seeds are polyphenols, included rosmarinic acid and luteolin, etc. In present study, we investigated the anticancer potential of proteins purified from fennel seeds against human breast cancer MCF 7 and pancreatic cancer AsPC‐1 cell lines. The crude fennel extract was partially purified using anion‐exchange and size exclusion chromatography techniques to have a comparative analysis for activity guided fractionation. For anion‐exchange chromatography (HiTrap‐Q FF 5ml column in 20mM Tris/HCl, pH 8.00 buffer) and for gel filtration (HiPrep Sephacryl S‐200 HR16/20 column in 20mM, pH 8.00 buffer ) were used. The crude extract and fractions collected were judged for their purity by 10% Tris‐tricine SDS‐PAGE gel electrophoresis. The MTT assay results exhibited that both anion‐exchange and gel filtration fractions demonstrated significant cytotoxic activity against MCF‐7 breast cancer cell line with cell inhibition in a range of 65–80% at 100μg/ml. In contrast, evaluation againist AsPC‐1pancreatic cancer cell line revealed non‐significant inhibition by fractions obtained from both methods. Further work is in progress to identify and fully characterized the primary structure of cytotoxic proteins by mass spectrometry and Edman protein sequencing.