Search Results (239)

Cardiac amyloidosis (CA) is an infiltrative cardiomyopathy caused by extracellular deposition of misfolded proteins, most commonly immunoglobulin light chains (AL) or transthyretin (ATTR). Once considered a rare disease, CA is increasingly recognised due to improved diagnostic strategies and the availability of disease-modifying therapies. Early diagnosis is crucial, as treatment efficacy and clinical outcomes are strongly influenced by the stage of cardiac involvement. Multimodality cardiac imaging plays a central role in the diagnostic pathway, risk stratification, and evaluation of therapeutic response in CA. Echocardiography represents the first-line imaging modality and is essential for raising clinical suspicion through the identification of characteristic structural and functional abnormalities, including ventricular wall thickening, diastolic dysfunction, and distinctive strain patterns. Bone scintigraphy has revolutionised the non-invasive diagnosis of ATTR-CA, allowing accurate identification of transthyretin-related disease in the absence of monoclonal gammopathy, which needs to be excluded via serum and urinary immunofixation. Cardiovascular magnetic resonance provides advanced tissue characterisation through late gadolinium enhancement and quantitative mapping techniques, enabling detection of early myocardial involvement and robust prognostic stratification. Emerging imaging modalities, including dual-energy (spectral) computed tomography and positron emission tomography tracers, show promise in myocardial amyloid quantification and subtype differentiation, although their role is still evolving. Integration of imaging findings with clinical and laboratory parameters allows comprehensive disease assessment, facilitating early diagnosis, guiding therapeutic decisions, and improving risk stratification. This review summarises the current role of multimodality imaging in CA, highlighting its contribution from early detection to prognostic evaluation and monitoring of treatment efficacy, with particular emphasis on the emerging role of quantitative imaging in monitoring treatment response. Full article

Mass spectrometry-based retina proteomics is an emerging method applied to a better understanding of glaucomatous neurodegeneration. This study reports the identification of retinal proteins and processes associated with elevated intraocular pressure using the magnetic microbead model of glaucoma in mice with consideration of sex as a biological variable. The harvested retinas were analyzed by shotgun proteomics via nanoflow liquid chromatography–electrospray ionization tandem mass spectrometry and label-free quantifications. Ingenuity Pathway Analysis^® was used for biological interpretation of the resulting dataset. Sex had a statistically significant influence on elevated intraocular pressure-induced changes of retinal protein expressions, and principal component analysis of all identified, verified, and quantified proteins indicated a clear separation of the ocular hypertensive versus normotensive clusters in males, while this separation was far less pronounced in females. Metabolic functions were among the most predominant biological features affected by the increased intraocular pressure. While our results overlapped with many previous findings regarding disease pathology, they have also revealed previously unreported proteins, as well as molecular and physiological functions potentially implicated in glaucomatous neurodegeneration of the retina in a sex-dependent manner. Full article

Tomato yellow leaf curl virus (TYLCV) is a major threat to tomato production, creating a need for sensitive, low-cost detection methods that can be applied to early symptomatic or low-viral-load samples. Recombinant antigen configuration may influence serological assay development, although the specific contribution of multiple-cloning-site (MCS)-derived intermediate sequences remains uncertain. In this study, a recombinant Trx-His-coat protein (CP) fusion antigen was produced using an MCS-free direct-fusion construct that retained the Trx-His tag while removing the MCS-derived intermediate sequence, followed by gradient refolding. No direct comparison with linker-containing, tag-cleaved, or tag-free antigen constructs was performed. The purified antigen was used to immunize rabbits and generate a high-titre polyclonal antibody (pAb). The resulting indirect enzyme-linked immunosorbent assay (ELISA) achieved a theoretical limit of detection of 1.8 ng/mL and an estimated pre-dilution equivalent the limit of detection (LOD) of 72 ng/mL after sample dilution. The assay showed favourable tolerance to crude tomato leaf matrices, with spike-recovery rates of 95.45–100.40%. In a preliminary evaluation using a balanced panel of 32 field-collected samples, ELISA absorbance correlated with droplet digital PCR quantification (R² = 0.9819) and plant disease index values (R² = 0.9774). Liquid chromatography–tandem mass spectrometry (LC-MS/MS) peptide mapping and AlphaFold2-based modelling were used only to provide preliminary computational context for antigen interpretation. The assay showed cross-recognition toward Tobacco curly shoot virus (TbCSV), indicating that it should not be considered strictly TYLCV species-specific. Therefore, this assay may support preliminary serological screening under the tested conditions, whereas molecular confirmation remains necessary when species-level identification is required. Full article

Background/Objectives: Oxidative and electrophilic stress-response pathways contribute to cellular resilience across various chronic diseases, including neurodegenerative, hepatic, and metabolic disorders. Pharmacological activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is a validated strategy to counteract oxidative damage, as evidenced by the clinical approval of dimethyl fumarate (DMF) and monomethyl fumarate (MMF) for relapsing forms of multiple sclerosis; both electrophilic compounds activate Nrf2 via covalent Keap1 (Kelch-like ECH-associated protein 1) modification. Cynara scolymus L. contains structurally related electrophilic metabolites; however, their contribution to Nrf2-associated signaling remains undefined. This study aimed to identify and characterize the constituents responsible for Nrf2-inducing activity and benchmark their potency against DMF and MMF. Methods: Bioactivity-guided fractionation combining Soxhlet extraction, Nrf2/ARE luciferase reporter screening, semi-preparative HPLC, and spectroscopic identification was employed. Functional validation included extracellular thiol quantification, H₂O₂ cytoprotection assays, and Western blot analysis of heme oxygenase-1 (HO-1). Results: The dichloromethane extract exhibited the highest Nrf2-inducing activity (54.4-fold). Fractionation yielded five guaianolide sesquiterpene lactones (1–5), four of which were active. The α-methylene-γ-lactone moiety was essential for activity. Aguerin B (3) exhibited the highest activity (39.14 ± 11.13-fold), while TBA analysis identified cynaropicrin (2) as the dominant extract-level contributor (62.9% of total activity). Notably, aguerin B (3) and cynaropicrin (2) induced greater reporter activity than DMF and MMF. Downstream pathway induction was confirmed by concentration- and time-dependent HO-1 upregulation, elevated extracellular glutathione and cysteinylglycine levels, and significant protection against H₂O₂-induced cytotoxicity without intrinsic toxicity. Conclusions: Guaianolide sesquiterpene lactones are the primary mediators of Nrf2-associated antioxidant signaling in C. scolymus. Cynaropicrin (2) exhibited stronger in vitro ARE-reporter induction than fumarates, supporting its relevance for further pharmacological investigation. Full article

Background/Objectives: Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of renal cancer, characterized by frequent metastasis and poor prognosis. Epithelial–mesenchymal transition (EMT) plays a pivotal role in tumor progression. Protocadherin 9 (PCDH9) has emerged as a potential tumor suppressor, but its relationship with EMT markers in ccRCC remains unclear. This study aimed to investigate the expression patterns and prognostic significance of PCDH9, β-catenin (CTNNB1), Snail (SNAI1), and Vimentin (VIM) in ccRCC. Methods: Immunofluorescence analysis was performed on formalin-fixed paraffin-embedded tissue sections from 48 ccRCC patients (31 low-grade, 17 high-grade) and adjacent normal renal cortex. Findings were validated using The Cancer Genome Atlas (TCGA-KIRC) dataset via GEPIA2/GEPIA3 platforms, including differential expression, correlation, and survival analyses. Results:PCDH9 mRNA was significantly downregulated in ccRCC tumors (TCGA-KIRC), while VIM was upregulated at the transcriptomic level. Tissue-level immunofluorescence quantification revealed discordant patterns, highlighting the influence of cellular heterogeneity on bulk protein assessment. The strong positive correlation between PCDH9 and CDH1 observed in normal kidney was completely lost in tumor tissue. Unexpectedly, PCDH9 showed positive correlations with EMT transcription factors (ZEB1, SNAI1) in tumors. In univariate survival analysis, high PCDH9 and CTNNB1 expression were associated with improved overall survival. Multivariate Cox regression revealed endpoint-specific prognostic signatures: VIM independently predicted disease progression, while SNAI1 predicted overall mortality. CTNNB1 was consistently protective across both endpoints. Conclusions: Our findings support a tumor-suppressive role for PCDH9 in ccRCC and reveal disruption of epithelial adhesion molecule co-regulation during tumorigenesis. The identification of endpoint-specific prognostic signatures has implications for patient stratification and suggests that ccRCC exhibits a partial EMT phenotype rather than classical EMT. Full article

Background: Maize is a vital crop, supporting 19.5% of global calorie intake. However, maize is vulnerable to even brief periods of drought which substantially reduces seed set and therefore yield. Methods: To identify proteins involved in responses of maize to drought, soluble proteins were extracted from leaf and silk tissues of Zea mays and protein abundance and phosphorylation status were quantified relative to well-watered controls. Label-free quantification and phosphopeptide enrichment were applied to the same biological samples and over 300 proteins were identified with significantly different changes. Results: Proteins known to be involved in drought responses were identified, such as the abscisic acid pathway and transcription factors. Of particular interest is a group of dehydrins quantified at both total protein and phosphopeptide levels, permitting insight into stoichiometry. The biological function of dehydrins in the model plant Arabidopsis thaliana is known to be regulated by phosphorylation. Conclusions: Translation of protein function from model plant to crops remains highly challenging because genome duplication has created complex sets of orthologous and homologous proteins. By focusing on proteomic changes during crop stress responses, this work enables the identification of known and novel proteins, substantially aiding the transfer of knowledge from model plants to crops. Full article

Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa rank among the most challenging pathogens due to increasing prevalence of multidrug-resistant (MDR) strains. These pathogens pose major risks to public health and food safety, earning their inclusion on the World Health Organization (WHO) priority list of MDR bacteria. While available conventional antibiotics are becoming less effective, natural products from plant extracts offer promising alternative and synergetic effects that can restore efficacy and lower required doses. Their antimicrobial activity is attributed to phytochemicals such as phenolic compounds and terpenoids acting via membrane disruption, efflux pump inhibition, biofilm interference, and cell protein disruption. Furthermore, phytochemicals in essential oils, such as carvacrol, thymol, and cinnamaldehyde, also exhibit antimicrobial and antioxidant activities. Their broad antimicrobial effects extend shelf life and enhance food safety, making them effective natural alternatives to synthetic preservatives. Moreover, advances in extraction and characterization techniques, including green solvents, spectrometry and hyphenated chromatographic methods, have improved recovery, identification and quantification. In addition, artificial intelligence (AI) emerges as a transformative tool to accelerate discovery, optimize compound screening, and predict synergistic interactions. Notwithstanding these advances, challenges persist in standardization, bioavailability, and clinical translation. Further studies are needed to isolate active compounds, elucidate mechanisms of action, validate combined use with conventional antibiotics and overcome formulation, delivery, sensory and regulatory hurdles. This review examines current knowledge of opportunities and limitations of plant-based antimicrobials against MDR pathogens supported by advances in extraction, characterization, and AI. Full article

Traditional edible plants such as quelites are an important component of the Mexican diet due to their nutritional and functional value; however, the effects of postharvest and culinary processing on their phytochemical composition remain poorly understood. This study evaluated the impact of oven-drying and freeze-drying, as well as thermal preparation (raw vs. boiled), on the proximal chemical composition, phenolic profile, and antioxidant capacity of leaves and inflorescences of Chenopodium berlandieri subsp. nuttalliae (huauzontle), using an integrated metabolomic approach. Proximal analysis showed that major macronutrients (protein, dietary fiber, lipids, and carbohydrates) were largely preserved across drying methods, whereas moisture and ash contents differed significantly among tissues and treatments (p < 0.05). Raw freeze-dried inflorescences exhibited the highest total phenolic content and antioxidant capacity. UPLC-DAD-ESI-QToF/MS enabled the identification and quantification of 26 phenolic compounds, predominantly glycosylated flavonols derived from quercetin, kaempferol, and isorhamnetin, while naringin was identified as the main flavanone glycoside present. Quercetin glucuronide was the most abundant compound, particularly in inflorescences. Multivariate analyses (principal component analysis [PCA], permutational multivariate analysis of variance [PERMANOVA], and partial least squares discriminant analysis [PLS-DA]) suggested that the drying method was a major source of variability, followed by thermal treatment and tissue type, although these patterns should be interpreted as indicative rather than conclusive. Overall, freeze-drying appeared to be the most effective method for preserving the phytochemical quality of huauzontle under the conditions evaluated, highlighting its potential as a valuable source of bioactive compounds within the genus Chenopodium. Full article

Background: Untargeted proteomics enables quantitative host cell protein (HCP) determination in biotherapeutics, yet no workflow has been validated under ICH Q2(R2) for regulated quality control. Methods: A prospective total-error (TE) validation of label-free ddaPASEF proteomics was performed. A stable isotope-labeled whole-proteome standard was spiked into NISTmAb at seven levels (20–80 ng) and analyzed in four independent assays (198 injections), supporting one-way random-effects ANOVA with Welch–Satterthwaite adjustment. Peptide-level identification error was evaluated by dual entrapment. Results: Empirical false-discovery proportions were below 1% at q = 0.01. Weighted least-squares regression (R² = 0.993) confirmed stable proportional compression with 81–85% recovery. Repeatability dominated the variance structure (median CV 2.7%); intermediate precision SD ranged from 0.69% to 3.81%. Both 95% β-expectation and 95/95 content tolerance intervals were contained within ±30% at all levels, defining a validated range of 20–80 ng. Abundance-stratified TE profiling revealed concentration-dependent calibration heterogeneity, with stratum-specific intervals within ±35% defining an abundance-aware LLOQ of 3.6 ppm (P95 = 3.87 ppm). Robustness under independent search software (FragPipe v24.0, CCC = 0.998) and cross-platform acquisition (Astral, CCC = 0.980) remained within ±30% limits. Conclusions: This constitutes the first prospective ICH Q2(R2)-aligned validation of untargeted proteomics for HCP quantification, with a transferable statistical framework for high-dimensional analytical methods. Full article

Background: Dental caries remains one of the most prevalent oral diseases worldwide, largely driven by the virulence of Streptococcus mutans. Although plant phenolics from green tea and pomegranate are known for their antimicrobial properties, their molecular mechanisms of action against key S. mutans virulence targets remain insufficiently characterized. Aim: This study investigated the antibacterial and anti-virulence properties of Viroelixir, a phenolic-rich formulation derived from green tea (Camellia sinensis) and pomegranate (Punica granatum), against S. mutans, with particular emphasis on predictive molecular docking interactions with critical virulence-associated proteins. Methods: Viroelixir phytochemical composition was characterized by LC–MS using a C18 reverse-phase column and negative electrospray ionization mode. Antibacterial activity was evaluated using growth kinetics, agar plating, and crystal violet assays. Acidogenicity, hemolytic activity, and biofilm formation were assessed using pH modulation, hemolysis assays, SEM, and biofilm biomass quantification. Virulence gene expression was analyzed by RT-qPCR. In silico molecular docking was performed to explore potential interactions between major LC–MS-supported phenolic constituents and S. mutans virulence proteins, including glucosyltransferase B (GtfB), LuxS, and SpaP. Biocompatibility was evaluated in human gingival epithelial cells. Results: The LC-MS analysis revealed a complex mixture of phenolic compounds consistent with catechins and ellagitannins. Compound identification was considered tentative and based on mass spectral range and chromatographic behavior. Viroelixir significantly inhibited S. mutans growth, acid production, hemolytic activity, and biofilm formation in a concentration-dependent manner. Key virulence genes were markedly downregulated. Docking analyses suggested stable binding of selected phenolics—particularly punicalagin, catechin, and epigallocatechin—within the active sites of GtfB, LuxS, and SpaP. Importantly, Viroelixir showed no cytotoxic effects on gingival epithelial cells. Conclusions: Viroelixir exerts potent antibacterial and anti-virulence effects against S. mutans through a multi-target mechanism combining transcriptional suppression and predictive molecular inhibition of virulence proteins, supporting its potential as a safe, natural therapeutic for caries prevention. Full article

Tubulin is a heterodimeric protein composed of α- and β-subunits, which polymerize to form the cell’s microtubules. The latter are key components in mitotic spindle formation and essential targets in anticancer therapy. Compounds such as paclitaxel, tubulysins, dolastatins and synthetic analogues of these latter compounds, including cemadotin, exert their cytotoxic effects by disrupting microtubule dynamics. Previously, we reported the production and anticancer activity of a library of cemadotin analogues featuring a C-terminal tertiary amide functionalized with a variety of N-substituents, thus resulting in compounds occurring as a mixture of amide rotamers. Here we describe a comprehensive NMR and conformational study that provides new insights into the effect of the conformational equilibrium on the binding mode of the novel cemadotin analogues to the tubulin target. The conformational behavior of the isomer equilibrium of cemadotin’s terminal amide bond was investigated by TOCSY and ROESY NMR experiments, which allowed the identification and quantification of individual rotamer populations. A slow interconversion between the s-cis and s-trans amide rotamers was observed under standard NMR conditions (25 °C), indicating a significant energy barrier and conformational rigidity. Molecular docking and saturation transfer difference (STD) NMR experiments were performed with a representative analogue and tubulin to assess the binding mode. The results revealed that the s-trans rotamer is the predominant conformer in solution and exhibits a more favorable interaction with tubulin compared to the s-cis isomer, thus helping to understand the conformational requirements for an improved tubulin binding and the inhibition of the polymerization process. Full article

Type 2 diabetes (T2D) causes systemic metabolic and inflammatory changes that affect the oral cavity, but salivary molecular markers remain poorly characterized. A peptide-centric reanalysis of salivary proteomics data was performed using DIA-NN for peptide-level quantification, without collapsing peptide signals into protein-level summaries. Although the qualitative peptide repertoire was largely conserved between T2D and control samples (>96% overlap), T2D showed coordinated quantitative changes in specific peptide subsets. Differentially abundant peptides primarily originated from complement C3, alpha-2-macroglobulin, serotransferrin, mucins, apolipoproteins, and hemoglobin, with a significant enrichment of oxidized cysteine-containing peptides, indicating redox imbalance and low-grade inflammation. Structural analysis with AlphaFold showed that T2D-associated peptides are located in solvent-exposed and conformationally dynamic regions of proteins. These findings suggest that disease specificity in diabetic saliva occurs mainly at the peptide level, offering mechanistic insight into non-invasive biomarker identification and longitudinal disease monitoring. Full article

Nitrofurans are banned veterinary medicinal products due to their carcinogenic and mutagenic properties; however, their protein-bound metabolites (AOZ, AMOZ, AHD, SEM, and DNSAH) may persist in food-producing animals, particularly in eggs. Reliable confirmatory methods are therefore essential for residue monitoring under the stringent requirements of Commission Implementing Regulation (EU) 2021/808. This study reports the development and validation of a sensitive and selective LC–MS/MS method combining acid hydrolysis, 2-nitrobenzaldehyde derivatization, and QuEChERS extraction for the determination of nitrofuran metabolites in eggs. Chromatographic separation was carried out using a phenyl-hexyl column, and detection using a tandem mass spectrometer, supported by isotope-labeled internal standards, ensured robust identification and quantification. Linearity was satisfactory over the investigated concentration range (R² > 0.99), with recoveries between 82 and 109%. The method’s precision was acceptable, with repeatability RSD values below 10% and within-laboratory reproducibility RSD values below 22%. Matrix effects were effectively controlled, remaining within ±20% following internal standard normalization. Decision limits (CCα) ranged from 0.29 to 0.37 µg/kg, well below the EU reference point for action of 0.5 µg/kg. The method’s performance was further confirmed through participation in an accredited proficiency test scheme. Overall, the validated method provides a reliable analytical tool for routine official control laboratories, enabling the sensitive confirmatory detection of banned nitrofuran residues in eggs and supporting food safety and regulatory compliance. Full article

Background: Aneurysmal subarachnoid hemorrhage (aSAH) induces complex systemic inflammatory and metabolic responses that may influence clinical outcome. DSC provides an integrative biophysical readout of proteome-level thermodynamic behavior rather than protein-specific identification or quantification; however, its applicability in neurocritical conditions remains largely unexplored. This pilot study aimed to explore whether serum DSC profiles show preliminary associations with clinical severity and neurological outcomes after aSAH. Methods: Serum samples collected on day 1 after aSAH were analyzed by DSC and compared with healthy control samples. A small patient cohort was stratified according to clinical severity and neurological outcome. Thermograms were evaluated based on melting temperatures (Tm), calorimetric enthalpy (ΔHcal), heat capacity changes (ΔCp), and the relative contributions of major serum protein components. Results: Healthy controls exhibited characteristic DSC profiles dominated by a cooperative albumin transition at approximately 65–66 °C. In this limited cohort, patients with severe clinical conditions and unfavorable outcomes displayed marked thermogram reorganization, including increased albumin Tm, reduced unfolding cooperativity, decreased ΔCp, and enhanced high-temperature immunoglobulin-related contributions. Patients with mild condition and favorable outcome showed profiles more similar to those of the controls. Notably, patients with severe conditions but favorable outcomes demonstrated heterogeneous albumin-related thermal domains, which may reflect individual-level variability and suggesting dynamic proteomic heterogeneity at the early post-ictus phase. Given the small group sizes, these patterns should be interpreted as exploratory and hypothesis-generating. Conclusions: This pilot exploratory study suggests that serum DSC may capture preliminary thermoanalytical patterns associated with clinical outcomes after aSAH. While the findings indicate the potential of DSC as a systems-level tool in neurocritical care, larger, well-powered studies are required to validate these observations and assess their robustness and generalizability. Full article

Background: The aim of this study was to evaluate the diagnostic and prognostic performance of albumin-based inflammatory–nutritional indices in hyperemesis gravidarum (HG) and to determine their associations with disease severity and risk of re-hospitalization. Methods: This retrospective case–control study included 246 women with HG and 246 gestational-age-matched healthy pregnant controls at 6–16 weeks of gestation. Disease severity was classified as mild, moderate, or severe using the Pregnancy-Unique Quantification of Emesis (24 h scale) (PUQE-24) score. A comprehensive panel of albumin-based inflammatory indices—including C-reactive protein-to-albumin ratio (CAR), fibrinogen-to-albumin ratio (FAR), neutrophil-to-albumin ratio (NAR), leukocyte-to-albumin ratio (LAR), neutrophil percentage-to-albumin ratio (NPAR), monocyte-to-albumin ratio (MAR), hemoglobin–albumin–lymphocyte–platelet (HALP) score, modified HALP (m-HALP) score, prognostic nutritional index (PNI) score, systemic immune-inflammation index-to-albumin (SII/Alb), and systemic inflammatory response index-to-albumin (SIRI/Alb)—was calculated from routine complete blood count and serum biochemistry results obtained at diagnosis. Receiver operating characteristic analysis, along with univariate and multivariate logistic regression models, was performed to evaluate diagnostic performance and identify predictors of severe HG and re-hospitalization. Results: Albumin-based indices exhibited severity-associated alterations, with an overall trend toward worsening immuno-nutritional status across increasing HG severity. Among these, m-HALP score demonstrated the strongest inverse correlations with PUQE-24 score, ketonuria grade, length of hospital stay, and re-hospitalization risk (r = −0.74 to −0.52; all p < 0.001) and achieved the highest discriminative accuracy for both severe HG (AUC 0.864, 95% CI 0.836–0.892, p < 0.001) and re-hospitalization (AUC 0.722, 95% CI 0.675–0.766, p < 0.001). In multivariable analysis, higher HALP, m-HALP, and PNI were independently associated with a lower likelihood of severe HG. For re-hospitalization, higher m-HALP and HALP were independently associated with a lower risk, whereas higher NPAR, higher ketonuria grade, and higher PUQE-24 score were independently associated with an increased risk of re-hospitalization. Conclusions: Albumin-based indices, particularly m-HALP, demonstrated robust diagnostic and prognostic performance in HG compared with conventional biomarkers. These readily available, cost-neutral composite biomarkers enable objective severity stratification and accurate identification of patients at elevated risk of recurrent hospitalization, offering immediate potential to guide personalized, evidence-based clinical management. Full article