Search Results (236)

Sperm capacitation is essential for fertilization and involves coordinated changes in membrane organization, ion fluxes, and intracellular signaling. However, commonly used detection methods may reflect different biological events, which can be strongly influenced by experimental methodology. This study critically evaluated fluorescence-based approaches for assessing capacitation in boar spermatozoa, focusing on their specificity, interpretative limits, and methodological sensitivity. Ejaculated boar spermatozoa were incubated under in vitro capacitating conditions in TALP medium. Selected samples were subsequently treated with calcium ionophore to induce the acrosome reaction (AR). Phosphotyrosine (PTyr) immunofluorescence was assessed using five fixation and labeling protocols, acrosin redistribution was evaluated with the ACR.2 antibody, calcium ion redistribution was assessed using chlortetracycline (CTC) fluorescence, and acrosomal responsiveness was monitored by peanut agglutinin (PNA) lectin labeling. PTyr immunofluorescence was highly dependent on fixation protocol, indicating marked methodological sensitivity. Acrosin immunodetection revealed a clear capacitation-associated redistribution from weak or diffuse staining to a well-defined acrosomal pattern, whereas ionophore treatment caused a pronounced signal loss consistent with acrosomal exocytosis. PNA labeling confirmed that capacitation alone did not increase spontaneous acrosome loss, whereas ionophore treatment induced a robust AR. CTC staining showed a significant shift from whole-head pattern to acrosome in TALP-treated spermatozoa, indicating capacitation-associated Ca²⁺ redistribution. Together with CTC and Western blot data, these findings show that sperm capacitation status should be evaluated using multiple complementary markers rather than a single gold-standard assay. Full article

The microsporidian parasite Enterocytozoon hepatopenaei (EHP) is a major pathogen causing severe growth retardation in shrimp, leading to substantial economic losses in global aquaculture. To address the urgent need for accurate, rapid, and field-deployable diagnostic tools for EHP, this study developed a novel one-pot detection platform by integrating asymmetric Enzymatic Recombinase Amplification (aERA) with a PAM-independent CRISPR/Cas12a system (AYERA-Cas12a) based on ssDNA activation. This design circumvents the compatibility challenge between isothermal amplification and CRISPR activity in a single tube by generating single-stranded DNA amplicons that activate Cas12a without requiring a PAM sequence. The assay operates at a constant temperature of 46 °C and completes detection within 15 min. It achieves a sensitivity of 10 copies/μL, equivalent to qPCR, and shows no cross-reactivity with six other prevalent shrimp pathogens. Validation using 56 clinical shrimp (Litopenaeus vannamei, L. vannamei) samples demonstrated complete agreement with qPCR results. With its simple procedure, isothermal conditions, and clear endpoint fluorescence readout under blue light, the AYERA-Cas12a platform is suitable for point-of-care testing (POCT). This work provides a user-friendly tool for the on-site surveillance and early diagnosis of EHP, offering significant potential for improving disease management in shrimp farming. Full article

Background/objectives: Epithelial ovarian cancer (EOC) is the deadliest gynaecologic malignancy, largely due to late-stage diagnosis and ineffective therapy. EOC commonly spreads through the peritoneal cavity as multicellular spheroids, which are metastatic structures that enhance survival under detachment stress, promote dissemination, and contribute to therapeutic resistance. We previously showed that ULK1, a serine/threonine kinase classically linked to macroautophagy initiation, supports EOC progression, suggesting non-canonical roles in spheroid biology and pathogenesis. Methods: CRISPR/Cas9 ULK1 knockout (ULK1KO) models were generated in OVCAR8, HEYA8, and ES2 cells. Mitochondrial degradation phenotypes were assessed in spheroids by immunoblotting and fluorescence microscopy. Label-free proteomics with bioinformatic pathway analysis identified ULK1-associated programs in EOC spheroids. Bioenergetic consequences were quantified using Seahorse ATP-Rate assays. Therapeutic interactions were evaluated using multi-dose combination matrices testing the ULK1 inhibitor DCC-3116 with metformin. Results: ULK1 modulated mitochondrial degradation in a cell-line-specific manner, either promoting or protecting against mitochondrial loss through mechanisms that were uncoupled from canonical autophagy machinery. Proteomic and bioinformatic analyses revealed significant alterations in mitochondria-related processes, aligning with emerging ULK1 functions in mitochondrial homeostasis. ULK1 loss broadly reduced OXPHOS complex proteins in EOC spheroids and consistently decreased hexokinase 2 (HK2), indicating coordinated metabolic remodeling. Seahorse profiling mirrored these shifts: OVCAR8 ULK1KO spheroids showed reduced OCR and ATP production, whereas HEYA8 and ES2 ULK1KO spheroids exhibited increased mitochondrial ATP production. Combination matrices showed potential synergy between DCC-3116 and metformin. Conclusions: These data show that ULK1 differentially regulates mitochondrial degradation across EOC spheroid models through potential mechanisms alternative to canonical autophagy machinery, while reshaping spheroid metabolism and revealing potential therapeutic vulnerabilities in advanced EOC. Full article

Rapid detection of Pseudomonas aeruginosa and its virulence factor ExoU is essential for improving patient outcomes. In this study, a CRISPR–Cas13a-based diagnostic assay combined with recombinase polymerase amplification (RPA) was developed to detect P. aeruginosa and the exoU gene in blood samples. The assay demonstrated robust amplification, with detection limits of 6 log₁₀ and 8 log₁₀ CFU/mL in Luria–Bertani medium and blood, respectively, and a 100% specificity, without cross-reactivity against four Gram-negative bacilli and Staphylococcus aureus reference strains. The utilisation of a fluorescence-based readout facilitated unambiguous discrimination between P. aeruginosa and P. aeruginosa/exoU⁺ isolates vs. negative controls. In conclusion, these results support the potential of RPA/CRISPR-Cas13a diagnostics for the rapid identification of P. aeruginosa and its ExoU virulence factor. Further optimisation and clinical validation are required to confirm its utility as a bedside diagnostic test, where its application would speed up clinical decisions in the treatment of these infections. Full article

Background/Objectives: Rapid identification of foodborne pathogens is of high practical significance because it enables prompt epidemiological response, timely patient management, and effective sanitary control of food products. In this study, we developed an integrated molecular platform combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology for rapid, sensitive, and specific detection of Salmonella enterica. Methods: Four virulence genes (sirA, stn, siiD, and pagN) were selected as targets to ensure reliable pathogen identification. Reaction conditions were optimized using the Moraxella bovoculi Cas12a (MbCas12a) nuclease. The study focused on integrating isothermal amplification with a custom-engineered hardware solution for visual fluorescence detection. Results: The developed method demonstrated sensitive and specific detection, with no cross-reactivity to non-target microorganisms. Optimization allowed for a substantially reduced assay time of approximately 30 min. As a result, a portable fluorescence visualization approach was developed, featuring a 3D-printed housing and an integrated ultraviolet light source for direct visual fluorescence detection. This allows rapid differentiation of samples without specialized laboratory equipment, making it suitable for field applications. Conclusions: The combination of isothermal amplification, MbCas12a-based detection, and the portable fluorescence visualization approach provides a versatile platform for rapid diagnostics and food safety monitoring. This approach has strong potential to improve public health outcomes and enhance the resilience of food supply chains by enabling accessible, field-deployable pathogen detection. Full article

Background: Terahertz (THz) waves exhibit both photon-like and electron-like properties, showing emerging potential in biomedical applications. Cutaneous squamous cell carcinoma (CSCC) is one of the most common skin tumors. Studies have reported that THz waves can induce apoptosis in cancer cells or ablate tumor tissues. Our previous studies also confirmed that 0.1 THz radiation could significantly promote apoptosis in cutaneous melanoma cells, while it had no apparent effect on fibroblast viability, proliferation, migration, and apoptosis. However, the effects of 0.1 THz radiation on CSCC cells have not yet been explored. Furthermore, there remains a lack of investigation into the structural and functional effects on fibroblasts. Therefore, it is necessary to conduct a systematic study to evaluate the influence of 0.1 THz radiation on both CSCC cells and fibroblasts in order to better understand its potential therapeutic applications in the treatment of skin cancer. Purpose: This study aims to explore the biological effects of 0.1 THz radiation on SCC-7 cells and to uncover the molecular mechanisms underlying THz-induced apoptosis, as well as its potential effect on L-929 cells. Methods: Cell viability was evaluated through the CCK-8 assay, while cell cycle distribution was analyzed with the DNA content detection kit. Wound healing assays were performed to assess cell migration, and Annexin V-FITC staining was used to detect apoptosis. Caspase-3 activity was measured using the caspase-3 activity assay kit. Cell morphology was observed using the Atomic Force Microscope (AFM) and the Transmission Electron Microscopy (TEM). Alterations in membrane potential were detected with the M09 membrane potential probe kit, and intracellular Ca²⁺ levels were quantified using the Fluo-8 AM fluorescent probe. Mitochondrial permeability transition pore (mPTP) opening was assessed with the MPTP detection kit, mitochondrial membrane potential changes were measured using the JC-1 probe kit, and cellular ATP levels were measured with the enhanced ATP assay kit. Subsequently, proteomic analysis was performed. Intracellular reactive oxygen species (ROS) levels were quantified with the ROS detection kit, and cytochrome c (Cyt c) release was quantified using the mouse Cyt c ELISA kit. Apoptosis-inducing factor (AIF) expression was analyzed at both mRNA and protein levels by quantitative real-time PCR (qPCR) and Western blot. AIF expression in CSCC tissues was further evaluated based on the GSE42677 and GSE45164 databases. Finally, cyclosporin A (CsA) was used to inhibit mPTP, and in combination with the iMAC inhibitor, the Aifm1 expression and Cyt c release were examined. Results: Our results showed that THz waves significantly disrupted the membrane integrity of SCC-7 cells and induced mitochondrial structural and functional damage. This resulted in a significant increase in ROS levels and the activation of mPTP and the mitochondrial apoptosis channel (MAC). THz radiation promoted the release of Cyt c and AIF from mitochondria, triggering a noncanonical caspase-3-dependent apoptosis pathway. Notably, L-929 cells did not show significant phenotypic or apoptotic changes under the same irradiation conditions. Bioinformatics analysis of the Gene Expression Omnibus (GEO) database revealed that AIF expression was significantly altered in CSCC tissues compared to normal skin tissues. Conclusions: These findings indicated that 0.1 THz radiation effectively induced apoptosis in SCC-7 cells by triggering mitochondrial dysfunction and ROS generation, which led to the release of AIF. Furthermore, the dysregulation of AIF in CSCC tissues suggested its potential as a promising biomarker. These results provided important molecular insights into the therapeutic potential of THz radiation, particularly for the treatment of cutaneous squamous cell carcinoma. Full article

Capsicum annuum is a Solanaceae crop that is sensitive to cold, which affects its growth and development upon prolonged exposure and ultimately reduces yield. In response, a complex regulatory network of cold-responsive genes is activated. Earlier studies have shown that SnRKs play a positive role in enhancing cold tolerance in different crops, including peppers; however, the underlying molecular mechanisms and downstream targets have yet to be fully elucidated. In this study, yeast hybrid screening using CaSnRK2.4 identified a potential interacting partner CaPDX1. The interaction between CaPDX1 and CaSnRK2.4 was further confirmed through Y2H, luciferase complementation, and bimolecular fluorescence complementation assays. Subcellular localization showed that CaPDX1 and CaSnRK2.4 are localized in the nucleus as well as in the cell membrane. Silencing of CaPDX1 through VIGS showed increased susceptibility of peppers to cold stress, negatively influenced antioxidant enzymatic activities, and increased relative electrolyte leakage and malondialdehyde levels. Conversely, transient overexpression of CaPDX1 in peppers enhanced cold tolerance by reducing the accumulation of REL and MDA. Ectopic expression of CaPDX1 in Arabidopsis thaliana significantly improved its cold tolerance, accompanied by enhanced activity of antioxidant enzymes and increased chlorophyll content. In summary, these results indicate that CaPDX1 is a positive regulator of cold tolerance in pepper, and its mechanism of action involves interaction with CaSnRK2.4 and the regulation of physiological and molecular responses in pepper under cold stress. 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

Lactococcus garvieae is a major bacterial pathogen responsible for lactococcosis outbreaks in aquaculture, resulting in substantial economic losses worldwide. Accurate identification of L. garvieae remains challenging because of its genetic similarity to other Lactococcus species and the limited field applicability of many existing molecular diagnostic methods. Therefore, there is an urgent need for a rapid, highly specific, and field-deployable analytical method that enables accurate identification of L. garvieae outside conventional laboratory settings. In this study, a one-pot analytical strategy integrating enzymatic recombinase amplification (ERA) with CRISPR/Cas12a detection was developed, enabling fluorescence or lateral flow dipstick (LFD) readouts within a single closed reaction tube. The one-pot ERA-CRISPR/Cas12a assays achieved a detection limit of 10 copies/reaction. When combined with a rapid DNA release protocol, qualitative detection could be completed within 50 min without the need for sophisticated instrumentation. In parallel, a TaqMan quantitative PCR assay was established as an analytical benchmark, exhibiting a detection limit of 20 copies/reaction with high linearity and good reproducibility. Clinical evaluation using 136 diseased fish samples demonstrated full concordance between the one-pot ERA-CRISPR/Cas12a and qPCR assays, with both methods achieving a positive detection rate of 23.5% (32/136). In addition, the ERA-CRISPR/Cas12a platform was successfully validated under simulated field conditions using a portable reaction device. This study presents a rapid and field-deployable CRISPR-based platform for the early detection and epidemiological surveillance of lactococcosis. Full article

Fascioliasis, a globally prevalent zoonosis, severely threatens public health and livestock security. Current diagnostic approaches, hindered by the need for sophisticated instrumentation and specialized expertise, are inadequate for on-site surveillance in resource-constrained settings. This study developed a rapid, visual detection assay for Fasciola hepatica via recombinase-aided amplification (RAA) integrated with CRISPR/Cas12b, addressing critical equipment and operational constraints. Targeting a specific mitochondrial DNA fragment of F. hepatica, recombinant plasmid standards were constructed, RAA primers and sgRNA optimized, and three detection modalities (real-time fluorescence, UV lamp, test strip) integrated. Clinical validation against PCR demonstrated 45 min turnaround time, F. hepatica-specific positivity, and real-time fluorescence sensitivity of 2.6 copies/μL. Results showed high concordance with PCR and qPCR, with substantially reduced assay duration and streamlined workflow. This highly sensitive, specific, multi-visualized method overcomes limitations of conventional techniques, offering an efficient, field-deployable tool for fascioliasis surveillance and control in grassroots and pastoral regions. Full article

Background: Biodegradable magnesium-based alloys are increasingly explored as emerging biomaterials for dental and maxillofacial applications due to their osteoconductive properties and potential to reduce long-term implant-related complications. However, early-stage evaluation requires predictive diagnostic screening methods capable of assessing cytocompatibility and cellular response under clinically relevant extract conditions. Objectives: In this study, Mg–0.5Ca alloys modified with increasing strontium concentrations (0.5–3 wt.%) were investigated through an in vitro diagnostic framework using MG-63 osteoblast-like cells. Methods: Cell viability was quantitatively assessed via MTT assays after 24 and 72 h of exposure, while fluorescence-based live-cell imaging provided complementary morphological insights. Results: demonstrated a composition-associated cytocompatibility profile, with Sr-enriched compositions showing improved cellular metabolic activity and adhesion patterns compared to lower-Sr compositions. Conclusions: These findings support the role of strontium as a functional alloying element and highlight the importance of standardized diagnostic screening workflows for emerging dental biomaterials. Overall, this study proposes a simplified predictive platform for early biocompatibility diagnostics, contributing to the integration of biomaterial evaluation into future digitalized dental regeneration workflows. Full article

This study presents a comprehensive characterization of Argopecten purpuratus (AP) shells—a marine-derived natural bioceramic composed predominantly of calcium carbonate (CaCO₃)—to evaluate their potential as biomaterials for regenerative medicine. Structural and compositional analyses were performed using micro-computed tomography (MicroCT), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and X-ray diffraction (XRD). These techniques confirmed a high CaCO₃ content (>96 wt%) and revealed distinct microstructural features: the outer surface showed irregular grooves and rough textures, while the inner surface exhibited smoother, foliated morphologies with mixed calcite and aragonite phases. To assess biocompatibility, human gingival mesenchymal stem cells (hGMSCs) were cultured on both shell surfaces. Viability and adhesion were evaluated via MTS assays and fluorescence microscopy at time points ranging from 30 min to four weeks. Both surfaces supported robust early metabolic activity and long-term proliferation, with cells covering the entire surface area after four weeks. Morphometric analysis indicated time-dependent changes in cell shape, transitioning from rounded to elongated morphologies, with minor differences linked to surface topography. The integration of structural, compositional, and biological data demonstrates that AP shells provide a cytocompatible and sustainable natural material platform capable of supporting cell adhesion and proliferation. Their inherent micro- and nanoscale surface features may facilitate protein adsorption and cell–material interactions. These findings highlight the importance of correlating microstructural material properties with cellular responses and support the future exploration of marine-derived bioceramics for regenerative medicine applications. 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

Taxifolin, a natural flavonoid, consistently exerts cytoprotective effects against various oxidative stresses. In this study, we systematically evaluated its photoprotective efficacy and underlying mechanisms against ultraviolet B (UVB)-induced injury in human immortalized keratinocytes (HaCaT). Cell viability and apoptosis were assessed by MTT, fluorescence staining, and flow cytometry, while integrative transcriptomic and proteomic analyses were employed to identify core pathways and key mediators. Taxifolin exhibited antioxidant capacity comparable to that of ascorbic acid under identical in vitro radical-scavenging assays. Moreover, it displayed a strong absorption peak at 289 nm that overlaps the UVB spectrum (280–320 nm), enabling it to act as a chemical sunscreen. In UVB-challenged HaCaT cells, taxifolin markedly reduced intracellular reactive oxygen species (ROS) and attenuated JNK/p38 MAPK activation, as evidenced by Western blot, thereby breaking the ROS-MAPK vicious cycle. Multi-omics revealed that taxifolin was associated with attenuation of UVB-imposed G1/S arrest concomitant with restored Cyclin expression, while up-regulating MYC, FOXQ1, HMOX1 and AP-1 components c-Jun/c-Fos and thereby switching on a pro-survival transcriptional program. Consequently, apoptosis was suppressed and survival was significantly improved. Collectively, taxifolin integrated chemical filtration, ROS scavenging and signaling modulation to support a multi-target photoprotective network, which provides mechanistic insight into taxifolin-mediated cytoprotection and identifies candidate molecular nodes for further validation. Full article

The fruits of Solanum lycopersicum L. cultivar “Microtom” are a powerful source of antioxidants. We investigated whether two light-quality regimes, i.e., fluorescent white (FL) and red-blue (RB), influenced the antioxidant composition in such fruits, and assessed the potential radioprotective properties of their extracts on normal human cells exposed to clinical photons as used in cancer radiotherapy (RT). Increasing normal-tissue tolerance to radiation is critical for reducing the risk of RT-associated sequelae. Biochemical characterization showed that RB enhanced the content of antioxidant phytochemicals (i.e., polyphenols, flavonoids, total carotenoids, lycopene), while FL promoted ascorbic acid synthesis. Initially tested at 200 µg/mL, RB-derived extracts decreased radiation-induced DNA damage as measured by the cytokinesis-block micronucleus (CBMN) assay in epidermal HaCaT cells. Both RB and FL regimes were subsequently studied in MCF-10A breast cancer (BC) cells, a model of normal-tissue radioresponse in BC RT, using extracts at 100 and 200 µg/mL and also evaluating oxidative stress by a ROS detection assay. Both FL and RB afforded radioprotection. However, RB suppressed radiation-induced MN formation and oxidative stress to a greater extent compared to FL. Therefore, modulation of light-quality regimes represents an innovative approach for developing radionutraceuticals with potential benefits for RT patients. Full article