Search Results (5,146)

Protein condensates and filaments are both intracellular structures characterized by their ability to facilitate specific biological functions. Their formation is primarily driven by phase separation, which can be elucidated by fluorescence microscopy or electron microscopy. Here we summarize the main studies on protein condensates and filaments organized according to the techniques used, including fluorescence methods like localization screening, fluorescence co-localization spectroscopy, methods based on photobleaching, super-resolution imaging, and electron methods including negative-stain electron microscopy and cryo-EM. We also discuss correlative light/electron microscopy (CLEM), which integrates fluorescence microscopy and electron microscopy to provide complementary insights. Collectively, these methods offer temporal and spatial insights into investigating the phase separation of protein condensates and filaments, and promote the discovery of unexplored structures and their yet-to-be-characterized biological roles. Full article

Background: Breast cancer brain metastases (BCBM) lack effective treatments, contributing to breast cancer-related morbidity and mortality. Integrating translational animal models and advanced non-invasive imaging can accelerate the development of urgently needed therapies. Method: In this study, we developed an intracarotid method mimicking BCBM and compared it to the stereotactic model in terms of animal welfare, tumour establishment, and blood–brain barrier (BBB) permeability. BCBM was established through intracarotid or stereotactic inoculation of BT474 and MDA-MB-231.Luc2 cells in NMRI nude mice. We utilised magnetic resonance imaging (MRI) and bioluminescence imaging (BLI) to monitor tumour growth and BBB permeability, supported by fluorescent immunohistochemistry for validation. Finally, light sheet microscopy (LSM) was employed to visualise tumour establishment in intact brains. Results: Both inoculation methods achieved a survival rate >70%, with animals recovering within a week post-surgery. MRI and BLI effectively visualised tumour growth with stereotactic implantation, resulting in single tumours, while intracarotid inoculation led to micro-seeding of up to seven tumours in one brain. Tumour growth was rapid and homogenous in the stereotactic model, whereas the intracarotid model exhibited slower, heterogenous growth. Notably, BBB permeability was significantly higher in small tumours in the stereotactic model when compared to the intracarotid model (p = 0.003). Ex vivo analyses validated these findings with the identification of multiple metastasis in the intracarotid model and single tumours in the stereotactic model. Conclusion: We developed an animal model that closely mimics BCBM, highlighting extravasation and micro-seeding while maintaining animal welfare. Our established imaging protocols enable longitudinal evaluations of BBB permeability and treatment response, creating a translational platform for testing novel anti-cancer therapies. Full article

Accurate cell and nuclei segmentation remains challenging due to the sensitivity of classical graph-cut methods to parameter tuning. While deep learning models like U-Net offer strong performance, they require large annotated datasets and substantial GPU resources. This work presents a cost-effective alternative: a genetic programming (GP) framework that jointly optimizes unary cost functions and regularization parameters for graph-cut segmentation, coupled with automatic seed selection. Evaluation is conducted under two distinct protocols: (1) oracle-guided per-image optimization, establishing upper-bound performance (mean Dice 0.822, IoU 0.733), and (2) true generalization via train/test split, where expressions learned on 50 images are applied to 50 unseen images (mean Dice 0.695, IoU 0.588). The fixed-model generalization still significantly outperforms the baseline graph cut ($+0.158$ Dice, $p<0.001$). Cross-dataset validation on MoNuSeg (H&E histopathology) achieves a Dice score of 0.823 with the fixed GP model, significantly outperforming the baseline (+0.272). This result uses a single fixed model—the best-performing expression from BBBC038 training—applied in a zero-shot manner to MoNuSeg without any retraining or domain adaptation. All 100 images showed non-negative improvement under oracle optimization in the experiments. The method requires no GPU training, runs in 550 s per image for oracle search, and offers interpretable symbolic cost functions. Code and annotations are provided to ensure reproducibility. This approach offers a practical, interpretable alternative in resource-constrained biomedical imaging settings. Full article

This study proposes a four-dimensional Historical Building Defect Information Modeling (HBDIM) framework designed to support the documentation, diagnosis, and conservation of deteriorated historic stucco elements. The framework integrates multi-source digital documentation techniques, including terrestrial laser scanning (TLS), high-resolution photogrammetry, and automated total station measurements with laboratory-based material diagnostics to create a unified digital environment for defect detection and conservation assessment. The approach was applied to the Baron Empain Palace in Egypt as a representative case study of complex architectural heritage affected by material deterioration. Within the HBDIM workflow, point cloud processing and defect-oriented information modeling were used to identify and spatially localize deterioration features such as cracking, erosion, and material loss. Laboratory investigations—including computed tomography (CT), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray fluorescence (XRF)—were conducted to evaluate the effectiveness of calcium hydroxide nanoparticle consolidation treatments and to relate microstructural material behavior to spatially mapped defects within the digital model. Mechanical testing demonstrated a significant improvement in material performance, with treated stucco samples exhibiting an average compressive strength increase of approximately 69.06% compared to untreated specimens. The results demonstrate that integrating digital documentation, defect-oriented modeling, and material diagnostics within a four-dimensional framework provides a robust platform for linking geometric deterioration patterns with material-level conservation performance. By embedding diagnostic data and treatment outcomes within a temporally structured digital model, the HBDIM approach supports preventive conservation strategies, long-term monitoring, and data-driven decision-making in sustainable heritage management. Full article

Helicobacter pylori (H. pylori) is the primary etiological agent for chronic gastritis, peptic ulcers, and gastric adenocarcinoma. The alarming rise in multidrug-resistant (MDR) strains, particularly against clarithromycin (CLR), metronidazole (MNZ), and levofloxacin (LVX), has severely compromised standard therapies. Thus, there is an urgent clinical need for novel antimicrobial agents that operate through distinct mechanisms to bypass resistance pathways and mitigate gastric cancer risk. We designed and synthesized a series of antimicrobial peptides, focusing on the proteolytically stable all-D-amino acid enantiomer, DT7-3, derived from a probiotic-sourced template. Minimum inhibitory concentrations (MICs) were determined against standard strains and 11 clinical MDR isolates via the broth microdilution method. Antimicrobial mechanisms were elucidated using scanning electron microscopy (SEM) for morphology, fluorescence-based assays for anti-adhesion activity, and real-time qPCR to quantify virulence gene expression (babA, ureA, and vacA). Biocompatibility was assessed using defibrinated sheep erythrocytes, gastric epithelial cells (GES-1), and representative beneficial gut microbiota. Analysis of the clinical isolates revealed resistance rates of 63.6% for CLR/LVX and 81.8% for MNZ, with 54.5% identified as MDR. DT7-3 exhibited superior potency (MIC 1–32 µg/mL) against all strains, significantly outperforming its L-enantiomer counterparts. Mechanistic studies unveiled a “triple-hit” mechanism: (1) rapid membrane disruption; (2) potent inhibition of bacterial adhesion to host cells (~60% reduction at 0.5 × MIC); (3) significant downregulation of critical virulence factors (babA, ureA, and vacA). Furthermore, DT7-3 showed an excellent safety profile, with negligible hemolysis (<5% at 32 µg/mL) and minimal cytotoxicity toward GES-1 cells, yielding a high selectivity index (SI, MHC/MIC) > 32 relative to mammalian cells. Crucially, DT7-3 showed high selectivity for the pathogen over beneficial gut microbiota (MIC > 128 µg/mL, SI > 16). Crucially, DT7-3 maintained potent bactericidal activity (MIC ≤ 16 µg/mL) even under cholesterol-enriched conditions. The engineered D-peptide DT7-3 is a potent candidate for combating MDR H. pylori. Its multifaceted mechanism, targeting bacterial viability while suppressing core virulence factors, positions it as a robust lead compound for next-generation eradication therapies aimed at reducing the burden of H. pylori-associated diseases. Full article

Abandoned mining areas provide valuable opportunities to investigate ore-forming processes, supergene mineral transformations, and the geochemical behaviour of metals. In this sense, the old Preguiça mine (Beja, Portugal), exploited for Fe–Zn–Pb, was studied providing new mineralogical and geochemical data aimed at improving the understanding of the secondary mineral assemblages of this deposit. A total of 70 samples collected from three accessible underground levels (first, second and third) and mine waste, complemented by 16 samples from a deeper level (fourth) previously collected, were analysed using X-ray diffraction (XRD), scanning electron microscopy (SEM), and a portable X-ray fluorescence (pXRF) equipment. Mineralogical phases are dominated by a wide range of secondary oxides, carbonates, arsenates, vanadates, silicates, phosphates and sulphates, but remnants of primary sulphides were also found. The following minerals can be emphasised: goethite, hematite, calcite, dolomite, descloizite, willemite, mimetite, cerussite, smithsonite and fraipontite. The presence of massicot in the Preguiça mine, is described for the first time. Bulk geochemical analyses show high concentrations of Fe, Ca, Zn and Pb, consistent with the observed mineralogy. The presence of vanadium- and arsenic-bearing minerals highlights the occurrence of critical raw materials, supporting the importance of reassessing other abandoned mining areas in the context of sustainable resource management and strategic raw-material planning. Full article

Canine endometritis is commonly associated with bacterial infections caused by Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Streptococcus canis (S. canis), leading to reproductive disorders in dogs. With increasing concern regarding antimicrobial resistance, alternative therapeutic strategies are needed. This study evaluated the in vitro antibacterial activity and underlying mechanisms of protocatechuic acid (PCA) against clinical isolates of these pathogens obtained from dogs diagnosed with endometritis. The antibacterial efficacy of PCA was assessed by determining minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and bacterial growth curves. PCA inhibited the growth of all three pathogens, with MIC values of 4 mg/mL for E. coli and S. aureus and 2 mg/mL for S. canis. The MBCs for E. coli and S. aureus were equal to their MICs, while the MBC for S. canis was twice the MIC, indicating bactericidal activity. Mechanistic analyses demonstrated that PCA disrupted bacterial membrane integrity, induced membrane depolarization, reduced intracellular ATP levels, and increased reactive oxygen species (ROS) accumulation. These effects were supported by SYTO9/PI fluorescence staining and scanning electron microscopy. In conclusion, PCA exhibits notable in vitro antibacterial activity against key pathogens associated with canine endometritis and represents a promising natural antimicrobial candidate. Full article

A sustainable way to recycle used tires and improve the functionality of asphalt pavements is through the use of crumb rubber modified asphalt (CRMA). However, its application during high-temperature construction raises environmental and occupational health concerns due to the release of significant quantities of odorous and potentially harmful gases. Therefore, this study selected α-Amyl cinnamic aldehyde (ACA) as a deodorant and added it to CRMA at proportions of 0.5%, 1.0%, 1.5%, and 2.0% to prepare DCRMA. A number of common tests, such as softening point, ductility, penetration, Brookfield rotational viscosity, and segregation analysis, were used to evaluate the basic characteristics of the modified asphalt. A self-developed asphalt fume monitoring device was used to quantitatively analyze the changes in VOCs, H₂S gas concentration, and solid particle content in the asphalt fumes to assess the deodorization effect of ACA on CRMA. Furthermore, the deodorization mechanism of ACA on CRMA was explored in depth using microscopic methods, such as fluorescence microscopy (FM) and Fourier transform infrared spectroscopy (FTIR). The findings demonstrated that ACA can increase the softening point and viscosity of CRMA while decreasing its penetration and ductility. The storage stability was optimal at a 1.0% ACA addition. Additionally, as the ACA content increased, the concentrations of VOCs, H₂S gas, and solid particles in the asphalt fumes continued to decrease. FM results indicated that when the ACA content did not exceed 1.0%, it promoted the swelling degree of CR in the asphalt. FTIR results showed that ACA can reduce the characteristic peak intensity of CRMA. This study offers important technical references and practical support for the environmentally friendly use of CRMA. Full article

Background: Persistent endodontic infections involving Enterococcus faecalis and Candida albicans are a major cause of root canal treatment failure. Although conventional irrigants, such as sodium hypochlorite and chlorhexidine (CHX), exhibit strong immediate antimicrobial activity, microbes may survive and recover from the initial antimicrobial effect, hence limiting their effectiveness, especially in complex root canal anatomies and in the apical terminus of the tooth. Antibacterial dressing techniques were not proven satisfactory due to depletion of the antibacterial component or difficulty in spreading it evenly along the entire root canal. This study aimed to develop and evaluate the antimicrobial efficacy and release characteristics of a novel sustained-release device (SRD), delivering CHX via gutta-percha points coated with a sustained-release formulation used as a temporary intracanal medicament. Methods: Gutta-percha points were coated with two sustained-release CHX varnishes (CHX1 and CHX2) or a placebo and assessed in vitro. Antimicrobial activity against E. faecalis and C. albicans was evaluated using agar diffusion assays over time. Release kinetics were analyzed using Rhodamine-labeled SRD in a 3D-printed acrylic molar tooth model via fluorescence microscopy. Additionally, biofilm-infected acrylic molar teeth were treated with a placebo, a single 2% CHX irrigation, or SRD-coated gutta-percha points placed as an intracanal dressing prior to obturation. Microbial viability was quantified by colony-forming unit (CFU/mL) analysis from root canals and gutta-percha points. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test (p < 0.05). Results: SRD-coated gutta-percha points demonstrated sustained antimicrobial activity for up to 21 days against E. faecalis and 19 days against C. albicans. Fluorescence analysis, in an acrylic tooth model, confirmed continuous release for up to 15 days, with pronounced diffusion in the isthmus and palatal canals. In biofilm-infected acrylic teeth models, SRD treatment resulted in a significant reduction of 2–3 log₁₀ CFU/mL compared to placebo groups (p < 0.001) and prevented microbial rebound over the 14-day observation period. In contrast, a single application of 2% CHX solution showed only transient reduction followed by regrowth. Conclusions: Sustained-release CHX delivery via polymer-coated gutta-percha points provided prolonged antimicrobial activity against bacterial and fungal biofilms compared to conventional single-dose CHX application in this in vitro model. These findings support the potential use of coated gutta-percha points as a removable intracanal drug delivery platform prior to final obturation, although further studies incorporating direct-release quantification and in vivo validation are required before clinical translation. Full article

Neurodegenerative diseases feature diverse pathological protein aggregates, including Lewy bodies in Alzheimer’s disease (AD) and skein-like filaments in amyotrophic lateral sclerosis (ALS). The physical mechanisms underlying this morphological diversity remain unclear. Here, we demonstrate that aggregation of the prion-like domain of hnRNPA1 (A1PrD), implicated in AD and ALS, is driven by solution composition and phase transition dynamics. Utilizing 3D timelapse and fluorescence lifetime imaging microscopy, we show that solution conditions modulate phase separation, gelation, and fibrillation, resulting in distinct structures such as fibril, gel, and starburst morphologies. Homotypic and heterotypic interactions between A1PrD and RNA were observed to shift the balance between pathological and physiological condensates. Importantly, amyloid-rich starbursts displayed prion-like infection capabilities toward amyloid-poor condensates. Our findings highlight how the interplay between solution composition and kinetic balances of liquid-liquid phase separation, gelation, and fibrillation shapes the diverse pathological aggregate morphologies characteristic of neurodegenerative diseases. Full article

Background/Objectives: Ovarian cancer is the most lethal gynecologic malignancy, largely due to late diagnosis and the high prevalence of malignant ascites, a hallmark of advanced disease that is difficult to control and contributes to immune suppression and treatment failure. Despite advances in standard care, durable responses are rare. This study investigates a novel immunotherapeutic strategy designed to overcome the suppressed peritoneal microenvironment using an oncolytic vaccinia virus engineered to express a decoy-resistant IL-18 mutein. Methods: We generated a vaccinia virus (vvDD-nsmDR-18) expressing a non-secreted, mature, decoy-resistant IL-18. Viral expression was validated via RT-qPCR and fluorescence microscopy, while cytotoxicity was confirmed using CCK-8 assays. The antitumor efficacy of vvDD-nsmDR-18 was evaluated in the aggressive murine ID8a ovarian cancer model. The underlying mechanisms of action were investigated using flow cytometry and transcriptional profiling. Results: Treatment with vvDD-nsmDR-18 significantly prolonged survival and was associated with reduced abdominal distension consistent with decreased ascites burden. Immune analyses indicated enhanced T cell activation across multiple anatomical compartments, including tumors, peritoneal cavity, and spleens, the latter recently suggested to serve as a reservoir for tumor-reactive T cells. This systemic activation was characterized by increased IFN-γ and perforin expression. In addition, vvDD-nsmDR-18 treatment was associated with expansion of CD39⁺CD103⁺CD8⁺ tumor-reactive T cells and a shift toward a lower PD-1 expression phenotype within this population. Conclusions: These findings demonstrate that nsmDR-18-expressing oncolytic viruses can remodel the immunosuppressive landscape of advanced ovarian cancer, suggesting this approach is a promising candidate for further clinical development. Full article

In order to achieve rapid and qualitative detection of soluble heavy metal ions, nitrogen-doped fluorescent carbon quantum dots (N-CQDs) were synthesized using chitin extracted from shrimp and crab shells as the carbon source. The structural, morphological, and optical properties of the synthesized N-CQDs were systematically characterized using transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), Fourier transform infrared spectroscopy (FTIR), Raman, X-ray photoelectron spectroscopies (XPS), ultraviolet-visible (UV-Vis) absorption spectroscopy and fluorescence spectroscopy. The resulting N-CQDs exhibited a carbonization yield of 54.46% and a fluorescence quantum yield of 34.33%. Their morphology, structure and optical properties were thoroughly characterized using a range of analytical techniques. The synthesized N-CQDs exhibited excellent fluorescence properties, and remarkable stability. When applied for metal ion detection, the N-CQDs displayed a distinct and selective fluorescence quenching response exclusively toward Fe³⁺ ions. The detection limit for Fe³⁺ at room temperature was 4.04 μmol/L. Furthermore, due to the inherent nitrogen present in the acetyl amino groups of chitin, nitrogen doping was achieved without the need for external dopants during the hydrothermal synthesis process. Owing to their high stability, low cost and low toxicity, the N-CQDs synthesized in this study provide a promising fluorescence sensing platform with excellent selectivity for Fe³⁺ detection, achieved through precise control of surface functional groups. Full article

Short-wavelength ultraviolet radiation can impair biological systems by causing DNA damage, oxidative stress, and disruption of photosynthetic processes. Although ultraviolet C (UVC) at 254 nm is widely used as a controlled laboratory stressor, the extent to which trophic condition influences repeated UVC tolerance in phototrophic protists remains unclear. Here, we examined the response of Euglena gracilis grown under photoautotrophic or ethanol-supported mixotrophic conditions and exposed to daily UVC pulses for five days. Cell growth, photosynthetic pigments, intracellular oxidative stress measured by 2′,7′ dichlorodihydrofluorescein diacetate fluorescence, and lipid peroxidation estimated as thiobarbituric acid reactive substances equivalent malondialdehyde were assessed, together with qualitative fluorescence microscopy. Repeated UVC exposure reduced cell density in both trophic conditions, with stronger inhibition under photoautotrophy. Photoautotrophic UVC-treated cultures showed the highest oxidative stress signal, whereas malondialdehyde displayed only a non-significant directional increase. Mixotrophic cultures maintained higher cell density under UVC and showed lower oxidative stress signals than photoautotrophic UVC-treated cultures. Pigment responses also differed between trophic conditions, with increased chlorophyll a and carotenoids per cell under photoautotrophic UVC treatment, while mixotrophic pigment levels remained comparatively stable. These findings show that trophic condition shapes repeated UVC stress responses in E. gracilis and that ethanol-supported mixotrophy is associated with improved physiological robustness under the present experimental conditions. Full article

Background/Objectives: Early embryonic arrest during the cleavage stage (days 2–4) accounts for a substantial proportion of developmental failure in in vitro fertilization. This phenomenon remains poorly understood at the molecular level, even in chromosomally normal embryos identified by preimplantation genetic testing. This review aims to redefine cleavage-stage arrest from a passive energy deficit to a checkpoint-regulated endpoint caused by inadequate coordination among metabolism, transcriptome integrity, and stress-response pathways. Methods: We integrate evidence from long-read transcriptomics, metabolomics, epigenetics, and immunobiology relevant to pre-blastocyst development. These data are assembled into a unifying mechanistic framework and a clinically oriented stratification model, together with candidate multimodal readouts for early classification. Results: We propose a three-axis model linking: (i) metabolic–epigenetic insufficiency, including defective histone lactylation and impaired alpha-ketoglutarate-dependent DNA demethylation; (ii) isoform-level abnormalities, including intron retention and retrotransposon activation within a hidden transcriptomic landscape better resolved by long-read sequencing; and (iii) stress-related immune signaling, in which NLRP7 links alternative splicing and DNA-damage-response dysfunction with mitochondrial stress and p53-associated arrest. Within this framework, we distinguish three molecular arrest states: an early transition failure marked by defective maternal-to-embryonic reprogramming and severe splicing disruption; a metabolically quiescent state that may retain a limited rescue window; and a later stress-associated state characterized by senescence-like features, oxidative stress, and broad transcriptomic and genomic instability. Conclusions: Early embryo arrest should no longer be viewed as a nonspecific developmental failure, but as a mechanistically stratifiable condition with distinct metabolic, transcriptomic, and stress-associated trajectories. A diagnostic platform combining fluorescence lifetime imaging microscopy, long-read sequencing, and digital polymerase chain reaction may improve early mechanistic classification, help identify embryos with possible reversibility, and reduce uncertainty in embryo selection during in vitro fertilization. Full article

The aim of this study was to investigate which physicochemical and structural properties of cationic peptides P1–P6 may determine their selective anticancer activity against melanoma cells and their interactions with tumor cell membranes. An integrated approach was applied, including characterization in solution (osmotic pressure, NaCl stability, surface tension); cytotoxicity evaluation against Me45, B16F10, and HaCaT cells; analysis of interactions with phosphatidylglycerol (POPG) model membranes using isothermal titration calorimetry and steady-state fluorescence spectroscopy; membrane permeability assays; and F-actin staining. Anticancer activity depended on positively charged residues, hydrophobic amino acids, and sequence arrangement. Tryptophan-rich peptides P2 and P5 exhibited strong membrane interactions and high efficacy after 72 h. Highly hydrophobic P4, containing long C₁₂ chains with a relatively low net charge, caused nonselective lysis. P3 showed reduced activity due to insufficient amphipathicity, whereas P6, with excessive WWW and KKKK motifs, exhibited weak or nonselective effects. Thermodynamic and fluorescence analyses indicated that P2 and P5 initially bind POPG membranes via entropy-driven electrostatic interactions, followed by hydrophobic insertion of tryptophan residues, evidenced by increased fluorescence intensity and a blue shift of the emission maximum. P2, P4, and P5 induced actin cytoskeleton reorganization and increased membrane permeability, emphasizing the role of balanced amphipathicity and charge–hydrophobicity in designing selective anticancer peptides. Full article