Search Results (24,191)

Breast cancer is classified into distinct molecular subtypes, including Luminal A, Luminal B, HER2-enriched, Basal-like, and Claudin-low. While traditional studies mostly use 2D cell cultures, 3D models better mimic in vivo tumor conditions. In this study, we generated and characterized 3D spheroids from breast cancer cell lines representing different molecular subtypes. Morphologically, spheroids were either compact (MCF-7/AZ, T47D, BT474, MDA-IBC-3, BT-20, SUM149PT) or loosely adhered (MDA-MB-468, SK-BR-3, MDA-MB-231), while retaining key parental subtype biomarkers. Cell viability decreased with increasing spheroid size, but apoptotic cCasp3 staining was restricted to Basal-like spheroids. Compact spheroids expressed E- and/or P-cadherin, indicating epithelial or epithelial–mesenchymal transition (EMT) hybrid traits, while loose spheroids showed vimentin expression linked to a mesenchymal phenotype. Overall, EMT status, rather than molecular subtype, primarily determined spheroid morphology. In conclusion, EMT-associated features, rather than intrinsic molecular subtype, may contribute to 3D spheroid architecture of breast cancer cell lines. Full article

Primary coenzyme Q10 (CoQ10) deficiency results from mutations in genes involved in the CoQ10 biosynthetic pathway. In humans, at least 10 genes (PDSS1, PDSS2 to COQ10) are required for the biosynthesis of functional CoQ10, a mutation in any one of which can result in a deficit in CoQ10 status and present as primary CoQ10 deficiency. Furthermore, the genes NDUFA9 and HPDL, whilst not part of the PDSS1, PDSS2 to COQ10 gene sequence, have also been shown to have a crucial role in CoQ10 biosynthesis. A major problem in treating primary CoQ10 deficiencies is the poor bioavailability of supplemental CoQ10, both in terms of lack of absorption from the digestive tract and inability to cross the human blood–brain barrier. Bypass strategies aim to circumvent this problem by using more bioavailable precursor analogues that can enter the cell and be incorporated into the CoQ10 synthesis pathway downstream of the affected enzyme, examples being 4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid or vanillic acid, which, in contrast to CoQ10, are small, water-soluble molecules. In this article, we have, therefore, reviewed potential bypass mechanisms for primary CoQ10 deficiencies, PDSS1, PDSS2 to COQ10, together with NDUFA9 and HPDL, using such precursors. Most of the published data relating to the bypass therapy of primary CoQ10 deficiency is derived from cell lines or animal models, and few human studies have so far been undertaken. In addition, further research is required to investigate the potential mechanisms by which bypass compounds such as 4-HB may access the human blood–brain barrier (BBB), for example, using in vitro co-culture BBB model systems incorporating CoQ10-deficient neurons. Overall, the objective of this article is, therefore, to systematically review the available data for each of the primary CoQ10 deficiencies, PDSS1, PDSS2 to COQ10 together with NDUFA9 and HPDL, in particular to identify the clinical potential of such studies. Full article

Background: Malaria parasites import essential nutrients from plasma into their host erythrocytes through the plasmodial surface anion channel (PSAC), a conserved ion and nutrient channel on the infected cell surface. A parasite-encoded ternary complex consisting of CLAG3, RhopH2, and RhopH3 determines PSAC activity, but the precise contributions of each member to formation of the nutrient uptake pore remains uncertain. Methods: Here, we devised a two-step CRIPSR transfection strategy to examine an amphipathic CLAG3 helix, termed α-helix 44 (α-H44), as a candidate pore-lining domain. Results: A CLAG3 truncation protein without α-H44 phenocopies a CLAG3 knockout line, suggesting a critical role of α-H44 in formation of the nutrient channel; CLAG3 restoration using a recodonized α-H44 restores PSAC activity fully. A saturation mutagenesis library that splits the helix into four sequential segments was devised and implemented. Two engineered mutants exhibit distinct PSAC phenotypes; their cultures failed to expand in a modified medium that approximates in vivo nutrient availability. Conclusions: These studies support a α-H44 role in channel permeation and block by a strain-specific inhibitor. Our strategy will enable saturation mutagenesis to determine how PSAC achieves its unique ion and nutrient selectivity and should help guide drug discovery against this antimalarial target. Full article

Background and Clinical Significance: Nodular fasciitis is a benign, self-limited myofibroblastic proliferation that frequently mimics malignant soft-tissue tumors both clinically and radiologically. Although it has been well described in the extremities, its uncommon occurrence in the submandibular region poses a diagnostic challenge. Case Presentation: We report the case of a 22-year-old male patient, presenting with a rapidly enlarging painless swelling in the left submandibular region. Ultrasound demonstrated a well-defined subcutaneous lesion, while magnetic resonance imaging revealed heterogeneous enhancement with diffusion restriction, suggesting inflammatory or neoplastic pathology. Fine-needle aspiration cytology showed spindle-cell proliferation with pseudosarcomatous features, warranting histological examination to exclude malignancy. Surgical resection was performed. Histopathological examination demonstrated a myofibroblastic proliferation with tissue culture-like morphology. Immunohistochemistry showed diffuse SMA positivity while many other immunohistological markers were negative, arguing against several histologic mimics. Fluorescence in situ hybridization confirmed USP6 gene rearrangement, establishing the diagnosis of nodular fasciitis. Conclusions: This case highlights the diagnostic challenges posed by nodular fasciitis in the head and neck region and emphasizes the importance of correlating imaging, cytology, histopathology, and molecular findings to avoid overtreatment. The literature review further supports the benign clinical course of this rare entity in the submandibular region and underscores the value of including it in the differential diagnosis of submandibular masses. Full article

Sickle cell disease (SCD) is a major public health concern in Tanzania where approximately 11,000 children are born with the condition annually. Newborn screening (NBS) enables early diagnosis and timely intervention. Despite the proven effectiveness of NBS in reducing early mortality from SCD, implementation in Tanzania remains limited to pilot programs at facilities such as Temeke and Amana Regional Referral Hospitals (RRHs) in the city of Dar-es-salaam. This study evaluated the implementation of NBS for the SCD Program at Temeke and Amana RRHs. An explanatory mixed-methods process evaluation was conducted between January 2022 and December 2024. Quantitative data were extracted from hospital registries and REDCap, while qualitative data were obtained from key informant interviews with 17 healthcare workers. Quantitative data were analyzed using SPSS v29.0, while qualitative transcripts were thematically analyzed using NVivo software version 15 to explore operational factors influencing implementation. A total of 10,711 newborns were screened across the two hospitals. Seventy-four (0.70%) newborns had homozygous SCD (HbS/S), whereas 1325 (12.53%) had sickle cell trait (HbA/S). Enrolment of infants diagnosed with SCD into comprehensive care declined substantially over time, from 65.6% in 2022 to 10.5% in 2024 at Temeke RRH, while Amana RRH recorded no enrolments beyond the first year of implementation. Qualitative findings highlighted facilitators for NBS such as maternal awareness, interdepartmental collaboration, and the availability of trained staff. However, implementation was hindered by inadequate refresher training, delayed staff incentives, supply shortages, and parental hesitancy influenced by cultural beliefs. This evaluation found a substantial decline in enrolment of newborns diagnosed with SCD into comprehensive care, driven by key operational challenges. Although early implementation benefited from trained, committed staff and interdepartmental collaboration, sustainability was limited by inadequate refresher training, delayed incentives, supply shortages, and parental hesitancy. Addressing these gaps through regular capacity building, strengthened supply chains, timely incentives, and culturally sensitive community education is critical to improving enrolment, continuity of care, and informing national scale-up of NBS for SCD in Tanzania. Full article

Harmful algal blooms (HABs) pose a serious threat to public health, aquaculture, and coastal ecosystems, making the development of tools for their rapid and specific detection a high priority. Laser-induced fluorescence (LIF) spectroscopy enables the assessment of characteristic photosynthetic pigments, offering a pathway to automated, high-throughput monitoring systems. Here, we investigate the temperature dependency of LIF spectra in the range of 20–80 °C to establish stable fluorescence fingerprints for the harmful microalgae Alexandrium catenella. Critically, we demonstrate that the relationship between temperature and both fluorescence intensity and spectral position remains consistent over 35 days of cultivation, independent of culture age. We performed complementary flow cytometric and pigment analyses (HPLC) to characterize the culture’s physiological state. Over the 35-day period, cell concentration increased 20-fold, while cell size, granularity, and fluorescence spectra remained stable. A transient decrease in fluorescence intensity observed on day 10 coincided with a drop in peridinin concentration, confirming the link between the spectral signal and pigment composition. Obtained results validate the use of this fluorescence fingerprint for the reliable identification of A. catenella without prior knowledge of the culture’s age—a key advantage for field applications. Furthermore, these fingerprints remained clearly distinguishable even when the culture was diluted with seawater to just 3% of its original volume, underscoring the potential sensitivity of this approach for early warning systems. Full article

(1): Endotoxins are components of Gram-negative bacteria. Uptake can induce allergies, nausea, or sepsis. These responses are triggered by an activation of the immune system. Endothelial cells, lining blood vessels, are the first to be exposed to circulating LPA. Activation can dramatically affect the blood system, such as the formation of thrombi. This study aimed to clarify whether the activation of primary human venous endothelial cells (HUVECs) by LPA could be reduced by the addition of an Arthrospira platensis (AP) extract. (2): HUVECs were cultured for 24 h in cell culture medium supplemented with different concentrations of AP (50, 100, 200 µg/mL). Then 2.5 µg/mL of LPA was added. Cell morphology, viability, cell proliferation, cell membrane integrity, cell metabolism, and cell function were examined after two and four days. (3): Treatment with LPA alone negatively affected HUVEC growth, viability, cell membrane integrity, and metabolic activity. Adding AP to the culture medium had a positive influence on these effects, with 100 µg/mL proving to be the most effective dose. (4): The results clearly revealed that an extract of AP has the potential to reduce the damage to the venous endothelium when exposed to lipopolysaccharides, in particular at a concentration of 100 µg/mL. Full article

Currently, poly- and monophenol antioxidants should be considered not only as inhibitors that interact with free radicals, but also take into account that they are biologically active substances that affect specific targets in cells and can induce the activity of certain genes or stimulate various signaling pathways. The phenols can directly influence different points of the apoptotic process, and/or the expression of regulatory proteins. In our present study the effect of two antioxidants, sterically hindered monophenols sodium anphene (ANa) and potassium phenosan (PhK), on cell apoptosis of splenocytes was studied by fluorescence and confocal microscopy. PhK has already been introduced into medical practice in the Russian Federation because it proved effective as an anticonvulsant and was useful in treating neonatal hypoxia. The study of ANa continues; it may be a promising anticancer drug for some types of tumors. The fluorescent and confocal microscopy methods demonstrate that ANa in combination with H₂O₂ enhances apoptosis in suspension of Lewis carcinoma cells and to a lesser extent in splenocyte culture. We also discovered that autofluorescence of FAD and immunofluorescence of NADPH enzymatic complexes (with the AV-FITC fluorophore) in splenocytes of normal cells increases symbatically. The autofluorescence of FAD in splenocytes of Lewis carcinoma cells significantly exceeded that of splenocytes of healthy animals. The exact distinctive result was obtained when using potassium phenozan. It turned out that PhK prevents the development of apoptosis in mouse splenocyte cell culture (F1(CBA×C57B)). The combined use of ANa and PhK had no effect on splenocyte apoptosis. We show that fluorescence and confocal microscopy allow observing and quantifying the apoptotic effect of ANa and hydrogen peroxide, and make it possible to visualize metabolic changes in the cell, increased FAD fluorescence in tumor cells and NADPH -oxidase complexes in splenocytes. The data obtained indicate the possibility of using ANa in combination with hydrogen peroxide as an antitumor drug acting on certain types of cells. The different effects of sterically hindered monophenols ANa and PhK on the level of the anti-apoptotic protein Bcl-2 in the cell were established. ANa acts to lower Bcl-2 levels, signaling apoptosis, while PhK prevents the development of apoptosis and induces repair processes. Full article

Shot-hole disease caused by Wilsonomyces carpophilus poses a significant threat to stone fruit species, including wild apricot (Prunus armeniaca L.). This study investigated pathogenic factors (cell wall-degrading enzymes and toxins) and metabolites produced by a highly pathogenic strain (CFCC 71544) and a weakly pathogenic strain (CFCC 71543) of W. carpophilus during infection of P. armeniaca (in planta conditions). Analysis using the 3,5-dinitrosalicylic acid colorimetric method revealed that polygalacturonase (CFCC 71544: 1367.02 U/g; CFCC 71543: 1264.00 U/g) and polymethylgalacturonase (CFCC 71544: 1898.71 U·g⁻¹; CFCC 71543: 1762.21 U·g⁻¹) were the most active cell wall-degrading enzymes, with higher activities observed in the highly pathogenic strain (CFCC 71544). Crude toxins from CFCC 71543 induced leaf lesions averaging 41.91 mm² and retained activity after exposure to 121 °C and UV treatment. Non-protein fractions of the toxins caused significantly larger lesions than protein fractions (15.93 mm² vs. 5.56 mm², respectively). Building on these in planta findings, we further characterized toxin properties under controlled laboratory conditions (in vitro). Optimal toxin production conditions were identified in Richard culture medium at pH 4, under a 12 h light/dark cycle, shaken for 12 days at 25 °C. Untargeted metabolomics identified 3244 compounds and 977 differential metabolites among mycelia, crude toxins, and the residual aqueous phase after organic solvent extraction; these metabolites were predominantly amino acids and derivatives and organic acids. These findings indicate that the main pathogenic factors of W. carpophilus are highly active polygalacturonase and heat/UV-stable, water-soluble, non-protein toxins, providing a theoretical basis for shot-hole disease prevention and control. Full article

Photoaging is an extrinsic skin aging process caused by chronic ultraviolet (UV) radiation. A core pathological feature of photoaging is excessive oxidative stress, which can further induce ferroptosis. The HSP70 family plays a critical role in this stress response by protecting the key antioxidant enzyme GPX4. In this study, we established UV-induced photoaging models in cultured cells and 3D skin organoids. UPLC-MS/MS analysis of Chenpi transdermal permeate (prepared by in vitro transdermal penetration of Chenpi extract through mouse skin) identified hesperidin as the primary bioactive compound of Chenpi (dried peel of the plant Citrus reticulata Blanco after the aging process). The efficacy of hesperidin was validated in human keratinocytes (HaCaTs), fibroblasts (HSFs), and skin organoids. Mechanistically, transcriptomic and metabolomics analysis indicated that ferroptosis is a key pathway through which hesperidin ameliorates photoaging. Limited proteolysis mass spectrometry (LiP-MS), transcriptomics, and molecular dynamics simulation results demonstrated that hesperidin directly binds to the molecular chaperone HSPA1L. By upregulating HSPA1L expression, hesperidin enhanced the stability of GPX4 and suppressed UV-triggered ferroptosis. Our findings identify the HSPA1L/GPX4 axis as a critical redox regulatory pathway targeted by hesperidin, providing a mechanistic foundation for anti-photoaging therapies. Full article

Circulating tumor cells (CTCs) are essential biomarkers for cancer prognosis, yet their extreme rarity and biological heterogeneity pose significant challenges for label-free detection. This study presents an automated, non-invasive classification framework integrating a self-assembly cell array (SACA) microfluidic chip with hyperspectral imaging (HSI) and deep learning. By utilizing the SACA chip’s 5 µm gap design, patient-derived blood samples were organized into a flattened monolayer, ensuring high-purity spectral acquisition by minimizing cell overlapping. We implemented two deep-learning pipelines: an Attention-Based Adaptive Spectral–Spatial Kernel ResNet (A²S²K-ResNet) for pixel-level feature extraction and a modified ResNet50 for structural image analysis. While spectral classification achieved ~80% accuracy for cultured cell lines, its performance on patient-derived CTCs was hindered by subtle spectral overlap with white blood cells (WBCs). To overcome this, a multi-band ensemble strategy using majority voting across seven optimized spectral bands (470–900 nm) was developed. This hybrid approach significantly enhanced detection robustness, achieving an overall accuracy of >93.5% and precision exceeding 92%. These results demonstrate that combining microfluidic spatial control with multi-band deep learning offers a reliable, label-free pipeline for clinical liquid biopsy and real-time cancer monitoring. Full article

Background: Despite pharmacological advances, many cancer therapies provide only limited clinical benefits while often inducing significant toxicity. Therefore, the search for more effective and safer anticancer drugs remains an urgent priority. This study aimed to identify plant extracts from the Andalusian flora (Southern Spain) with selective anticancer potential. Methodology: A total of 67 extracts from 59 plant species were screened for selective cytotoxicity using A549 lung adenocarcinoma and HaCaT non-malignant cells. The most promising candidates, extracts from Thymelaea lanuginosa and Daphne oleoides, were further evaluated through fluorescence-based co-cultures, cell cycle analysis, and redox-mechanism assay. These extracts were also tested against a panel of cancer cells derived from different tissues (MDA-MB-231, T24, KATO-III, SK-OV-3, and MeWo). Results: Several extracts exhibited selective activity against A549 cancer cells, including extracts from Chamaeiris foetidissima (L.) Medik. (=Iris foetidissima L.), Daphne oleoides Schreb, Iberodes linifolia (L.) M. Serrano, R. Carbajal & S. Ortiz, Reseda media Lag., Saxifraga hirsuta L., Seseli montanum subsp. granatense (Willk.) C. Pardo, Thymelaea lanuginosa (Lam.), and Tordylium officinale L. The extracts from D. oleoides and T. lanuginosa were over 1000 times more active against lung cancer cells than non-malignant cells. These extracts induced a specific G1-phase arrest in A549 cells. Both extracts showed also selective activity against triple-negative breast cancer cells (MDA-MB-231) and bladder cancer cells (T24). Conclusions: These findings highlight Daphne and Thymelaea species as valuable sources for discovering novel selective anticancer agents. Future research should focus on bio-guided fractionation and in vivo validation to fully delineate their therapeutic potential. Full article

Background: Infective endocarditis (IE) affects both native and prosthetic heart valves, the endocardial surface, as well as cardiac implantable electronic devices. Identifying specific IE biomarkers for its early risk stratification remains crucial, particularly in cases with blood culture-negative endocarditis. Methods: Eleven native heart valves obtained from IE and calcific aortic valve disease (CAVD) patients were analyzed. Immunohistochemical analysis of a pan-leukocyte marker (CD45), macrophage marker (CD68), T-lymphocyte marker (CD3), B-lymphocyte marker (CD19), neutrophil myeloperoxidase (MPO), and marker of vascular endothelial cells (CD31) was performed. Differentially expressed genes (DEGs) were identified by whole-transcriptome sequencing; proteomic profiling was performed by dot-blotting. Results: The immunophenotyping demonstrates the infiltration of macrophages and neutrophils, as well as occasional T-lymphocytes in the IE-affected aortic valves, and the CAVD-affected heart valves were characterized by the absence of neutrophils. For the whole-transcriptome sequencing, 157 DEGs were identified: 124 DEGs were upregulated, and 33 genes were downregulated in the IE-affected heart valves compared to the CAVD-affected ones. According to the dot-blotting, 35 cytokines were identified in the studied heart valves, but only 21 molecules were expressed in both IE and CAVD-affected heart valves. Analysis of proteases and their inhibitors allowed the identification of 13 protease molecules and 18 enzyme inhibitor molecules in all examined heart valves. Conclusions: The results of the present study can help to improve our understanding of the IE pathogenesis. In addition, we identified the candidate cellular and molecular-genetic features of IE-affected native heart valves. Full article

Understanding human’s responses to extreme environments holds significant importance for space exploration, deep-sea research, and environmental adaptation. Traditionally, human subjects were used to study humans’ responses to extreme environments. The main limitations of this approach include the inability to independently investigate specific cellular mechanisms, ethical and safety constraints, limited experimental controllability, and inter-individual variability that complicates mechanistic interpretation. Another approach is to study humans’ responses at the cellular level using 2D culture. This approach often exhibits limited reproducibility due to its inability to recapitulate physiologically relevant microenvironments. Bioprinting can enable studies on human’s responses at the cellular level and within 3D environments. One way is to study human cells’ responses to localized and transient extreme environments created during printing. Another way is to expose 3D printed samples (embedded with human cells) to extreme environments. However, the literature does not contain comprehensive review papers to discuss the prospects and limitations of bioprinting for investigating human cells’ responses to extreme environments. This review paper aims to fill this gap in the literature. It begins with a brief description of the effects of extreme environments on human health and summarizes reported studies on cells’ responses to extreme environments. Afterward, it discusses the prospects and limitations of the two ways of using bioprinting to investigate cells’ responses to extreme environments. Finally, it concludes with identifying knowledge gaps and proposing research directions in the application of bioprinting to study human cells’ responses to extreme environments. Full article

Osteoporosis is a major global health challenge, particularly among aging populations, underscoring the need for safe and effective nutritional interventions. Probiotics and their metabolites have emerged as promising candidates for modulating bone health via the gut-bone axis. In this study, we investigated the effects of a cell-free culture supernatant (CFS) from the food-grade bacterium Limosilactobacillus fermentum GBE18 on the proliferation, differentiation, and mineralization of MC3T3-E1 pre-osteoblasts. GBE18 CFS exhibited no cytotoxicity at concentrations ranging from 1% to 4% (v/v). Notably, 2% (v/v) CFS significantly enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization (p < 0.05). Transcriptomic profiling revealed that differentially expressed genes were enriched in osteoblast-related processes and two key signaling pathways: Wnt/β-catenin and PI3K/Akt. Subsequent qRT-PCR and Western blot analyses confirmed the upregulation of critical regulators (Rspo2, Pdpk1, Malat1) and demonstrated coordinated activation of Akt phosphorylation, β-catenin stabilization, and Runx2 protein expression. Our findings indicate that GBE18 CFS promotes osteogenic differentiation through coordinated modulation of the PI3K/Akt and Wnt/β-catenin pathways. Consequently, this study provides mechanistic evidence supporting the potential application of L. fermentum GBE18-derived metabolites as functional food ingredients or dietary interventions for bone health and osteoporosis management. Full article