Gel Filtration Research Articles

A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies

Background: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. Methods: We analyzed the quality of such biomolecules by a combination of gel filtration and SDS-PAGE to identify artefacts due to aggregation or material degradation. Results: In the SDS-PAGE run, unexpected bands corresponding to separate fluobodies were evidenced and characterized as either degradation products or artefacts that systematically resulted in the presence of specific FPs and some experimental conditions. The elimination of N-terminal methionine from FPs did not impair the appearance of FP fragments, whereas the stability and migration characteristics of some FP constructs were strongly affected by heating in loading buffer, which is a step samples undergo before electrophoretic separation. Conclusions: In this work, we provide explanations for some odd results observed during the quality control of fluobodies and summarize practical suggestions for the choice of the most convenient FPs to fuse to antibody fragments.

Impact on Quality during In-Use Preparation of an Antibody Drug Conjugate with Eight Different Closed System Transfer Device Brands.

The aim of this study was to investigate the in-use compatibility of eight commercially available closed system transfer device brands (CSTDs) with a formulated model antibody drug conjugate (ADC). Overall, in-use simulated dosing preparation applying the CSTD systems investigated raised concerns for several product quality attributes. The incompatibilities observed were mainly associated with increased visible and subvisible particles formation as well as significant changes in holdup volumes. Visible and subvisible particles contained heterogeneous mixtures of particle classes, with the majority of subvisible particles associated with silicone oil leaching from CSTD systems during simulated dose preparation upon contact with the ADC formulation. These observations demonstrate that CSTD use may adversely impact product quality and delivered dose which could potentially lead to safety and efficacy concerns during administration. Other product quality attributes measured including turbidity, color, ADC recovery, and purity by size exclusion HPLC, did not show relevant changes. It is therefore strongly recommended to test and screen the compatibility of CSTDs with the respective ADC, in a representative in-use simulated administration setting, during early CMC development, i.e., well before the start of clinical studies, to include information about compatibility and to ensure that the CSTD listed in the manuals of preparation for clinical handling has been thoroughly assessed before human use.

Interaction of heparin with human cardiac troponin complex and its influence on the immunodetection of troponins in human blood samples.

Heparin is a highly charged polysaccharide used as an anticoagulant to prevent blood coagulation in patients with presumed myocardial infarction and to prepare heparin plasma samples for laboratory tests. There are conflicting data regarding the effects of heparin on the measurement of cardiac isoforms of troponin I (cTnI) and troponin T (cTnT), which are used for the immunodiagnosis of acute myocardial infarction. In this study, we investigated the influence of heparin on the immunodetection of human cardiac troponins. Gel filtration (GF) techniques and sandwich fluoroimmunoassay were performed. The regions of сTnI and cTnT that are affected by heparin were investigated with a panel of anti-cTnI and anti-cTnT monoclonal antibodies, specific to different epitopes. Heparin was shown to bind to the human cardiac full-size ternary troponin complex (ITC-complex) and free cTnT, which increased their apparent molecular weights in GF studies. Heparin did not bind to the low molecular weight ITC-complex and to binary cTnI-troponin С complex. We did not detect any sites on cTnI in the ITC-complex that were specifically affected by heparin. In contrast, cTnT regions limited to approximately 69-99, 119-138 and 145-164 amino acid residues (aar) in the ITC-complex and a region that lies approximately between 236 and 255 aar of free cTnT were prone to heparin influence. Heparin binds to the ITC-complex via cTnT, interacting with several sites on the N-terminal and/or central parts of the cTnT molecule, which might influence the immunodetection of analytes in human blood.

(Thermo)mechanical and chemical characteristics of photochemically crosslinked acrylates from bio‐ and fossil‐based origin

AbstractThe acrylates with oligomers and monomers from (partially) bio‐based feedstock become available at (semi‐)industrial scale, which can be processed through photochemical crosslinking for applications in coatings, additive manufacturing, electronics, or inks. Although fossil‐ and bio‐based acrylates may have a similar chemical composition, it requires good understanding of processing and structure–property relationships as minor changes in microstructure may strongly alter the performance. A comparative study on mechanical properties and chemical structure of bio‐ and fossil‐based acrylates with different functionalities and backbone structures reveals higher ductility of bio‐based acrylates, in relation with a more complex organization of the intrinsic molecular structure. The latter is confirmed by mechanical testing and visco‐elastic characteristics (dynamic mechanical analysis) yielding lower stiffness and higher dampening of bio‐based acrylates, in parallel with a lower glass transition temperature (differential scanning calorimetry). The complex molecular arrangements include a nanoscale morphology with ordered structure (X‐ray diffraction), conformational changes (infrared spectroscopy), and a residual high‐molecular weight fraction (size exclusion chromatography). The visco‐elastic calculations indicate only 4% to 5% lower crosslinking density and around 10% higher mean molar mass of the polymer chains segments between chemical crosslinks and trapped chain entanglements, which explain the unique structure and performance of bio‐based acrylates.

Defining albumin as a glycoprotein with multiple N-linked glycosylation sites

BackgroundGlycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated.MethodsWe enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography–tandem mass spectrometry (LC–MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC–MS/MS.ResultsHuman albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated.ConclusionsAlbumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.

Interfacial Super‐Assembly of Vacancy Engineered Ultrathin‐Nanosheets toward Nanochannels for Smart Ion Transport and Salinity Gradient Power Conversion

Ion‐selective nanochannel membranes assembled from two‐dimensional (2D) nanosheets hold immense promise for power conversion using salinity gradient. However, they face challenges stemming from insufficient surface charge density, which impairs both permselectivity and durability. Herein, we present a novel vacancy‐engineered, oxygen‐deficient NiCo layered double hydroxide (NiCoLDH)/cellulose nanofibers‐wrapped carbon nanotubes (VOLDH/CNF‐CNT) composite membrane. This membrane, featuring abundant angstrom‐scale, cation‐selective nanochannels, is designed and fabricated through a synergistic combination of vacancy engineering and interfacial super‐assembly. The membrane shows interlayer free‐spacing of ~3.62 Å, which validates the membrane size exclusion selectivity.This strategy, validated by DFT calculations and experimental data, improves hydrophilicity and surface charge density, leading to the strong interaction with K+ ions to benefit the low ion transport resistance and exceptional charge selectivity. When employed in an artificial river water|seawater salinity gradient power generator, it delivers a high‐power density of 5.35 W/m2 with long‐term durability (20,000s), which is almost 400% higher than that of the pristine NiCoLDH membrane. Furthermore, it displays both pH‐ and temperature‐sensitive ion transport behavior, offering additional opportunities for optimization. This work establishes a basis for high‐performance salinity gradient power conversion and underscores the potential of vacancy engineering and super‐assembly in customizing 2D nanomaterials for diverse advanced nanofluidic energy devices.

Interfacial Super-Assembly of Vacancy Engineered Ultrathin-Nanosheets toward Nanochannels for Smart Ion Transport and Salinity Gradient Power Conversion.

Ion-selective nanochannel membranes assembled from two-dimensional (2D) nanosheets hold immense promise for power conversion using salinity gradient. However, they face challenges stemming from insufficient surface charge density, which impairs both permselectivity and durability. Herein, we present a novel vacancy-engineered, oxygen-deficient NiCo layered double hydroxide (NiCoLDH)/cellulose nanofibers-wrapped carbon nanotubes (VOLDH/CNF-CNT) composite membrane. This membrane, featuring abundant angstrom-scale, cation-selective nanochannels, is designed and fabricated through a synergistic combination of vacancy engineering and interfacial super-assembly. The membrane shows interlayer free-spacing of ~3.62 Å, which validates the membrane size exclusion selectivity.This strategy, validated by DFT calculations and experimental data, improves hydrophilicity and surface charge density, leading to the strong interaction with K+ ions to benefit the low ion transport resistance and exceptional charge selectivity. When employed in an artificial river water|seawater salinity gradient power generator, it delivers a high-power density of 5.35 W/m2 with long-term durability (20,000s), which is almost 400% higher than that of the pristine NiCoLDH membrane. Furthermore, it displays both pH- and temperature-sensitive ion transport behavior, offering additional opportunities for optimization. This work establishes a basis for high-performance salinity gradient power conversion and underscores the potential of vacancy engineering and super-assembly in customizing 2D nanomaterials for diverse advanced nanofluidic energy devices.

Purification, Characterization and Antifungal Activity of the Aspergillus niveus Chitinase Produced Using Shrimp Shells

Chitinases are biotechnologically relevant enzymes that can be applied in such different sectors as pharmaceutical, food, environmental management, the biocontrol of pests and in the paper and cellulose industry. Microorganisms as filamentous fungi are the most important source of these biomolecules. The fungus Aspergillus niveus produces extracellular chitinase when cultured under submerged fermentation using shrimp shells, a residue generated by the fish industry, as a carbon source, for 96 h at 30 °C and 100 rpm. The particle size and concentration of the shrimp shells affected enzyme production. The chitinase was purified until electrophoretic homogeneity through the use of a Sephadex G-100 chromatographic column. It is a monomeric glycoprotein with a molecular mass of 47 kDa estimated using SDS-PAGE and 49.3 kDa determined using gel filtration. The carbohydrate content was 22.8%. The best temperature and pH for enzyme activity were 65 °C and 6.0, respectively. Approximately 80% of the enzymatic activity was preserved at pH 4.0 and 5.0 for 48 h, and the half-life (t50) was maintained for 48 h at 40 °C. Salts, EDTA and β-mercaptoethanol did not affect chitinase activity significantly, but organic solvents reduced it. The kinetic parameters determined using p-NPGlycNac were Km of 2.67 mmol L−1, Vmax of 12.58 U mg of protein−1, Kcat of 2.47 s−1 and K cat/Km of 0.93 s−1 mmol L−1. The A. niveus chitinase inhibited the growth of all fungal strains used, especially Trichoderma harzianum (MIC = 22.4 μg mL−1) and Penicillium purpurogenum (MIC = 11.2 μg mL−1). The chitinase produced by A. niveus presented interesting characteristics that indicate its potential of application in different areas.

Phytochemical evaluation of Ziziphus mucronata and Xysmalobium undulutum towards the discovery and development of anti-malarial drugs

BackgroundThe development of resistance by Plasmodium falciparum is a burdening hazard that continues to undermine the strides made to alleviate malaria. As such, there is an increasing need to find new alternative strategies. This study evaluated and validated 2 medicinal plants used in traditional medicine to treat malaria.MethodsInspired by their ethnobotanical reputation of being effective against malaria, Ziziphus mucronata and Xysmalobium undulutum were collected and sequentially extracted using hexane (HEX), ethyl acetate (ETA), Dichloromethane (DCM) and methanol (MTL). The resulting crude extracts were screened for their anti-malarial and cytotoxic potential using the parasite lactate dehydrogenase (pLDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. This was followed by isolating the active compounds from the DCM extract of Z. mucronata using silica gel chromatography and structural elucidation using spectroscopic techniques (NMR: 1H, 12C, and DEPT). The active compounds were then targeted against P. falciparum heat shock protein 70–1 (PfHsp70-1) using Autodock Vina, followed by in vitro validation assays using ultraviolet–visible (UV–VIS) spectroscopy and the malate dehydrogenase (MDH) chaperone activity assay.ResultsThe extracts except those of methanol displayed anti-malarial potential with varying IC50 values, Z. mucronata HEX (11.69 ± 3.84 µg/mL), ETA (7.25 ± 1.41 µg/mL), DCM (5.49 ± 0.03 µg/mL), and X. undulutum HEX (4.9 ± 0.037 µg/mL), ETA (17.46 ± 0.024 µg/mL) and DCM (19.27 ± 0.492 µg/mL). The extracts exhibited minimal cytotoxicity except for the ETA and DCM of Z. mucronata with CC50 values of 10.96 and 10.01 µg/mL, respectively. Isolation and structural characterization of the active compounds from the DCM extracts revealed that betulinic acid (19.95 ± 1.53 µg/mL) and lupeol (7.56 ± 2.03 µg/mL) were responsible for the anti-malarial activity and had no considerable cytotoxicity (CC50 > µg/mL). Molecular docking suggested strong binding between PfHsp70-1, betulinic acid (− 6.8 kcal/mol), and lupeol (− 6.9 kcal/mol). Meanwhile, the in vitro validation assays revealed the disruption of the protein structural elements and chaperone function.ConclusionThis study proves that X undulutum and Z. mucronata have anti-malarial potential and that betulinic acid and lupeol are responsible for the activity seen on Z. mucronata. They also make a case for guided purification of new phytochemicals in the other extracts and support the notion of considering medicinal plants to discover new anti-malarials.

From laboratory to industrial use: Understanding process variation during enzymatic protein hydrolysis with dry film fourier-transform infrared spectroscopy

Industries are continuously looking for dedicated sensor solutions for evaluating the chemical composition of products that can provide valuable insights into process control, process optimization, and product quality. A rapid and robust analytical method, Fourier-Transform Infrared (FTIR) spectroscopy, holds significant potential in this regard as it provides detailed compositional values of food nutrients. The main aim of the present study was to use dry film FTIR spectroscopy as an analytical tool to characterize products from an industrial enzymatic protein hydrolysis process and link this to understand industrial process variations. For this purpose, 463 protein hydrolysate samples were obtained from an industrial enzymatic protein hydrolysis plant. In the same period, systematic variations in process parameters such as raw material composition, enzyme type, and water addition, were performed. All samples were analyzed using dry film FTIR spectroscopy. Two subsets containing 200 and 68 hydrolysate samples were chosen for measuring average molecular weight (AMW) and collagen content respectively, providing reference data for constructing calibration models based on partial least squares regression (PLSR). The percentage of low molecular weight constituents was derived from the molecular weight distribution of size exclusion chromatograms of protein hydrolysates and also used in the modeling. Calibration models for the prediction of AMW, low molecular weight constituents, and collagen content were obtained with a good fit and lower estimation errors. Principal component analysis (PCA) of protein hydrolysates' FTIR spectra effectively differentiated process variations (enzyme types and raw materials) without extensive reference analysis. One-factor analysis of variance (ANOVA) tests was used to observe the impact of process variation on product quality. FTIR proved to be a sensitive method for liquid protein analysis and process control with a significant potential in process optimization approaches. To the authors’ knowledge, this is the first time dry film FTIR spectroscopy has been used to evaluate an industrial bioprocess with designed process variations.

Investigation of long-term pressure on primary packaging materials and a biologic drug product for injection with a novel autoinjector concept.

Gerresheimer and Midas Pharma have developed a novel cartridge-based autoinjector concept in which the cartridge as primary packaging is under constant pressure. In this article standard cartridge primary packaging material of five different companies were analyzed for their behavior under long-term pressure. Materials of 3 glass manufacturers and 2 manufacturers for cartridge rubber parts were considered. Within the test program septum stability, septum piercing, glide forces (GF), break-loose forces (BLF), glass breaking as well as a regulatory approved and marketed antibody drug product under pressure were subject to analysis. Under pressure the cartridge septum bulge grew within the first 14 days and then relevantly slowed down. An accelerated study in different atmospheric conditions allowed to extrapolate values for 24 months storage, not showing any signs of decay or problematic septum bulge increase. Pierce forces were in normal ranges and septum rupture could not be observed at the end of 42 days of pressurization. GF and BLF were within acceptable ranges and changes due to pressure could not be observed. Lowest glass breaking pressures at 4922kPa turned out to be at least 3.5 times higher than pressures used in the autoinjector concept. Degradation of the Adalimumab antibody drug product due to pressure or device fluid pathway could not be observed with size exclusion chromatography, electrophoresis or sub-visible particles tested as a release testing in a GMP setting.

Chain branching during the polycondensation of isophthalic aldehyde and 1,1′‐diacetylferrocene by the Claisen–Schmidt reaction

AbstractN‐methyl‐2‐pyrrolidone‐soluble ferrocene‐containing polychalcones, which are promising as a platform for the formation of ferromagnetic coatings after pyrolysis in an inert atmosphere is synthesized through the polycondensation of isophthalic aldehyde and 1,1′‐diacetylferrocene by the Claisen–Schmidt reaction. The structure and the molecular weight distribution of the polychalcones is characterized by NMR, IR spectroscopy, and size exclusion chromatography. It is shown that the formation of polychalcones is accompanied by chain branching via the Michael reactions and the aldol‐croton condensation. The structure of the linear fragments of the polychalcone chains is simulated with a tailored low‐molecular model compound, that is 1,3‐bis(3‐ferrocenyl‐1‐propen‐2‐one)benzene, which is characterized by the NMR and IR spectroscopy as well as the X‐ray diffraction analysis. The obtained results indicate an increasing tendency for polychalcone chain branching with the temperature growth during the Claisen–Schmidt reaction. The weight loss and the increase in magnetization under the conditions of pyrolysis of polychalcones at different temperatures is studied. The assumptions regarding the possible mechanisms of thermal destruction of ferrocene‐containing macromolecules are put forward.

In-Depth Endogenous Phosphopeptidomics of Serum with Zirconium(IV)-Grafted Mesoporous Silica Enrichment.

Detection of endogenous peptides, especially those with modifications (such as phosphorylation) in biofluids, can serve as an indicator of intracellular pathophysiology. Although great progress has been made in phosphoproteomics in recent years, endogenous phosphopeptidomics has largely lagged behind. One main hurdle in endogenous phosphopeptidomics analysis is the coexistence of proteins and highly abundant nonmodified peptides in complex matrices. In this study, we developed an approach using zirconium(IV)-grafted mesoporous beads to enrich phosphopeptides, followed by analysis with a high resolution nanoRPLC-MS/MS system. The bifunctional material was first tested with digests of standard phosphoproteins and HeLa cell lysates, with excellent enrichment performance achieved. Given the size exclusion nature, the beads were directly applied for endogenous phosphopeptidomic analysis of serum samples from pancreatic ductal adenocarcinoma (PDAC) patients and controls. In total, 329 endogenous phosphopeptides (containing 113 high confidence sites) were identified across samples, by far the largest endogenous phosphopeptide data set cataloged to date. In addition, the method was readily applied for phosphoproteomics of the same set of samples, with 172 phosphopeptides identified and significant changes in dozens of phosphopeptides observed. Given the simplicity and robustness of the proposed method, we envision that it can be readily used for comprehensive phosphorylation studies of serum and other biofluid samples.

Comparative characterisation of extracellular vesicles from canine and human plasma: a necessary step in biomarker discovery.

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.

Biochemical characterization, stability, and kinetics of three substrates of the recombinant TMPRSS2 serine protease domain

Transmembrane serine protease 2 (TMPRSS2) is a membrane-bound protease belonging to the type II transmembrane serine protease (TTSP) family. It is a multidomain protein, including a serine protease domain responsible for its self-activation. The protein has been implicated as an oncogenic transcription factor and for its ability to cleave (prime) the SARS-CoV-2 spike protein. In order to characterize the TMPRSS2 biochemical properties, we expressed the serine protease domain (rTMPRSS2_SP) in Komagataella phaffii using the pPICZαA vector and purified it using immobilized metal affinity (Ni Sepharose™ excel) and size exclusion (Superdex 75) chromatography. We explored operational fluorescence resonance energy transfer FRET peptides as substrates. We chose the peptide Abz-QARK-(Dnp)-NH2 (Abz = ortho-aminobenzoic acid, the fluorescence donor, and Dnp = 2,4-dinitrophenyl, the quencher group) as a substrate to find the optimal conditions for maximum enzymatic activity. We found that metallic ions such as Ca2+ and Na+ increased enzymatic activity, but ionic surfactants and reducing agents decreased catalytic capacity. Finally, we determined the rTMPRSS2_SP stability for long-term storage. Altogether, our results represent the first comprehensive characterization of TMPRSS2’s biochemical properties, providing valuable insights into its serine protease domain.

Chinese medicine Di-long (Pheretima vulgaris) and its active fraction exhibit anti-rheumatoid arthritis effects by inhibiting CXCL10/CXCR3 chemotaxis in synovium

Ethnopharmacological relevanceDi-Long (Pheretima vulgaris) is a classic animal sourced traditional Chinese medicine. It has been used for the treatment of joint inflammation and arthralgia for over two thousand years due to its effects of Tong-Luo-Zhi-Tong (dredging collaterals and alleviating pain). Our previous study showed that Chinese medicine Di-Long has significant anti-rheumatoid arthritis (RA) effects. Aim of the studyConsidering Di-Long as a potential source of active compounds with specific anti-RA therapeutic effects, this research was to obtain the anti-RA target-specific active fraction from Di-Long extracts (DL), and to further explore the chemical basis and verify the anti-RA mechanism of this active fraction. Materials and methodsTranscriptomic was applied to obtain the main anti-RA targets of DL on human RA fibroblast-like synoviocytes (FLS) and validated by qPCR. The target-corresponding active fraction was isolated from DL by ethanol precipitation and gel chromatography, and analyzed by nanoliter chromatography-mass spectrometry. Anti-RA effects of this active fraction was investigated by collagen-induced arthritis (CIA) in mice, and anti-RA mechanisms were verified in cocultured model of rat FLS and peripheral blood lymphocytes. ResultsWe confirmed that CXCL10/CXCR3 was the main anti-RA target of DL. The active fraction - A (2182 - 890 Da) was isolated from DL based on its CXCL10 inhibiting effects in RA-FLS. Fraction A contains 195 peptides (192 were newly discovered), 26 of which might be bioactive and were considered to be the chemical basis of its anti-RA effects. Fraction A significantly ameliorated the joint destruction and overall inflammation in CIA mice, and downregulated CXCR3 expression in mice joint. Fraction A inhibited the chemotaxis of Th-cells in rat peripheral blood lymphocytes towards the TNF-α-induced rat FLS through CXCL10/CXCR3 pathway. ConclusionsOur work indicated that active fraction from DL containing small peptides exhibits promising therapeutic effects for RA through inhibiting CXCL10/CXCR3 chemotaxis.

Effect of the 35 nm and 70 nm Size Exclusion Chromatography (SEC) Column and Plasma Storage Time on Separated Extracellular Vesicles.

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.

Identification and characterization of a new potent inhibitor targeting CtBP1/BARS in melanoma cells

BackgroundThe C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with “cancer hallmarks” and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT).Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions.Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities.MethodsStructured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model.ResultsWe identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models. ConclusionsThis study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.

Characteristics of fibrin/fibrinogen degradation products in multiple sclerosis following SARS-CoV-2 infection

Background. The purpose of this study was to investigate plasma levels of fibrinogen and products of its degradation in patients with multiple sclerosis (MS) with and without a history of coronavirus disease 2019 (COVID-19). Materials and methods. We examined 97 patients with MS. Based on the presence of COVID-19, all cases were divided into two groups. MS group included 56 patients who did not suffer from COVID-19 previously. MS + COVID group consisted of 41 cases who had a laboratory-verified diagnosis of COVID-19. The group of healthy controls included 30 healthy volunteers. Spectrophotometric techniques were used to measure the concentrations of fibrinogen, D-dimer, and soluble fibrin monomer complexes (SFMCs). Size-exclusion chromatography was applied to analyze the composition of SFMC fractions. Results. We found that concentrations of fibrinogen, D-dimer, and SFMCs were remarkably increased in plasma of all MS patients compared with healthy controls. The levels of D-dimer, and SFMCs did not differ between two MS groups, while plasma fibrinogen concentration was significantly increased in MS + COVID patients compared to MS group. Moreover, the development of MS was accompanied by the changes in both quantity and quality of SFMC composition compared to that of healthy controls. Our results demonstrated accumulation of high-molecular-weight SFMCs in plasma of MS patients. Conclusions. The findings indicated that MS patients had changed hemostasis characteristics; however, more research is required to determine the connection between particular hemostatic factors, namely fibrinogen, D-dimer, and SFMCs, and the pathophysiology of MS.

Cytotoxic Secondary Metabolites from the Ascomycetous Fungus Pseudogymnoascus roseus

Background: Fungi found in unique and competitive environments are abundant in bioactive compounds. Objective: The objective of this study is to isolate and identify secondary metabolites from fungi of unique ecological niches and evaluate their cytotoxicity. Methods: The compounds were isolated and purified using silica gel column chromatography, Sephadex LH-20 gel chromatography, and semi-preparative high-performance liquid chromatography (HPLC). The structures of the isolated compounds were determined using NMR and MS. The cytotoxic activities of these compounds were tested by the MTS assay. Results: Three diphenyl ethers, dechlorodihydromaldoxin (1), violaceol-I (2) and violaceol-II (3), one quinolinone compound, 2-(2-heptenyl)-3-methyl-4(1H)-quinolinone (4), and one α-pyrone nafuredin (5) were isolated from the fermented extracts of Pseudogymnoascus roseus S161. Compound 1 showed modest cytotoxicity against two human tumor cell lines A549 and MCF-7, with IC50 values of 87.12 and 51.07 µM, respectively. Conclusion: Five compounds were isolated from the fungus P. roseus S161. Compound 1 showed moderate cytotoxicity. This study provided a basis for the development of antitumor drugs.