Search Results (1,953)

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

Objectives: The patient presented various neurological symptoms in her 50s, such as memory issues, insomnia, depression, and motor impairment. Diverse investigations were performed to identify the underlying causes on her neurological symptoms and understand her neuro- deteriorations. Methods: Clinical neurological and brain imaging analyses: CT, MRI and PET were performed on the patient. Blood was drawn for the whole-exome sequencing and functional studies with biomarker for amyloid-beta oligomers and SLC20A2 protein in plasma. Results: Brain imaging revealed calcifications in multiple regions, including the subcortical white matter, basal ganglia, thalami, and dentate nuclei. Genetic analysis revealed a c.1152_1153delCA, p.Asn384Lysfs*30 variant in SLC20A2 gene. The decreased SLC20A2 protein levels in plasma in comparison to healthy controls suggested a loss-of-function mechanism from the mutation. The patient had a positive AlzOn result, indicating an increased tendency for amyloid oligomerization and suggesting a potential indirect link between SLC20A2 and amyloid-beta pathways. Conclusions: A novel frameshift mutation, Asn384Lysfs*30, in the SLC20A2 gene was identified in a patient with Primary Brain Calcification (PBC). This mutation was located in a critical large loop region of the protein, where other similar mutations (e.g., Gly366fs89, Ser385Ilefs*70) were previously reported. These findings indicated that mutations in SLC20A2 caused the reduced protein expressions, potentially resulting PBC through haploinsufficiency. Full article

Liver disease encompasses a wide range of conditions, each requiring tailored therapeutic approaches. This review describes and critically discusses treatments with robust evidence for improving liver health. Ursodeoxycholic acid (UDCA) is a drug approved by the Food and Drug Administration of the USA to treat primary biliary cholangitis (PBC). In addition, UDCA has been demonstrated to protect against metabolic dysfunction-associated steatohepatitis, fibrosis, and drug-induced liver injury (DILI). The mechanism of action of UDCA has been attributed not only to decreasing the effects of toxic bile acids but also to protecting mitochondrial integrity and function, as well as to antioxidant, anti-inflammatory, and anti-apoptotic activities. UDCA can scavenge reactive oxygen species (ROS) and activate the nuclear factor-E2-related factor-2 (Nrf2) pathway, thereby exerting antioxidant activity. The anti-inflammatory activity of UDCA is associated with its ability to inhibit the nuclear factor-κB pathway. Pirfenidone is a well-recognized antifibrotic drug for the treatment of idiopathic pulmonary fibrosis; its effects on liver fibrosis have also been demonstrated. Pirfenidone exerts anti-inflammatory effects by attenuating the nucleotide-binding oligomerization domain-like receptor 3 inflammasome signaling pathway. The antioxidant actions of pirfenidone are associated with its ability to upregulate the Nrf2 pathway. Both the anti-inflammatory and antioxidant properties of pirfenidone act together to attenuate lung and liver fibrosis, decreasing transforming growth factor-β levels, inhibiting profibrogenic hepatic stellate cell activation, and increasing extracellular matrix degradation. Methyltransferases utilize S-adenosyl-L-methionine (SAM) as a methyl donor for most transmethylation reactions in the body. SAM increases reduced glutathione (GSH) levels, exerting important antioxidant effects. Evidence indicates that SAM prevents fibrosis and attenuates hepatocellular carcinoma development, improving patient survival. N-acetylcysteine (NAC) is a precursor to L-cysteine and GSH and is used in clinical settings to treat cancer, nephropathy, heart disease, pulmonary fibrosis, polycystic ovary syndrome, and influenza. Regarding the liver, NAC is the most accepted treatment for DILI, especially after paracetamol overdose. Owing to its antioxidant and anti-inflammatory actions, NAC has been successfully used to treat chronic liver injuries, including hepatosteatosis and fibrosis. Therefore, ursodeoxycholic acid, pirfenidone, S-adenosyl-L-methionine, and N-acetylcysteine could represent therapeutic strategies for the treatment of liver pathologies. Full article

Late-life Alzheimer’s disease (AD) is increasingly defined by biomarkers, yet in adults aged ≥65 years the relationship between amyloid positivity and near-term cognitive decline is often discordant. Amyloid PET robustly detects fibrillar plaque burden, but it incompletely captures dynamic and potentially neurotoxic amyloid processes, particularly soluble assemblies and oligomer-related “activity.” This review rethinks the late-life AD spectrum by integrating four clinical lenses that frequently drive real-world interpretive uncertainty: (1) amyloid PET positivity as a measure of fibrillar plaque presence and magnitude; (2) plasma amyloid-β oligomerization tendency measured by the multimer detection system (MDS-OAβ) as an activity-oriented (i.e., a dynamic readout hypothesized to reflect ongoing processes rather than cumulative lesion burden), process-linked readout that may decouple from plaque burden; (3) postoperative delirium (POD) as a time-anchored stress-test phenotype revealing vulnerability and reduced resilience under systemic insults; and (4) drug-linked biomarker trajectories, contrasting rapid plaque removal by anti-amyloid monoclonal antibodies with observational signals raising the hypothesis that Ginkgo biloba may be associated with oligomer-related biology and, in some contexts, differences in longitudinal amyloid accumulation trajectories in the absence of observed immediate plaque reduction. We propose a pragmatic multi-axis framework—plaque burden, amyloid activity, downstream engagement, and vulnerability/resilience—to contextualize late-life discordances such as PET positivity without decline, PET negativity with elevated MDS-OAβ, delirium-associated decompensation, and clinical change without rapid PET decline. This synthesis highlights testable predictions and prioritizes longitudinal, multi-marker studies to determine whether activity-oriented biomarkers and stress phenotypes can refine late-life risk stratification beyond plaque-centered models. Full article

Background/Objectives: Astrocytes play a critical role in maintaining brain homeostasis and are increasingly recognized as active contributors to neurodegenerative processes. Metabolic dysfunction in astrocytes has been implicated in the onset and progression of Alzheimer’s disease (AD), yet the underlying metabolic alterations remain poorly characterized. Methods: We used an optimized protocol for untargeted metabolomic profiling of both intracellular and extracellular compartments of primary human astrocytes derived from AD patients and healthy subjects (HS) using ¹H nuclear magnetic resonance (NMR) spectroscopy. Cells were treated with oligomeric Aβ_1-42 to model pathological conditions. Results: Aβ_1-42 treatment induced intracellular metabolic alterations in both AD and HS astrocytes, including a consistent reduction in phosphocreatine, potentially indicating impaired energy-buffering capacity. Notably, a decrease in β-alanine was observed only in AD astrocytes, suggesting alterations in carnosine-related antioxidant defence. Analysis of conditioned media revealed differential responses between groups: AD astrocytes showed increased extracellular levels of 2-oxoglutarate, citrate, and glycine, whereas HS astrocytes exhibited reduced extracellular levels of leucine and isoleucine, suggesting distinct adaptive metabolic responses to Aβ-induced stress. However, none of these differences remained statistically significant after correction for multiple testing. Conclusions: These findings suggest that NMR-based metabolomics can detect subtle metabolic shifts in human astrocyte models of AD and HS exposed to amiloidogenic challenge. Given the limited sample size and the exploratory design adopted, the results should be interpreted as preliminary and require validation in larger, better-matched cohorts. Nevertheless, this study provides a methodological framework and generates biologically plausible hypotheses regarding astrocyte metabolic responses relevant to AD pathophysiology. Full article

The environmental degradation of plastics results not only in their mechanical fragmentation into microplastics (MPs), but also in polymer main-chain scission processes, causing continuous leaching and/or volatilization of low-molecular-weight species, often characterized by a hazardous profile. In this study, we investigated the hydrophilic photooxidation products (HyPOPs) generated upon UV irradiation of polystyrene (PS) and their transformation catalyzed by the enzyme laccase from the fungus Trametes versicolor. Through a series of enzymatic tests, the enzyme was found to promote coupling and conjugation reactions of HyPOPs into poorly soluble compounds mimicking natural humic acids. The enzymatic activity of laccase was studied under different experimental conditions to simulate those found in environmental matrices. Due to their oligomeric nature, these humic acid-like products of metabolic transformation by the fungal laccase are here nicknamed “mycoplastics” (i.e., polymers from fungi). This enzymatic biodegradation and biotransformation of xenobiotic HyPOPs highlights the role of specific enzymes as biological tools for environmental self-repair of polluted ecosystems. Moreover, it opens new perspectives for remediation strategies targeting elusive micro- and nanoplastics and their continuously generated hazardous molecular degradation by-products. Humic acid-like products resulting from laccase conversion of HyPOPs could contribute to the rehabilitation of contaminated sites by promoting the removal of toxic contaminants from soil and water. Full article

Octa(3,3,3-trifluoropropyl) polyhedral oligomeric silsesquioxane (8F-POSS) was synthesized via a vertex-capping method and incorporated into natural rubber (NR) and deproteinized natural rubber (DPNR) to fabricate inorganic–organic vulcanizates. Curing characteristics, crosslink density, and the filler–rubber interaction parameter (α) were evaluated. We found that 8F-POSS retarded vulcanization kinetics but eventually enhanced network integrity. Two-dimensional infrared (2D-IR) spectroscopy indicated a hydrogen-bond shielding effect between siloxane cages and protein hydroxyl groups in NR. This interaction governed morphology development: proteins in NR acted as compatibilizers to improve initial POSS dispersion, though at high loadings they compromised reinforcement efficiency (α fell from 18.12 to 9.04). In contrast, DPNR vulcanizates showed stronger direct filler–rubber interactions, with higher α values (25.66–35.58) and a more constrained physical network. Despite a denser physical network, the 8F-POSS cages increased fractional free volume and promoted interfacial frictional slippage, leading to a synergistic “reinforcement–dissipation” effect. As a consequence, 8F-POSS/DPNR vulcanizates exhibited an enhanced damping performance (e.g., a loss factor of 1.26) alongside a depressed Tg, reduced equilibrium swelling in oil from 324% to 147%, high hydrophobicity (water contact angle above 120°), and distinctive multi-stage thermal stability. These findings demonstrate a strategy to manipulate the protein network in NR using nanoscale hybrid fillers for the design of high-performance vulcanizates. Full article

Kynurenic acid (KynA), a tryptophan metabolite that regulates immune homeostasis via G protein-coupled receptor 35 (GPR35), has an undefined role in post-myocardial infarction (MI) immune responses. To clarify this role, we established a murine MI model and administered KynA intraperitoneally to evaluate cardiac function and ventricular remodeling. Macrophage infiltration was assessed, and macrophages were depleted via clodronate liposomes to confirm their contribution to KynA-mediated cardioprotection. In bone marrow-derived macrophages (BMDMs), GPR35-targeted siRNA verified the receptor-dependent action of KynA. KynA improved cardiac function, reduced infarct scarring and fibrosis, and suppressed pro-inflammatory macrophage infiltration in MI mice, with these cardioprotective effects abrogated by macrophage depletion. Mechanistically, KynA inhibited voltage-dependent anion channel 1 oligomerization, prevented mitochondrial DNA leakage, and downregulated the cGAS/STING/TBK1/IκBα/P65 pathway in macrophages, while exogenous mitochondrial DNA counteracted this inhibition. Collectively, the KynA/GPR35 axis exerts cardioprotective effects against MI by attenuating macrophage pro-inflammatory responses, highlighting its potential as a novel therapeutic target. 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

In low Earth orbit (LEO), special environments such as atomic oxygen (AO), alternating high and low temperatures, and high vacuum can seriously affect the reliability and service lifetime of moving parts of space equipment. Therefore, there is an increasingly urgent demand for long-life, high-performance lubricating protective coatings with the rapid evolution of astronautical technology. In this study, polyimide (PI) was modified by polyhedral oligomeric silsesquioxane (POSS) with different numbers of functional groups to fabricate PI-based self-stratifying gradient composite lubricating coatings. The coating exhibited significantly enhanced AO resistance, and its vacuum tribological properties under alternating high and low temperature conditions were investigated. Results show that the mass loss of the gradient coating under AO exposure was significantly reduced by 78%, and the tribological properties of the coating under high and low temperature alternating conditions were significantly different. The friction coefficient was more stable and was smaller than that at high temperatures, and the wear rates of the POSS-modified coating also decreased by 77.5% and 50% for both high and low temperatures compared with that of the PI/PTFE coating. Full article

Nucleation remains one of the least understood steps during protein crystallization, although it strongly impacts product quality attributes, including total crystal numbers, final crystal size distributions, and thus downstream processing. In this work, the nucleation behavior of Lactobacillus brevis alcohol dehydrogenase (LbADH) wild type (WT) and five mutants (Q207D, Q126H, K32A, D54F, and T102E) is investigated in a stirred 7 mL crystallizer monitored by in situ multi-angle dynamic light scattering (MADLS). Nucleation was studied with highly pure homotetrameric LbADHs by establishing a crystallization, lyophilization, and re-solubilization protocol combined with size exclusion chromatography (SEC) and size exclusion high-performance liquid chromatography (SE-HPLC), yielding tetramer purities above 94% and removing low molecular weight impurities. During stirred batch crystallizations initiated by the addition of polyethyleneglycol 550 monomethyl ether (PEG 550 MME), SEC and SE-HPLC revealed decreasing tetramer peak areas but essentially constant peak apex positions, indicating that no long-lasting oligomeric intermediates accumulate at detectable levels. Time-resolved MADLS measurements using a custom-made flow-through cuvette in a bypass to the stirred crystallizer uncovered transient cluster populations. All protein variants exhibited an initial tetramer peak, followed by the formation of larger aggregates and a rapid rise in signal above a hydrodynamic diameter of 1000 nm, coinciding with the onset of macroscopic turbidity. A simple mesoscale nucleation model was formulated, yielding end-of-nucleation times, crystallized fractions, critical soluble concentrations, and apparent nucleation rate constants. The crystal contact mutations modulate both the timing and magnitude of the nucleation burst (rapid build-up of nuclei/cluster populations). The mutant Q207D showed strongly attenuated nucleation compared to the WT, whereas the other mutants (K32A, D54F, and particularly T102E) display markedly accelerated nucleation at nearly invariant critical concentrations. The combined workflow demonstrates how in situ MADLS, together with a tailored kinetic description, can provide mechanistic insight into protein nucleation in stirred batch crystallizers. Full article

Background: In advanced non-small cell lung cancer (NSCLC) with sensitizing EGFR mutations, EGFR tyrosine kinase inhibitors (EGFR-TKIs) improve progression-free survival (PFS). However, clinical outcomes vary according to EGFR mutation subtype and TP53 co-mutations. Most prior studies have evaluated TP53 status as binary, and the clinical relevance of domain-specific TP53 alterations remains insufficiently defined. Methods: We retrospectively analyzed patients with advanced NSCLC harboring sensitizing EGFR mutations who received first-line EGFR-TKI therapy at the National Cancer Centre Singapore between 22 November 2007, and 17 February 2022. EGFR mutations were classified as common (exon 19 deletion or L858R) or uncommon (all others). TP53 alterations were categorized into three groups: (i) DNA-binding domain (DBD)-involved mutations, including DBD-only mutations and those with additional oligomerization domain (OD) involvement; (ii) other TP53 mutations not involving the DBD or OD; and (iii) TP53 wild type (TP53-WT). The primary endpoint was PFS. Survival analyses were performed using the Kaplan–Meier method and Cox proportional hazards models. Results: TP53 alterations were identified in approximately half of the cohort and were predominantly concentrated within the DBD. In the overall cohort, patients treated with third-generation EGFR-TKIs had longer PFS than those treated with first- or second-generation EGFR-TKIs, with this difference being more pronounced among patients with TP53-mutant tumors; no clear PFS difference by TKI generation was observed in the TP53-WT subgroup. Patients with common EGFR mutations experienced significantly longer PFS than those with uncommon mutations, particularly in the presence of TP53 co-mutations. Across multiple analyses, TP53 DBD-involved mutations were associated with shorter PFS compared with other TP53 mutations and TP53-WT, especially in patients treated with first- or second-generation EGFR-TKIs and in those with common EGFR mutations. Conclusions: In EGFR-mutant NSCLC treated with EGFR-TKIs, TP53 functional domain involvement provides prognostic information beyond TP53 mutation status alone. TP53 DBD-involved alterations define a high-risk subgroup with inferior PFS, particularly in treatment settings using first- or second-generation EGFR-TKIs. Incorporation of TP53 domain-based classification, together with EGFR mutation subtype, may improve risk stratification and help guide treatment planning in EGFR-mutant NSCLC. Full article

α2-Macroglobulin (A2M), a large tetrameric glycoprotein with a molecular weight of approximately 720 kDa, is a key member of the α-macroglobulin superfamily. Its origin dates back 600–700 million years, positioning A2M as an evolutionary link within the α-macroglobulin family and complement components C3, C4, and C5. Structural predictions of A2M across different species reveal a remarkably high degree of conservation between invertebrates and vertebrates. A2M is abundantly present in the body fluids of both vertebrates and invertebrates, and its diverse biological functions are governed by five key functional domains within its molecular structure. The most well-established role of A2M is the entrapment and inhibition of proteases. Beyond that, it interacts with cytokines, growth factors, and membrane receptors, thereby playing a broad role in immune and inflammatory responses, hemostasis and coagulation, as well as in disease mechanisms and therapeutic processes. This review summarizes the origin and evolution of A2M, its molecular structure and functional domains, principal mechanisms of action, and research progress regarding its functions in both invertebrates and vertebrates. Our goal is to provide new insights and directions for further exploring the functional potential of A2M and its future applications in the treatment of clinical diseases. Full article

Antimicrobial resistance (AMR) was associated with 4.95 million deaths in 2019 and may cause 10 million deaths annually by 2050. We synthesize evidence on how the black soldier fly (Hermetia illucens) has evolved an expanded antimicrobial peptide (AMP) repertoire, which structural features drive family-specific activity, what mechanisms are directly demonstrated in H. illucens, and how AI contributes. PubMed, Web of Science, and Scopus (plus targeted Google Scholar) were searched from inception to 1 February 2026; studies were included when they reported BSF peptide identities, expression/proteomics, evolutionary analyses, quantitative activity, mechanistic assays, or BSF-focused computation, and claims were tiered as predicted, expression-supported, or experimentally supported. The literature supports 50–80 BSF AMP genes, plausibly shaped by gene duplication and balancing/diversifying selection in microbe-rich substrates, with marked induction plasticity across tissues, development, diet, and challenge. SAR is family-dependent: defensin-like peptides rely on disulfide-stabilized CSαβ folds and cationic surface topology; cecropin-like peptides on amphipathic α-helices with selectivity trade-offs; attacin-like peptides on β-architecture where charge-based heuristics are weak; and diptericin/proline-rich peptides remain largely inference-driven in BSF. Mechanistic evidence is strongest for membrane/envelope-centered killing by DLP4 and pore-associated envelope disruption by a recombinant attacin-like peptide, whereas pore geometry, oligomerization, intracellular targets, and broad “resistance-proof” claims remain unresolved. Key gaps include assay heterogeneity, salt/serum stability, selectivity/toxicity, resistance-risk testing, and limited in vivo validation, which must be addressed for credible AMR-relevant translation. Full article

The increasing energy demand and global dependence on conventional fuels have resulted in severe greenhouse gas (GHG) emissions, necessitating the development of sustainable bioenergy alternatives. Algal is recognized as a promising feedstock for the production of fourth-generation biofuels. This study optimizes catalytic pyrolysis of Arthrospira platensis for bio-oil production via a dual-bed catalyst system of iron-impregnated dolomite (Fe/DM) and a copper-impregnated spent fluid catalytic cracking catalyst (Cu/sFCC). A face-central composite design (FCCD) and response surface methodology (RSM) were used for the delineation of optimal conditions, ensuring that all experimental tests remained within feasible operating conditions of 500–600 °C, a reaction time of 45–75 min, a N₂ flow rate of 50–200 mL/min, and a catalyst loading of 5–20 wt%. The bio-oil yield was maximized at 39.73 ± 2.86 wt% at 500 °C for 45 min, a N₂ flow of 50 mL/min, and 5 wt% catalyst loading to feedstock with a 0.4:0.6 mass ratio of Fe/DM: Cu/sFCC. The dual-catalysts combined Brønsted and Lewis acid sites enhanced the catalytic activity, which promotes the cleavage of carbon–carbon and carbon–hydrogen bonds, including the mechanism of catalytic pathways such as dehydration, decarboxylation, oligomerization, aromatization, and further cracking reactions, and was successful in converting high-molecular-weight molecules into lighter hydrocarbons and significantly improving product selectivity, demonstrating a highly effective pathway for producing high-quality sustainable biofuel. Full article