Search Results (279)

Bone formation requires a substantial energy supply to sustain extracellular matrix production and mineralization, yet the temporal contribution of lipid metabolism during osteoblast maturation remains incompletely characterized. This study investigated the molecular and transcriptional remodeling of lipid metabolism. Intracellular lipid distribution was analyzed by confocal microscopy using Nile Red staining. Transcriptional modulation of lipid synthesis, storage, lipolysis, genes associated with mitochondrial fatty acid oxidation, and osteogenic markers were assessed by quantitative real-time PCR, and the biochemical composition was evaluated by Raman spectroscopy. Early stages of spheroid development showed higher expression of genes involved in lipid synthesis and storage (FASN, DGAT2, and PLIN2) together with intracellular lipid accumulation, whereas later stages displayed increased expression of lipolytic and β-oxidation markers (PNPLA2/ATGL, CPT1A, and HADHA), accompanied by the redistribution of lipid droplets. The Raman analysis revealed a time-dependent variation of lipid-associated CH₂/CH₃ bands and modulation of protein-related Amide I–III signals, consistent with biochemical remodeling during maturation. Overall, the data indicate a coordinated transcriptional shift from lipid accumulation-associated pathways toward lipid mobilization during osteogenic progression in a 3D culture. This model provides a controlled experimental platform for investigating metabolic regulation during bone formation and for studying metabolic alterations associated with skeletal disorders. Full article

Graphene oxide (GO) is a promising nanocarrier for the delivery of oligonucleotides. It offers a high loading capacity, efficient cellular uptake, and surface functionalization. MicroRNA-21 (miR-21) is a well-characterized oncomiR commonly overexpressed in hepatocellular carcinoma (HCC). In HCC, miR-21 contributes to tumor progression, inflammation, and angiogenesis. In a previous in vitro study, we showed that GO alone induces the upregulation of pro-inflammatory and tumor-related genes in HepG2 cells. However, conjugation with an antisense miR-21 (GO-antisense miRNA 21) reverses this effect, suggesting a potential therapeutic application. This study aims to evaluate the antitumor and anti-angiogenic efficacy of the GO-antisense miR-21 nanosystem in ovo using the chick embryo chorioallantoic membrane (CAM) model. Fertilized chicken eggs (n = 4 per group) were randomized into untreated, GO-treated, and GO–antisense miR-21-treated cohorts. A dose of 200 μL (GO 10.0 µg/mL: antisense miR-21 5.0 pmol/mL) was administered intratumorally. Tumor size, volume, and vascularization were monitored through stereomicroscopy and histological analysis. The expression of inflammatory and tumor-associated genes (IL-8, MCP-1, TIMP-2, ICAM-1 and NF-kB) was assessed by quantitative PCR. Given its prominent response, IL-8 protein expression was further analyzed via immunofluorescence. To evaluate tumor-specific delivery, FITC-labeled GO was tracked by confocal microscopy. Our data revealed that treatment with unfunctionalized graphene oxide (GO) unexpectedly promoted tumor vascularization and led to a significant increase in tumor weight. This was accompanied by upregulation of inflammatory markers. In contrast, GO-antisense miR-21 significantly reduced the tumor volume and vessel density. It also successfully downregulated all target genes. Confocal imaging demonstrated preferential accumulation of the nanosystem within the tumor mass. Our results highlight the dual anti-inflammatory and anti-angiogenic effects of GO-antisense miRNA 21 in ovo and support its potential as a targeted nanoplatform for HCC treatment. Full article

Long-term adapted cell wall-deficient (L-forms) bacteria show unique cell shapes and growth patterns compared to wild-type bacteria. However, quantitative analysis to assess the morphological heterogeneity of existing L-forms is limited. In this study, we validated that two stable L-form strains of Escherichia coli (NC-7 and LWF+) hold spherical or pleomorphic morphology in confocal and electron microscopy. Using imaging flow cytometry, we further reported that the variations in cell size distribution and cell viability between L-forms and walled cells are statistically significant. Moreover, freeze-fracture electron microscopy observations revealed a clear presence of an outer membrane in NC-7 but not in LWF+, suggesting that E. coli L-form strains could survive in both spheroplastic and protoplastic forms after adaptive evolution. Accordingly, the mutations in genes associated with cell envelope and outer membrane components are more prevalent in the LWF+ genome, potentially leading to outer membrane depletion. Notably, experimental evidence derived from E. coli LWF+ cells exhibiting a monoderm phenotype may support the diderm-to-monoderm transition hypothesis, implying the monoderm phenotype arose from the evolutionary loss of the outer membrane in diderm ancestors. Taken together, our findings offer insights into quantification analysis and the cell envelope status of two E. coli L-forms, facilitating future investigations into genotype-phenotype associations in these two L-form bacteria models. Full article

Staphylococcus epidermidis is a leading opportunistic pathogen in medical device-associated infections due to its ability to adhere to abiotic materials and develop biofilms that are difficult to eradicate. This study investigated the antibiofilm potential of an ethanolic extract of the red seaweed Gracilaria fisheri and its purified constituent, N-benzylcinnamamide, against S. epidermidis. Antibacterial activity was determined, and antibiofilm effects were assessed using the crystal violet assay and confocal laser scanning microscopy (CLSM). Early bacterial adhesion on glass and polyurethane (PU) surfaces was measured. The effect on catheter-associated biofilms was evaluated by scanning electron microscopy (SEM). Transcripts of biofilm- and quorum-sensing-associated genes (icaA and luxS) were assessed by semi-quantitative RT-PCR. Cytotoxicity was evaluated by MTT assay. At 200 µg/mL, biofilm biomass decreased to 48.21 ± 5.52% with the extract and to 36.65 ± 6.82% with N-benzylcinnamamide. CLSM time-course imaging showed delayed biofilm maturation and less consolidated, discontinuous structures. Surface exposure to the extract markedly reduced early attachment on both materials. On PU catheter segments, SEM demonstrated that N-benzylcinnamamide markedly reduced surface coverage and disrupted three-dimensional biofilm architecture. At the molecular level, transcription of icaA and luxS was reduced. Both the extract and N-benzylcinnamamide showed minimal cytotoxicity in HeLa cells. These findings support further evaluation of these marine-derived agents as candidates for antibiofilm surface treatments to reduce early medical device colonization. Full article

Background and Objectives: The use of St. John’s wort (Hypericum perforatum) in periodontal therapy remains underexplored despite its anti-inflammatory, antimicrobial, and potential osteoregenerative effects. This was the first study aiming to determine the in vitro efficacy of ultrasonic debridement combined with a H. perforatum extract against dental biofilms. Materials and Methods: A multispecies biofilm model comprising Streptococcus mutans, Porphyromonas gingivalis, Fusobacterium nucleatum, and Tannerella forsythia was established on bovine dentin discs. Biofilms were treated with saline solution (control), ultrasonic debridement alone, ultrasonic debridement combined with H. perforatum extract (0.5%), and ultrasonic debridement combined with chlorhexidine (0.12%). Biofilm biomass was quantified with the crystal violet assay, and total viable counts were determined by colony-forming unit (CFU) analysis. Quantitative PCR was used to assess the genomic load of P. gingivalis. Biofilm architecture and bacterial viability were further examined using confocal laser scanning microscopy (CLSM). Results: Ultrasonic debridement combined with H. perforatum extract significantly reduced biofilm biomass compared to saline irrigation (p < 0.001) and ultrasonic debridement alone (p < 0.01). Similar reductions were observed for viable bacterial counts and P. gingivalis genomic load. The antimicrobial effect of the plant extract was comparable to that of chlorhexidine, with only minor differences in efficacy. Confocal microscopy confirmed marked disruption of biofilm architecture and decreased bacterial viability following treatment with the plant extract. Conclusions: Within the limitations of this in vitro model, H. perforatum extract demonstrated measurable antibiofilm activity when used as an adjunct to ultrasonic debridement. These findings provide proof-of-concept evidence supporting the antimicrobial potential of this plant-derived extract under controlled laboratory conditions. Further preclinical studies and well-designed clinical investigations are required to determine its therapeutic relevance in periodontal treatment. Full article

Acute and chronic wounds are a major clinical burden, with persistent inflammation, impaired fibroblast function, defective angiogenesis, and disordered extracellular matrix deposition. The translational potential of existing in vitro models is limited by their poor durability and physiological relevance. The present paper aims to develop a robust in vitro organotypic model to simulate the early phases of both acute and chronic wounds and to validate it by testing the biocompatibility of clinically available wound dressings. Human fibroblasts and vascular endothelial cell lines were cultured at a ratio of 1:1 for 48 h, either on uncoated tissue culture plastic or on tissue culture plastic coated with a synthetic substrate (PhenoDrive-Y) that biomimics the extracellular matrix and promotes cell organization into tissue-like structures on a 2D plane (i.e., angiogenesis sprouting and fibroblast organization around it). Wound conditions were then created by damaging the formed structures using a conventional scratch procedure and introducing U937 human macrophage cells to the model to simulate either the onset of an acute wound or that of a chronic wound through the simultaneous spiking of the culture with relevant cytokines, i.e., IL-6 and TNF-α. The formation of new tissue-like structures in the scratch area was quantified by the extent of scratch closure after a further 24 h of incubation. Morphological analysis of wound healing was performed by light microscopy, while angiogenesis was assessed by CD31 immunostaining by confocal microscopy. The deposition of components of the extracellular matrix was determined both qualitatively and quantitatively by Picrosirius Red staining for collagen production and by Alcian Blue staining for glycosoaminoglycan synthesis on the adhering cells and their supernatants. Macrophage polarization into either M1 or M2 phenotype was studied by immunostaining with iNOS (M1) and CD206 (M2) antibodies by confocal microscopy. The model was validated by studying the gap closure areas in simulated acute and chronic wound-like conditions when incubated with clinically available wound dressings, N-A Ultra and Kaltostat. PhenoDrive-Y allowed the formation of tissue-like structures on the 2D tissue culture plane as opposed to the formation of cell monolayers on the uncoated tissue culture plastic. Upon mechanical damage, cell migration was significantly different; uncoated control co-cultures achieved complete closure as an indistinct monolayer by 24 h, while the organotypic wound models showed a slower percentage of damage closure. A further delay in the closure of the damaged area was observed when chronic wound-like conditions were simulated. Angiogenesis in chronic wound conditions was considerably impaired compared to the acute conditions. The analysis of the extracellular matrix component synthesis, specifically collagen and polysaccharides, revealed the deposition of dense, organized collagen fibers in the acute wound model, in contrast to the thin, fragmented collagen fibers and intracellular polysaccharides observed under chronic wound-like conditions. This corresponded to a statistically significant increase in the levels of both collagen and polysaccharides detected as soluble molecules in the supernatants. Macrophage polarization showed no statistically significant differences in the acute and chronic wound models, though iNOS did significantly decrease after N-A application in acute and chronic models. However, acute wound-like conditions showed a restoration of the vascularized tissue-like structures after treatment with these types of dressings, albeit through different organizational pathways, whereas only minimal improvement was noted under chronic wound conditions, particularly in the case of the N-A dressing. The organotypic dermis model for the onsets of acute and chronic wounds emerges as a highly versatile tool to understand healing mechanisms in the absence or presence of co-morbidities and to assess the biocompatibility of wound dressings as well as the safety, efficacy and dosage of drugs. Full article

Background: Brugada syndrome (BrS) is a genetic cardiac arrhythmia disorder inherited in an autosomal dominant manner, characterized by ST-segment elevation in the right precordial leads (V1–V3) on electrocardiograms (ECGs). This syndrome predominantly affects young individuals with structurally normal hearts and significantly increases the risk of ventricular arrhythmias and sudden cardiac death (SCD). The most common genotype found among BrS patients is caused by variants in the SCN5A gene, which lead to a loss of function of the cardiac sodium channel Nav1.5 by different mechanisms. Methods: Plasmids containing SCN5A were constructed using PCR and site-directed mutagenesis to create the D372H variant. HEK293 cells were cultured and transfected with the WT, D372H, or a combination of both plasmids. Patch-clamp recordings assessed sodium current characteristics. Confocal microscopy visualized channel localization. Quantitative RT-PCR was used to analyze mRNA expression levels, while Western blot evaluated protein expression using specific antibodies. Results: In HEK293 cells expressing the D372H mutant, functional assays revealed a near-complete loss of sodium currents. Co-transfection of WT and D372H plasmids resulted in a significant reduction in current density compared with WT alone, while activation, inactivation, and recovery kinetics were unaffected. In addition, both the mutant protein and protein expressed in co-transfected cells exhibited reduced fluorescence intensity, indicating decreased expression levels. These findings were further supported by Western blot and RT-qPCR analyses. Conclusions: In summary, our findings indicate that the D372H variant produces a marked reduction in Nav1.5 function through reduced sodium current density and decreased channel expression. Given its critical position within the DI-pore loop, this defect is expected to markedly diminish the inward sodium current necessary for normal depolarization. Such impaired excitability—particularly relevant in the right ventricular outflow tract—may accentuate regional differences in repolarization and create conditions that favor reentrant activity. These findings provide mechanistic insights into how the p.D372H variant alters Nav1.5 channel function in vitro and offer functional evidence that may assist in interpreting its potential relevance to Brugada syndrome. Full article

Background and Aims: Neurovascular abnormalities, such as aberrant nerve migration in Hirschsprung’s disease and reduced vascular density in necrotizing enterocolitis, are frequently observed in intestinal diseases. Traditional 2-dimensional (2D) staining methods are complicated, time-consuming and fail to comprehensively visualize the intricate neurovascular structures and morphology of the intestine. This study focuses on evaluating a novel 3D staining technique that promises simpler, faster, and more effective visualization of intact neurovascular structures in the colon. Additionally, it aims to compare the strengths and limitations of this 3D method against traditional 2D techniques for analyzing neuronal and vascular changes in two prevalent pathological conditions. Methods: A novel tissue-clearing approach was used to render mouse and patient distal colon tissues transparent. Neural structures and blood vessels were stained. 2D and 3D imaging were performed with laser confocal or tiling light sheet microscopy. Parameters include total imaging time, imaging range, image quality, operational complexity, and post-processing were compared between 2D and 3D methods. Results: Compared to 2D imaging, 3D imaging reveals the complete morphology and trajectory of neurovascular structures. Confocal 3D imaging offers superior clarity, higher transparency, and faster workflow efficiency, whereas light-sheet microscopy provides broader coverage at the expense of lower image quality. Post-processing facilitated spatial modeling and quantitative analyses. Applications included Hirschsprung’s disease, where 3D imaging revealed abnormal nerve distribution, and congenital heart disease, where hypoperfusion impacted vascular development in the colon. Conclusions: Confocal 3D staining and imaging offered a more streamlined workflow and enabled comprehensive visualization of neurovascular architecture, supporting efficient assessment of intestinal neurovascular phenotypic features. Full article

Background: To assess longitudinal changes in the in vivo confocal microscopy (IVCM) features during Acanthamoeba keratitis (AK) treatment and develop a prognostic model. Methods: This retrospective study included 59 AK patients who underwent IVCM at baseline and 1 and 3 months. Fourteen morphological features covering pathogen-related characteristics, cyst arrangement patterns, and inflammatory markers were compared between good and poor prognosis groups, which were defined based on clinical outcomes including corneal perforation, the need for therapeutic keratoplasty, or final best-corrected visual acuity (BCVA) ≤ 0.05. Prognostic modeling was performed exclusively using baseline IVCM features and applied univariable and Firth-corrected multivariable logistic regression with collinearity assessment and clinical filtering, followed by 5-fold cross-validation. Results: Among 59 AK patients, 45 (76.3%) had a good prognosis and 14 (23.7%) had a poor prognosis. Poor prognosis eyes showed a higher prevalence of double-walled cysts, trophozoites, and clustered cysts, along with higher cyst density and deeper stromal invasion. In contrast, good-prognosis eyes had more target-like cysts, immature dendritic cells, and mature dendritic cells. Clustered cysts independently predicted poor prognosis (OR = 2.98), whereas target-like cysts (OR = 0.26) and mature dendritic cells (OR = 0.37) were protective (AUC = 0.883; all p < 0.05). Conclusions: IVCM provides a quantitative tool for early outcome prediction and individualized management. Higher cyst burden, clustered cysts, and persistent stromal involvement indicated poorer prognosis, whereas target-like cysts and mature dendritic cells indicated better prognosis. Full article

Accurate quantification of spatial distances between fluorescent signals in multi-channel 3D microscopy is essential for understanding genomic organization and gene regulation. However, chromatic aberration introduces systematic spatial offsets between channels that significantly bias distance measurements, particularly at short genomic distances. We present FISH-Dist, an automated computational pipeline for quantitative distance measurements in 3D fluorescence in situ hybridization (FISH) experiments acquired on standard confocal microscopes. Our method combines deep learning-based spot segmentation, 3D Gaussian fitting for sub-pixel localization, and two complementary chromatic aberration correction approaches: affine (ACC) and linear (LCC). We validated the pipeline by measuring the lengths of DNA origami nanorulers and systematically evaluated FISH probe design parameters, including probe spacing, density, and target sequence length. FISH-Dist achieves sub-pixel accuracy in signal detection and substantially reduces inter-channel distance measurement errors. This enables a reproducible quantification of spatial relationships in 3D FISH datasets. Unlike existing tools optimized for long-range chromosomal interactions or requiring super-resolution microscopy, FISH-Dist specifically addresses the technical challenges of standard confocal imaging at short genomic distances, where chromatic aberration has a proportionally greater impact on measurement accuracy. Full article

Polyethylene glycol (PEG) is a widely used water-soluble polymer (WSP) whose properties such as crystallinity depend on molecular weight. This study explores whether Raman spectroscopy, combined with supervised machine learning, can differentiate PEG samples of defined molecular weights within the investigated molecular weight range. Eight PEG materials with molecular weights ranging from 1000 to 35,000 g/mol were analyzed by confocal Raman microscopy under standardized conditions. A Support Vector Machine (SVM) classifier achieved 93.4% accuracy in five-fold cross-validation and 72.6% on an independent test set, confirming that molecular-weight-dependent vibrational signatures are present in the Raman spectra. Principal component analysis followed by linear discriminant analysis (PCA–LDA) models supported these findings, revealing that discriminative information arises mainly from line-shape and shoulder regions rather than from peak centers, consistent with gradual increases in conformational order. Although sample morphology and drying behavior introduce variability, the results demonstrate that Raman spectroscopy provides a reproducible, non-destructive means of distinguishing between PEG samples of different molecular weights. The established workflow provides a foundation for future quantitative evaluations of spectral trends, cross-polymer generalization, and adaptation to variable measurement conditions to enhance applicability in analytical and industrial contexts. Full article

Background: In actinic keratosis (AK), clinical clearance after field-directed therapies does not necessarily correspond to histological resolution, resulting in subclinical persistence and risk of recurrence. Objective: To provide a practical, up-to-date framework for non-invasive monitoring of treatment response in AK, integrating clinical assessment and dermoscopy with high-resolution imaging techniques, reflectance confocal microscopy (RCM), line-field confocal optical coherence tomography (LC-OCT), and high-frequency ultrasound (HFUS), and to discuss emerging optical biomarkers based on Raman spectroscopy. Results: For each modality, we summarize pre- and post-treatment imaging patterns, proposed response criteria, recommended follow-up timing, and correlations with clinical outcomes (including clearance and AKASI) and, when available, histological findings. The available evidence is derived from a limited number of observational studies, predominantly involving RCM and LC-OCT, whereas data on HFUS and Raman spectroscopy remain comparatively scarce. RCM and LC-OCT allow in vivo assessment of epidermal architectural normalization and reduction of intraepidermal keratinocyte atypia. HFUS captures quantitative trajectories of superficial dermal remodeling, including changes in the subepidermal low-echogenic band (SLEB) and dermal echogenicity after photodynamic therapy and other field treatments. Dermoscopy remains the first-line tool for routine follow-up but may fail to detect minimal subclinical persistence. Finally, we discuss the potential role of in vivo Raman spectroscopy for dynamic molecular endpoints and its possible integration with artificial intelligence–based analytical approaches. Conclusions: A standardized multimodal follow-up strategy improves the accuracy of treatment-response assessment compared with clinical evaluation alone. We propose a technique-specific checklist of minimal response criteria and a pragmatic temporal assessment scheme, and outline a research roadmap to support validation and clinical implementation of non-invasive imaging-guided monitoring in actinic keratosis. Full article

Corneal nerves (CNs) are essential to maintain corneal epithelial integrity and ocular surface homeostasis. In vivo confocal microscopy (IVCM) enables the acquisition of high-resolution visualization of CNs, allowing visualization on a microscopic level. Traditionally, CN images must be analyzed by manual examination, which is time consuming and labor intensive. Artificial intelligence (AI) has facilitated reliable analysis of CN parameters, allowing for automatic and semiautomatic analysis of CNs. These include the identification, segmentation, and quantitative analysis of various CN parameters. This review summarizes the applications of AI-driven, automatic, and semiautomatic models in the CN analysis of IVCM images while also focusing on their diagnostic relevance in dry eye disease (DED) and neuropathic corneal pain (NCP). Recent advancements in AI have transformed IVCM image analysis by improving reproducibility and reducing operator dependency and time. The AI-based algorithm has been demonstrated to have good performance and sensitivity to identify and quantify the CN metrics. AI has also been utilized to improve the diagnostic accuracy of DED with IVCM scans, involving multiple portions of the CNs, such as the inferior whorl region. When employed with IVCM images of patients with NCP, AI-assisted identification of microneuromas and changes in CN metrics has provided an improvement in diagnostic accuracy. Despite promising advances and outcomes, the widespread implementation of these AI models in CN image analysis requires large-scale validation. Future integration of multimodal AI algorithms remains a promising endeavor to enhance diagnostic accuracy and disease stratification. Full article

The condition of technical surfaces strongly influences the functionality and lifetime of many components. In particular, the performance of aero-engines can be impaired by increased roughness of the turbine blade surfaces. In this work, an LED- and camera-based illumination sensor is presented for reflection-based characterisation of turbine blade surfaces, with a focus on rapid, wide-area assessment rather than direct roughness measurement. Traditional roughness measurements (e.g., profilometry, confocal microscopy) provide micrometre-scale height information but are limited in working distance and measurement volume, making complete surface coverage time-consuming. The proposed sensor acquires multi-illumination image data, from which an anisotropic BRDF (bidirectional reflectance distribution function) model is fitted on a per-pixel basis to obtain reflectance parameters. Independently, surface roughness parameters (Sa, Sq, Sz, Ssk, Sku) are measured using a confocal laser scanning microscope in accordance with ISO 25178 and used as reference data. Using two turbine blades with contrasting surface conditions (comparatively smooth vs. visibly rough), the study qualitatively investigates whether there are indications of relationships between BRDF model parameters and roughness characteristics. The results show weak relationships with height-based parameters (Sa, Sq, Sz), but clearer trends for distribution parameters (Ssk, Sku) and a good qualitative agreement between directional BRDF parameters and texture orientation. These findings indicate that the illumination sensor provides a complementary, reflectance-based approach for surface condition triage in MRO and QA contexts, highlighting regions that warrant more detailed roughness measurements. Extension of the approach to other component geometries and a comprehensive quantitative analysis of BRDF–roughness relationships are planned for follow-up studies. Full article

Solid lipid nanoparticles (SLNs) are submicron colloidal systems widely investigated as drug carriers; however, their intrinsic biodistribution properties are also critical when SLNs are considered for diagnostic imaging. In the present proof-of-concept study, drug-free SLNs were evaluated exclusively as a radiolabeled imaging agent rather than as a drug delivery system. SLNs were radiolabeled with Technetium-99m (^99mTc), and their in vivo biodistribution was investigated using gamma camera imaging, ex vivo organ counting, and confocal microscopy. SLNs were prepared by a microemulsion–low-temperature solidification method and characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC). Radiolabeling efficiency was determined by instant thin-layer chromatography (ITLC) and exceeded 95%. Following intravenous administration in a rabbit model, dynamic scintigraphic imaging demonstrated predominant uptake in the liver and spleen. These findings were quantitatively confirmed by ex vivo biodistribution analysis at 4 h post-injection and qualitatively supported by confocal microscopy of liver and spleen tissues. The results indicate that ^99mTc-labeled SLNs behave as RES-targeting radiocolloids and may serve as potential agents for liver–spleen scintigraphy. Full article