Search Results (483)

Biological manipulation via physical stimuli such as light and magnetism has become a central goal in modern biotechnology. Among these modalities, magnetic fields offer unique advantages, including deep tissue penetration and untethered interventions in living systems. An ideal platform for such a magnetogenetic toolkit would be a genetically encodable protein with tunable magnetic features under physiological conditions. However, the development of such tools has been hindered by the lack of robust and stable protein scaffolds with strong intrinsic magnetic properties. Inspired by animal magnetoreception in nature, here, we rationally designed and systematically screened single-chain variants of the magnetoreceptor MagR. Through nine iterative rounds of design and experimental validation, we generated 25 constructs and ultimately identified a stable single-chain-dimer-based-tetramer, SDT-MagR, as the optimal magnetic molecular platform. This engineered protein exhibits exceptional structural stability and state-dependent magnetic behavior, showing ferrimagnetic-like characteristics in the solid state and paramagnetic behavior in solution. With enhanced magnetic susceptibility, purified SDT-MagR can be directly attracted by a magnet in vitro, establishing it as a promising new platform for future biomagnetic manipulation and magnetogenetics applications. Full article

Glioblastoma (GBM) is one of the most aggressive and therapy-resistant primary brain tumors, mainly due to its pronounced intratumoral heterogeneity and highly invasive phenotype. Patient-derived three-dimensional (3D) culture models, including tumor spheroids, represent valuable tools to preserve the cellular complexity, phenotypic plasticity, and microenvironmental features of GBM ex vivo. However, standardized and reproducible protocols for the generation and maintenance of GBM spheroids from surgical specimens are still limited. Here, we describe a detailed and robust protocol for the isolation, 3D cultures, and expansion of primary GBM cells obtained from patient biopsies, leading to the formation of stable and morphologically consistent spheroids. The protocol provides step-by-step instructions for tissue dissociation, cell seeding under low-adhesion conditions, optimization of culture density, and long-term spheroid maintenance. In addition, we include guidelines for the morpho-phenotypical characterization of the resulting 3D structures. This methodological workflow offers a reproducible platform for modeling GBM in vitro, enabling the study of tumor biology and supporting translational applications such as drug screening, biomarker validation, and patient-specific therapeutic testing in a 3D context. Full article

Background/Objectives: Cryptosporidium parvum is a major causative agent of cryptosporidiosis; however, progress in anti-cryptosporidial drug discovery has been hindered by the lack of robust and reproducible in vitro evaluation systems. In this study, we developed and optimized a luciferase-based in vitro assay to quantitatively monitor C. parvum growth in HCT-8 cells. Methods: Key experimental parameters affecting infection efficiency were systematically examined, including sodium taurocholate treatment, timing of medium replacement, and serum concentration. Results: Sodium taurocholate significantly enhanced parasite infectivity, and removal of non-invaded parasites at 3 h post-infection (hpi) resulted in approximately 2-fold and 3.7-fold increase in luciferase activity at 24 and 48 hpi, respectively, compared with untreated controls. In contrast, removal at 24 hpi led to only an approximately 2.5-fold increase at 48 hpi, consistent with stage-dependent differences in parasite development. Morphological analyses confirmed parasite differentiation from trophozoites to meronts, followed by progression toward sexual stages. Using the optimized assay system, we evaluated several anticoccidial compounds and demonstrated potent in vitro activity of monensin and its structural analog kijimicin, whereas diclazuril and toltrazuril exhibited limited efficacy. Conclusions: Collectively, this luciferase-based platform provides a reliable and quantitative tool for anti-cryptosporidial drug screening and will facilitate future therapeutic development against C. parvum. Full article

Aging is a complex biological process characterized by progressive loss of cellular homeostasis, impaired regenerative capacity, and accumulation of senescent cells that collectively predispose tissues to disease. Traditional two-dimensional culture systems and animal models have provided valuable insights but fail to fully recapitulate the spatial organization, cellular heterogeneity, and microenvironmental cues of aging human tissues. Organoid technology—three-dimensional self-organizing structures derived from adult stem cells or pluripotent stem cells has emerged as a transformative platform to model aging in vitro. These mini-tissues retain the architecture, signaling dynamics, and lineage hierarchy of native organs, making them powerful systems to interrogate age-associated cellular phenotypes, DNA damage responses, and senescence programs. This review discusses how organoid models are advancing our understanding of aging biology across multiple organ systems, from the intestines and liver to the brain and lung. We highlighted key molecular pathways driving cellular senescence within organoids—including p16INK4a/p21CIP1 signaling, SASP activation, mitochondrial dysfunction, and epigenetic drift—and how these can be targeted to restore tissue homeostasis. We further discussed how organoids derived from aged tissues, induced pluripotent stem cells, and engineered oncogene systems reveal new therapeutic opportunities to modulate senescence in age-related disorders, cancer, and regenerative medicine. Finally, we discussed emerging integrative tools such as organoid co-cultures, single-cell omics, and senolytics drug screening that are expanding the potential of organoids as translational platforms for anti-aging and disease intervention. Full article

Fungal diseases represent one of the major threats to citrus production, such as anthracnose caused by Colletotrichum gloeosporioides and Fungal Trunk Diseases (FTDs) associated with Botryosphaeriaceae, with Neofusicoccum parvum being the most prevalent species. In response to the need to reduce chemical fungicide use, this study evaluated the antifungal activity of essential oil-based nanoemulsions (N-EOs) as alternative management methods. Seven N-EOs (citronella, clove, fennel, garlic, laurel, lavender and peppermint) were first screened in vitro against multiple isolates of both pathogens through mycelial growth and conidial germination assays. Based on estimated EC₅₀ and EC₉₀ values, clove and garlic N-EOs exhibited the highest inhibitory activity, while lavender displayed intermediate but promising efficacy, particularly against N. parvum. These N-EOs were subsequently evaluated in vivo on lemon fruits inoculated with C. gloeosporioides and on detached lemon twigs inoculated with N. parvum. In vivo assays largely confirmed the in vitro trends, with clove and garlic significantly reducing lesion development. In contrast, lavender displayed limited efficacy under in vivo conditions. The phytotoxic effects at higher concentrations limited the range of applicable doses. Overall, the results suggest that N-EOs, particularly those based on clove and garlic, may offer potential as alternative tools for citrus disease management. However, host tissue interactions, formulation stability, volatility, and validation under field conditions remain critical aspects requiring further investigation. Full article

Alveolar osteitis (AO) and postoperative pain are common complications after dental extractions. Excessive fibrinolysis leading to premature clot loss contributes to AO. Tranexamic acid (TXA), an antifibrinolytic agent, may stabilize post-extraction blood clots and reduce AO, although evidence from randomized controlled trials (RCTs) remains inconsistent. This systematic review and meta-analysis evaluated the effectiveness of topical TXA in preventing AO and reducing postoperative pain following dental extractions. PubMed, Embase, Scopus, and CENTRAL were searched from inception to June 2025 using terms related to “dental extraction” and “tranexamic acid”. Only English-language human studies were included. Eligible studies were RCTs assessing topical TXA versus placebo, saline, or plain gauze, reporting AO and/or pain outcomes. Non-RCTs, in vitro or animal studies, and trials lacking relevant outcomes or controls were excluded. Two reviewers independently screened and selected studies. Following PRISMA guidelines, two reviewers extracted data and assessed risk of bias with the Cochrane RoB-2 tool. Pooled analyses used random-effects models, with risk ratios (RRs) for AO and standardized mean differences (SMDs) for pain. AO was defined as exposed bone, foul odor, or persistent pain after day 3. Pain was measured on the Visual Analogue Scale (VAS) on days 3 and 7. Five RCTs (378 patients) were included. TXA significantly reduced AO incidence compared with controls (RR = 0.49; 95% CI: 0.32–0.76; p = 0.001; I² = 0%), indicating a ~50% risk reduction. Pain outcomes showed no significant differences on day 3 (SMD = −0.36; 95% CI: −0.95 to 0.24; p = 0.24; I² = 84%) or day 7 (SMD = −0.43; 95% CI: −1.34 to 0.48; p = 0.36; I² = 93%). Topical TXA significantly reduces the risk of AO after dental extraction, while its effect on postoperative pain remains inconsistent. Its safety, accessibility, and low cost support its use as a preventive adjunct in dental extractions. Further standardized, high-quality RCTs are needed to clarify its role in pain management. Full article

Background/Objectives: Metabolic dysfunction-associated steatotic liver disease (MASLD) involves the interplay of hepatic lipid accumulation and immune-mediated inflammatory signaling, yet human-relevant in vitro systems that capture both processes simultaneously in a scalable format remain limited. The objective of this study was to develop and characterize a matrix-free 3D hepatocyte–macrophage co-culture model enabling simultaneous assessment of lipid accumulation and NF-κB-mediated inflammatory activation under glucolipotoxic stress. Methods: A 3D liver co-culture model was established by combining HepG2 hepatocyte-like cells with phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophage-like cells stably expressing a NF-κB–Luc2 reporter. Spheroids were generated using a hanging-drop method in standard 96-well plates and matured for 8–10 days. Mature spheroids were subjected to acute 24 h glucolipotoxic challenge combining high glucose and palmitic acid and assessed for neutral lipid accumulation, NF-κB reporter activation (luciferase), and macrophage marker expression (qPCR). Results: Time-course characterization demonstrated progressive hepatocyte marker remodeling (albumin, alpha-fetoprotein, CYP3A4) and dynamic macrophage phenotype shifts (CD14, CD206, MARCO, TREM2). Acute glucolipotoxic challenge induced dose-dependent increases in neutral lipid accumulation and NF-κB reporter activation, accompanied by coordinated macrophage-associated transcriptional changes consistent with lipid-handling and tissue-remodeling programs. Post-challenge metabolic activity was retained under the selected stress conditions. As a proof-of-concept demonstration, three botanical extracts showed distinct attenuation profiles across the lipid and inflammatory endpoints. Conclusions: This 3D hepatocyte–macrophage co-culture model provides orthogonal readouts of steatosis and NF-κB-mediated inflammatory activation under glucolipotoxic stress, offering a reproducible, fit-for-purpose screening tool for investigating early glucolipotoxic hepatic responses and evaluating candidate compounds in a defined in vitro setting. Full article

Ebola virus disease remains one of the most serious viral infections with no approved small-molecule treatments. The Ebola virus glycoprotein (EBOV-GP), which enables the virus’s entry to host cells, is a promising target for drug discovery. In this study, a multistage computer-aided drug discovery approach was used to identify new specific EBOV-GP inhibitors. A reliable QSAR model was built using 55 terpenoid derivatives. This model was able to predict the activity of newly designed compounds with good accuracy and validated statistical metrics ($R_{tr}^2$= 0.70; $R_{ext}^2$ = 0.73). It was subsequently applied to screen over 15,500 newly generated compounds from three lead molecules by fragment-based design tools. Predicted activity, binding affinity toward EBOV-GP, and good ADMET drug-like properties prioritized the eleven most promising hits. Through 150 ns molecular dynamics simulations, these compounds remained stable in the EBOV-GP binding site. Further binding free energy analysis (MM/PBSA) showed strong binding affinities, especially for the compounds L-60, L-832, M-1618, and L-1366. This study showed how combining QSAR, fragment-based design, docking, ADMET, and molecular dynamics could help in identifying potent and safe small molecules against the EBOV-GP. The top compounds are ready for further experimental and in vitro biological testing. Full article

Background and Objectives: Advances in digital dentistry, particularly CAD-CAM, have improved the efficiency and precision of crown design and fabrication. Recently, artificial intelligence (AI)-integrated CAD-CAM systems have enabled automated tooth morphology generation, margin detection, and occlusal analysis, enhancing consistency and accuracy. This systematic review evaluates the accuracy of AI-assisted crown design compared with conventional and CAD-CAM workflows. Materials and Methods: A systematic search was conducted across PubMed/MEDLINE, Scopus, Web of Science, Cochrane, and LILACS for studies published between January 2010 and December 2025 that assessed the marginal fit, internal adaptation, and occlusal contact accuracy of single crowns. Screening, full-text assessment, and data extraction followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Methodological quality and risk of bias were evaluated using the Modified CONSORT checklist for in vitro studies and the Joanna Briggs Institute tools for clinical studies. Results: Of 887 records identified, 12 studies met the inclusion criteria. Nine studies showed a moderate risk of bias, two moderate-to-high, and one low-to-moderate. AI-assisted crown design demonstrated clinically acceptable internal fit and marginal adaptation, comparable or superior to CAD-CAM systems. Occlusal contact accuracy was generally comparable to CAD-CAM and technician-designed crowns, though variability was observed across AI models. Conclusions: AI-assisted crown design provides a reliable fit and marginal adaptation, with occlusal accuracy approaching conventional CAD-CAM and technician workflows. While not a replacement for clinical expertise, AI serves as a valuable adjunct, enhancing reproducibility, precision, and overall quality in restorative dentistry. Further standardized clinical studies are needed to validate long-term outcomes and optimize occlusal performance. Full article

Hepatocellular carcinoma (HCC) is a highly malignant tumour with a poor prognosis and few effective treatment options. Development of resistance to conventional therapies and occurrence of severe side effects highlight the urgent need for novel, low-toxicity interventions. Natural products are promising candidates for HCC drug development thanks to their multi-target activity and favourable safety profiles. Previous studies reported that Lingonberry extract, a bioactive natural product, inhibits proliferation of HepG₂ cells. However, the key molecular targets and underlying anticancer mechanisms remain unclear. In this study, we analysed gene chip data from Lingonberry extract-treated HepG₂ tumour-bearing mice using bioinformatics tools, employing a cross-species, multi-level screening strategy to identify PSMB8 as the core regulatory gene. In vitro functional validations (Western blotting, RT-PCR, CCK-8 assay, colony formation assay, flow cytometry and TUNEL staining) confirmed these findings. Downregulating PSMB8 was found to effectively induce late apoptosis in HepG₂ cells, and Lingonberry extract was found to significantly reduce PSMB8 protein expression. This study identifies PSMB8 as a key mediator of the anticancer effect of Lingonberry extract in HepG₂ cells. It provides a reliable methodological reference for screening anticancer targets of natural products and supports further exploration of Lingonberry extract as a potential adjuvant/lead compound for HCC. Full article

Physiologically relevant in vitro intestinal models are essential for studying key physiological processes, including barrier function, drug screening and gut-microbiota interactions. However, conventional 2D culture systems often fail to recapitulate structural and functional complexity. Here, we aimed to validate a 3D bioelectronic transmembrane platform, previously used for monitoring human intestinal epithelium and vascular endothelium, for modeling the rat small intestinal barrier in vitro. The device integrates a poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) scaffold supporting co-cultures of rat intestinal epithelial cells (IEC-6) and rat fibroblasts (208F), enabling real-time monitoring of barrier formation through electrical measurements using electrochemical impedance spectroscopy (EIS). Barrier formation was monitored over 21 days and exhibited a time-dependent increase in barrier resistance. The 3D platform was compared with traditional 2D insert-based cultures and ex vivo rat tissue using an Ethylene Glycol Tetraacetic Acid (EGTA)-induced calcium switch assay to evaluate barrier disruption and recovery. EGTA treatment and removal induced reversible barrier disruption in the 3D in vitro and ex vivo models, whereas 2D in vitro cultures showed limited recovery. These findings demonstrate that the 3D platform more faithfully recapitulates native tissue architecture and function, closely paralleling ex vivo responses. Our study highlights the importance of validating advanced 3D in vitro models and establishes this bioelectronic platform as a robust tool for drug screening, barrier studies, and preclinical gastrointestinal research. Full article

Antibiotic resistance among several bacteria is a warning sign that reinforces the need for research to identify new compounds that are effective against resistant strains. In this sense, bioinformatics stands out as an excellent tool for identifying drug candidates by using computational methodologies to detect compounds with potential biological activity. Two pivot compounds (QNZ and 0Y5) with biological activity against Staphylococcus aureus were selected. A virtual screening was performed in the MolPort database with a Tanimoto index of 0.5, resulting in 20,000 compounds, 10,000 compounds for each template. Then, methodologies were applied to calculate pharmacokinetic and toxicological parameters using Discovery Studio software; molecular docking via DockThor; lethal dose via ProTOX; lipophilicity, solubility, and Lipinski parameters via SwissADME; in silico prediction of bacterial activity via Way2Drug; theoretical synthetic accessibility via SwissADME and AMBIT-SA; and, finally, molecular dynamics simulations via AMBER 18. After the entire methodological process, 10 compounds were identified with potential results according to the criteria adopted in this study and with possible antimicrobial activity against resistant bacterial strains of S. aureus. Our theoretical findings suggest 10 potential candidates with possible antimicrobial activity against S. aureus and other genera and species of bacteria as these compounds presented excellent results using the proposed methodology. Certainly, more in vitro and in vivo study steps are necessary. Full article

Background: An emerging tool to better simulate the complexity of tumour biology in vitro is 3D culture models. Several approaches have been introduced, yet many face challenges such as technical complexity or limited ability to reproduce critical tumour traits. Modular tissue engineering is a well-known method in tissue transplantation, where it has been used to develop various healthy tissue constructs. In this study, we set out to adapt this established approach to fabricate cancer microtissues and to assess their effectiveness as a tumour model that can capture essential features of cancer biology and drug-treatment response. Methods: Two triple-negative breast cancer (TNBC) cell lines, HCC1806 and MDA-MB-231, were cultured in microtissues and assessed for viability, cell death, generation of hypoxia and response to chemotherapy. To benchmark our model, we utilized flow cytometry to analyze the CD44⁺/CD24⁻ cancer stem cell (CSC) phenotype across microtissues, 2D monolayers, and established 3D models, including spheroids, collagen domes, and laminin-rich domes. Results: The cells showed sustained cell viability with minimal cell death, along with natural development of tumour properties, such as hypoxia. Crucially, flow cytometry revealed a cell-line-dependent regulation of the CD44⁺/CD24⁻ phenotype, underscoring the complex influence of the 3D microenvironment on stem cell regulation. Furthermore, by screening the model with standard anti-breast cancer chemotherapeutics, we observed drug resistance at concentrations comparable to those used in the clinic. Conclusions: Our model offers the unique ability to spontaneously reproduce fundamental features of tumours in vitro, capturing the cellular heterogeneity and reprogramming that drive clinical drug resistance. Full article

In vitro fecal inoculation coupled with gas chromatography–mass spectrometry (GC-MS) has been used for evaluating fecal deodorants. However, high cost and complex data interpretation limit its routine application. An electronic nose (eNose) offers a rapid, cost-effective alternative. This study aimed to evaluate the eNose as a screening tool for fecal odor compared with solid-phase microextraction gas chromatography–mass spectrometry (SPME GC-MS) and to examine the in vitro effects of fecal deodorant supplements on fecal odor profiles. Feces from ten healthy cats were serially diluted (1:1 to 1:8) and analyzed using both instruments. Four dietary supplements—Yucca schidigera extract (YSE), Quillaja saponaria extract (QSE), fructooligosaccharides (FOS), and oat beta-glucans (OBG)—were tested at concentrations of 0.0, 0.2, 0.4, and 0.8 g/100 mL. The eNose showed comparable performance to GC-MS in discriminating among sample dilutions. In vitro fermentation showed that FOS and OBG significantly increased volatile fatty acid (VFA)-related sensor responses while signals linked to ammonia and sulfur compounds were reduced. QSE had minimal effect, whereas YSE produced moderate changes. The total sensor response intensities did not differ between treatments. These findings indicate that prebiotic supplements exert stronger effects than saponin-based supplements and highlight the potential of eNoses with in vitro fermentation for rapid screening of fecal deodorants. Full article

Red blood cells represent a widely used cellular model in cytotoxicity studies, particularly in hemocompatibility assessments. As enucleated cells, which are abundant and easily accessible in both humans and animals, red blood cells allow for rapid, reproducible, and low-cost evaluation of the toxicity of bioactive compounds, whether natural, synthetic, or nanoparticulate. From a functional perspective, the red blood cell membrane is highly sensitive to physical and chemical environmental changes (osmolarity, temperature, pH, and the presence of oxidizing agents). This sensitivity makes red blood cells an effective biosensor for detecting membrane damage, hemolysis, oxidative stress, methemoglobin formation, and aggregation processes. Therefore, in vitro tests using red blood cells allow for the preliminary evaluation in preclinical development, particularly for the early screening of cytotoxicity, membrane-disruptive effects, and hemocompatibility of small molecules, nanomaterials, and blood-contacting biomaterials. These techniques include hemocompatibility tests, evaluation of oxidative and osmotic damage, and evaluation of erythrocyte aggregation and function. However, the use of red blood cells as a cytotoxicity model also has significant limitations. As anucleate cells, erythrocytes lack organelles such as nuclei, mitochondria, or lysosomes, which prevents the evaluation of their effects on key intracellular processes such as protein synthesis, cell signaling, apoptosis, or endoplasmic reticulum stress. This lack of cellular complexity limits their usefulness as a sole model in studies of systemic toxicity or tissue-specific cytotoxicity. These tools offer an effective preliminary approach to anticipating risks in biomedical and pharmacological research. Full article