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Due to its versatility in formulation and manufacturing, self-emulsifying drug delivery systems (SEDDS) can be used to design parenteral formulations. Therefore, it is necessary to understand the effects of excipients on the behavior of SEDDS formulations upon parenteral administration, particularly their interactions with blood plasma and cell membranes. In this study, we prepared three neutrally charged SEDDS formulations composed of medium-chain triglycerides as the oil phase, polyoxyl-35 castor oil (EL35) and polyethylene glycol (15)-hydroxystearate (HS15) as the nonionic surfactants, medium-chain mono- and diglycerides as the co-surfactant, and propylene glycol as the co-solvent. The cationic surfactant, didodecyldimethylammonium bromide (DDA), and the anionic surfactant, sodium deoxycholate (DEO), were added to the neutral SEDDS preconcentrates to obtain cationic and anionic SEDDS, respectively. SEDDS were incubated with human blood plasma and recovered by size exclusion chromatography. Data showed that SEDDS emulsion droplets can bind plasma protein to different extents depending on their surface charge and surfactant used. At pH 7.4, the least protein binding was observed with anionic SEDDS. Positive charges increased protein binding. SEDDS stabilized by HS15 can adsorb more plasma protein and induce more plasma membrane disruption activity than SEDDS stabilized by EL35. These effects were more pronounced with the HS15 + DDA combination. The addition of DDA and DEO to SEDDS increased plasma membrane disruption (PMD) activities, and DDA (1% w/w) was more active than DEO (2% w/w). PMD activities of SEDDS were concentration-dependent and vanished at appropriate dilution ratios. [ABSTRACT FROM AUTHOR]

Background: Canine mammary tumours (CMT) are among the most common types of tumours in female dogs. Diagnosis currently requires invasive tissue biopsies and histological analysis. Tumour cells shed extracellular vesicles (EVs) containing RNAs and proteins with potential for liquid biopsy diagnostics. We aimed to identify CMT subtype-specific proteome profiles by comparing the proteomes of EVs isolated from epithelial cell lines derived from morphologically normal canine mammary tissue, adenomas, and carcinomas. Methods: Whole-cell protein lysates (WCLs) and EV-lysates were obtained from five canine mammary cell lines: MTH53A (non-neoplastic); ZMTH3 (adenoma); MTH52C (simple carcinoma); 1305, DT1406TB (complex carcinoma); and their proteins identified by LC-MS/MS analyses. Gene Ontology analysis was performed on differentially abundant proteins from each group to identify up- and down-regulated biological processes. To establish CMT subtype-specific proteomic profiles, weighted gene correlation network analysis (WGCNA) was carried out. Results: WCL and EVs displayed distinct protein abundance signatures while still showing the same increase in adhesion, migration, and motility-related proteins in carcinoma-derived cell lines, and of RNA processing and RNA splicing factors in the adenoma cell line. WGCNA identified CMT stage-specific co-abundant EV proteins, allowing the identification of adenoma and carcinoma EV signatures not seen in WCLs. Conclusions: EVs from CMT cell lines exhibit distinct protein profiles reflecting malignancy state, allowing us to identify potential biomarkers for canine mammary carcinomas, such as biglycan. Our dataset could therefore potentially serve as a basis for the development of a less invasive clinical diagnostic tool for the characterisation of CMT. [ABSTRACT FROM AUTHOR]

During the last years, there has been an increasing research interest in the analysis of biological fluids requiring non-invasive sampling for biomedical and clinical applications. In this work, we have focused on the nasal exudate with the aim of investigating the potential use of this fluid to know the role of iron in stroke and also for diagnosis. Potential differences in the nasal exudate, collected in swabs, from diagnosed hemorrhagic stroke, ischemic stroke, and control groups were investigated with regard to total iron by inductively coupled plasma-mass spectrometry, iron fractionation studies by size exclusion chromatography together with post-column isotope dilution analysis, and four proteins containing iron (ferritin, transferrin, lactoferrin, and ferroportin) with ELISA kits. All these analyses represent an analytical challenge, considering the rather limited amount of sample (10–40 mg) available, being the nasal exudate extracted from the swab with 300 µL 10 mM Tris/HCl, pH = 7.4. Studies to obtain reliable analytical information, such as the blank contribution of the sampling step, evaluation of the extraction efficiency of the nasal exudate from the swab, and normalization strategies for data treatment, have been carried out. Results showed that despite the limited number of investigated samples, fractionation studies as well as the concentrations of ferritin and ferroportin obtained with ELISA kits showed a differential behavior between the different cohorts. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are valuable targets for liquid biopsy. However, attempts to introduce EV‐based biomarkers into clinical practice have not been successful to the extent expected. One of the reasons for this failure is the lack of reliable methods for EV baseline purification from complex biofluids, such as cell‐free plasma or serum. Because available one‐step approaches for EV isolation are insufficient to purify EVs, the majority of studies on clinical samples were performed either on a mixture of EVs and lipoproteins, whilst the real number of EVs and their individual specific biomarker content remained elusive, or on a low number of samples of sufficient volume to allow elaborate 2‐step EV separation by size and density, resulting in a high purity but utmost low recovery. Here we introduce Fast Protein Liquid Chromatography (FPLC) using Superose 6 as a matrix to obtain small EVs from biofluids that are almost free of soluble proteins and lipoproteins. Along with the estimation of a realistic number of small EVs in human samples, we show temporal resolution of the effect of the duration of postprandial phase on the proportion of lipoproteins in purified EVs, suggesting acceptable time frames additionally to the recommendation to use fasting samples for human studies. Furthermore, we assessed a potential value of pure EVs for liquid biopsy, exemplarily examining EV‐ and tumour‐biomarkers in pure FPLC‐derived fractions isolated from the serum of patients with pancreatic cancer. Consistent among different techniques, showed the presence of diseases‐associated biomarkers in pure EVs, supporting the feasibility of using single‐vesicle analysis for liquid biopsy. [ABSTRACT FROM AUTHOR]

Hemp (Cannabis sativa L.) seeds are an interesting raw material for malting regarding its relatively high bioactive compounds concentration and proven advantageous properties in different food products and dietary supplements. In the first stage of the study, important seeds properties relevant to the malting process including moisture content, seed viability, and water absorption capacity were determined. However, a few parameters determining the seeds' usability for malt preparation, such as germination ability and water sensitivity, are different in comparison to typical malting raw materials such as barley or wheat. However, they make it possible to obtain high-quality hemp malt. In the next stage of research, spectroscopic and chromatographic analyses, including measurements of antioxidant activity and protein separation by SEC-HPLC, were conducted. The results showed that the malting process improved the total antioxidant potential of hemp seeds by 15%, leading to an increase in the concentration of lower molecular weight proteins and oligopeptides—below molecular mass of 10 kDa—responsible for this high antioxidant activity. The processing of hemp seeds reduced the phytate content while increasing phosphate fractions with fewer phosphate groups, which may have a beneficial effect on nutritional value. These results suggest that malting hemp seeds needs optimalization of the process but can increase its nutritional value as a promising raw material in the food industry. [ABSTRACT FROM AUTHOR]

Purposes: Size exclusion chromatography (SEC) is widely used to characterize molecular size variants of antibody drugs. However, SEC analysis is hindered by secondary interactions (or nonspecific interactions) between proteins and stationary phase packing, which result in poor column efficiency. Previous studies have reported that chaotropic salt can inhibit these interactions, but the corresponding applications of this aspect are relatively rare. Therefore, this study introduces a novel approach using sodium iodide (NaI) as a mobile-phase component in SEC and investigates the influence of the mobile-phase composition on secondary interactions. Methods: SEC analysis was performed on one antibody–drug conjugate and four monoclonal antibodies (mAbs) using three different mobile-phase systems (i.e., sodium chloride/L-arginine hydrochloride/NaI mobile phases system) to compare the column efficiency. Subsequently, mAb-1 was used as a model to investigate the effects of these factors on secondary interactions by adjusting the ionic strength (salt concentration) and pH of the NaI mobile-phase system. Results: NaI exhibits superior column efficiency performance in the SEC analysis of most products. The ionic strength will affect nonideal electrostatic and hydrophobic interaction. An appropriate ionic strength can inhibit electrostatic interactions, while an excessive ionic strength increases hydrophobic interactions. pH primarily influences electrostatic interactions. Determining the appropriate pH necessitates consideration of the isoelectric point of the protein and the pH tolerance of the column. Conclusions: In SEC analysis, using NaI as the salt component in the mobile phase reduces secondary interactions and improves column efficiency. This approach is advantageous for samples with intense secondary interactions and is a suitable alternative. [ABSTRACT FROM AUTHOR]

In this study, spherical silica with pore size varied from 30 to 200 Å was synthesized by pseudomorphic transformation at atmospheric pressure. 40–80 Å silica particles with a narrow pore distribution were obtained by using quaternary amine cationic surfactants and different kinds of swelling agents, including polypropylene glycol, 1,3,5‐trimethylbenzene, alkanes, and alkanols. Alkyl imidazolium ionic liquid surfactants were used to synthesize large pore size distribution silica spheres with pore sizes in the range of 110–200 Å. All these silica particles can be synthesized under mild conditions within 12 h, which provides a facile synthesis method for the preparation of a chromatographic matrix with tunable pore size. The method is reproducible and the relative standard deviation of silica sphere pore structure parameters in scaled‐up preparations is less than 6%. The pore size on the fraction of low‐molecular‐weight heparin (LMWH) was investigated in size exclusion chromatography. Matrixes with different pore size distributions have various size exclusion regions. By using UPS‐60‐Diol columns in a twin‐column recirculation separation process, LMWH with >85% heparin with molecular weight within the range of 3000–8000 Da were separated in five‐column volumes. [ABSTRACT FROM AUTHOR]

Abstract Background Canine mammary tumours (CMT) are among the most common types of tumours in female dogs. Diagnosis currently requires invasive tissue biopsies and histological analysis. Tumour cells shed extracellular vesicles (EVs) containing RNAs and proteins with potential for liquid biopsy diagnostics. We aimed to identify CMT subtype-specific proteome profiles by comparing the proteomes of EVs isolated from epithelial cell lines derived from morphologically normal canine mammary tissue, adenomas, and carcinomas. Methods Whole-cell protein lysates (WCLs) and EV-lysates were obtained from five canine mammary cell lines: MTH53A (non-neoplastic); ZMTH3 (adenoma); MTH52C (simple carcinoma); 1305, DT1406TB (complex carcinoma); and their proteins identified by LC-MS/MS analyses. Gene Ontology analysis was performed on differentially abundant proteins from each group to identify up- and down-regulated biological processes. To establish CMT subtype-specific proteomic profiles, weighted gene correlation network analysis (WGCNA) was carried out. Results WCL and EVs displayed distinct protein abundance signatures while still showing the same increase in adhesion, migration, and motility-related proteins in carcinoma-derived cell lines, and of RNA processing and RNA splicing factors in the adenoma cell line. WGCNA identified CMT stage-specific co-abundant EV proteins, allowing the identification of adenoma and carcinoma EV signatures not seen in WCLs. Conclusions EVs from CMT cell lines exhibit distinct protein profiles reflecting malignancy state, allowing us to identify potential biomarkers for canine mammary carcinomas, such as biglycan. Our dataset could therefore potentially serve as a basis for the development of a less invasive clinical diagnostic tool for the characterisation of CMT.

Abstract This work deals with the influence of different heat treatment temperatures (140, 150, 160, 170, 180, 190, 200, and 210 °C) on changes in sapwood and red heartwood of European beech (Fagus sylvatica L.). According to the results of wet chemistry methods, HPLC (high-performance liquid chromatography), FTIR (Fourier transform infrared spectroscopy), SEC (size exclusion chromatography), the wood constituents in sapwood and red heartwood behaved similarly to heat treatment, but the individual proportions were different. The loss of hemicelluloses and the increase in extractives with increasing temperature were more pronounced in sapwood. The amount of cellulose in sapwood and red heartwood showed similar behaviour with increasing temperature. Thermal treatment causes changes in cellulose crystallinity, and the formation of aromatic structures, mainly in beech sapwood. However, the increase in the lignin content of red heartwood was significantly lower than that of sapwood due to its auto condensation, and formation of pseudo-lignin. Among the carbohydrates, the most significant changes were observed in xylose content, which was almost twice as high in red heartwood as in sapwood. Other carbohydrates (glucose, mannose, galactose, and arabinose) reached similar values in sapwood and red heartwood.

Abstract Background The lack of standardized protocols for isolating extracellular vesicles (EVs), especially from biobank-stored blood plasma, translates to limitations for the study of new biomarkers. This study examines whether a combination of current isolation methods could enhance the specificity and purity of isolated EVs for diagnosis and personalized medicine purposes. Results EVs were isolated from healthy human plasma stored for one year by ultracentrifugation (UC), size exclusion chromatography (SEC), or SEC and UC combined (SEC + UC). The EV isolates were then characterized by transmission electron microscopy imaging, nanoparticle tracking analysis (NTA) and western blotting. Proteomic procedures were used to analyze protein contents. The presence of EV markers in all isolates was confirmed by western blotting yet this analysis revealed higher albumin expression in EVs-UC, suggesting plasma protein contamination. Proteomic analysis identified 542 proteins, SEC + UC yielding the most complex proteome at 364 proteins. Through gene ontology enrichment, we observed differences in the cellular components of EVs and plasma in that SEC + UC isolates featured higher proportions of EV proteins than those derived from the other two methods. Analysis of proteins unique to each isolation method served to identify 181 unique proteins for the combined approach, including those normally appearing in low concentrations in plasma. This indicates that with this combined method, it is possible to detect less abundant plasma proteins by proteomics in the resultant isolates. Conclusions Our findings reveal that the SEC + UC approach yields highly pure and diverse EVs suitable for comprehensive proteomic analysis with applications for the detection of new biomarkers in biobank-stored plasma samples.

Background: In recent years, lytic polysaccharide monooxygenases (LPMOs) that oxidatively cleave cellulose have gained increasing attention in cellulose fiber modification. LPMOs are relatively small copper-dependent redox enzymes that occur as single domain proteins but may also contain an appended carbohydrate-binding module (CBM). Previous studies have indicated that the CBM "immobilizes" the LPMO on the substrate and thus leads to more localized oxidation of the fiber surface. Still, our understanding of how LPMOs and their CBMs modify cellulose fibers remains limited. Results: Here, we studied the impact of the CBM on the fiber-modifying properties of NcAA9C, a two-domain family AA9 LPMO from Neurospora crassa, using both biochemical methods as well as newly developed multistep fiber dissolution methods that allow mapping LPMO action across the fiber, from the fiber surface to the fiber core. The presence of the CBM in NcAA9C improved binding towards amorphous (PASC), natural (Cell I), and alkali-treated (Cell II) cellulose, and the CBM was essential for significant binding of the non-reduced LPMO to Cell I and Cell II. Substrate binding of the catalytic domain was promoted by reduction, allowing the truncated CBM-free NcAA9C to degrade Cell I and Cell II, albeit less efficiently and with more autocatalytic enzyme degradation compared to the full-length enzyme. The sequential dissolution analyses showed that cuts by the CBM-free enzyme are more evenly spread through the fiber compared to the CBM-containing full-length enzyme and showed that the truncated enzyme can penetrate deeper into the fiber, thus giving relatively more oxidation and cleavage in the fiber core. Conclusions: These results demonstrate the capability of LPMOs to modify cellulose fibers from surface to core and reveal how variation in enzyme modularity can be used to generate varying cellulose-based materials. While the implications of these findings for LPMO-based cellulose fiber engineering remain to be explored, it is clear that the presence of a CBM is an important determinant of the three-dimensional distribution of oxidation sites in the fiber. [ABSTRACT FROM AUTHOR]

This work deals with the influence of different heat treatment temperatures (140, 150, 160, 170, 180, 190, 200, and 210 °C) on changes in sapwood and red heartwood of European beech (Fagus sylvatica L.). According to the results of wet chemistry methods, HPLC (high-performance liquid chromatography), FTIR (Fourier transform infrared spectroscopy), SEC (size exclusion chromatography), the wood constituents in sapwood and red heartwood behaved similarly to heat treatment, but the individual proportions were different. The loss of hemicelluloses and the increase in extractives with increasing temperature were more pronounced in sapwood. The amount of cellulose in sapwood and red heartwood showed similar behaviour with increasing temperature. Thermal treatment causes changes in cellulose crystallinity, and the formation of aromatic structures, mainly in beech sapwood. However, the increase in the lignin content of red heartwood was significantly lower than that of sapwood due to its auto condensation, and formation of pseudo-lignin. Among the carbohydrates, the most significant changes were observed in xylose content, which was almost twice as high in red heartwood as in sapwood. Other carbohydrates (glucose, mannose, galactose, and arabinose) reached similar values in sapwood and red heartwood. [ABSTRACT FROM AUTHOR]

Extracellular Vesicles (EV) have become an interesting focus as novel biomarkers of disease and are increasingly reported upon in humans and other species. The Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) guidelines were published to improve rigor and standardisation within the EV field and provide a framework for the reliable isolation and characterisation of EV populations. However, this rigor and standardisation has been challenging in the area of comparative medicine. Herein we present the successful isolation of EVs from human and canine plasma using Size Exclusion Chromatography and characterise these EVs according to best international practice. This study provides evidence for the reliable comparison of human and canine EVs isolated by this approach, and a baseline description of the EVs from healthy dogs to inform future biomarker studies. This work also demonstrates that the MISEV2018 guidelines can be successfully applied to EVs isolated from canine plasma. [ABSTRACT FROM AUTHOR]

Background: Extracellular vesicles (EVs) are amongst the most promising candidates for developing blood-based biomarkers. However, patient sample availability is a key barrier to translational research whilst most biobanks store samples of 1.5mL volume or less. To date, there is no consensus on the most suitable method of EV separation and current techniques frequently require large volumes of biofluids, complicated technology, technical expertise, or significant operating costs, which prevents their widespread adoption by less EV-focussed laboratories. Therefore, there is a need for an easy and reproducible method that separates representative EVs from clinically relevant 1mL volumes of plasma prior to subsequent biomarker identification. Methods: In this study, EVs were separated from a clinically relevant 1mL volume of human plasma using four different separation techniques: size exclusion chromatography (SEC), differential ultracentrifugation, precipitation, and immunoaffinity magnetic bead capture. The EVs were characterised using several orthogonal techniques (protein quantification, nanoparticle tracking analysis, transmission electron microscopy, Western blot, single particle interferometric reflectance imaging sensing, and mass spectrometry-based proteomics) to comprehensively compare the separated samples. Results: We provide examples of anticipated results highlighting that SEC-processed samples have greater protein quantification yield, greater particle yield of the expected size for EVs, and sufficient EV purity, which facilitates effective EV cargo assessment by proteomics. Moreover, we confirm significant overlap with known EV-related proteins within the Vesiclepedia database. Additionally, using single particle interferometric reflectance imaging sensing (Leprechaun®), we identify that SEC has the most representative surface tetraspanin distribution of the separated EV population compared to unprocessed plasma. Discussion: Given that SEC requires minimal expertise, no complicated technology and can separate EVs within 90 min, this comparison reinforces SEC as a clinically relevant EV separation method from 1mL of plasma making it suitable for widespread implementation. [ABSTRACT FROM AUTHOR]

Background: The lack of standardized protocols for isolating extracellular vesicles (EVs), especially from biobank-stored blood plasma, translates to limitations for the study of new biomarkers. This study examines whether a combination of current isolation methods could enhance the specificity and purity of isolated EVs for diagnosis and personalized medicine purposes. Results: EVs were isolated from healthy human plasma stored for one year by ultracentrifugation (UC), size exclusion chromatography (SEC), or SEC and UC combined (SEC + UC). The EV isolates were then characterized by transmission electron microscopy imaging, nanoparticle tracking analysis (NTA) and western blotting. Proteomic procedures were used to analyze protein contents. The presence of EV markers in all isolates was confirmed by western blotting yet this analysis revealed higher albumin expression in EVs-UC, suggesting plasma protein contamination. Proteomic analysis identified 542 proteins, SEC + UC yielding the most complex proteome at 364 proteins. Through gene ontology enrichment, we observed differences in the cellular components of EVs and plasma in that SEC + UC isolates featured higher proportions of EV proteins than those derived from the other two methods. Analysis of proteins unique to each isolation method served to identify 181 unique proteins for the combined approach, including those normally appearing in low concentrations in plasma. This indicates that with this combined method, it is possible to detect less abundant plasma proteins by proteomics in the resultant isolates. Conclusions: Our findings reveal that the SEC + UC approach yields highly pure and diverse EVs suitable for comprehensive proteomic analysis with applications for the detection of new biomarkers in biobank-stored plasma samples. [ABSTRACT FROM AUTHOR]

Biopharmaceutical products, in particular messenger ribonucleic acid (mRNA), have the potential to dramatically improve the quality of life for patients suffering from respiratory and infectious diseases, rare genetic disorders, and cancer. However, the quality and safety of such products are particularly critical for patients and require close scrutiny. Key product-related impurities, such as fragments and aggregates, among others, can significantly reduce the efficacy of mRNA therapies. In the present work, the possibilities offered by size exclusion chromatography (SEC) for the characterization of mRNA samples were explored using state-of-the-art ultra-wide pore columns with average pore diameters of 1000 and 2500 Å. Our investigation shows that a column with 1000 Å pores proved to be optimal for the analysis of mRNA products, whatever the size between 500 and 5000 nucleotides (nt). We also studied the influence of mobile phase composition and found that the addition of 10 mM magnesium chloride (MgCl2) can be beneficial in improving the resolution and recovery of large size variants for some mRNA samples. We demonstrate that caution should be exercised when increasing column length or decreasing the flow rate. While these adjustments slightly improve resolution, they also lead to an apparent increase in the amount of low-molecular-weight species (LMWS) and monomer peak tailing, which can be attributed to the prolonged residence time inside the column. Finally, our optimal SEC method has been successfully applied to a wide range of mRNA products, ranging from 1000 to 4500 nt in length, as well as mRNA from different suppliers and stressed/unstressed samples. [ABSTRACT FROM AUTHOR]

In the last few years, several studies have emphasized the existence of injury-specific EV "barcodes" that could have significant importance for the precise diagnosis of different organ injuries in polytrauma patients. To expand the research potential of the NTF (network trauma research) biobank of polytraumatized patients, the NTF research group decided to further establish a biobank for EVs. However, until now, the protocols for the isolation, characterization, and storage of EVs for biobank purposes have not been conceptualized. Plasma and serum samples from healthy volunteers (n = 10) were used. Three EV isolation methods of high relevance for the work with patients' samples (ultracentrifugation, size exclusion chromatography, and immune magnetic bead-based isolation) were compared. EVs were quantified using nanoparticle tracking analysis, EV proteins, and miRNAs. The effects of different isolation solutions; the long storage of samples (up to 3 years); and the sensibility of EVs to serial freezing–thawing cycles and different storage conditions (RT, 4/−20/−80 °C, dry ice) were evaluated. The SEC isolation method was considered the most suitable for EV biobanking. We did not find any difference in the quantity of EVs between serum and plasma-EVs. The importance of particle-free PBS as an isolation solution was confirmed. Plasma that has been frozen for a long time can also be used as a source of EVs. Serial freezing–thawing cycles were found to affect the mean size of EVs but not their amount. The storage of EV samples for 5 days on dry ice significantly reduced the EV protein concentration. [ABSTRACT FROM AUTHOR]

ABSTRACT Chitin degradation is a keystone process in the oceans, mediated by marine microorganisms with the help of several enzymes, mostly chitinases. Sediment, seawater, and filter-feeding marine invertebrates, such as sponges, are known to harbor chitin-degrading bacteria and are presumably hotspots for chitin turnover. Here, we employed an artificial selection process involving enrichment cultures derived from microbial communities associated with the marine sponge Hymeniacidon perlevis, its surrounding seawater and sediment, to select bacterial consortia capable of degrading raw chitin. Throughout the artificial selection process, chitin degradation rates and the taxonomic composition of the four successive enrichment cultures were followed. To the best of our knowledge, chitin degradation was characterized for the first time using size exclusion chromatography, which revealed significant shifts in the numbered average chitin molecular weight, strongly suggesting the involvement of endo-chitinases in the breakdown of the chitin polymer during the enrichment process. Concomitantly with chitin degradation, the enrichment cultures exhibited a decrease in alpha diversity compared with the environmental samples. Notably, some of the dominant taxa in the enriched communities, such as Motilimonas, Arcobacter, and Halarcobacter, were previously unknown to be involved in chitin degradation. In particular, the analysis of published genomes of these genera suggests a pivotal role of Motilimonas in the hydrolytic cleavage of chitin. This study provides context to the microbiome of the marine sponge Hymeniacidon perlevis in light of its environmental surroundings and opens new ground to the future discovery and characterization of novel enzymes of marine origin involved in chitin degradation processes.IMPORTANCEChitin is the second most abundant biopolymer on Earth after cellulose, and the most abundant in the marine environment. At present, industrial processes for the conversion of seafood waste into chitin, chitosan, and chitooligosaccharide (COS) rely on the use of high amounts of concentrated acids or strong alkali at high temperature. Developing bio-based methods to transform available chitin into valuable compounds, such as chitosan and COS, holds promise in promoting a more sustainable, circular bioeconomy. By employing an artificial selection procedure based on chitin as a sole C and N source, we discovered microorganisms so-far unknown to metabolize chitin in the rare microbial biosphere of several marine biotopes. This finding represents a first important step on the path towards characterizing and exploiting potentially novel enzymes of marine origin with biotechnological interest, since products of chitin degradation may find applications across several sectors, such as agriculture, pharmacy, and waste management.

Polydioxanone (PPDX) has gained significant attention as a biocompatible and absorbable polymer used in various medical applications, such as sutures and tissue scaffolds. This research presents a thorough investigation into the degradation mechanisms of PPDX under low pH conditions, simulating physiological environments, e.g., esophagus and stomach. It mainly focuses on the dependence of the PPDX degradation rate on various ambient pH values (7.4 and below), which is a substantial knowledge for successful gastrointestinal treatment. The PPDX suture samples were degraded for up to 6 weeks and analyzed using size exclusion chromatography, differential scanning calorimetry, Raman spectroscopy, scanning electron microscopy, X-ray microtomography, and mechanical property measurements. The results show that the PPDX degradation is significantly accelerated at pH below 1.67. Correlations of the molecular weight, crystallinity, glass transition temperature, Young's modulus, shear modulus, tensile strength, and the 1733 cm−1 Raman peak shoulder area (RPSA1733) indicate that the degradation mechanism does not change with increasing acidity. Measurements of tensile strength, shear modulus, and RPSA1733 were found to be the most suitable parameters for characterizing the PPDX filament's macroscopic integrity. Raman spectroscopy is of particular interest in this regard due to its rapidity and practically no requirements on the sample preparation.

The technical difficulty of separating extracellular vesicles (EVs) from plasma proteins in human blood presents a significant hurdle in EV research, particularly during nano ultra-high-performance liquid chromatography–tandem mass spectrometric (UHPLC-MS/MS) analysis, where detecting “vesicular” proteins among abundant plasma proteins is challenging. Standardisation is a pressing issue in EV research, prompting collaborative global efforts to address it. While the MISEV guidelines offer valuable recommendations, unanswered questions remain, particularly regarding sample storage. We compared size exclusion chromatography (SEC) columns with pore sizes of 35 nm and 70 nm to identify fractions with minimal contaminating proteins and the highest concentration of small EVs (sEVs). Following column selection, we explored potential differences in the quality and quantity of sEVs isolated from platelet-free plasma (PFP) after long-term storage at −80 °C (>2.5 years) compared to freshly drawn blood. Our methodologically rigorous study indicates that prolonged storage, under correct storage and processing conditions, does not compromise sEV quality. Both columns effectively isolated vesicles, with the 70 nm column exhibiting a higher abundance of “vesicular” proteins. We propose a relatively rapid and moderately efficient protocol for obtaining a comparatively pure sEV fraction from plasma, facilitating sEV processing in clinical trials.

Geosynthetics are widely used in various civil engineering applications, such as geotextiles in coastal protection, and display a sustainable alternative to natural mineral materials. However, the full benefits of using geosynthetics can only be gained with a long service lifetime of the products. With the use of added stabilizers to the polymers, service lifetimes can be achieved in the range of 100 years. Therefore, accelerated aging methods are needed for the assessment of the long-term performance of geotextiles. In the present study, the behavior of geosynthetic materials made of polypropylene was investigated under artificial aging conditions involving elevated temperatures ranging from 30 to 80 °C, increased oxygen pressures ranging from 10 to 50 bar in water-filled autoclaves, and UV irradiation under atmospheric conditions. ATR-IR spectroscopy was employed to detect the increase in the carbonyl index over various aging durations, indicating the oxidative degradation of the geotextile. The most pronounced increase was observed in the case of aging through UV irradiation, followed by thermal aging. Elevated pressure, on the other hand, had a lower impact on oxidation. High-temperature size exclusion chromatography was utilized to follow the reduction in molar mass under different degradation conditions, and the results were consistent with those obtained from ATR-IR spectroscopy. In polyolefins such as polypropylene, Hindered Amine Stabilizers (HAS) are used to suppress oxidation caused by UV radiation. The quantitative analysis of HAS was carried out using a UV/Vis method and HPLC. The degradation of UV stabilizers during the aging of geotextiles is responsible for the oxidation and the reduction in the molar mass of polypropylene. From the results, it can be concluded that applications of PP geotextile without soil or sand cover might cause the risk of the formation of microplastic particles. Material selection, design, and maintenance of the construction must follow best practices, including the system's removal or replacement at end-of-life. Otherwise, a sustainable use of geotextiles in civil engineering is not possible. [ABSTRACT FROM AUTHOR]

[Display omitted] The competitive interaction of oxyanions and humic nanoparticles (HNPs) with metal (hydr)oxide surfaces can be used to trace the ligand and charge distribution of adsorbed HNPs in relation to heterogeneity, fractionation, and conformational change. Batch adsorption experiments of HNPs on goethite were performed in the absence and presence of phosphate. The size of HNPs was measured with size exclusion chromatography. The Ligand and Charge Distribution (LCD) model framework was further developed to describe the simultaneous interaction of HNPs and phosphate with goethite. Preferential adsorption decreases the mean molar mass of adsorbed HNPs, independent of the phosphate presence, showing a linear dependency on the adsorbed HNPs fraction. Phosphate ion can be used as a probe to trace the distribution of functional groups and the variation in affinity of HNPs. The spatial distribution of adsorbed HNPs is driven by the potential gradients in the electrical double layer, which changes the conformation of the adsorbed HNPs. At the particle level, the adsorption of heterogeneous HNPs has an affinity distribution, which can be explained by the variation in molar mass (kDa) and density of the functional groups (mol kg−1) of the HNPs. The presented model can simultaneously describe the competitive adsorption of HNPs and phosphate in a consistent manner. [ABSTRACT FROM AUTHOR]

The two major challenges in synchrotron size‐exclusion chromatography coupled in‐line with small‐angle x‐ray scattering (SEC‐SAXS) experiments are the overlapping peaks in the elution profile and the fouling of radiation‐damaged materials on the walls of the sample cell. In recent years, many post‐experimental analyses techniques have been developed and applied to extract scattering profiles from these problematic SEC‐SAXS data. Here, we present three modes of data collection at the BioSAXS Beamline 4–2 of the Stanford Synchrotron Radiation Lightsource (SSRL BL4‐2). The first mode, the High‐Resolution mode, enables SEC‐SAXS data collection with excellent sample separation and virtually no additional peak broadening from the UHPLC UV detector to the x‐ray position by taking advantage of the low system dispersion of the UHPLC. The small bed volume of the analytical SEC column minimizes sample dilution in the column and facilitates data collection at higher sample concentrations with excellent sample economy equal to or even less than that of the conventional equilibrium SAXS method. Radiation damage problems during SEC‐SAXS data collection are evaded by additional cleaning of the sample cell after buffer data collection and avoidance of unnecessary exposures through the use of the x‐ray shutter control options, allowing sample data collection with a clean sample cell. Therefore, accurate background subtraction can be performed at a level equivalent to the conventional equilibrium SAXS method without requiring baseline correction, thereby leading to more reliable downstream structural analysis and quicker access to new science. The two other data collection modes, the High‐Throughput mode and the Co‐Flow mode, add agility to the planning and execution of experiments to efficiently achieve the user's scientific objectives at the SSRL BL4‐2. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) play a pivotal role in various biological pathways, such as immune responses and the progression of diseases, including cancer. However, it is challenging to isolate EVs at high purity from blood plasma and other biofluids due to their low abundance compared to more predominant biomolecular species such as lipoprotein particles and free protein complexes. Ultracentrifugation‐based EV isolation, the current gold standard technique, cannot overcome this challenge due to the similar biophysical characteristics of such species. We developed several novel approaches to enrich EVs from plasma while depleting contaminating molecular species using multimode chromatography‐based strategies. On average, we identified 716 ± 68 and 1054 ± 35 protein groups in EV isolates from 100 µL of plasma using multimode chromatography‐ and ultracentrifugation‐based techniques, respectively. The developed methods resulted in similar EV isolates purity, providing significant advantages in simplicity, throughput, scalability, and applicability for various downstream analytical and potential clinical applications. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms have established photoprotective mechanisms in order to dissipate excess light energy into heat, which is commonly known as non-photochemical quenching. Cyanobacteria utilize the orange carotenoid protein (OCP) as a high-light sensor and quencher to regulate the energy flow in the photosynthetic apparatus. Triggered by strong light, OCP undergoes conformational changes to form the active red state (OCPR). In many cyanobacteria, the back conversion of OCP to the dark-adapted state is assisted by the fluorescence recovery protein (FRP). However, the exact molecular events involving OCP and its interaction with FRP remain largely unraveled so far due to their metastability. Here, we use small-angle neutron scattering combined with size exclusion chromatography (SEC-SANS) to unravel the solution structures of FRP-OCP complexes using a compact mutant of OCP lacking the N-terminal extension (∆NTEOCPO) and wild-type FRP. The results are consistent with the simultaneous presence of stable 2:2 and 2:1 FRP-∆NTEOCPO complexes in solution, where the former complex type is observed for the first time. For both complex types, we provide ab initio low-resolution shape reconstructions and compare them to homology models based on available crystal structures. It is likely that both complexes represent intermediate states of the back conversion of OCP to its dark-adapted state in the presence of FRP, which are of transient nature in the photocycle of wild-type OCP. This study demonstrates the large potential of SEC-SANS in revealing the solution structures of protein complexes in polydisperse solutions that would otherwise be averaged, leading to unspecific results. [ABSTRACT FROM AUTHOR]

Background Major depressive disorder (MDD) is a leading cause of disability with significant mortality risk. Despite progress in our understanding of the etiology of MDD, the underlying molecular changes in the brain remain poorly understood. Extracellular vesicles (EVs) are lipid-bound particles that can reflect the molecular signatures of the tissue of origin. We aimed to optimize a streamlined EV isolation protocol from postmortem brain tissue and determine whether EV RNA cargo, particularly microRNAs (miRNAs), have an MDD-specific profile. Methods EVs were isolated from postmortem human brain tissue. Quality was assessed using western blots, transmission electron microscopy, and microfluidic resistive pulse sensing. EV RNA was extracted and sequenced on Illumina platforms. Functional follow-up was performed in silico. Results Quality assessment showed an enrichment of EV markers, as well as a size distribution of 30 to 200 nm in diameter, and no contamination with cellular debris. Small RNA profiling indicated the presence of several RNA biotypes, with miRNAs and transfer RNAs being the most prominent. Exploring miRNA levels between groups revealed decreased expression of miR-92a-3p and miR-129-5p, which was validated by qPCR and was specific to EVs and not seen in bulk tissue. Finally, in silico functional analyses indicate potential roles for these 2 miRNAs in neurotransmission and synaptic plasticity. Conclusion We provide a streamlined isolation protocol that yields EVs of high quality that are suitable for molecular follow-up. Our findings warrant future investigations into brain EV miRNA dysregulation in MDD. [ABSTRACT FROM AUTHOR]

Size exclusion chromatography (SEC) has become a powerful tool for analysing size variants of biologic drugs in their native form. Modern SEC can be defined by the use of chromatographic columns packed with sub-3 µm particles, allowing an increase in method throughput compared to that of conventional SEC.

Hemp (Cannabis sativa L.) seeds are an interesting raw material for malting regarding its relatively high bioactive compounds concentration and proven advantageous properties in different food products and dietary supplements. In the first stage of the study, important seeds properties relevant to the malting process including moisture content, seed viability, and water absorption capacity were determined. However, a few parameters determining the seeds’ usability for malt preparation, such as germination ability and water sensitivity, are different in comparison to typical malting raw materials such as barley or wheat. However, they make it possible to obtain high-quality hemp malt. In the next stage of research, spectroscopic and chromatographic analyses, including measurements of antioxidant activity and protein separation by SEC-HPLC, were conducted. The results showed that the malting process improved the total antioxidant potential of hemp seeds by 15%, leading to an increase in the concentration of lower molecular weight proteins and oligopeptides—below molecular mass of 10 kDa—responsible for this high antioxidant activity. The processing of hemp seeds reduced the phytate content while increasing phosphate fractions with fewer phosphate groups, which may have a beneficial effect on nutritional value. These results suggest that malting hemp seeds needs optimalization of the process but can increase its nutritional value as a promising raw material in the food industry.

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Extracellular vesicles (EVs) are cell derived membranous nanoparticles. EVs are important mediators of cell–cell communication via the transfer of bioactive content and as such they are being investigated for disease diagnostics as biomarkers and for potential therapeutic cargo delivery to recipient cells. However, existing methods for isolating EVs from biological samples suffer from challenges related to co‐isolation of unwanted materials such as proteins, nucleic acids, and lipoproteins. In the pursuit of improved EV isolation techniques, we introduce multimodal flowthrough chromatography (MFC) as a scalable alternative to size exclusion chromatography (SEC). The use of MFC offers significant advantages for purifying EVs, resulting in enhanced yields and increased purity with respect to protein and nucleic acid co‐isolates from conditioned 3D cell culture media. Compared to SEC, significantly higher EV yields with similar purity and preserved functionality were also obtained with MFC in 2D cell cultures. Additionally, MFC yielded EVs from serum with comparable purity to SEC and similar apolipoprotein B content. Overall, MFC presents an advancement in EV purification yielding EVs with high recovery, purity, and functionality, and offers an accessible improvement to researchers currently employing SEC. [ABSTRACT FROM AUTHOR]

This study compares the impact of two isolation methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), on small extracellular vesicles (sEVs) from primary human cardiac mesenchymal‐derived progenitor cells (CPCs). sEV_UC and sEV_SEC exhibit similar size, marker expression, and miRNA cargo, but sEV_UC contains notably higher total protein levels. In vitro assays show that sEV_UC, despite an equal particle count, induces more robust ERK phosphorylation, cytoprotection, and proliferation in iPS‐derived cardiomyocytes (iPS‐CMs) compared to sEV_SEC. sEV_UC also contains elevated periostin (POSTN) protein levels, resulting in enhanced focal adhesion kinase (FAK) phosphorylation in iPS‐CMs. Importantly, this effect persists with treatment with soluble free‐sEV protein fraction from SEC (Prote_SEC), indicating that free proteins like POSTN in sEV_UC enhance FAK phosphorylation. In vivo, sEV contamination with soluble proteins doesn't affect cardiac targeting or FAK phosphorylation, underscoring the intrinsic tissue targeting properties of sEV. These findings emphasize the need for standardized sEV isolation methods, as the choice of method can impact experimental outcomes, particularly in vitro. [ABSTRACT FROM AUTHOR]

Small extracellular vesicles (EVs) contain various signaling molecules, thus playing a crucial role in cell-to-cell communication and emerging as a promising source of biomarkers. However, the lack of standardized procedures impedes their translation to clinical practice. Thus, we compared different approaches for high-throughput analysis of small EVs transcriptome. Small EVs were isolated from 150 μL of serum. Quality and quantity were assessed by dynamic light scattering, transmission electron microscopy, and Western blot. Comparison of RNA extraction efficiency was performed, and expression of selected genes was analyzed by RT-qPCR. Whole transcriptome analysis was done using microarrays. Obtained data confirmed the suitability of size exclusion chromatography for isolation of small EVs. Analyses of gene expression showed the best results in case of samples isolated by Monarch Total RNA Miniprep Kit. Totally, 7,182 transcripts were identified to be deregulated between colorectal cancer patients and healthy controls. The majority of them were non-coding RNAs with more than 70 % being lncRNAs, while protein-coding genes represented the second most common gene biotype. We have optimized the protocol for isolation of small EVs and their RNA from low volume of sera and confirmed the suitability of Clariom D Pico Assays for transcriptome profiling. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are highly sought after as a source of biomarkers for disease detection and monitoring. Tumor EV isolation, processing, and evaluation from biofluids is convoluted by EV heterogeneity and biological contaminants and is limited by technical processing efficacy. This study rigorously compares common bulk EV isolation workflows (size exclusion chromatography, SEC; membrane affinity, MA) alongside downstream RNA extraction protocols to investigate molecular analyte recovery. EV integrity and recovery is evaluated using a variety of technologies to quantify total intact EVs, total and surface proteins, and RNA purity and recovery. A comprehensive evaluation of each analyte is performed, with a specific emphasis on maintaining user (n = 2), biological (n = 3), and technical replicates (n≥3) under in vitro conditions. Subsequent study of tumor EV spike‐in into healthy donor plasma samples is performed to further validate biofluid‐derived EV purity and isolation for clinical application. Results show that EV surface integrity is considerably preserved in eluates from SEC‐derived EVs, but RNA recovery and purity, as well as bulk protein isolation, is significantly improved in MA‐isolated EVs. This study concludes that EV isolation and RNA extraction pipelines govern recovered analyte integrity, necessitating careful selection of processing modality to enhance recovery of the analyte of interest. [ABSTRACT FROM AUTHOR]

Molar mass correction coefficients (cc) were determined for calculating the absolute molar masses of polylactide (PLA), polycaprolactone (PCL), and poly(2,2‐dimethyltrimethylene carbonate) (PDTC) in the range of 2,5–190 kg/mol, using calibration based on polystyrene standards (PS). Linear PLAs, PCLs, and PDTCs with a wide range of molar masses were synthesized using tin (II) octoate and diol as a catalyst/initiator system. Size Exclusion Chromatography (SEC) coupled with a Refractive Index detector (SEC‐RI) or a Multi‐Angle Laser Light Scattering detector (SEC‐MALLS) was utilized to determine the relative molar mass or absolute molar mass of the samples under investigation. Additionally, for comparison, the absolute molar mass of low molar mass PLA, PCL, or PDTC was calculated based on end‐group analysis using the 1H NMR method. Correction coefficients were subsequently calculated by comparing the molar mass obtained from SEC‐MALLS and SEC‐RI. The calculated correction coefficients ranged from 0.40 to 0.87 and increased with the rising molar mass of the polymers. The derived equation could be used to evaluate the absolute molar masses from PS standards over a broad range for the analyzed polymers. [ABSTRACT FROM AUTHOR]

Lignin, the second most abundant biopolymer on earth and with a predominantly aromatic structure, has the potential to be a raw material for valuable chemicals and other bio‐based chemicals. In industry, lignin is underutilized by being used mostly as a fuel for producing thermal energy. Valorization of lignin requires knowledge of the structure and different linkages in the isolated lignin, making the study of structure of lignin important. In this article, lignin samples isolated from two types of reactors (autoclave reactor and displacement reactor) were analyzed by FT‐IR, size exclusion chromatography, thermogravimetric analysis (TGA), and Py‐GC‐MS. The average molecular mass of the organosolv lignins isolated from the autoclave reactor decreased at higher severities, and FT‐IR showed an increase in free phenolic content with increasing severity. Except for molecular mass and molecular mass dispersity, there were only minor differences between lignins isolated from the autoclave reactor and lignins isolated from the displacement reactor. Carbohydrate analysis, Py‐GC–MS and TGA showed that the lignin isolated using either of the reactor systems is of high purity, suggesting that organosolv lignin is a good candidate for valorization. [ABSTRACT FROM AUTHOR]

The cement industry is coming into focus, as the annual production of around 4 Gt of cement is responsible for the emission of 1.5 Gt of CO2 and thus for over 8 % of anthropogenic CO2 emissions. This leads to the search for alternative binders. Such binders are calcined clays, which are available worldwide but vary greatly in their chemical and mineralogical composition. In many studies, particularly low‐calcium metakaolin is used as calcined clay, which reacts to form a low‐calcium aluminosilicate binder when mixed with a calcium‐free alkaline activator. The adjustment of the properties in the fresh state, especially regarding the consistency of these binders, is almost exclusively achieved by the addition of water, since commercially available superplasticizers are usually ineffective in low calcium geopolymer systems. The objective of this study was to investigate various PCE superplasticizers (MPEG‐, IPEG‐, HPEG‐PCE) with respect to their stability in different alkaline activators (NaOH, KOH, sodium, and potassium silicate solutions). The effectiveness of superplasticizers in low calcium geopolymer binders was verified by rheological tests. Size exclusion chromatography was used to investigate if structural degradation of the superplasticizers occurs. The investigated PCE superplasticizers showed no liquefying effect in the low calcium geopolymer system. This is due to a degradation process, i.e., the hydrolysis of the PEG side chains depending on the alkalinity of the activator. [ABSTRACT FROM AUTHOR]

Abstract Extracellular vesicles (EVs) play a pivotal role in various biological pathways, such as immune responses and the progression of diseases, including cancer. However, it is challenging to isolate EVs at high purity from blood plasma and other biofluids due to their low abundance compared to more predominant biomolecular species such as lipoprotein particles and free protein complexes. Ultracentrifugation‐based EV isolation, the current gold standard technique, cannot overcome this challenge due to the similar biophysical characteristics of such species. We developed several novel approaches to enrich EVs from plasma while depleting contaminating molecular species using multimode chromatography‐based strategies. On average, we identified 716 ± 68 and 1054 ± 35 protein groups in EV isolates from 100 µL of plasma using multimode chromatography‐ and ultracentrifugation‐based techniques, respectively. The developed methods resulted in similar EV isolates purity, providing significant advantages in simplicity, throughput, scalability, and applicability for various downstream analytical and potential clinical applications.

Biopharmaceutical products, in particular messenger ribonucleic acid (mRNA), have the potential to dramatically improve the quality of life for patients suffering from respiratory and infectious diseases, rare genetic disorders, and cancer. However, the quality and safety of such products are particularly critical for patients and require close scrutiny. Key product-related impurities, such as fragments and aggregates, among others, can significantly reduce the efficacy of mRNA therapies. In the present work, the possibilities offered by size exclusion chromatography (SEC) for the characterization of mRNA samples were explored using state-of-the-art ultra-wide pore columns with average pore diameters of 1000 and 2500 Å. Our investigation shows that a column with 1000 Å pores proved to be optimal for the analysis of mRNA products, whatever the size between 500 and 5000 nucleotides (nt). We also studied the influence of mobile phase composition and found that the addition of 10 mM magnesium chloride (MgCl2) can be beneficial in improving the resolution and recovery of large size variants for some mRNA samples. We demonstrate that caution should be exercised when increasing column length or decreasing the flow rate. While these adjustments slightly improve resolution, they also lead to an apparent increase in the amount of low-molecular-weight species (LMWS) and monomer peak tailing, which can be attributed to the prolonged residence time inside the column. Finally, our optimal SEC method has been successfully applied to a wide range of mRNA products, ranging from 1000 to 4500 nt in length, as well as mRNA from different suppliers and stressed/unstressed samples.

Purpose: Teduglutide is the first and only FDA-approved drug for long-term treatment of short bowel syndrome (SBS). The current study aimed to present an approach for production of teduglutide using recombinant DNA technology. Methods: The coding gene for teduglutide was cloned into pGEX-2T vector, where coding sequence for factor Xa cleavage site was added between GST and teduglutide coding genes. The GST-teduglutide protein was overexpressed in E. coli BL21 (DE3) strain and affinity purified using glutathione sepharose affinity column. Results: On-column proteolytic activity of factor Xa followed by size exclusion chromatography resulted in the pure teduglutide. Circular dichroism (CD) spectropolarimetry showed that the produced teduglutide folds into mainly α-helical structure (>50%), as expected. In mass spectroscopy analysis, the fragments of teduglutide resulted by cyanogen bromide cleavage as well as those expected theoretically due to mass fragmentation were identified. The functionality of the produced peptide was evaluated by measuring its proliferative effect on Caco2 intestinal epithelial cells, and the results indicated that produced teduglutide induces cell proliferation by 19±0.30 and 33±7.82 % at 1.21 and 3.64 µM concentrations, respectively, compared to untreated cells. Conclusion: Teduglutide was successfully expressed and purified and its functionality and structural integrity were confirmed by in vitro experiments. We believe that the experimental-scale method presented in the current study can be useful for pilot-scale and also industrial-scale production of teduglutide.

Abstract Extracellular vesicles (EVs) are cell derived membranous nanoparticles. EVs are important mediators of cell–cell communication via the transfer of bioactive content and as such they are being investigated for disease diagnostics as biomarkers and for potential therapeutic cargo delivery to recipient cells. However, existing methods for isolating EVs from biological samples suffer from challenges related to co‐isolation of unwanted materials such as proteins, nucleic acids, and lipoproteins. In the pursuit of improved EV isolation techniques, we introduce multimodal flowthrough chromatography (MFC) as a scalable alternative to size exclusion chromatography (SEC). The use of MFC offers significant advantages for purifying EVs, resulting in enhanced yields and increased purity with respect to protein and nucleic acid co‐isolates from conditioned 3D cell culture media. Compared to SEC, significantly higher EV yields with similar purity and preserved functionality were also obtained with MFC in 2D cell cultures. Additionally, MFC yielded EVs from serum with comparable purity to SEC and similar apolipoprotein B content. Overall, MFC presents an advancement in EV purification yielding EVs with high recovery, purity, and functionality, and offers an accessible improvement to researchers currently employing SEC.

Size exclusion chromatography (SEC) is a type of liquid chromatography used for separating molecules based on their size. The pipeline for converting a raw chromatogram to a molecular weight distribution involves multiple steps and require various parameters to be defined for each step. Commercial software lack transparency in terms of methods and algorithms, and it may be cumbersome to explore effects of different parameter settings. We have therefore developed a MATLAB toolbox that reproduces the main functionality of commercial software in a transparent and flexible manner. The toolbox consists of seven main functions, each representing a step in the calculation pipeline. The modular architecture makes it easy to modify or replace individual steps of the pipeline if necessary.

Yeastolate is often used as a media supplement in industrial mammalian cell culture or as a major media component for microbial fermentations. Yeastolate variability can significantly affect process performance, but analysis is technically challenging because of its compositional complexity. However, what may be adequate for manufacturing purposes is a fast, inexpensive screening method to identify molecular variance and provide sufficient information for quality control purposes, without characterizing all the molecular components. Here we used Size Exclusion Chromatography (SEC) and chemometrics as a relatively fast screening method for identifying lot-to-lot variance (with Principal Component Analysis, PCA) and investigated if Partial Least Squares, PLS, predictive models which correlated SEC data with process titer could be obtained. SEC provided a relatively fast measure of gross molecular size hydrolysate variability with minimal sample preparation and relatively simple data analysis. The sample set comprised of 18 samples from 12 unique source lots of an ultra-filtered yeastolate (10 kDa molecular weight cut-off) used in a mammalian cell culture process. SEC showed significant lot-to-lot variation, at 214 and 280 nm detection, with the most significant variation, that correlated with process performance, occurring at a retention time of ∼6 min. PCA and PLS regression correlation models provided fast identification of yeastolate variance and its process impact. The primary drawback is the limited column lifetime (<300 injections) caused by the complex nature of yeastolate and the presence of zinc. This limited long term reproducibility because these age-related, non-linear changes in chromatogram peak positions and shapes were very significant. • Shows how to use Size Exclusion Chromatography (SEC) for screening lot-to-lot hydrolysate variance. • Uses Multivariate Data Analysis to correlate variance with process performance. • Identifies the critical limitation for method implementation. [ABSTRACT FROM AUTHOR]

Simple Summary: Cetaceans are species of scientific interest for many reasons. First, they can be useful to assess environmental health and, second, they have peculiar features which also make them interesting for human comparative pathology. In the last decades, extracellular vesicles have been studied as important carriers in cell-to-cell communication, and many studies in human and veterinary medicine have focused on their role in pathophysiological mechanisms or as biomarker to diagnose diseases. In vitro studies are good models to explore extracellular vesicles. However, cell lines have been poorly used and investigated in these species. For these reasons, here we describe for the first time the isolation of extracellular vesicles from two cetacean cell lines established from bottlenose dolphin and Cuvier's beaked whale. We also compare two different techniques to isolate extracellular vesicles, reporting the difference in the yield and quality of the obtained sample. This preliminary study on extracellular vesicles isolated in vitro aims to be the basis for future research to deepen our understanding on cetacean pathophysiology. Cetaceans are of scientific interest because they are good candidates as environmental bioindicators. However, in vivo research is arduous and in vitro studies represent a rarely used valid alternative. Extracellular vesicles (EVs) are membrane-bound structures playing roles in cell-to-cell communication. Despite being a promising investigative tool in different fields of science, EVs have been poorly studied in cetaceans. To fill this gap, we describe the preliminary characterization of EVs isolated from a bottlenose dolphin and a Cuvier's beaked whale cell line. EVs have been isolated with ultracentrifugation (UC) or size exclusion chromatography (SEC) and characterized with nanoparticle tracking analysis (NTA), Western blotting (WB), and scanning transmission electron microscopy (STEM). UC and SEC allowed the isolation of mainly small EVs (<200 nm). A higher number of particles were isolated through UC compared to SEC from both cell lines. At WB, all EVs expressed the EV-markers CD9 and integrin-β. Only EVs isolated with UC were positive for TSG101. In conclusion, we isolated for the first time EVs from a bottlenose dolphin and a Cuvier's beaked whale cell line using two different techniques. Further studies on cell-derived EVs will be useful to deepen our knowledge on cetacean pathophysiology and health status assessment. [ABSTRACT FROM AUTHOR]

Chemical exchange saturation transfer (CEST) MRI has been identified as a novel alternative to classical diagnostic imaging. Over the last several decades, many studies have been conducted to determine possible CEST agents, such as endogenously expressed compounds or proteins, that can be utilized to produce contrast with minimally invasive procedures and reduced or non‐existent levels of toxicity. In recent years there has been an increased interest in the generation of genetically engineered CEST contrast agents, typically based on existing proteins with CEST contrast or modified to produce CEST contrast. We have developed an in silico method for the evolution of peptide sequences to optimize CEST contrast and showed that these peptides could be combined to create de novo biosensors for CEST MRI. A single protein, superCESTide, was designed to be 198 amino acids. SuperCESTide was expressed in E. coli and purified with size exclusion chromatography. The magnetic transfer ratio asymmetry generated by superCESTide was comparable to levels seen in previous CEST reporters, such as protamine sulfate (salmon protamine) and human protamine. These data show that novel peptides with sequences optimized in silico for CEST contrast that utilize a more comprehensive range of amino acids can still produce contrast when assembled into protein units expressed in complex living environments. [ABSTRACT FROM AUTHOR]

Owing to a supposed quantitative transformation, oximation of dialdehyde cellulose (DAC) with hydroxylamine hydrochloride is commonly employed in chemical DAC analysis, e.g., for the determination of the degree of oxidation (DO) by titration or elemental analysis. In this study, this modification was utilized for the indirect determination of molecular weight distributions (MWD) by gel permeation chromatography (GPC). The presumably quantitative conversion of aldehyde groups in DAC to the corresponding oxime also breaks up the intermolecular and intramolecular hemiacetal crosslinks, which were associated with solubility issues in the DMAc/LiCl solvent system in previous studies. The limits of the procedure and the material's stability during oximation were investigated. For samples with a DO up to approximately 9% a good applicability was observed, before at higher DO values residual crosslinks led to solubility problems. The oximation/GPC protocol was used to examine the development of the MWD in the early stages of DAC formation under different reaction conditions. The time-dependent partial depolymerization of the polymer backbone was observed. Furthermore, the stability of DAC towards different pH conditions ranging from strongly acidic to strongly alkaline was tested. The depolymerization of DAC in alkaline media occurred with concomitant degradation of aldehyde moieties. In turn, DAC proved to be remarkably stable in acidic and neutral solutions up to a pH of 7. [ABSTRACT FROM AUTHOR]

Modern analytical size exclusion chromatography (SEC) is a suitable technique to separate venom toxin families according to their size characteristics. In this study, a method was developed to separate intact venom toxins from Bungarus multicinctus and Daboia russelii venoms via analytical SEC using volatile, non-salt-containing eluents for post-column mass spectrometry, coagulation bioassaying and high-throughput venomics. Two venoms were used to demonstrate the method developed. While the venom of Bungaurs multicinctus is known to exert anticoagulant effects on plasma, in this study, we showed the existence of both procoagulant toxins and anticoagulant toxins. For Daboia russelii venom, the method revealed characteristic procoagulant effects, with a 90 kDa mass toxin detected and matched with the Factor X-activating procoagulant heterotrimeric glycoprotein named RVV-X. The strong procoagulant effects for this toxin show that it was most likely eluted from size exclusion chromatography non-denatured. In conclusion, the separation of snake venom by size gave the opportunity to separate some specific toxin families from each other non-denatured, test these for functional bioactivities, detect the eluting mass on-line via mass spectrometry and identify the eluted toxins using high-throughput venomics. [ABSTRACT FROM AUTHOR]

Scorpion venoms have long captivated scientific researchers, primarily due to the potency and specificity of the mechanism of action of their derived components. Among other molecules, these venoms contain highly active compounds, including antimicrobial peptides (AMPs) and ion channel-specific components that selectively target biological receptors with remarkable affinity. Some of these receptors have emerged as prime therapeutic targets for addressing various human pathologies, including cancer and infectious diseases, and have served as models for designing novel drugs. Consequently, extensive biochemical and proteomic investigations have focused on characterizing scorpion venoms. This review provides a comprehensive overview of the key methodologies used in the extraction, purification, analysis, and characterization of AMPs and other bioactive molecules present in scorpion venoms. Noteworthy techniques such as gel electrophoresis, reverse-phase high-performance liquid chromatography, size exclusion chromatography, and "omics" approaches are explored, along with various combinations of methods that enable bioassay-guided venom fractionation. Furthermore, this review presents four adapted proteomic workflows that lead to the comprehensive dissection of the scorpion venom proteome, with an emphasis on AMPs. These workflows differ based on whether the venom is pre-fractionated using separation techniques or is proteolytically digested directly before further proteomic analyses. Since the composition and functionality of scorpion venoms are species-specific, the selection and sequence of the techniques for venom analyses, including these workflows, should be tailored to the specific parameters of the study. [ABSTRACT FROM AUTHOR]