Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 μm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.
- Keywords
- High-throughput lipidomics, Mass spectrometry, Plasma, Quantitation, Reversed-phase, Serum, Ultrahigh-performance liquid chromatography,
- MeSH
- Chromatography, Reverse-Phase * methods MeSH
- Mass Spectrometry methods MeSH
- Liquid Chromatography-Mass Spectrometry MeSH
- Humans MeSH
- Lipidomics * methods MeSH
- Lipids * analysis MeSH
- High-Throughput Screening Assays methods MeSH
- Software MeSH
- Tandem Mass Spectrometry methods MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Lipids * MeSH
The enzymatic degradation of polyethylene terephthalate (PET) by PETases has gained significant attention as a potential solution for plastic waste management. However, the absence of a standardized protocol for PETase production across studies presents a challenge for consistent enzyme characterization and activity comparison. Variations in production methods, including expression systems and purification techniques, may contribute to discrepancies in reported PETase activities. Here, we present the development of a unified protocol for the production of wild-type and engineered IsPETase variants. This protocol comprises standardized expression, purification, and quality control steps to ensure reproducibility and reliability. By enabling more accurate comparisons of PETase variants and addressing inconsistencies in PETase production, this approach facilitates collaborative efforts to advance plastic degradation technologies and lays the groundwork for accelerating research in enzymatic PET degradation and its applications in plastic waste management.
- Keywords
- Enzyme activity assay, IsPETase, Optimization, PET degradation, Standardized protocol,
- MeSH
- Escherichia coli genetics metabolism MeSH
- Polyethylene Terephthalates * metabolism chemistry MeSH
- Workflow MeSH
- Recombinant Proteins genetics isolation & purification chemistry biosynthesis metabolism MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
- Names of Substances
- Polyethylene Terephthalates * MeSH
- Recombinant Proteins MeSH
Current standards in vascular reconstruction imply the use of autologous or synthetic material. Despite being standard, autologous grafts are limited by pathologies already affecting the patient and possible complications at the site of explantation, while synthetic grafts carry increased infection risks. Decellularized tissues have gained significant attention due to their potential for improving integration and functionality. The decellularization process removes cellular components while retaining the extracellular matrix, providing a scaffold that supports endothelial cell growth and minimizes immune rejection. Porcine decellularized vena cava is a promising candidate for vascular graft applications due to its structural similarity to human blood vessels and biocompatibility. In this study, we decellularized porcine vena cava with a combination of Triton X-100 and sodium dodecyl sulfate in four hours. We subsequently characterized the wall structure through histological stainings and proteomic analysis. Parameters such as wall thickness, intima-media layers thickness, collagen and elastin area fraction were quantified and compared. Moreover, decellularized veins were repopulated in vitro with human endothelial cells in static and dynamic conditions to verify the adhesion of human cells to the porcine scaffold and fully functionalize the lumen. An in-house-designed bioreactor was developed to seed endothelial cells on the lumen, mimicking the in vivo blood flow.
- Keywords
- Bioreactor repopulation, Decellularization, Histological analysis, Porcine vena cava, Proteomics,
- MeSH
- Bioreactors MeSH
- Decellularized Extracellular Matrix * chemistry MeSH
- Human Umbilical Vein Endothelial Cells cytology MeSH
- Endothelial Cells cytology MeSH
- Extracellular Matrix chemistry MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Swine MeSH
- Tissue Engineering methods MeSH
- Tissue Scaffolds * chemistry MeSH
- Venae Cavae * cytology chemistry MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Decellularized Extracellular Matrix * MeSH
The layer-by-layer (LbL) assembly of polyelectrolyte multilayer films offers a versatile approach to construct ultrathin films with controlled nanostructures and functionalities. The properties of LbL assemblies are strongly influenced by the intrinsic properties of the polyelectrolytes and the assembly conditions. In this study, we investigated the effect of charge content, concentration, deposition time, and molecular weight of polyelectrolytes on the formation and stability of LbL films composed of quaternized dextran (QDex) with varying degrees of substitution (DS) (30%-90%) and heparin (Hep). Surface plasmon resonance analysis revealed that the introduction of a QDex/tannic acid anchoring bilayer effectively reduced the desorption occurring during the deposition of both strong polyelectrolytes, resulting in continuous, exponential growth of QDex/Hep LbL films. The mass deposition increased with increasing DS of QDex, particularly when the QDex concentration and deposition time were optimized. The quartz crystal microbalance with dissipation (QCM-D) monitoring revealed that increasing DS of QDex led to LbL films with progressively higher apparent elastic modulus and viscosity, indicating a transition from soft, water-rich networks to more rigid, cohesive, and less dissipative structures due to enhanced electrostatic interactions (proved by isothermal titration calorimetry) and reduced chain mobility. Furthermore, spectroscopic ellipsometry analysis of 20-bilayer QDex/Hep assemblies deposited on real silica substrates confirmed the increase in film thickness with increasing DS of QDex, especially after the formation of nine QDex/Hep bilayers, where the film structure became more stable. The obtained findings provide detailed insights into the precise control of film growth and stability, which are essential for potential applications in tissue engineering and biomaterial field.
- Keywords
- Charge content, Heparin, Layer-by-layer films, Molecular weight, Polyelectrolytes, QCM-D, Quaternized dextran,
- Publication type
- Journal Article MeSH
Antimicrobial resistance (AMR) is a mounting global health challenge projected to cause up to 10 million deaths annually by 2050. Despite advances in antibiotic discovery, the rapid emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) pathogens undermines modern medicine, threatening procedures such as surgery, chemotherapy, and organ transplantation. Conventional antibiotics face increasing limitations due to target-site mutations, efflux mechanisms, enzymatic degradation, and biofilm-associated tolerance, underscoring the urgent need for novel antimicrobial strategies. Phenazines, particularly 1-hydroxyphenazine (1-HP), represent promising alternatives owing to their redox activity, broad-spectrum antimicrobial properties, and ecological roles in microbial competition. Recent advances highlight the potential of 1-HP as both a virulence factor and a therapeutic scaffold, with applications spanning agriculture, biotechnology, and medicine. Synthetic biology, metabolic engineering, and nanocarrier-based delivery systems have enabled scalable production and reduced toxicity, while structural modifications such as halogenation have expanded therapeutic potential. This review consolidates historical, mechanistic, and translational insights into 1-HP, emphasizing its dual role as a pathogenic metabolite and a lead compound for future antimicrobial and anticancer development.
- Keywords
- 1-Hydroxyphenazine, Antimicrobial resistance, Biofilm, Nanocarriers, Phenazines, Synthetic biology,
- Publication type
- Journal Article MeSH
- Review MeSH
Cardiovascular diseases, including thrombotic events such as ischemic stroke, pulmonary embolism, and myocardial infarction, are among the leading causes of morbidity and disability worldwide. The application of clot-dissolving thrombolytic enzymes is a cost-effective therapeutic intervention for these life-threatening conditions. However, the effectiveness and safety profiles of current drugs are suboptimal, necessitating the discovery of new medicines or the engineering and enhancement of the existing ones. Here, we present a set of optimized biochemical protocols that allow robust screening and the therapeutic potential assessment of thrombolytic biomolecules. The assays provide information on multiple characteristics such as enzymatic activity, fibrinolysis rate, fibrin and fibrinogen stimulation, fibrin selectivity, clot binding affinity, and inhibition resistance. Such detailed characterization enables a rapid and reliable evaluation of candidate effectiveness and provides an indication of biological half-life, associated bleeding complications, and other side effects. We demonstrate the credibility of the methodology by applying it to the two most widely used thrombolytic drugs: alteplase (Activase®/Actilyse®) and tenecteplase (Metalyse®/TNKase®). Consistent with previous studies, tenecteplase exhibited increased fibrin selectivity and inhibition resistance, which explains its extended half-life. Our findings reinforce the growing consensus that tenecteplase may be a superior alternative to alteplase for thrombolytic treatment.
- Keywords
- PAI‐1 inhibition resistance, alteplase, fibrinolysis, plasminogen activator, staphylokinase, tenecteplase,
- Publication type
- Journal Article MeSH
Poly(1,2,3-triazole)-based polymers remain underexplored as a versatile platform for biomedical soft materials. Here, we report the modular synthesis of amphiphilic poly(1,2,3-triazole)-co-polytriazolium copolymers (qH-PETH) via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) polyaddition and post-polymerization quaternization with a degree of quaternization equal to (0.85). These polymers exhibit unusual visible colouration in the absence of chromophores-appearing almost colourless in N-methyl-2-pyrrolidone (NMP), orange in DMSO, and pale orange in water-accompanied by a pronounced red shift in non-polar solvents such as dichloromethane (DCM) or chloroform. This optical behaviour is attributed to solvent-dependent supramolecular aggregation. Dynamic light scattering (DLS) measurements reveal hydrodynamic diameters of <20 nm in water and approximately 500 nm in chloroform for quaternized polymers (qH-PETH), indicating strong aggregation in low-polarity environments. UV-vis spectroscopy and atomic force microscopy (AFM) further support aggregation-induced optical effects modulated by solvent polarity and hydrogen-bonding capacity. Both neutral and quaternized polymers display excellent cytocompatibility toward human cell lines (HeLa, HEK293, THP-1) at concentrations up to 100 μg mL-1, with no detectable immunogenic activation. These findings position poly(1,2,3-triazole)-co-polytriazolium copolymers as a promising, tunable class of biocompatible soft materials for biointerface engineering and responsive biomedical applications.
- Publication type
- Journal Article MeSH
Monitoring cognitive load is critical in diverse, demanding environments, yet conventional assessment methods face limitations in real-time applicability. While machine learning approaches using physiological signals show promise, they often require long data segments, exhibit high computational complexity, or neglect underlying causal dynamics. This paper proposes an efficient framework for cognitive load decoding using causal spatiotemporal patterns derived from multimodal peripheral biosignals. We introduce a novel feature engineering pipeline that transforms short signal segments into image-like representations capturing temporal dynamics via Gramian Angular Difference Fields and Motif Difference Fields, alongside causal interdependencies assessed using forward-backward copula Granger causality networks. These fused multimodal features are classified using a lightweight capsule neural network employing a self-attention routing mechanism. To evaluate the proposed solution, we conducted experiments on two widely used benchmark datasets, WESAD and CLAS. Our model achieved up to 94% accuracy using only 5-second signal segments, and maintained robust performance (84% accuracy) even with 1-second windows, a configuration rarely addressed in prior research. The proposed architecture includes only 323K trainable parameters, offering a favorable balance between model complexity and classification performance. The results confirm the framework's potential for computationally efficient, real-time cognitive load assessment suitable for resource-constrained environments and biofeedback applications.
- Keywords
- Capsule network, Cognitive load, Electrocardiogram, Electrodermal activity, Machine learning, Pattern recognition, Physiology,
- MeSH
- Cognition * physiology MeSH
- Humans MeSH
- Neural Networks, Computer * MeSH
- Signal Processing, Computer-Assisted * MeSH
- Machine Learning MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
Poly-(vinyl alcohol) (PVA) has been widely explored as a model material for articular cartilage (AC) in biotribological evaluations. However, PVA hydrogels prepared by freeze-thawing or cast-drying methods have limitations in precisely controlling their elasticity parameters and may require reinforcement to enhance their mechanical performance and change their transparency, required in some tribological measurement setups by using fluorescence methods. To overcome these issues, poly-(hydroxyethyl methacrylate) (pHEMA) hydrogels have been introduced as alternatives. In our study, pHEMA hydrogels synthesized using free-radical polymerization with blue light under two different atmospheres (nitrogen N2 and air) were compared with natural samples of articular bovine cartilage. The optical, mechanical, swelling, and tribological properties demonstrate the superior properties of pHEMA, which may result in the replacement of the currently used PVA-based model in future studies. Synthesis under a nitrogen atmosphere (pHEMA N 2) resulted in the formation of smooth-surfaced hydrogels, whereas synthesis under a laboratory atmosphere (pHEMA air) resulted in the formation of wrinkled-surfaced hydrogels. The swelling of both the hydrogels and AC followed first-order kinetics. Pin-on-plate biotribology measurements showed that the coefficient of friction of the wrinkled-surface hydrogels resembled that of AC. Our results showed that pHEMA-based hydrogels are suitable biotribological AC models for a better understanding of the biological functions of bovine AC. This knowledge brings new insights into cartilage complex mechanisms and might be applied in both biomedical and engineering applications.
- Publication type
- Journal Article MeSH
Mass spectrometry-based lipidomics and metabolomics generate extensive data sets that, along with metadata such as clinical parameters, require specific data exploration skills to identify and visualize statistically significant trends and biologically relevant differences. Besides tailored methods developed by individual labs, a solid core of freely accessible tools exists for exploratory data analysis and visualization, which we have compiled here, including preparation of descriptive statistics, annotated box plots, hypothesis testing, volcano plots, lipid maps and fatty acyl chain plots, unsupervised and supervised dimensionality reduction, dendrograms, and heat maps. This review is intended for those who would like to develop their skills in data analysis and visualization using freely available R or Python solutions. Beginners are guided through a selection of R and Python libraries for producing publication-ready graphics without being overwhelmed by the code complexity. This manuscript, along with associated GitBook code repository containing step-by-step instructions, offers readers a comprehensive guide, encouraging the application of R and Python for robust and reproducible chemometric analysis of omics data.
- MeSH
- Mass Spectrometry MeSH
- Humans MeSH
- Lipidomics * methods MeSH
- Metabolomics * methods MeSH
- Programming Languages MeSH
- Software * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
