Search Results (240)

Monitoring peptide and protein self-association is essential for understanding biological function, formulation stability, and aggregation mechanisms. While size-exclusion chromatography (SEC) is routinely used to quantify protein-size variants under native conditions, its hyphenation to high-resolution mass spectrometry (HRMS) for simultaneous structural characterization remains limited. Here, we report the development and validation of a robust SEC-UV/HRMS method optimized for native-like analysis of bovine serum albumin (BSA) monomers and higher-order oligomers using standard-flow electrospray ionization. Systematic evaluation of source parameters, mobile-phase composition, and chromatographic conditions enabled retention of native BSA structure, minimized in-source unfolding, and enhanced MS sensitivity, allowing detection of oligomers up to the heptamer. A short, narrow-bore 200 Å UHPLC SEC separation column was used. Low-flow separations (~0.05 mL/min) enabled efficient ionization and 10 min run times. An accelerated 60 °C stress-testing protocol demonstrated that SEC-MS can semi-quantitatively monitor oligomerization dynamics, complementing UV-based quantification and revealing transient species not resolved by UV alone. The method showed acceptable linearity, precision, and sample stability, and comparison with SEC-RALS/LALS confirmed molecular-weight trends across aggregation states. Overall, the developed SEC-UV/HRMS workflow provides a rapid, sensitive, and widely accessible approach for UV-based quantification of monomer- and HRMS-based characterizing protein aggregation in research and quality control in pharmaceutical laboratories. Full article

α-Synuclein (α-syn) aggregation underlies synucleinopathies, yet the physicochemical determinants that govern which assembly states form under defined solution conditions remain incompletely resolved. Here, we examine how pH and metal ions reshape α-syn self-assembly. Across acidic and physiological pH conditions, α-syn populates monomeric, nanoscale oligomeric, and mesoscale aggregate states whose relative abundances evolve over time. Fluorescence microscopy reveals robust mesoscale assembly at pH 5, minimal aggregation at pH 7, and transient assemblies at pH 3, highlighting the limitations of imaging-based detection alone. Therefore, we use dynamic light scattering (DLS) to resolve oligomeric populations and quantify pH-dependent redistribution of assembly mass. Toxicity-mitigating modulators altered α-syn assembly in a strongly pH-dependent manner. Anle138b increased the abundance of oligomeric species at low pH, whereas EGCG produced divergent effects at pH 5 and pH 3. We further examined the effects of metal ions, finding that Fe³⁺ stabilized higher-order assemblies under acidic conditions, Cu²⁺ delayed assembly at pH 5 while enhancing aggregation at pH 3, and Zn²⁺ increased oligomerization primarily at low pH. Overall, these results demonstrate that α-syn assembly is highly sensitive to coupled effects of pH, metal chemistry, and time. Full article

The p53 tumor suppressor, a key transcription factor, acts as a cellular stress sensor that regulates hundreds of genes involved in responses to DNA damage, oxidative stress, and ischemia. Through these actions, p53 can arrest cell cycle, initiate DNA repair, or trigger cell death. In addition to its nuclear functions, p53 can translocate to mitochondria to promote apoptosis. Studies using isolated mitochondria have suggested that p53 drives the voltage-dependent anion channel (VDAC1) into high molecular mass complexes to mediate apoptosis. VDAC1 is a central regulator of cellular energy production and metabolism and also an essential player in apoptosis, induced by various apoptotic stimuli and stress conditions. We previously demonstrated that VDAC1 oligomerization, induced by various apoptosis stimuli and stress conditions, forms a large pore that enables cytochrome c release from mitochondria, thereby promoting apoptotic cell death. In this study, we show that p53 interacts with VDAC1, modulates its expression levels, and promotes VDAC1 oligomerization-dependent apoptosis. Using purified proteins, we found that p53 directly binds VDAC1, as revealed by microscale thermophoresis and by experiments using bilayer-reconstituted VDAC1, in which p53 reduced VDAC1 channel conductance. Furthermore, overexpression of p53 in p53-null cells or in cells expressing wild-type p53 increased VDAC1 expression and induced VDAC1 oligomerization even in the absence of apoptotic stimuli. Together, these findings identify VDAC1 as a direct p53 target whose expression, oligomerization, and pro-apoptotic activity are regulated by p53. They also reinforce the central role of VDAC1 oligomerization in apoptosis. Full article

Plasmodesmata (PDs) are enriched in sphingolipids and sterols, creating a specialized environment for regulatory proteins like plasmodesmata-localized proteins (PDLPs). How PDLPs regulate PD function in a specific lipid environment remains poorly understood. Here, we provide a unique insight from the interaction network of two different PDLPs together with sphingolipids and propose a concept that PDLPs form homo- or hetero-dimers only in the presence of sphingolipids. Located in the detergent resistance region, PDLP7 demonstrated the ability to influence the sphingolipid composition in PD-enriched fraction, particularly the GIPC content, and finally, modulating the membrane order. The presence of sphingolipids, in turn, affected the oligomeric state of PDLP7 in membranes. The PDLP7 recombinant protein existed as a monomer in vitro, but it formed self-aggregates in yeast and plant cells. We further examined PDLP5, another known phytosphinganine (t18:0)-specific binding PDLP, alongside PDLP7, and confirmed a similar interaction pattern: no direct interaction was observed in vitro, but interactions were noted in vivo. Co-overexpression of the two disrupted their PD localization and induced the upregulation of pathogenesis-related protein 1 (PR1). In summary, we gained insights into the network of PDLPs with lipids and propose that PDLPs were under precise regulation during plant development and stress responses. Full article

Proteins play an important role in living organisms, and, for most of them, the function depends on their structure. There are some proteins that have similar properties but different structures. An example of this is ice-binding proteins (IBPs), which have different structures but share the ability to bind to ice. Many organisms have evolved such proteins to help them survive in cold environments. Therefore, it is important to study the oligomeric state of the active form in solutions. The activity of IBP is related to the area of their ice-binding site. We have demonstrated the presence of oligomeric forms of protein in solution using multiple techniques, such as mass spectrometry, native gel electrophoresis, atomic force microscopy (AFM), isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). It is noteworthy that, to date, there have been no reports of the oligomerization of ice-binding protein from Longhorn sculpin. Additionally, our findings suggest that larger molecules may influence the ability of proteins to bind to ice. In our study, the ice-binding protein forms elongated assemblies with limited intermonomer interfaces. The combination of SAXS and AFM data indicates a structure that combines compactness and flexibility and probably consists of four monomeric units. The employment of molecular modelling methodologies resulted in the attainment of a tetrameric complex that is in alignment with AFM data. Details of oligomers observed using the methods in our study emphasize the importance of different techniques that complement each other in resolving structural features. Additionally, we suggest that the protein particles, which were dispersed on the surface, exhibit softness or the form planar complexes with loose quaternary structures. It is conceivable that, depending on ionic strength and/or temperature, the various oligomeric forms of the ice-binding protein form thermodynamically more favorable complexes than their monomeric forms. Full article

Antibiotic resistance is an increasing concern in modern healthcare. Therefore, it is important to identify novel antimicrobial agents and new molecular targets for such compounds. Here, we describe the identification of an N-pyridylpyrazolone derivative, 4-(2-aminoethyl)-2-(pyridin-2-yl)-1,2-dihydro-3H-pyrazol-3-one dihydrochloride (compound 1), which is effective against Gram-positive and Gram-negative bacteria and inhibits the enzymatic activity of Escherichia coli L-threonine dehydrogenase (TDH). To characterize its interaction with compound 1, TDH was overexpressed in E. coli. The recombinant enzyme was shown to exist in dilute solution in equilibrium between dimeric and tetrameric forms, with a K_d value for the dimer/tetramer transition of 3 ± 1 nM, and to bind L-threonine cooperatively with a Hill coefficient of 1.4. Compound 1 acted as a partial mixed inhibitor of TDH with an EC₅₀ value of 47 ± 16 µM and did not affect the equilibrium between oligomeric states. Altogether, these findings identify compound 1 as a promising starting point for the development of novel antibiotics and as a tool compound for studying the functional properties of TDH. Full article

Fluorescence fluctuation spectroscopy (FFS) techniques rely on determination of monomeric molecular brightness, i.e., the fluorescence intensity of a single, non-aggregated fluorophore, as a critical reference for estimating protein oligomer size. By comparing measured molecular brightness of fluorescently labeled proteins of interest to this monomeric brightness benchmark, FFS enables inference of oligomerization states. However, widely used fluorescent proteins often exhibit self-association, compromising monomeric brightness calibration and introducing errors in brightness-derived oligomer-size estimates. This study compares two strategies for determining monomeric brightness: the conventional fluctuation-based method and a more recently proposed average-intensity-based alternative. The comparison uses two model fluorophores, a fluorescent protein (mCitrine) and the small-molecule dye Janelia Fluor 525 (JF₅₂₅) conjugated to HaloTag. Our results show strong agreement between intensity- and fluctuation-derived brightness values only in the minimally aggregating JF₅₂₅–HaloTag benchmark. In contrast, in the mCitrine samples, where aggregation is more prevalent, only the intensity-based method maintains a consistent estimate across sample preparation, while the fluctuation-based method overestimates brightness when aggregation effects become pronounced. This robustness makes the intensity-based approach a valuable cross-check for monomeric brightness calibration. Our results support a combined strategy, using both methods to improve the reliability of monomeric brightness calibration and protein oligomerization analysis in FFS. Full article

Background/Objective: Timely and successful treatments for Alzheimer’s disease (AD) depend on early detection. The Multimer Detection System (MDS-OAβ) for quantifying plasma oligomeric amyloid-β (OAβ) has shown promise as a biomarker of amyloid disease. The theta-to-alpha ratio (TAR) and theta-to-beta ratio (TBR) are two examples of spectral power metrics that can be used in resting-state quantitative EEG (qEEG) to evaluate brain function non-invasively. This study used resting-state EEG (rEEG) recordings obtained while the subjects were both eyes-open (EO) and eyes-closed (EC) to investigate the relationship between regional qEEG power ratios and plasma MDS-OAβ levels in older adults. Methods: The analysis comprised 174 patients between the ages of 60 and 85, with 2 in the low-MDS-OAβ group and 82 in the high-MDS-OAβ group. The clinical plasma cutoff was 0.78 ng/mL. All participants underwent rEEG recordings and plasma OAβ quantification. EEG pre-processing included bandpass filtering (0.5–100 Hz), average re-referencing, artifact rejection using independent component analysis (ICA), and spectral power estimation using Welch’s method. The TAR and TBR were calculated across five lobar regions (frontal, central, parietal, occipital, and temporal) during both EO and EC conditions. To normalize data distributions, EEG ratio variables were log-transformed prior to statistical analysis. Group comparisons and linear regression analyses were conducted to evaluate the associations between EEG power ratios and MDS-OAβ levels. Adjusted regression models included age, years of education, and neuropsychological test scores as covariates. Statistical significance was set at p < 0.05. Results: No significant associations were found between TAR and plasma MDS-OAβ levels across any lobar regions under either EO or EC conditions. In contrast, TBR exhibited consistent and significant negative associations with MDS-OAβ levels, particularly under EC conditions. Adjusted regression models revealed that higher MDS-OAβ levels were associated with lower TBR values in the central (β = −0.059, p = 0.015), parietal (β = −0.072, p = 0.006), occipital (β = −0.067, p = 0.040), and temporal (β = −0.053, p = 0.018) lobes, with the strongest inverse relationship observed in the parietal lobe. A similar, though slightly weaker, pattern was observed during EO conditions, with significant inverse associations in the frontal, central, and temporal lobes. Conclusions: Our findings indicate that, after adjusting for covariates, increased plasma MDS-OAβ levels are significantly associated with a reduced TBR, particularly in the parietal and central lobes, under both EO and EC resting-state conditions. In contrast, no significant associations were observed with TAR. These results suggest that a lower TBR may reflect an increased peripheral amyloid burden and highlight its potential as a sensitive qEEG biomarker for early amyloid-related brain changes in older adults. Full article

Background: Early diagnosis of Alzheimer’s disease (AD) is constrained by invasive and costly tests. Aggregation of $\beta$-amyloid and the A$\beta$42/A$\beta$40 ratio in cerebrospinal fluid (CSF) and blood are key biomarkers. Fluorescent probes can report aggregate states, and artificial intelligence (AI) can extract subtle patterns from spectral and blood data. This review synthesizes how probes and AI can identify aggregates and assess the A$\beta$42/A$\beta$40 ratio in body fluids to facilitate earlier AD diagnosis. Methods: PRISMA-compliant searches were conducted in Scopus, PubMed, Web of Science, and IEEE Xplore. Results: Twenty-eight studies met inclusion criteria. Plasma A$\beta$42/A$\beta$40 was lower in PET-positive individuals by ∼7–18%, with higher performance for mass spectrometry (mean AUC ≈ 0.80) than immunoassays (AUC ≈ 0.71). CSF A$\beta$42/A$\beta$40 showed larger group differences (∼50% reductions in PET+) and stronger PET concordance, outperforming plasma. Fluorescent probes—including AN-SP and CRANAD-28—were sensitive to early aggregates and showed in vivo imaging potential, but evidence is largely preclinical or from small cohorts. AI/ML approaches frequently achieved within-study accuracies >90% (e.g., 94–100% in spectral tasks), yet external validation and head-to-head tests of ratio alone versus ratio + AI remain scarce. Conclusions: Plasma A$\beta$42/40 —particularly by mass spectrometry—currently provides the most reproducible fluid approximation to amyloid PET (mean AUC ≈ 0.80). Fluorescent probes sensitively detect oligomeric A$\beta$ species and show in vivo potential, but evidence remains largely preclinical or from small cohorts. AI/ML methods can extract additional signal from spectral and multivariate blood data, yet consistent incremental gains over optimized Aβ42/40 assays have not been demonstrated due to limited external validation and head-to-head comparisons. Full article

The coordination chemistry of benzimidazole ligands combines σ donation and π backbonding. Owing to this electronic flexibility, benzimidazole ligands stabilize both electron deficient and electron-rich transition states in the catalytic cycle of Ziegler-Natta polymerizations. In this study, Fe(III) and Ni(II) complexes of 2-substituted-benzimidazoles were tested as catalysts for ethylene and 1-butene oligomerization. The tests realized in toluene yielded mainly butenes and minor amounts of hexenes. When dichloromethane was used as solvent, a tandem reaction took place and 1-butene produced by ethylene dimerization was further oligomerized, yielding octenes and dodecenes as main products. All tested catalysts exhibited moderate selectivity for 1-octene, indicating 1-ω enchainment in 1-butene dimerization. Beyond catalytic tests, a theoretical study of the ligand 2,2′-(furan-2,5-diyl)bis(1H-benzimidazole) confirmed the planar structure of this compound as evidenced by NMR spectroscopy. Full article

The development of a straightforward strategy for preparing organic fluorescent materials, fine-tuning white-light emission, and subsequently constructing white light-emitting diodes (LEDs) is of great significance. Herein, we report on the modulation of white-light emission and the fabrication of white LEDs using polyhedral oligomeric silsesquioxane (POSS)-based fluorescent hybrid porous polymers (HPPs) through simple physical blending. Two HPPs, namely HPP-1 and HPP-2, which emit blue and red light, respectively, were synthesized via the efficient Heck reactions of octavinylsilsesquioxane with 4,4′-dibromobiphenyl and 1,3,6,8-tetrabromopyrene. By physically doping of HPP-1 and HPP-2 in variable ratios in solvent suspensions, it was discovered that white-light emission is significantly influenced by the concentrations of the materials and the excitation wavelength. Similar findings were also observed in the solid-state physical doping. An ideal white light emission with a CIE coordinate of (0.33, 0.33) can be achieved when excited at 380 nm with a mass ratio of HPP-1 to HPP-2 of 1:2. Finally, the two HPPs were dispersed in polysiloxane matrices, and a white LED with a CIE coordinate of (0.42, 0.36) was obtained. The LED exhibited a color rendering index of up to 90 and a correlated color temperature of 2858 K, realizing warm white light emission. This simple and convenient white-light regulation strategy holds great promise for application in the development of novel white LEDs based on organic fluorescent porous materials. Full article

Chlorophylls, which are associated with carotenoids and photosynthetic protein complexes, acquire optical properties that enable the absorption of sunlight, necessary for the synthesis of energy and redox equivalents, necessary for photosynthetic absorption of CO₂ and the production of oxygen as an intermediate product. These processes are important for plants, but also for the biosphere. In stressful situations, when photosynthesis is limited, the production of reactive oxygen and other species increases, and the activation of various protective systems is necessary to remove the aforementioned reactive species or reduce the excessive reduction in photosynthetic electron transport, as the cause of the production of the reactive species. A review of studies where the content and physiological state of chlorophyll are monitored, using destructive and non-destructive methods, such as various optical methods for monitoring its content and physiological activity, is given. Polyphenolic compounds belong to non-enzymatic systems for quenching the reactive species. In addition to their presence in monomeric and oligomeric forms of polyphenols, polymerization of this type of compound can occur. In addition to having a protective effect on the plants that synthesize them, polyphenolic compounds also have a beneficial effect on the health of animals and humans who consume them from plants. Full article

In the polyurethane industry, catalytically generated carbodiimides can modify the properties of isocyanate and, thus, the resulting foams. In this work, a kinetic reaction study was carried out to investigate the formation of a simple, bifunctional carbodiimide from a widely used polyurethane raw material: 4,4′-methylenediphenyl diisocyanate (MDI). The experimental section outlines a catalytic process, using a 3-methyl-1-phenyl-2-phospholene-1-oxide (MPPO) catalyst in ortho-dichlorobenzene (ODCB) solvent, to model industrial circumstances. The reaction produces carbon dioxide, which was observed using gas volumetry at between 50 and 80 °C to obtain kinetic data. A detailed regression analysis with linear and novel nonlinear fits showed that the initial stage of the reaction is second-order, and the temperature dependence of the rate constant is $k(T)=(3.4\pm 3.8)\mathrm{‧}{10}^6\cdot e^{{\displaystyle \frac{-7192\pm 389}{T}}}$. However, the other isocyanate group of MDI reacts with new isocyanate groups and the reaction deviates from the second-order due to oligomer (polycarbodiimide) formation and other side reactions. A linearized Arrhenius equation was used to determine the activation energy of the reaction, which was E_a = 60.4 ± 3.0 kJ mol⁻¹ at the applied temperature range, differing by only 4.6 kJ mol⁻¹ from a monoisocyanate-based carbodiimide. In addition to experimental results, computationally derived thermochemical data (from simplified DFT and IRC calculations) were applied in transition state theory (TST) for a comprehensive prediction of rate constants and Arrhenius parameters. As a result, it was found that the activation energy of the carbodiimide bond formation reaction from theoretical and experimental results was independent of the number and position of isocyanate groups, which is consistent with the principle of equal reactivity of functional groups. Full article

Phylogenetic studies of the pyruvate kinase family reveal two clusters: the K⁺-dependent and -independent enzymes. Thermoplasma acidophilum pyruvate kinase belongs to the latter but possesses the conserved signature of those K⁺-dependent. Recently, we found two distinct ways for these groups to catalyze. It is interesting to elucidate how the T. acidophilum enzyme achieves its active conformation. A structural model of this enzyme revealed the presence of an extra C-terminal sequence (ECTS). To understand its role, an enzyme lacking this sequence from T. acidophilum was constructed. We then compared the kinetic parameters, far-UV CD spectra, thermal stability, molecular dynamics simulations, and oligomeric states of both the wild-type and truncated enzymes. We found that the truncated enzyme is aggregated and almost inactive, with residual 20% of the total interactions, and it exhibits a soluble fraction of smaller oligomeric states than the wild-type enzyme. These findings suggest that ECTS plays a crucial role in maintaining its active tetrameric state. This sequence is the first reported in an archaeal pyruvate kinase and is also found in other archaea and bacteria. Phylogenetic analysis of ECTS in pyruvate kinases exhibits a sparse distribution that might be explained if ECTS represents an ancient domain prone to loss. Full article

Ultra-high-molecular-weight polyethylene (UHMWPE) is a pivotal material in engineering and biomedical applications due to its exceptional mechanical strength, wear resistance, and impact performance. However, its extreme melt viscosity, caused by extensive chain entanglements, severely limits processability via conventional melt-processing techniques. Recent advances in catalytic synthesis have enabled the production of disentangled UHMWPE (dis-UHMWPE), which exhibits enhanced processability while retaining superior mechanical properties. Notably, heterogeneous catalytic systems, utilizing supported fluorinated bis (phenoxy-imine) titanium (FI) catalysts, polyhedral oligomeric silsesquioxanes (POSS)-modified Z-N catalysts, and other novel catalysts, have emerged as promising solutions, combining structural control with industrial feasibility. Moreover, optimizing polymerization conditions further enhances chain disentanglement while maintaining ultra-high molecular weights. These systems utilize nanoscale supports and ligand engineering to spatially isolate active sites, tailor the chain propagation/crystallization kinetics, and suppress interchain entanglement during polymerization. Furthermore, characterization techniques such as melt rheology and differential scanning calorimetry (DSC) provide critical insights into chain entanglement, revealing distinct reorganization kinetics and bimodal melting behavior in dis-UHMWPE. This development of hybrid catalytic systems opens up new avenues for solid-state processing and industrial-scale production. This review highlights recent advances concerning interaction between catalyst design, polymerization control, and material performance, ultimately unlocking the full potential of UHMWPE for next-generation applications. Full article