Search Results (14,262)

Background/Objectives: Sacituzumab govitecan (SG, IMMU-132) is a TROP2-directed antibody–drug conjugate carrying SN-38. Patients with colorectal liver metastasis (CRLM) still have limited treatment options after first-line systemic therapy, and the preclinical pharmacological value of TROP2-directed SN-38 delivery in CRLM remains insufficiently defined. Methods: Public single-cell RNA-sequencing data were reanalyzed to explore the distribution of TACSTD2/TROP2-positive epithelial-associated cells in adjacent normal tissues, primary colorectal cancer, and CRLM. The prognostic relevance of TACSTD2 was evaluated using the Kaplan–Meier Plotter database. TROP2-knockdown and TROP2-overexpressing colorectal cancer models were used to assess IMMU-132 response in vitro and in vivo. Pharmacological antitumor activity was further evaluated using subcutaneous xenografts, syngeneic intrasplenic liver metastasis models, and CRLM patient-derived organoids (PDOs). Transcriptomic profiling and γ-H2AX immunofluorescence were used to explore treatment-associated molecular changes. Results: At the patient/sample level, TACSTD2/TROP2-positive epithelial-associated cells showed numerically higher proportions in primary colorectal tumors and liver metastases than in adjacent normal tissues, and high TACSTD2 expression was associated with inferior overall survival in a public survival database. TROP2 knockdown attenuated IMMU-132-induced growth inhibition, apoptosis, and suppression of colony formation. In vivo, IMMU-132 suppressed colorectal tumor growth and reduced liver metastatic burden, with more evident activity in human TROP2-overexpressing models. In a limited exploratory CRLM PDO cohort established after first-line therapy, liver metastasis-derived PDOs showed lower normalized AUC values than primary tumor-derived PDOs. Transcriptomic analysis and representative γ-H2AX immunofluorescence suggested that IMMU-132 treatment was associated with changes in adhesion/cytoskeletal-related pathways, Wnt/cancer-associated transcriptional programs, and DNA damage-associated signals. Conclusions: These in vitro, in vivo, and PDO-based findings support further preclinical pharmacological evaluation of TROP2-directed SN-38 delivery by IMMU-132 in biomarker-annotated CRLM models, particularly in the post-first-line systemic therapy setting. Full article

Apical periodontitis (AP), a localized inflammatory bone disease, and osteoporosis (OP), a systemic metabolic bone disorder, are both characterized by chronic inflammation and dysregulated bone metabolism, yet the molecular mechanisms underlying their association remain unclear. This study aimed to identify shared biomarkers and pathogenic pathways linking AP and OP. Gene expression datasets for AP and OP were obtained from the Gene Expression Omnibus database, and shared differentially expressed genes were identified and analyzed using functional enrichment, Gene set enrichment analysis (GSEA), Gene set variation analysis GSVA, and single-sample gene set enrichment analysis (ssGSEA)-based immune infiltration approaches. A random forest model was applied to prioritize candidate genes, followed by validation using single-cell RNA sequencing datasets, clinical samples, and in vitro assays. Forty-three shared differentially expressed genes were identified, among which FAM87B, ASCL2, and PACS1 were prioritized as candidate genes. PACS1 showed consistent upregulation in both AP and OP and was further validated in clinical samples. Integrated analyses suggested that shared molecular alterations were associated with NF-κB signaling and immune-metabolic dysregulation, while single-cell analysis supported PACS1 expression in cell subsets involved in inflammatory bone remodeling. Functionally, PACS1 knockdown promoted osteoblast differentiation, accompanied by increased ALP, RUNX2, and OPN expression. These findings suggest that PACS1 may serve as a shared candidate biomarker linking AP and OP and provide insight into immune-bone crosstalk in inflammatory bone loss. Full article

Alzheimer’s disease (AD) is a complex neurodegenerative disorder with limited effective therapies. In this study, a series of 2-(arylamino)thiazol-4(5H)-one derivatives was synthesized using an efficient microwave-assisted protocol, and evaluated for neuroprotective effects against 6-hydroxydopamine (6-OHDA)-induced oxidative stress (OS) in SH-SY5Y neuronal cells and an Aβ42-expressing Drosophila melanogaster model of AD. Among the synthesized compounds, 3n, 3i, and 3b exhibited low cytotoxicity and significant neuroprotection, as evidenced by increased cell viability, reduced reactive oxygen species (ROS) production, and preserved mitochondrial membrane potential. Notably, compound 3n demonstrated the highest activity and effectively ameliorated Aβ42-induced behavioral deficits in vivo. Molecular docking studies predicted favorable binding affinity within the acetylcholinesterase (AChE) active site, with the thiazol scaffold and aryl substituents stabilizing ligand binding through π–π stacking and hydrophobic interactions; compound 3n also formed additional hydrogen bonds enhancing its affinity. Consistent with the docking predictions, in vitro AChE inhibition studies demonstrated that compounds 3n, 3i, and 3b inhibited AChE in a concentration-dependent manner. Network pharmacology predicted seven potential core targets implicated in AD pathogenesis and related pathways, including OS, neuroinflammation, and synaptic dysfunction. Furthermore, in silico pharmacokinetic analysis indicated compliance with Lipinski’s and Veber’s rules, supporting favorable drug-like properties. While further experimental validation is essential, these findings highlight 2-(arylamino)thiazol-4(5H)-one derivatives, particularly compound 3n, as promising multifunctional candidates for further preclinical development against AD. Full article

Background/Objectives: The chemokine receptor CXCR4 plays a pivotal role in tumor progression, metastasis, and therapy resistance and is frequently overexpressed in hematologic malignancies, including multiple myeloma and lymphoma. This study investigates a CXCR4-targeted theranostic platform comprising a PET imaging agent and two therapeutic radioconjugates derived from the high-affinity CXCR4 antagonist LY2510924. Methods: The PET tracer [¹⁸F]AlF-NOTA-SC and therapeutic radioconjugates [¹⁷⁷Lu]Lu-BL02 and [¹⁶¹Tb]Tb-BL02 were synthesized and evaluated. Radiolabeling efficiency, molar activity, and in vitro binding affinity were assessed. Specificity and uptake were evaluated in CXCR4-expressing U87.CD4.CXCR4 and MM.1S cells, with cytotoxic potential being analyzed via clonogenic survival assays. In vivo biodistribution and pharmacokinetics were evaluated in MM.1S xenograft mouse models, supported by longitudinal SPECT imaging. Results: All radioconjugates were obtained with high radiochemical purity (>98%). The constructs showed nanomolar affinity for human CXCR4; in vitro assays confirmed specific uptake in CXCR4-positive cells, and both therapeutic agents demonstrated dose-dependent cytotoxicity. In vivo, all compounds displayed comparable tumor uptake with low off-target accumulation. Co-injection studies confirmed consistent pharmacokinetics across agents, while SPECT/CT imaging demonstrated gradual tumor clearance of [¹⁷⁷Lu]Lu-BL02 over seven days. Conclusions: The radiopharmaceutical trio [¹⁸F]AlF-NOTA-SC, [¹⁷⁷Lu]Lu-BL02, and [¹⁶¹Tb]Tb-BL02 demonstrates the feasibility of a CXCR4-targeted theranostic approach for imaging and treating hematologic malignancies. The observed tumor washout highlights the need for further structural optimization to enhance tumor retention and therapeutic efficacy, providing a clear direction for future development toward clinical translation. Full article

Background/Objectives: Psoriasis is a chronic, immune-mediated inflammatory skin disorder affecting millions of individuals worldwide. It remains a therapeutic challenge due to the limited skin penetration of many drugs, adverse systemic effects, and the need for long-term management. Transferosomal nanoformulations containing natural products were proposed as a potential therapeutic tool. Methods: Transferosomes containing 20-hydroxyecdysone and resveratrol were prepared using the thin-film hydration method followed by probe ultrasonication, generating several formulation variants differing in composition. The vesicles were characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) to determine size, polydispersity index (PDI), and zeta potential. Furthermore, time-domain nuclear magnetic resonance (TD-NMR) relaxation measurements were employed to evaluate local molecular dynamics and membrane fluidity, providing deeper insights into the structural integrity and elasticity of the transferosomal systems. The stability of the nanoformulations was assessed for one month in water and phosphate-buffered saline (PBS). Viability was evaluated in vitro using the MTS assay on human epidermal keratinocyte (HEK) and psoriasis-patient derived human epidermal keratinocyte (PHEK) cell lines, alongside antimicrobial profiling against four representative human skin microbiome strains. Acute toxicity was further examined in vivo using the Danio rerio FET test. Results: The formulations demonstrated high physicochemical stability over the tested period, maintained desirable particle sizes within 100–200 nm, and showed no cytotoxicity toward skin-associated bacteria or in the zebrafish model. Conclusions: The developed nanoformulations present suitable properties in terms of safety and stability and can be considered for potential use in psoriasis treatment. Full article

Cardiac fibroblasts (cFbs) are important in vitro models for studying myocardial fibrosis, extracellular matrix homeostasis, and tissue remodeling. However, establishing primary cultures from adult mouse hearts remains technically demanding due to the limited proliferative capacity of freshly isolated cells, their susceptibility to enzymatic and mechanical stress during tissue dissociation, and the resulting difficulty in obtaining sufficient numbers of viable fibroblasts for downstream experiments. Even small variations in digestion and early culture conditions can affect cell recovery, purity, and expansion, highlighting the need for optimized and reproducible isolation protocols. Here, we compared three enzymatic strategies for establishing primary adult mouse cardiac fibroblast cultures: sequential digestion with collagenase I and trypsin, retrograde coronary perfusion with collagenase II, and continuous digestion with Liberase DH. The three protocols were evaluated by assessing post-isolation cell viability, the composition of recovered non-myocyte populations by flow cytometry, and the ability of isolated cells to establish and expand during the first five days of culture. Retrograde coronary perfusion generated viable fibroblasts but yielded relatively few cells and is most advantageous when fibroblasts are isolated simultaneously with cardiomyocytes from the same heart. Liberase DH digestion recovered a broader spectrum of non-myocyte populations, making it suitable for immediate phenotypic analyses, although it produced less efficient fibroblast expansion in culture. Sequential collagenase I/trypsin digestion showed the best overall performance, with low mortality, negligible CD31⁺ endothelial contamination, and robust expansion into confluent spindle-shaped monolayers within five days. Overall, these findings identify sequential collagenase I/trypsin digestion as an effective and reproducible approach for establishing viable adult mouse cardiac fibroblast cultures for downstream functional and molecular studies. Full article

Background/Objectives: Sarcopenia involves a gradual decline in skeletal muscle mass that may occur during aging or in association with chronic pathological conditions. It markedly reduces muscle strength and mobility, thereby impairing quality of life. Because sarcopenia’s severity directly correlates with frailty, it represents an important predictor of prognosis and disease risk. Current preventive and therapeutic strategies rely mainly on physical activity, which is not feasible for all patients. This study investigated the biological effects of two independently prepared Tuber borchii (T. borchii) extracts in an in vitro model of sarcopenic stress. Methods: The activity of T. borchii extracts was investigated in a cell-based model of sarcopenia, following previous observations that these preparations influence proliferation-related pathways, including ERK1/2 phosphorylation. Specifically, differentiated myotubes were exposed to D-galactose to reproduce atrophy-associated cellular changes, and the impact of T. borchii extracts on protein synthesis, turnover, and cell morphology was assessed. Results: T. borchii extracts enhanced protein synthesis and turnover in myotubes. Furthermore, the treatment significantly reduced the expression of key galactose-induced sarcopenia and atrophy markers, such as MuRF1. Morphological analysis confirmed this protective effect, showing that treated myotubes maintained greater thickness and exhibited a larger cross-sectional area despite exposure to the sarcopenic stimulus. Conclusions: These results indicate that T. borchii extracts can attenuate selected cellular alterations associated with muscle aging. Future identification of the most active components may support their development as nutraceutical supplements. Full article

Disease variants in GABR genes encoding γ-aminobutyric acid type A receptor (GABA_AR) subunits are major causes of developmental and epileptic encephalopathies (DEEs). There is no effective treatment for these DEEs, although the GABA_AR is a major target for antiseizure drugs. We previously identified the therapeutic effect of 4-phenylbutyrate (PBA) in Gabrg2^+/Q390X knockin DEE mice and in this study tested the effect of the drug in GABRA1 variants that encode the α1 subunit of GABA_AR. We used a multidisciplinary approach including in silico structural modeling, flow cytometry, patch-clamp recordings and biochemistry in conjunction with differential tagging of the wildtype (WT) and the mutant alleles to evaluate the effect of PBA on rescue of GABA_AR subunit expression, surface trafficking, and function in vitro in a heterologous HEK293T cell model and in vivo in Gabra1^+/A322D mice. We found that the α1 subunit expression at both the total level and the cell surface was reduced when the variant α1 protein was present, suggesting reduced functional receptor availability on the cell membrane and synapse. Patch-clamp recordings identified that α1 variants reduced GABA-evoked current amplitude. In silico prediction indicated reduced protein stability for GABRA1 variants by negative ∆∆G values. PBA increased both total and surface expression of WT α1 and α1 variants and improved expression of both WT and variant α1 alleles when these were co-expressed. Importantly, PBA also increased the GABA_AR expression in the cortex and thalamus of the Gabra1^+/A322D mice. This study indicates that PBA is a promising treatment option for DEEs associated with GABRA1 mutations. Our previous work has demonstrated that PBA improves proteostasis by enhancing expression of the WT allele, repairing the mutant allele, and reducing endoplasmic reticulum stress in other DEEs associated with GABRG2 and SLC6A1 mutations. Importantly, it can mitigate seizures and improve neurobehavioral phenotypes at behavioral levels. Based on this and our previous work on GABRG2 and SLC6A1 mutations, we propose that PBA holds promise as a common medicine for multiple genetic neurologic disorders that share the proteostasis pathology with a broad clinical application in DEEs. Full article

Poor adherence to prolonged antibiotic regimens remains a major challenge in the treatment and prevention of chronic infectious diseases such as tuberculosis. Transdermal drug delivery systems capable of sustained antibiotic release may improve therapeutic compliance while reducing the need for frequent oral administration. In this study, electrospun polymeric membranes based on poly(lactic acid) (PLA) and poly(ε-caprolactone) (PCL) were developed as transdermal rifampicin delivery platforms. Homogeneous nanofibrous membranes with average fiber diameters of approximately 250 nm were successfully fabricated and exhibited efficient drug incorporation while preserving the structural integrity of the polymeric matrix. The electrospun membranes retained sufficient tensile strength and dimensional stability after accelerated temperature–humidity aging, supporting their stability during storage, handling, and application. In vitro cytotoxicity and biocompatibility assays using primary human peripheral blood mononuclear cells (PBMCs) demonstrated that the developed systems did not induce significant cytotoxic or pro-inflammatory responses. Transdermal permeation studies using an in vitro mouse skin model demonstrated sustained rifampicin diffusion for at least 72 h. Importantly, the antibiotic recovered after skin permeation preserved antimycobacterial activity against Mycobacterium tuberculosis H37Ra and Mycobacterium bovis BCG, confirming that rifampicin maintained its biological functionality after electrospinning and transdermal migration. Overall, these findings demonstrate the potential of electrospun PLA/PCL membranes as stable and biocompatible transdermal antibiotic delivery systems capable of sustained release and preservation of antimicrobial activity. This proof-of-concept study supports the translational potential of electrospun polymeric platforms for controlled antibiotic delivery in long-term infectious disease therapies. Full article

Colorectal cancer (CRC) accounted for nearly 2 million new cases and over 900,000 deaths worldwide in 2020. Current treatment strategies include surgery, chemotherapy, radiotherapy, and targeted therapies, including monoclonal antibodies and other forms of immunotherapy. Despite these options, high mortality rates and frequent treatment-related adverse effects underscore the urgent need for novel therapeutic approaches. Epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, exhibits potent antioxidant, anti-inflammatory, and anticancer activities. This review summarizes evidence from the past decade on the in vitro and in vivo effects of EGCG in CRC. In vitro studies demonstrate that EGCG inhibits cell proliferation and migration while inducing apoptosis through multiple molecular mechanisms, including modulation of signaling pathways such as Wnt/β-catenin, PI3K/Akt, and NF-κB. In vivo studies in mouse xenograft models indicate that EGCG reduces tumor growth, volume, and mass. Nevertheless, additional in vivo investigations are needed to fully define EGCG’s therapeutic potential and its translational relevance for human clinical trials. Full article

Caspase-8 and the NF-κB essential modulator (NEMO, also referred to as IKKγ) play critical roles in controlling TNF-α-induced cell death and survival in hepatocytes. The aim of this study was to generate a hepatocyte-derived cell line in which the Casp8 and Nemo genes can be conditionally inactivated simultaneously to investigate the significance of the corresponding signaling pathways. To this end, we induced hepatocellular carcinoma in Casp8^f/fNemo^f/f mice using diethylnitrosamine and established an immortalized hepatoma cell line from explanted liver tumors, which is subsequently referred to as ΔCN60. ΔCN60 cells still retain floxed Casp8 and Nemo alleles, allowing for efficient Cre-mediated deletion to generate Casp8^ΔNemo^Δ derivatives. Loss of both Caspase-8 and NEMO inhibits cell proliferation, increases the expression of tumor and progenitor markers (AFP, CD133), reduces albumin expression, and blocks TNF-α-induced NF-κB p65 nuclear translocation. ΔCN60 Casp8^ΔNemo^Δ cells display altered sensitivity to prolonged TNF-α exposure, suggesting a potential shift in necroptosis-associated signaling pathways, although necroptotic cell death was not directly demonstrated. ΔCN60 serves as a versatile hepatoma model for investigating Caspase-8/NEMO-dependent TNF-α signaling and hepatocyte plasticity and may help prioritize future in vivo experiments by enabling selected mechanistic questions to be addressed first in vitro. Full article

Background: Global aging has led to a rise in neurodegenerative diseases. Neuroinflammation constitutes a core pathological mechanism in these disorders. The Kongsheng Zhenzhong Pill (KSZZP), a modern preparation derived from a classical Chinese herbal formula, are reputed for their sedative and cognition-enhancing effects. However, the molecular mechanism underlying its modulation of microglia-mediated neuroinflammation remains unclear. The present study was designed to assess the intervention effects of KSZZP on LPS-induced neuroinflammation in BV-2 microglial cells and to preliminarily explore the potential molecular mechanisms involved. Methods: An in vitro neuroinflammation model was established in LPS-induced BV-2 microglial cells. The chemical components of KSZZP were identified using UPLC-Q-Exactive HFX technology. The pharmacological effects of KSZZP were evaluated by assessing cell activation, inflammatory response, oxidative stress, and apoptosis. Molecular docking and Western blotting were used to explore the specific mechanism of its action on the cGAS-STING pathway. Results: Chemical analysis identified 67 components in KSZZP, primarily flavonoids, prenyl lipids, and isoflavones. KSZZP treatment dose-dependently inhibited LPS-induced BV-2 microglial activation and significantly reduced pro-inflammatory factor release. Furthermore, it alleviated oxidative stress, mitigated mitochondrial ultrastructural damage, and inhibited apoptosis induced by LPS. Molecular docking revealed that key active components of KSZZP exhibit strong binding potential to cGAS and STING proteins. Western blotting further confirmed that KSZZP dose-dependently suppressed the expression of key cGAS-STING pathway proteins (cGAS, STING) and downstream proteins associated with M1 polarization (iNOS, TNF-α, COX-2). Conclusions: This study indicates that KSZZP alleviates LPS-induced microglial activation, neuroinflammation, oxidative stress, mitochondrial damage, and apoptosis, and these effects may involve the modulation of the cGAS-STING signaling pathway. Collectively, these findings provide a preliminary experimental basis for understanding the anti-neuroinflammatory mechanism of KSZZP and support its potential application in the prevention and treatment of neurodegenerative diseases. Full article

Schwann cells (SC) myelinate peripheral axons and orchestrate nerve regeneration after injury by switching between myelinating, proliferative and repair states. Evidence suggests that Botulinum Neurotoxin Type A (BoNT/A) influences SC biology, potentially supporting nerve repair and pain relief in peripheral neuropathic pain (PNP) models. However, BoNT/A receptor and target expression in human SC (hSC) remains poorly explored. Here, this translational gap was addressed by transcriptionally profiling genes encoding BoNT/A-relevant receptors and targets in primary hSC and testing whether paclitaxel evokes hSC phenotype plasticity in vitro based on changes in gene expression. Primary hSC were isolated, cultured, and treated with paclitaxel or vehicle, followed by RT-qPCR profiling of hSC markers and BoNT/A receptor/targets genes. Untreated hSC expressed moderate NGFR and S100β, with low MBP levels, suggesting a non-myelinating state profile. Transcripts encoding the BoNT/A receptor machinery (SV2A, SYT1) and the target SNAP25 were detectable at moderate levels. Paclitaxel induced changes in gene expression: SV2A and SYT1 decreased (up to two-fold), whereas SNAP25 and MBP increased, accompanied by reduced NGFR, indicating a shift toward a more differentiated transcriptional state. These data indicate hSC transcriptional plasticity in vitro and provide transcriptional evidence for the expression of BoNT/A-related molecular components in non-neuronal human cells. Full article

Background/Objectives: Human tendon stem/progenitor cells (hTSPCs) isolated from tendinopathic tendon tissue were used to identify key inflammatory biomarkers, including reactive oxygen species (ROS), pro-inflammatory cytokine release, and type III collagen expression. This cellular model was employed as an in vitro platform to investigate the effects of a semiconductor-nanoparticles embedded fabric (hereafter named fabric) on inflammatory marker regulation. Methods: hTSPCs were cultured in both conventional monolayer conditions and within a three-dimensional (3D) bioplotted methacrylated collagen (ColMA) scaffold under perfusion. The cells were exposed to a microenvironment enriched with negative ions and far-infrared radiation generated by fabric to assess its modulatory effects on native inflammatory and fibrotic pathways. Results: Fabric exposure significantly reduced ROS levels and modulated cytokine signaling pathways. These changes were associated with observed enhanced cell viability and proper extracellular matrix composition profile, characterized by reduced type III collagen and increased type I collagen deposition. Consistent findings were observed at both the protein and gene expression levels. Notably, these effects were more evident in the 3D culture system, likely due to its greater biomimetic relevance and ability to more accurately reproduce the native cellular microenvironment. Conclusions: Overall, these preliminary findings suggest that fabric promotes a transition from a fibrotic toward a more regenerative tendon-like phenotype, likely mediated by redox balance which probably improved ECM remodeling. Further studies should evaluate the potential of semiconductor-based fabrics to support tendon regeneration in vivo. Full article

Right ventricular (RV) failure secondary to pulmonary hypertension manifests in significant biomechanical alterations of myocardial tissue, including capillary rarefaction and enhanced stiffness and anisotropy. However, how the changes in mechanical cues affect the angiogenic potential of endothelial cells (ECs)—and thereby impact the RV failure progression—remains unclear. The aim of this study is to investigate the effects of RV-relevant substrate stiffness and anisotropy on different EC types using polyurethane urea scaffolds engineered to mimic RV tissues. We find that substrate anisotropy increased EC number but reduced metabolic activity in both cell types. In contrast, the two cell types exhibit divergent responses in their angiogenic potential and angiogenic protein secretome. For HUVECs, neovessel formation is suppressed by substrate stiffening and anisotropy, whereas for HCMECs, it is increased by stiffening and suppressed by anisotropy. Our results highlight the mechanobiological regulation of ECs in a tissue- and cell-type-dependent manner, which is critical for new biomaterial or in vitro model development for cardiac diseases. Full article