Search Results (106)

Plant bioreactors offer low production costs, high biosafety, and facile scalability, making tomato an excellent platform for the expression of bioactive proteins. Lactoferrin (LF) possesses a wide range of biological activities; however, its production using microbial expression systems is limited by inherent drawbacks. These limitations can potentially be overcome through tomato-based heterologous expression. Nevertheless, studies investigating the biological activity of LF produced in tomato expression systems remain scarce, and validation using animal cell models has been relatively limited. In this study, the human lactoferrin (hLF) gene was codon-optimized, and the recombinant plasmid pSlhLF was constructed and introduced into tomato plants via Agrobacterium-mediated transformation to generate positive transgenic lines. Molecular analyses confirmed that hLF was expressed in all examined tomato tissues, with the highest expression level detected in fruits. Most transgenic lines exhibited significantly increased transcript levels of genes encoding antioxidant enzymes. Furthermore, in vitro cell assays demonstrated that tomato-derived hLF promoted the proliferation of H₂O₂-treated human fetal lung fibroblasts (HFL-1 cells) and reduced intracellular reactive oxygen species (ROS) accumulation. These findings suggest that hLF expressed in tomato may exert its antioxidant activity, at least in part, through the direct scavenging of ROS. Full article

Background/Objectives: Intrinsic genetic instability and the marked heterogeneity of malignant cell populations represent significant clinical challenges in oncology, often limiting the efficacy of conventional receptor-targeted and antigen-based therapies. To overcome these limitations, [⁶⁴Cu]CuCl₂ has emerged as a particularly promising theranostic agent because it combines PET imaging (β⁺ emission) with therapeutic effects (β⁻ particles and Auger electrons). In particular, Auger electrons, when delivered to the cell nucleus, induce severe DNA damage due to their high linear energy transfer and very short tissue range. This work aimed to deepen existing preclinical knowledge by providing a comprehensive in vitro analysis of the interactions of [⁶⁴Cu]CuCl₂ with various human cancer cell lines—specifically, the breast adenocarcinoma (MDAf-MB-231) and gastric carcinoma (NCI-N87) cell lines—and a healthy control (IMR-90 normal human fetal lung fibroblasts). Methods: We focused on evaluating cellular uptake, subcellular localization, impact on metabolic activity, and induction of apoptosis. Cell lines (MDA-MB-231, NCI-N87, IMR-90) were exposed to increasing activities of [⁶⁴Cu]CuCl₂ (10, 100, and 250 µCi/mL). Uptake was assessed in both nuclear and cytoplasmic compartments after 4 h. Metabolic activity and apoptosis/necrosis were evaluated at 96 and 120 h post-treatment. Results: Tumor cell lines demonstrated significantly higher [⁶⁴Cu]CuCl₂ uptake, particularly at the nuclear level, compared to healthy controls. A marked decrease in metabolic activity and an increase in apoptosis were observed in MDA-MB-231 and NCI-N87 cells (from 50% to 90% and 5% to 60% apoptosis, respectively). In contrast, IMR-90 cells exhibited minimal cytotoxic response (≤20%), suggesting a preferential response in the malignant cell models tested. Conclusions: [⁶⁴Cu]CuCl₂ induced distinct patterns of intracellular accumulation and biological response among the investigated cell models, with cancer cells displaying greater nuclear uptake and apoptotic susceptibility than non-malignant cells. These findings provide a high-resolution radiobiological baseline and microdosimetric validation, supporting the rigorous design of future, dedicated in vivo preclinical investigations to evaluate the translational potential of ionic [⁶⁴Cu]CuCl₂. Full article

Newborn piglets in intensive pig farms are often prone to pulmonary diseases due to underdeveloped fetal lung neural systems, and ATRA is often regarded as an important morphogen that displays pleiotropic functions during embryonic development. However, information about the effect of maternal administration with ATRA on the peripheral neural system of fetal lungs is still scare. Fifteen pregnant sows were assigned to the ATRA0 (0 mg/kg diet), ATRA4, ATRA8, ATRA16 and ATRA32 treatment groups and offered their own feed from d12 to d95 after artificial insemination; then two neonatal pigs with birth weights similar to the average birth weight were taken out from each litter for the collection of lung samples, and samples were subjected to immunofluorescence staining, RNA-seq and RT-qPCR assays. Results indicated that compared to newborn piglets from the ATRA0 treatment group, newborn piglets from the ATRA4 treatment group had higher percentages of GFAP-positive astrocyte cells (p < 0.05) and GFAP-TUBB3 colocalization (p < 0.05) in the lungs. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that differential expressed genes are mainly enriched in the pathways of neuroactive ligand–receptor interaction, GABAergic synapses and cell adhesion molecules. Addition of ATRA at 4 mg/kg to the diet of pregnant sows can enhance the healthy development of the pulmonary nervous systems of fetal pigs. Full article

Organoid culture represents a sophisticated biological model that surpasses traditional two-dimensional (2D) methods and animal models in physiological relevance and cost-effectiveness. Current organoid systems derive from adult, fetal, and induced pluripotent stem cells, providing innovative platforms for studying organ development, disease pathogenesis, and drug discovery. Recent technological advances now enable respiratory organoids to significantly contribute to respiratory disease research. This review comprehensively synthesizes the development of respiratory organoid models and their applications in studying major respiratory diseases, including pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), and lung cancer. It further evaluates the transformative potential of these models in advancing respiratory disease research. Respiratory organoids uniquely model disease mechanisms and drug responses in human-specific microenvironments, enabling pathogenesis studies of respiratory diseases. They serve as functional platforms for drug screening and personalized therapy development. Future integration of multi-organoid systems with precision medicine promises to redefine respiratory disease research paradigms. Full article

Marine invertebrates are a prime source of biologically active peptides due to their role in humoral immunity. These peptides typically exhibit broad-spectrum functions, including antibacterial, antifungal, anticancer, and immunomodulatory activities. In this report, we describe the identification and biological characterization of five novel bioactive peptides from the marine mollusk Pisania pusio. An extract of P. pusio was analyzed using nanoLC-ESI-MS-MS, and five peptides (PP1–5) were selected via bioinformatic screening as potential antimicrobial and anticancer peptides and subsequently validated experimentally. Among these, PP1, PP2, and PP4 were identified as cryptides derived from the proteolytic cleavage of actin, while PP3 and PP5 are novel peptides with no known protein precursors. All peptides exhibited moderate activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae with minimum inhibitory concentrations (MICs) predominantly at 100 µM. In contrast, only PP1 and PP5 were active against cancer cells, with PP1 being the most effective against A375 melanoma cells (IC₅₀ = 17.08 µM). This experimental validation confirmed the utility of the integrated in silico/peptidomic pipeline for lead identification. None of these peptides showed significant hemolytic activity or toxicity on fetal lung fibroblasts over 800 μM, demonstrating promising in vitro selectivity. These results highlight the multifunctional nature of P. pusio-derived peptides and their potential as lead compounds for further optimization and development into therapeutic agents against microbial infections and cancer, subject to more comprehensive safety evaluations in relevant models Full article

Salivary gland carcinomas (SGCs) are aggressive malignancies with limited treatment options, primarily due to the complexity of the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) remodel the extracellular matrix (ECM), enhance cancer cell stemness, and drive drug resistance. This study introduces a decellularized CAF-derived spheroid system as a biomimetic platform to study tumor–stromal interactions in SGC. Multicellular spheroids were generated by co-culturing Medical Research Council cell strain 5 (MRC-5) fibroblasts (fetal lung-derived) with A253 salivary gland cancer cells, producing distinct spatial architecture, with fibroblasts at the core and cancer cells at the periphery. Compared with A253-only spheroids, A253/MRC-5 spheroids exhibited enhanced proliferation and elevated expression of stemness markers (aldehyde dehydrogenase 1 [ALDH1], CD133, cytokeratin 19 [CK19]). MRC-5 spheroids displayed robust ECM and growth factor expression that persisted after decellularization. Decellularized spheroids retained biological activity, enabling A253 cells to develop invasive phenotypes, metabolic reprogramming, and stemness-associated signatures. Transcriptomic analysis revealed a transition from proliferative pathways to stress-adaptive survival programs, mirroring in vivo tumor behavior. Moreover, A253 cells cultured with decellularized fibroblast spheroids exhibited altered cisplatin sensitivity, highlighting the critical role of stromal ECM in therapeutic response. In conclusion, this study establishes decellularized CAF spheroids as a simplified yet biologically relevant TME-mimetic platform. By recapitulating tumor–stromal crosstalk without live co-culture, this system provides a powerful tool for mechanistic studies of salivary gland cancer, preclinical drug screening, and development of stroma-targeted therapies. Full article

The role of mesenchymal-to-endothelial transition in the angiogenic response remains controversial. In this study, we investigated whether human fetal lung fibroblasts (MRC-5 cells) exhibit morphological plasticity in a biomimetic extracellular matrix environment. To this end, MRC-5 cells were first cultured on and within Matrigel hydrogel and then studied with tube formation assays, confocal/fluorescence microscopy, invasion assays, and transcriptomic profiling. In addition, quantitative assessment for cord formation and gene expression was conducted via qPCR and RNA sequencing. In this study, MRC-5 cells quickly self-organized into cord-like networks, resembling early stages of vascular patterning, and at higher densities, invaded the hydrogel and formed spheroid-like aggregates. Transcriptomic analysis revealed upregulation of genes related to nervous system development and synaptic signaling in Matrigel-grown MRC-5 cultures. Collectively, these findings suggest that MRC-5 fibroblasts display structural and transcriptional plasticity in 3D Matrigel cultures, forming vascular-like cords that are more likely to resemble early developmental morphologies or neuroectodermal-like transcriptional signatures than definitive endothelial structures. This work underscores the potential of fibroblasts as an alternative cell source for vascular tissue engineering and highlights a strategy to overcome current limitations in autologous endothelial cell availability for regenerative applications. Full article

Umbilical cord mesenchymal stem cells (UCMSCs)-derived small extracellular vehicles (sEVs) are reported to offer therapeutic effects in regenerative medicine, but they lack safety and biodistribution profiles to support smooth translation at the clinical stage and regulatory requirements. Our study aimed to determine the safety and biodistribution profile in a healthy animal model before application in the metabolic syndrome model. Method: Healthy male Sprague Dawley (SD) rats were given an intravenous (IV) injection of normal saline (control group) or pooled fetal UCMSCs-derived sEVs (treated group) every three weeks for 90 days. Morbidity and mortality observation (daily), physical measurements (weekly), selected serum biochemistry (every three weeks), and hematology (every three weeks) were performed for 90 days. Acute toxicity (on day 14) and sub-chronic toxicity (on day 90) were assessed for gross necropsy, relative organ weight, and histopathological assessment of lungs, liver, spleen, kidney, and lymph nodes. Separately, a biodistribution study was conducted with the sEVs preparations labeled with PKH26 fluorescent dye, given intravenously to the rats. The organs were harvested 24 h post-injection. There were no drastic changes in either group’s morbidity or mortality, physical, hematological, and biochemistry evaluation. The histopathological assessment concluded moderate (focal) inflammation in the treated group’s kidneys and signs of recovery from the inflammation and vascular congestion in the liver. A biodistribution study revealed a higher accumulation of sEVs in the spleen. Multiple IV injections of the pooled fetal UCMSCs-derived sEVs in healthy male SD rats were deemed safe. The sEVs were abundantly distributed in the spleen 24 h post-injection. Full article

Collective cell migration is crucial in various biological processes, including tumor progression and metastasis. The widely used scratch assay (wound healing assay) has limitations in throughput, reproducibility, and data analysis. To overcome these challenges, we previously developed the Transient Agarose Spot (TAS) assay, which enhanced assay precision and reproducibility. In this study, we present an improved microplate-based TAS assay. By using a microplate reader, we automated data acquisition, enabling the detection of cell migration in a 96-well plate format with greater throughput and accuracy. The new method applies Hoechst staining to label viable cells, providing a stable signal for kinetic analysis without compromising cell viability. We validated this approach with fluorophore-expressing cancer cells and demonstrated its ability to monitor dose-dependent effects of fetal bovine serum on cell migration. Additionally, we applied the microplate-based TAS assay to assess the anti-migratory effects of kinase inhibitors and mesenchymal stem cell-derived extracellular vesicles (EVs) on lung cancer cells. The assay accurately quantified migration inhibition and revealed the concentration-dependent effects of EVs, highlighting their potential as therapeutic agents. This microplate-based TAS assay provides a scalable, efficient, and cost-effective platform for high-throughput screening of cell migration and drug discovery, offering a robust alternative to traditional microscopy-based methods. Full article

CEA (carcinoembryonic antigen), which belongs to the acidic glycoproteins, is primarily produced during the fetal period. Following this stage, low levels of CEA are considered physiological, while elevated concentrations are associated with a range of both benign and malignant pathologies. The liver plays a key role in CEA metabolism. The most common material used to determine CEA concentrations by various techniques is blood, and measuring CEA in peritoneal fluid holds clinical value. CEA has been found to contribute to carcinogenesis, metastasis, and treatment resistance. Therefore, its serum concentration is widely used in oncology for prognosis, disease monitoring, and recurrence detection, despite its limited sensitivity and specificity, which prevent it from serving as a standalone diagnostic tool. Elevated serum CEA levels are linked to worse outcomes in lung, liver, breast, colorectal, and pancreatic cancers. Imaging and multi-marker panels that include CEA enhance diagnostic accuracy, but its role remains context-dependent and varies by cancer type. CEA levels in peritoneal fluid have been explored as a potential marker for detecting malignancy and predicting recurrence, particularly in gastric, gynecological, and colorectal cancers. Peritoneal fluid CEA has also been proven useful in differentiating the etiology of ascites. While cytology remains the standard for the detection of tumor cells in body fluids, its limited sensitivity provides a strong rationale for incorporating peritoneal fluid CEA measurements as a complementary diagnostic tool, potentially alongside other markers. Additionally, the lack of standardized measurement techniques and cut-off values underlines the methodological challenges that still need to be addressed in future research for both serum and peritoneal CEA levels. Full article

Background/Objectives: The oncofetal membrane protein Claudin 6 (CLDN6) is an attractive target for T cell-based therapies. There is a lack of detailed analyses on the age-dependent expression of CLDN6 in normal tissues is lacking, which limits the expansion of CLDN6 CAR-T cell clinical trials to pediatric populations. Methods: We analyzed CLDN6 expression in extracranial solid tumors and normal tissues of children using RNA-sequencing data from over 500 pediatric solid tumor samples, qRT-PCR and immunohistochemistry (IHC) in more than 100 fresh-frozen tumor samples and, approximately, 250 formalin-fixed paraffin-embedded (FFPE) samples. We examined normal tissue expression via qRT-PCR in 32 different infant tissues and via IHC in roughly 290 tissues from donors across four age groups, as well as in fetal autopsy samples. Results: In fetal tissues, we detected CLDN6 expression primarily in the epithelial cells of several organs, including the skin, lungs, kidneys, intestinal tract, and pancreas, but not in undifferentiated blastemal cells. Postnatally, we found CLDN6-positive epithelial progenitors only during the first few weeks of life. In older-age groups, isolated clusters of CLDN6-positive progenitors were present, but in scarce quantities. In tumor tissues, we found strong and homogeneous CLDN6 expression in desmoplastic small round cell tumors and germ cell tumors. Wilms tumors demonstrated heterogeneous CLDN6 expression, notably absent in the blastemal component. Conclusions: These findings highlight an organ-specific presence of CLDN6-positive epithelial precursors that largely disappear in terminally differentiated epithelia within weeks after birth. Therefore, our data support CLDN6 as a viable therapeutic target in pediatric patients and justify their inclusion in basket studies for anti-CLDN6-based therapies. Full article

Although every cell biologist knows the importance of selecting the right growth conditions and it is well known that the composition of growth medium may vary depending on a product brand or lot affecting many cellular processes, still those effects are poorly systematized. We addressed this issue by comparing the effect of 12 fetal bovine sera (FBS) and eight growth media from different brands on the morphological and functional parameters of five cell types: lung adenocarcinoma, neuroblastoma, glioblastoma, embryonic kidney, and colorectal cancer cells. Using high-throughput imaging, we compared cell proliferation; performed morphological profiling based on the imaging of 561,519 cells; measured extracellular regulated kinases (ERK1/2) activity, mitochondria potential, and lysosome accumulation; and compared cell sensitivity to drugs, response to EGF stimulation, and ability to differentiate. We found that changes in cell proliferation and morphology were independent, and morphological changes were associated with differences in mitochondria potential or the cell’s ability to differentiate. Surprisingly, the most drastic differences were detected in serum-free conditions, where medium choice affected cell survival and response to EGF. Overall, our data may be used to improve the reproducibility of experiments involving cell cultures, and the effects of 28 growth conditions on proliferation and 44 morphological parameters can be explored through a Shinyapp. Full article

Congenital diaphragmatic hernia (CDH) is a relatively rare and severe developmental disease. Even with the most recent multidisciplinary therapies, the risk for neonatal mortality and morbidity remains high. Recent advancements in prenatal treatments, alongside experimental and clinical data, suggest that fetoscopic endoluminal tracheal occlusion (FETO) promotes lung development and offers a promising strategy against lung hypoplasia and pulmonary hypertension. It is the only existing direct mechanical therapy that intervenes in the regulation of pulmonary pressure. Its influence on lung development also interferes with tissue homeostasis and cell differentiation; it also enhances inflammation and apoptosis. Its physiopathology on cellular and molecular levels is still poorly understood. Unfortunately, the procedure also carries significant pregnancy-, maternal-, and fetus-related risks. Assessing a multifaceted intervention requires a collective view of all aspects. This scoping review uncovers potential materno-fetal procedure-related risks and highlights innovative solutions. Future research on lung development therapies in CDH may focus on the “dual hit” mechanism, combining molecular-targeting drugs and regenerative medicine with the mechanical nature of FETO for synergistic effects. Full article

Background/Objective: Metabolic syndrome (MetS) is characterized by abdominal obesity, increased blood pressure (BP), fasting blood glucose (FBG) and triglyceride levels, and reduced high-density lipoprotein (HDL) levels. This study aims to investigate the efficacy of the Wharton’s jelly mesenchymal stem cells (WJMSCs)-derived small extracellular vesicles’ (sEVs) preparations in managing MetS. Method: Twenty-four rats were fed with a high-fat and high-fructose diet to induce MetS for 16 weeks and randomized into three groups (n = 8/group): a MetS Control group treated with normal saline, MetS Low Dose (LD) group treated with a LD of sEVs preparations (3 × 10⁹ particle/rat), and MetS High Dose (HD) group treated with a HD of sEVs preparations (9 × 10⁹ particles/rat). The Control Non-Disease (ND) group was given a standard rat diet and autoclaved tap water with normal saline as treatment. Treatments were given via intravenous injection every three weeks for twelve weeks. Rats were assessed every six weeks for physical measurements, FBG, lipid profiles, CRP, leptin, adiponectin, and BP. Necropsy evaluation was performed on the lungs, liver, spleen, and kidney. Results: Significant reductions in FBG, triglycerides, BP, and increased HDL levels were observed in the treated groups compared to the control group. However, significant abdominal circumference (AC) improvement was not observed in the treated groups. Non-significant associations were found between fasting CRP, leptin, and adiponectin levels with MetS rats after treatment. In addition, sEVs preparations improved inflammation and hemorrhage in the lung and mineralisation in the renal of the treated group. Conclusions: Human fetal WJMSCs-derived sEVs preparations improve all the clusters of MetS in rats except AC and could be further explored as a treatment for MetS. Full article

Using non-classical polyfluorophenyl ligands in Pt(II) complexes and other transition metals such as silver is a promising approach in the search for more effective and safer antitumoral drugs. In this work, a series of chelating N-donor ligands with 1,10-phenanthroline and 4,5-diazafluorene backbones and ketone groups were synthesized (1,10-phenanthroline-5,6-dione, 1; (R/S)-6-hydroxy-6-(2-oxypropyl)-1,10-phenanthroline-5(6H)-one, 2; 4,5-diazafluoren-9-one, 3; 9-hydroxy-9-(2-oxypropyl)-4,5-diazafluorene, 4). The corresponding [Ag(N,N)₂]NO₃ (1Ag–4Ag) and [Pt(C₆F₅)₂(N,N)] (1Pt–4Pt) complexes were prepared. The stability of these complexes in DMSO solution was studied, showing no dissociation over 48 h for almost all complexes, except 3Pt. The compounds were characterized by NMR (¹H, ¹³C, and ¹⁹F), MS, and X-ray diffraction (2, 4, 1Ag, 3Ag, 1Pt, and 3Pt). A study of the cytotoxicity of the compounds in lung carcinoma (A-549) and fetal lung fibroblast (MRC-5) cell lines was performed. Compounds 1, 2, 1Ag, 2Ag, 3Ag, 1Pt, 3Pt, and 4Pt were more active against A-549 cells than cisplatin. Complexes 3Ag and 1Pt showed an acceptable SI and better selectivity than cisplatin, proving that silver(I) complexes and Pt(polyfluorophenyl) complexes are valuable options in searching for new antitumoral drugs. Full article