Search Results (872)

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with a desmoplastic, immunosuppressive tumor microenvironment enriched in extracellular adenosine. High adenosine levels disrupt anti-tumor immunity by engaging adenosine receptors on T cells, macrophages, and dendritic cells. In this context, strategies to reduce extracellular adenosine signaling within the TME are in focus. Among them, enzymatic degradation of extracellular adenosine levels has emerged as a promising approach to overcome immune suppression and restore anti-tumor immune responses. Methods: Here, we engineered an E. coli strain to express a surface-bound form of a bacterial ADA2 enzyme and used the outer membrane vesicles produced by this strain as nanoparticles carrying the functional enzyme. Our data indicate robust surface expression and enzymatic activity of ADA2 on OMV particles. Results: In a PDAC subcutaneous flank tumor model, intratumoral administration of OMV-ADA2 particles was associated with significantly increased infiltration of CD8⁺ T cells and Granzyme B expression and concomitant decreases in collagen deposition and α-smooth muscle actin (α-SMA) positive stromal cells, suggesting modulation of the desmoplastic stroma. Conclusions: These findings support further investigation of OMV–bADA2 as a potential platform to modulate tumor metabolism and stroma, activate anti-tumor immunity, and serve as a carrier for co-delivery of additional anticancer modalities in PDAC. Full article

Reliable reference genes are critical for ensuring the accuracy of RT-qPCR-based gene expression analysis, especially under environmental stress conditions. In this study, Leymus chinensis was used as the experimental material, and eight candidate reference genes—alpha-tubulin (TUA), beta-tubulin (TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-alpha (EF1α), 18S ribosomal RNA (18S rRNA), adenylyl cyclase-associated protein (CAP), adenine phosphoribosyl transferase (APRT), and actin (ACT)—were selected to evaluate their expression stability under cold, drought, heat, NaCl, high pH, wounding, abscisic acid (ABA) and jasmonic acid (JA) treatments. Primer specificity and amplification efficiency were first assessed, and the candidate genes were then comprehensively analyzed using geNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the amplification efficiencies of all primers ranged from 95.0% to 107.2%, and the Ct values of the candidate genes ranged from 17.31 to 30.99. Comprehensive analysis using RefFinder showed that ACTIN was the most stable gene under ABA and NaCl treatments, EF1α under heat and wounding treatments, CAP under JA and high pH treatments, APRT under cold treatment, and TUB under drought treatment. geNorm analysis indicated that two reference genes were sufficient for accurate normalization under each treatment condition. The reliability of the screening results was further confirmed by expression-level validation of LcbZIP46, LcWRKY5, and LcFIN1. This study provides a stable reference gene system for RT-qPCR-based expression analysis in Leymus chinensis under environmental stress conditions. Full article

Cell surface markers are essential for lineage tracing and for understanding cellular mechanisms of regeneration, yet they remain largely unavailable for echinoderms. Using the starfish Asterias rubens as a model for physiological coelomocyte renewal, we combined Percoll density-gradient fractionation with label-free LC-MALDI proteomics to identify candidate surface proteins that distinguish subpopulations of coelomocytes and coelomic epithelium (CE) cells. However, LC-MALDI is prone to signal variability, sample depletion and nonlinear response. To address the quantitative limitations of LC-MALDI, we implemented a chi-square test with an empirically derived probability density function, which we validated on control mixtures, achieving high precision. Four fractions enriched in distinct cell types were analyzed: coelomocyte fractions 1 (roundish cells) and 4 (large agranulocytes that form cell nets) and CE fractions 1 (type 2 small CE cells) and 4 (ciliated cells), together with a weakly attached CE subpopulation enriched in small putative progenitor cells. LC-MALDI identified 714 protein groups, of which 160 showed significant differential abundance across fractions. Functional enrichment revealed actin cytoskeleton and integrin signaling in coelomocytes, and catenin-related terms in CE-W. Among the 101 predicted membrane proteins, 31 candidate cell surface markers were selected. These include integrins α8β1 and α9β1, three-finger proteins, scavenger receptors, and low-density lipoprotein receptor-related proteins, as well as metallopeptidases. This work provides the first proteome-scale set of candidate surface markers for starfish cell subpopulations, enabling future lineage-tracing studies to resolve the origin of coelomocytes from the coelomic epithelium. Full article

Quantum Dots (QDs) are nanoparticles that are highly desirable for biomedical applications such as drug delivery, cellular tracking, and imaging due to their fluorescent and tunable optical properties. However, the biochemical mechanism of their interaction with intracellular proteins that regulate cytoskeletal organization remains poorly understood. While previous studies have shown QDs’ ability to interact with actin and alter actin dynamics, their impacts on actin-binding proteins have not been explored. In this study, we investigated the interaction between CdSe/ZnS QDs and the actin-binding protein, α-actinin, and assessed its impact on actin cytoskeletal organization. Our results demonstrated a strong interaction between QDs and α-actinin, which impeded an α-actinin-mediated filamentous actin (F-actin) bundling, as well as compromised the activity of α-actinin in preventing actin depolymerization. Furthermore, the physics-based modeling and simulations carried out at physiological temperatures supported these findings by identifying stable interaction surfaces between QDs and α-actinin. This study provides mechanistic insight into nanoparticle–protein interactions and highlights the potential cytoskeletal toxicity associated with it. Full article

The autism spectrum disorder-associated SCN2A, ANK2, and SHANK-family genes encode molecularly distinct proteins that converge functionally on dendritic integration. Recent work established that ankyrin-B, encoded by ANK2, acts as an obligate dendritic scaffold for NaV1.2, encoded by SCN2A, in neocortical pyramidal neurons. Loss of this module mislocalizes dendritic NaV1.2, reduces dendritic Na⁺ influx, weakens backpropagating action potentials, and impairs synaptic maturation and long-term potentiation. SHANK proteins organize a complementary postsynaptic receptor scaffold within dendritic spines, coupling N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and metabotropic glutamate receptor (e.g., mGluR5) signaling to the actin cytoskeleton through layered PSD-95/GKAP/Homer interactions. Disruption of this scaffold can destabilize excitatory transmission, spine morphology, and plasticity. We propose that these dendritic shaft and spine-associated modules jointly regulate dendritic input–output gain and that their disruption may contribute to autism spectrum disorder by destabilizing, rather than uniformly shifting, excitatory integration across cortico-subcortical circuits relevant to sensory reactivity, behavioral flexibility, and social-valence processing. Here, we review the cortical evidence for this layered dendritic convergence and evaluate its potential relevance beyond the cortex. We assess the striatum, thalamus, and amygdala as subcortical sites where related dendritic scaffolding mechanisms may operate. The striatum provides the strongest current test case, with established roles for both NaV1.2 and SHANK3 in medium spiny neuron physiology and corticostriatal connectivity. Thalamic and amygdalar extensions are supported mainly by SHANK-related circuit and channelopathy data but lack direct evidence for ANK2–SCN2A involvement. The framework is experimentally testable: conditional Ank2 deletion in striatal, thalamic, and amygdalar cell types; dendritic Na⁺/Ca²⁺ imaging across Scn2a, Ank2, and Shank3 models; adult rescue experiments; and genetic-interaction designs would determine whether ankyrin-B supports dendritic excitability beyond the cortex and whether these genes converge on, rather than merely parallel, dendritic input–output gain. Validation in human subcortical tissue would then establish whether this dendritic scaffolding logic represents a shared point of convergence through which genetically distinct autism spectrum disorder-risk variants alter circuit function. Full article

Background/Objectives: Accurate preoperative assessment of peritoneal dissemination is essential in ovarian cancer because it influences surgical strategy and the achievement of complete gross resection. However, [¹⁸F]FDG-PET may be limited in detecting lesions with low glycolytic activity and in differentiating malignancy from inflammatory changes. This case series evaluated the clinical relevance of [¹⁸F]FAPI-74 PET/CT for preoperative assessment of peritoneal dissemination in ovarian cancer. Methods: Four patients underwent [¹⁸F]FAPI-74 PET/CT as part of preoperative evaluation, with comparison to [¹⁸F]FDG-PET/CT when available. Imaging findings were correlated with intraoperative observations and histopathological results, including immunohistochemical assessment of fibroblast activation protein and α-smooth muscle actin. Results: FAPI-PET detected peritoneal dissemination not identified by FDG-PET in several cases, including occult metastasis confirmed histologically and additional lesions after neoadjuvant chemotherapy. FAPI-avid lesions showed stromal activation on immunohistochemistry, supporting the biological basis of FAPI uptake. In one case, additional FAPI uptake may have been partly influenced by inflammatory changes associated with bloody ascites. Conclusions: FAPI-PET may provide complementary information by visualizing stromal components of ovarian cancer and may support preoperative mapping of peritoneal dissemination, although interpretation should consider inflammatory conditions. Full article

Myogenic differentiation is a powerful mechanism for generating diverse cell types from fibroblasts. Here, we show that targeted suppression of β-actin by RNA interference in human fibroblasts triggers coordinated molecular and structural changes consistent with trans-differentiation toward SMC-like phenotype. This conversion is marked by upregulation of smooth muscle differentiation markers (α- and γ-smooth muscle actins, SM22, smooth muscle myosin, desmin, vinculin) at mRNA and protein levels, together with distinct morphological alterations: increased cell area, loss of polarity, and reorganization of the actin cytoskeleton. Notably, β-actin-downregulated fibroblasts exhibited a focal adhesion architecture that differed from parental fibroblasts. These findings indicate that β-actin downregulation may provide a novel in vitro method to induce SMC-like differentiation, with potential implications for vascular biology and tissue engineering. Full article

Although praziquantel (PZQ) is the main antischistosomal drug currently in use, concerns remain regarding incomplete reversal of schistosomiasis-induced pathology and the emergence of drug resistance. This study evaluates the combined effect of PZQ with safranal, a bioactive saffron constituent, on Schistosoma mansoni-induced pathology in mice. Male CD1 Swiss albino mice were exposed to 60 S. mansoni cercariae and, at week 9 post-infection, were treated with PZQ (500 mg/kg orally for two consecutive days), safranal (50 mg/kg/day), or both, for three weeks. The animals were sacrificed at week 11 post-infection. Worm and egg burdens, liver histopathology, fibrotic markers, oxidative stress, and inflammatory cytokines were assessed. Combined PZQ + safranal therapy significantly reduced adult worm counts and hepatic and intestinal egg loads compared to PZQ alone. All treatments decreased liver index (hepatomegaly), with the combination treatment providing the best intervention. Histological analyses revealed significantly reduced granuloma size and hepatic necrosis post-treatment, particularly in the combination group. The levels of proinflammatory cytokines (TNF-α, IL-1β) and Th2 cytokines (IL-4, IL-5, IL-6, IL-10) were significantly lowered in treated mice, most notably with the combination treatment. Oxidative stress was also markedly attenuated, and infected mice exhibited elevated malondialdehyde and depleted antioxidant enzymes (SOD, CAT, GSH). Interestingly, PZQ and/or safranal restored antioxidant status and reduced lipid peroxidation, with the combination being most effective. Furthermore, collagen deposition and expression of hepatic fibrotic markers α-smooth muscle actin (α-SMA), TGF-β1, and matrix metalloproteinase-9 were most effectively suppressed by combined therapy. To conclude, safranal enhances PZQ’s antischistosomal efficacy and confers additive protection against Schistosoma-induced liver fibrosis. Full article

Background: Solriamfetol, a dopamine and norepinephrine reuptake inhibitor widely used in narcolepsy management, has not been thoroughly investigated for its anti-fibrotic and anti-inflammatory properties. Herein, we investigated its potential therapeutic applications and underlying mechanisms in both cellular and murine models of pulmonary fibrosis. Methods: To induce fibrosis, C57BL/6 male mice (six-week-old) were administered bleomycin via the intratracheal route. These animals subsequently received solriamfetol orally once per day at dosages of 3 or 10 mg/kg. Histological and immunohistochemical techniques were employed to evaluate inflammatory cell infiltration, collagen accumulation, and α-smooth muscle actin (α-SMA) expression in bronchoalveolar lavage samples and lung tissue sections. Cytokine levels were measured by ELISA, and gene/protein expression of pro-fibrotic markers, A_2A/A_2B adenosine receptors (ARs), adenylate cyclases (ACs), Epac, K_Ca3.1, and adenosine deaminase (ADA) were assessed via quantitative PCR and Western blot. Electrophysiological recordings evaluated K_Ca3.1 channel activity. Purified ADA and normal human lung fibroblasts (NHLFs) were treated with solriamfetol to assess effects on ADA activity and levels of cAMP and adenosine, respectively. Results: Solriamfetol significantly reduced inflammatory cell infiltration, collagen accumulation, and α-SMA expression in fibrotic lungs. Solriamfetol restored downregulated A_2AAR, A_2BAR, ACs, and Epac, while suppressing ADA expression and activity, resulting in elevated extracellular adenosine and intracellular cAMP. The intervention potentiated Epac signaling and inhibited fibroblast activation. Solriamfetol inhibited the K_Ca3.1 current in fibroblasts and reduced K_Ca3.1 protein expression levels in TGFβ-treated fibroblasts and lung tissues from bleomycin-challenged mice. Notably, these effects were abolished by A_2AAR or A_2BAR antagonists, implying that they occur through AR-mediated pathways. Conclusions: Solriamfetol inhibits ADA and reinforces adenosine–cAMP signaling, suppressing pathological fibroblast activation. These findings suggest its therapeutic utility as a novel anti-fibrotic compound for various fibrotic diseases, including pulmonary fibrosis. Full article

Burn wound healing is a complex and dynamic process involving coordinated regulation of inflammation, oxidative stress, angiogenesis, and tissue remodeling. Polygonum cuspidatum, a traditional Chinese medicinal herb widely used for trauma- and inflammation-related disorders, represents an important source of bioactive compounds for tissue repair. Piceatannol (PIC), a naturally occurring stilbene constituent of P. cuspidatum, possesses potent anti-inflammatory and antioxidant activities; however, its therapeutic potential in burn wound healing remains insufficiently understood. In the present study, the therapeutic effects and underlying mechanisms of topical PIC were investigated using a murine deep second-degree burn model combined with multiple skin-related cellular models, including keratinocytes, fibroblasts, endothelial cells, and macrophages. PIC markedly accelerated wound closure and improved histological architecture, as evidenced by reduced inflammatory infiltration, enhanced collagen organization, and increased neovascularization. Mechanistically, PIC suppressed NF-κB activation and modulated KEAP1/NRF2-associated redox signaling, thereby alleviating inflammation–oxidative stress crosstalk during wound healing. In keratinocyte–fibroblast co-culture systems, PIC inhibited fibroblast-to-myofibroblast transition, reduced α-smooth muscle actin (α-SMA) expression, and attenuated excessive collagen deposition, suggesting anti-fibrotic activity. In addition, PIC promoted endothelial tube formation through activation of the STAT3–VEGF signaling axis. Collectively, these findings demonstrate that PIC facilitates burn wound repair through coordinated anti-inflammatory, antioxidative, pro-angiogenic, and anti-fibrotic effects. This study provides pharmacological support for the therapeutic potential of P. cuspidatum-derived compounds in burn management and highlights PIC as a promising candidate for topical treatment of burn injuries. Full article

Hyblaea puera is a major pest of teak and mangroves. Reliable RT-qPCR normalization requires stable reference genes, none of which have been validated in H. puera. In this study, we assessed the expression stability of ten candidate reference genes under different experimental conditions. Stability was evaluated using the ΔCt method, BestKeeper, NormFinder, and geNorm, and a comprehensive stability ranking was generated using the RefFinder online tool. Our results indicated that amplification efficiencies ranged from 91.67% to 100.82%, with R² values exceeding 0.9901. The optimal reference gene combinations varied by condition: Ribosomal Protein L27 (RPL27) and Ribosomal Protein L10 (RPL10) for temperature treatments; Actin and RPL10 for larval instars; Ribosomal protein S5 (RPS5) and Elongation factor-1α (EF-1a) for adult sexes; RPL10 and EF-1a for tall developmental stages; RPL10 and RPS5 for tissues; as well as EF-1α and Actin for all combined conditions. Finally, the expression profiles of target gene Lethal were evaluated, and the outcomes further confirm the importance of selecting fitting reference genes for normalization of qRT-PCR data. These results provide the evaluated reference gene sets for H. puera, facilitating more accurate RT-qPCR normalization in future molecular studies of host plant adaptation (teak vs. mangroves), temperature tolerance, and larval development in this pest. Full article

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) has emerged as a global metabolic disorder. As a non-pharmacological intervention, the effects of high-intensity interval training (HIIT) on MASLD and its molecular mechanisms remain poorly understood. This study aimed to investigate whether HIIT could ameliorate high-fat diet (HFD)-induced liver fibrosis by recalibrating the intrahepatic lactate metabolic axis. Methods: An HFD-induced murine MASLD model combined with HIIT intervention was utilized to evaluate the therapeutic efficacy and underlying mechanisms. Hepatosomatic indices, histological architecture and fibrosis severity were examined. Lactate concentrations within the systemic circulation and hepatic parenchyma, alongside comprehensive lipid profiles, were measured. The expressions of genes and proteins involved in hepatic lactate metabolism were delineated via qPCR and Western blotting. Results: The 8-week HIIT intervention effectively improved liver lipid accumulation, hepatocellular injury, and oxidative stress caused by a high-fat diet. Fibrotic expansion and suppressed hepatic stellate cell activation were restricted markedly, as evidenced by the downregulation of collagen type I alpha 1 chain and alpha-smooth muscle actin(α-SMA). HIIT reversed the HFD-induced accumulation of lactate in both systemic circulation and liver tissues, which was found to positively correlate with hepatic α-SMA. Mechanistically, HIIT regulated the expression of the lactate metabolism-related proteins lactate dehydrogenase A and monocarboxylate transporter 1, while selectively enhancing the expression of the gluconeogenic enzymes. Conclusions: Our findings indicate that HIIT effectively ameliorated MASLD and associated hepatic fibrosis by remodeling the hepatic lactate metabolic axis, specifically through the suppression of lactate production and the enhancement of its clearance. These results indicate that targeting lactate homeostasis might be a promising therapeutic strategy for MASLD. Full article

Melanoma is a highly aggressive and invasive form of skin cancer that arises from the uncontrolled growth of melanocytes. It is characterized by early spread through the lymphatic system and metastasis. The success of metastasis is linked to the ability of melanoma and other cancer cells to resist anoikis, a type of cell death that occurs when cells lose their adhesion to the extracellular matrix. Redox signaling plays an essential role in anoikis resistance. The balance between intracellular levels of nitric oxide (NO) and the reactive oxygen species (ROS) O₂⁻ and H₂O₂ stimulate signaling pathways related to proliferation and survival or cell death. A375 and SK-MEL-28 human melanomas cell lines, representing primary melanoma and lymph node metastatic melanoma cells, respectively, under suspension and adherent culture conditions were used to investigate the redox regulation of anoikis resistance. Both cell lines express the three isoforms of nitric oxide synthases (NOS) and NADPH oxidase 4 (NOX4) as endogenous sources of NO and ROS, respectively. When A375 cells in suspension were treated with the pan-NOS inhibitor L-NAME, their viability decreased. The treatment resulted in a decrease in FAK phosphorylation at Tyr397 and in ERK 1/2 phosphorylation. The expression of FAK, ERK 1/2, β-actin, and α-tubulin were significantly reduced. Treatment with L-NAME led to an increase in the expression of the metalloprotease MMP-9. SK-MEL-28 cells in suspension and treated with the NOX4 inhibitor, GKT36901, exhibited reduced viability. This was accompanied by the inhibition of FAK phosphorylation at Tyr397, ERK 1/2 phosphorylation, and a reduction in the expression of FAK, ERK 1/2, β-actin, and α-tubulin, with a slight elevation in the expression of MMP-9. Migration and invasion were strongly inhibited in A375 cells upon treatment with L-NAME, while treatment with GKT36901 had a marginal effect on the migration and invasion capacities of SK-MEL-28 cells. In summary, melanoma cells employ nitrosative and oxidative stress to shield themselves from anoikis. Nitric oxide was essential for melanoma cells at the primary site for resisting anoikis, while H₂O₂ contributed to anoikis resistance in metastatic melanoma cells. Full article

As the use of electronics and mobile devices increases, indium and its related compounds are increasingly prevalent in consumer products. However, the effects of the ionic form of indium on the mammalian renal cells are unclear. Understanding indium toxicity in these cells is important, as it relates to kidney health. Kidneys remove heavy metals, maintain electrolyte balance, and perform other vital functions. This in vitro study examines the effects of indium chloride (InCl₃) on Vero cells, focusing on cell morphology, viability, reactive oxygen species (ROS) production, and expression of key focal adhesion proteins. Cells were incubated in culture media with InCl₃ concentrations ranging from 0 to 3.2 mM for 24 h. Fluorescence confocal microscopy analyses revealed that concentrations above 0.8 mM caused the cells to become more compact and display decreased actin filament lengths, suggesting cellular degeneration, which was further supported by the AlamarBlue^® Cell Viability Reagent. Using a 2′,7′–dichlorofluorescin diacetate (DCFDA/H2DCFDA) assay, we show that ROS levels increase with InCl₃ concentration, accompanied by significant increases in focal adhesion kinase (FAK) and paxillin at InCl₃ concentrations above 0.8 mM. Interestingly, the level of α-actinin detected is not affected by exposure to InCl₃. Our findings demonstrate that InCl₃ has negative impacts on the growth and behaviour of Vero cells at concentrations exceeding 0.8 mM, underscoring the need for further investigation into the biological effects of indium-containing compounds. Full article

Corneal blindness remains a major clinical challenge, yet donor grafts are scarce. Bioprinting has emerged in recent decades to potentially overcome donor shortage. In bioprinting, collagen is a common biomaterial. However, one alternative biomaterial, which has shown promising results in corneal tissue engineering, is keratin. Therefore, human hair keratin was investigated in this study as a bioink component for stroma bioprinting. Two keratin preparations, an aqueous extraction and an alkaline extraction, were incorporated into a methacrylated hyaluronic acid bioink and compared with a collagen methacrylated hyaluronic acid bioink. Corneal stroma-like constructs were printed by extrusion bioprinting and evaluated for optical transmission, biomechanical properties, cell compatibility and protein expression of collagen type I and alpha-smooth muscle actin over a four-week period. Two different cell types, immortalized corneal keratocytes and human corneal fibroblasts, were used. The alkaline keratin dialysate-supplemented bioink showed similar optical transparency and biomechanical properties to the collagen-supplemented bioink. Cell viability was high in all formulations. Protein expression of collagen type I and α-smooth muscle actin remained low in all bioinks. Human corneal fibroblasts appeared to be in a quiescent state and were unable to produce large amounts of collagen. This comparative study between collagen and keratin is a first step towards establishing keratins in bioprinting, leading to more complex bioinks. Full article