A cascaded tapered seven-core fiber biosensor for rapid detection of the p53 protein in 293T cell lysate.

Reuterin integrated as dynamic imine crosslinks strengthens creatine modified chitosan hydrogel and boosts antibacterial efficacy.

Inhibition of agonist-induced M2 muscarinic receptor (M2R) activation by functional anti-M2R autoantibodies has been associated with cardiac parasympathetic dysfunction in patients with chronic Chagas disease (CD). This study explored the allosteric nature of that inhibitory effect by assessing the ability of serum IgG from patients with CD and dysautonomia (DCD IgG) to modulate the interaction between M2R and β-arrestins in HEK 293T cells using bioluminescence resonance energy transfer. DCD IgG alone did not stimulate arrestin-2 or arrestin-3 recruitment. When cells were preincubated with DCD IgG and then treated with carbachol, arrestin-2 translocation decreased in a concentration-dependent manner, while arrestin-3 recruitment remained unaffected. Inhibition curve analysis showed a submaximal inhibitory effect (68.1 ± 2.4%) and a Hill slope less than −1 (−4.03 ± 0.39). Carbachol concentration–response assays after preincubation with DCD IgG revealed a noncompetitive inhibition of arrestin-2 recruitment, with no change in arrestin-3 translocation. Unlikely, simultaneous exposure to DCD IgG and carbachol potentiated agonist-induced Arr-2 recruitment. We conclude that anti-M2R autoantibodies selectively inhibit agonist-induced arrestin-2 recruitment, acting as negative allosteric modulators of agonist efficacy. The direction of autoantibody-induced allosteric modulation depends on the timing of IgG application relative to the agonist and the duration of receptor exposure to autoantibodies.

Purpose: The present study evaluates the cytocompatibility of chitosan (CS)-modified glass ionomer cement (GIC) diluents for a Balb/c 3T3 fibroblast cell line. Methods: Three different commercially available hand-mix GIC materials were used in this experiment: Fuji IX GP, Ketac Universal, and Riva Self Cure. The diluents for cell viability tests were produced from DMEM exposed to sterile CS-modified glass ionomer material specimens for three different time periods (0–1, 1–7, and 7–21 days). The resultant diluents were exposed to a 3T3 fibroblast cell line using the indirect contact technique in 96-well plates. In order to assess the physical cell response, five material specimens (1 mm high and 3 mm in diameter) of each material (n = 45) were produced and 3T3 cells were seeded on the specimens. SEM evaluation of the cells was conducted. Results: All the Ketac Universal materials resulted in a decrease in cell viability on day 1. Fuji IX and the CS-modified GICs are the most consistent regarding cell viability. None of the CS-modified GICs exhibited improved cumulative cell biocompatibility. Conclusion: Two materials—Riva Self Cure modified with 5% and 10% CS—retained a decreased cell viability at day 21 compared to the viability of 3T3 cells exposed to the control DMEM.

BackgroundCoptisine (COP) is a natural compound extracted from Rhizoma Coptidis, and it represses the malignant biological behaviors of bladder cancer cells. However, the underlying molecular mechanism has not been fully elucidated. The aim of this study was to clarify the downstream mechanism by which COP treats bladder cancer.Materials and methodsSwissTargetPrediction, STITCH, SymMap, ETCM, TCMSP, CTD databases were used to collect the related targets of COP. GeneCards, DisGeNET, TTD and OMIM databases were used to obtain the related targets of bladder cancer. A Venn diagram was used to identify the potential targets of COP in bladder cancer treatment. The protein-protein interaction network was constructed using STRING database, and Cytoscape 3.9.0 software was used to screen the hub targets. The binding relationship between COP and the hub targets was verified by molecular docking and molecular dynamics simulation. After the bladder cell lines T24 and BIU-87 were treated with different doses of COP, the regulatory effects of COP on PI3K/AKT pathway were investigated with western blotting. Additionally, the tumor-suppressive properties of COP on bladder cancer cells were validated with tumorigenesis model and metastasis model in nude mice.ResultsRAC-alpha serine/threonine-protein kinase 1 (AKT1), glycogen synthase kinase 3 beta (GSK3B), caspase-3 (CASP3), tumor necrosis factor (TNF) and cyclin D1 (CCND1) were identified as the main hub targets of COP in bladder cancer treatment. PI3K/AKT pathway was predicted to be a crucial pathway regulated by COP. The binding affinities between COP and AKT1, GSK3B, CASP3, TNF and CCND1 were high. COP treatment markedly repressed the phosphorylation level of ERK1/2, AKT1, PI3K p85 and mTOR in T24 and BIU-87 cells, and repressed the tumorigenesis and lung/liver metastasis of T24 cells in vivo.ConclusionCOP may be a natural inhibitor for AKT1, GSK3B, CASP3, TNF and CCND1. COP represses PI3K/AKT pathway to suppress the progression of bladder cancer.Supplementary InformationThe online version contains supplementary material available at 10.1186/s41065-025-00600-7.

Juglone, a natural naphthoquinone compound, exhibits potent anticancer activity but faces clinical limitations due to poor solubility, low bioavailability, and systemic toxicity. While tumor microenvironment-responsive drug activation strategies offer a promising solution, achieving precise spatial control over the conversion of non-toxic precursors into cytotoxic agents remains a significant challenge. We developed DHN@Pt/PtMBCPsNSs, a platinum (Pt)-methylene blue (MB)- based nanoscale sonosensitizer, to enable ultrasound-triggered on-demand juglone synthesis within tumors. The system was evaluated in bladder cancer models including cell lines, patient-derived organoids (PDOs) and patient-derived tumor xenograft (PDX) models, assessing tumor uptake, intracellular distribution, hypoxia modulation ("H2O2-O2-¹O2" cascade), oxidative stress markers (SOD, GSH, MDA, GSSG), and cell death pathways (apoptosis/pyroptosis). Transcriptome sequencing was performed to elucidate molecular mechanisms. DHN@Pt/PtMBCPsNSs were internalized by UMUC-3 and T24 cells and primarily accumulated within the mitochondrial compartments. The platinum-based components catalytically exhausted GSH via redox cycling. DHN@Pt/PtMBCPsNSs triggered the production of O2 and ¹O2 after local US irradiation, alleviating hypoxia and selectively converting dihydroxynaphthalene (DHN) to juglone in tumors, which reducing the systemic toxicity of juglone. The nanosystem further disrupted cellular redox balance by triggering depletion of antioxidant defenses (SOD and GSH) and reducing the ratio of GSH/GSSG. In vitro cell experiments and PDOs experiments demonstrated that DHN@Pt/PtMBCPsNSs combined with US irradiation can significantly inhibit bladder cancer cell proliferation, induce apoptosis, and regulate pyroptosis via the ROS-mediated caspase 3/GSDME signaling pathway. In the PDX model, DHN@Pt/PtMBCPsNSs accumulated at the tumor site and significantly inhibited tumor growth under US irradiation. As a novel sonosensitizer and pyroptosis inducer, DHN@Pt/PtMBCPsNSs achieve a precise synergistic effect of chemotherapy and sonodynamic therapy within cells, while minimizing adverse effects on normal cells and overcoming the limitations associated with natural chemotherapeutic agents. This approach may advance clinical translation of natural anticancer agents.

ABSTRACT The regulation of macrophage polarization is an emerging strategy for biomaterials implanted in vivo to accelerate wound tissue regeneration. In this study, a self‐healing hydrogel was developed using a polysaccharide‐based system formed via a Schiff base reaction between proline‐functionalized chitosan (CS‐Pr) and oxidized dextran (Odex), with epigallocatechin gallate (EGCG) incorporated into the polymer network. EGCG in the hydrogels exhibited not only the capacity to facilitate the transformation of macrophage polarization toward the M2 phenotype but also excellent ROS‐scavenging activity. Additionally, the hydrogels demonstrated concentration‐dependent antibacterial activity against E. coli and S. aureus . The antibacterial ratio of hydrogels for S. aureus was higher than that of E. coli . The composite hydrogels also demonstrated tunable mechanical properties and favorable self‐healing properties. When co‐cultured with NIH 3 T3 cells, the hydrogels showed good biocompatibility. Based on the above results, the composite hydrogels showed promising potential as clinical materials for wound healing.

The influence of naturally occurring and in silico-informed mutations of MRP1/ABCC1 on the transport of arsenic triglutathione.

Mitochondrial DNA copy number (mtDNA-CN) is a metric of mitochondrial function that has been associated with a variety of diseases including cardiovascular disease and all-cause mortality. To investigate genes and pathways affected by mtDNA-CN variation, we perturbed HEK 293T cells with ethidium bromide to deplete mtDNA. Using RNASeq and methylation microarrays, we evaluated transcriptomic and methylomic changes in treated cell lines. We observed an 8-fold decrease in mtDNA-CN and compensatory shifts in mitochondrial transcription to support mtDNA replication. Nuclear transcriptomic and methylomic analysis highlighted changes in metabolic pathways, including oxidative phosphorylation and canonical glycolysis. Longitudinal analyses revealed that the identified genes and pathways have different response timing, with nuclear response lagging behind mitochondrial response. These findings further elucidate the mechanisms behind mtDNA maintenance and responses to cellular energetics as well as mitochondrial-nuclear crosstalk dynamics.

Abstract INTRODUCTION Glioblastoma poses a persistent challenge in oncology. IDH mutations were found in 73 % of clinical cases in some series. How IDH status affect the prognosis of the disease remained controversial. Some studies reported IDH interfered with tumor metabolism in which mutated IDH prefers NADPH and α-KG, instead of NADP+ and isocitrate, as its substrates. High affinity of NADPH and α-KG leads to consumption of NADPH, and in turn affects cellular redox homeostasis and reactive oxygen species (ROS) accumulation and oxidative damage. In addition, mutated IDH converted α-KG to the oncometabolite D-2-hydroxyglutarate (2-HG). The accumulation of 2-HG leads to epigenetic dysregulation via inhibition of αKG-dependent histone and DNA demethylases, and a block in cellular differentiation. Malic enzyme (ME) is a key enzyme in maintaining cellular redox homeostasis. This enzyme catalyzes an oxidative decarboxylation of L-malate (MAL) to pyruvate (PYR) concomitant with the reduction of NAD(P)+ to NAD(P)H. ME2 was also found to increase 2-HG production by adjusting glutaminolysis. This study aimed to determine the role of ME2 in Glioblastoma. METHODS We use glioma cell lines (T98G) to perform in vitro tumor metabolic studies. Silencing of ME2 was achieved by transfection with the 4D-Nucleofector (Lonza) using 2 μg of plasmid targeting ME2. The ATP production was measured using a CellTiter-Glo® 2.0 Cell Viability Assay and ROS was determined using a ROS Detection Assay Kit. RESULTS We observed that silencing ME2 in T98G cells resulted in noticeable alterations in cell metabolite patterns including ATP production and ROS level (decreased ATP level and increased ROS level). In addition, knockdown of ME2 in T98 cells increased the NAD/NADH ratio. CONCLUSION These findings suggest a potential role of ME2 in rewiring metabolic pathways in glioblastoma cells under different environmental conditions and also the importance of ME2 in fueling tumorigenesis via energy production and maintaining redox homeostasis.

Mammalian cells sense and respond to environmental changes using a complex and intelligent system that integrates chemical and mechanical signals. The transduction of mechanical cues into chemical changes modulates cell physiology, allowing a cell to adapt to its microenvironment. Understanding how the chemical and mechanical regulatory modules interact is crucial for elucidating mechanisms of mechanosensation and cellular homeostasis. In this study, we find that cells exhibit nonmonotonic changes in cell volume and intracellular pH when subjected to physical stimuli and varying degrees of actomyosin cytoskeleton disruption. We find that these nonmonotonic responses are mediated by a chemical compensation mechanism, where the attenuation of actomyosin activity stimulates the activity of PI3K/Akt pathway. This, in turn, activates sodium-hydrogen exchanger 1 (NHE1), resulting in elevated intracellular pH and increased cell volume. Furthermore, we identify a competitive interaction between the PI3K/Akt and MAPK/ERK pathways-two major regulators of cell proliferation and motility. This competition modulates the chemical compensation based on the relative activities of these pathways. Our mathematical modeling reveals the network structure that is essential for establishing the nonmonotonic response. Interestingly, this regulatory system is altered in HT1080 fibrosarcoma, highlighting a potential mechanistic divergence in cancer cells in contrast to their normal-like counterpart, such as NIH 3T3 and HFF-1 fibroblasts. Overall, our work reveals a compensatory mechanism between chemical and mechanical signals, providing an infrastructure to elucidate the integrated mechanochemical response to environmental stimuli.

CRISPR-Cas9-targeted sequencing can enrich DNA regions of interest by directing the Cas9 protein to bind and cleave specific DNA sequences via single-guide RNA (sgRNA). It is interesting to explore the efficacy of using CRISPR-Cas9-targeted nanopore sequencing (referred to as Cas9-seq), a polymerase chain reaction (PCR)-free workflow, for forensic short tandem repeats (STR) profiling, and to compare it with the amplification-based approach. In this pilot study, we constructed a Cas9-seq method for profiling seven STR loci, including D18S51, FGA, TPOX, D16S539, vWA, CSF1PO, and TH01. With 3µg DNA inputs from human NA12878 and 293T cell lines, we achieved 643.45- and 468.34-fold enrichment ratios of the sgRNA-targeted regions by using Cas9-seq, respectively. Compared to nanopore sequencing of PCR amplicon products (amplicon-seq) of the ForenSeq DNA Signature Prep kit, the Cas9-seq reads had an ultralow strand bias. However, surprisingly, Cas9-seq did not show advantages in allele balance and had higher noise in the reads. At the seven STR loci for the two samples, both Cas9-seq and amplicon-seq had three genotyping errors. Additionally, there were no false-positive single-nucleotide polymorphisms (SNPs) introduced by Cas9-seq, whereas amplicon-seq produced three. In sum, we conclude that the PCR-free Cas9-seq might not be favorable for forensic STR genotyping.

Advanced dual-channel fluorescent probes for simultaneous biomarker detection are in critical demand nowadays in biomedical research. Herein, a blue-green emissive ratiometric probe CD@Tb has been engineered by coupling pyridinedicarboxylate-functionalized carbon dots (CD) with Tb3+ ions. This probe exhibits ultrasensitive and selective detection of epinephrine (EP, LOD: 1.2 nM) and pyrophosphate (PPi, LOD: 0.25 μM) via distinct fluorescence responses: Epinephrine (EP) enhances the blue emission while suppressing the green emission by deactivating the excited state of terbium through energy transfer. In contrast, pyrophosphate (PPi) amplifies the green emission of terbium through bridging coordination by inducing carbon dot aggregation, which strengthens the antenna effect, while simultaneously diminishing the blue emission. The sensing efficiency of the probe was tested in real human blood serum samples to check its reliability. Moreover, after thorough testing of its biocompatibility, the CD@Tb probe was employed as an imaging tool for visualization of epinephrine in human neuroblastoma cells, SH-SY5Y, and pyrophosphate in mouse fibroblast cells, NIH 3T3.

Collective migration is a crucial mechanism guiding cell movement in developmental processes and disease progression. Understanding the migration behavior of cell clusters is key to advancing our knowledge of morphogenesis, wound healing, and collective cancer invasion. Despite the understanding of the response of single cells to environmental physical cues, the collective behavior of cells in response to different levels of extracellular matrix stiffness is yet to be fully understood. Here, we present a quantitative investigation into how substrate stiffness and cell cluster size modulate the collective behavior and migration dynamics of NIH 3T3 fibroblasts. With the variation of PDMS and curing agent concentrations, two contrasting soft and stiff substrates with different stiffness were developed. Using a combination of atomic force microscopy (AFM) to precisely characterize substrate elastic moduli and time-lapse microscopy for tracking migration parameters, we demonstrate that substrate mechanics and cluster geometry synergistically govern collective behavior. Fibroblast migratory characteristics were greatly improved with increased stiffness and cluster size. Large clusters on stiff substrates exhibited greater circularity (~ 0.8), migration distance, displacement (135.6 µm), directionality (0.81), and velocity (24 µm/h) compared to single cells and small clusters on soft and stiff substrates. Moreover, detailed analysis of cytoskeletal reorganization via actin staining revealed the mechanotransductive pathways that convert physical cues into migratory behavior. These findings provide important insights into how substrate stiffness influences collective cell migration, offering potential applications in elucidating the mechanisms of morphogenesis and the dynamics of collective cell invasion during tumor progression.

Methylparaben and propylparaben promote bladder cancer invasion via MMP2 and PPARG modulation.

Cellular and functional insights into FIH-mediated hydroxylation of TRPA1.

Effects of dibenzazepine compounds on Nav1.2 channels and neuronal network activity: A systematic comparison.

Deficiency of adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory disease manifested as polyarteritis nodosa, stroke, and bone marrow failure. Leveraging an international cohort of 200 DADA2 cases, we aimed to characterize the diagnostic utility of a plasma ADA2 enzyme activity assay and understand the implications of residual ADA2 activity. Data were collected from individuals who underwent ADA2 testing from 2018 to 2025. Plasma ADA2 activity was determined using an established spectrophotometric assay. ADA2 variants were analyzed in transfected cells by enzyme assay and western blotting. We determined that plasma ADA2 activity is 99.0%/96.0% sensitive, and 99.7%/98.8% specific in distinguishing genetically confirmed DADA2 cases from controls and carriers, respectively. Eighteen individuals with DADA2 (9%) possessed detectable ADA2 activity, including several cases with levels seen in carriers. Residual ADA2 activity was associated with the vasculitis / inflammatory phenotype but not with disease severity. Genotype analysis revealed that 14/18 cases with residual plasma activity possessed at least one hypomorphic missense variant with >20% residual ADA2 function when overexpressed in 293T cells, often occurring in trans with a more deleterious variant. In vitro analysis revealed that missense ADA2 variants exert variable dominant-negative effects by forming large intracellular protein aggregates via disulfide bond formation at a cysteine residue (Cys408). We confirmed the utility of plasma ADA2 activity as a diagnostic assay and showed that the inflammatory phenotype of DADA2 occurred in cases with residual activity. In vitro findings illustrate potential interactions of ADA2 variants to synergistically disrupt protein function.

Investigation of alternative cell models to BALB/c 3T3 for in vitro neutral red uptake phototoxicity test of pharmaceuticals: NIH 3T3 and HaCaT cells

The current study aimed to evaluate the in-vitro toxicity and in-vivo antidiabetic effects of a standardized extract from Eugenia uniflora (E. uniflora) fruit, comparing its efficacy to metformin (Met), a widely used anti-hyperglycemic drug. First, toxicity of the extract was determined by MTT assay in 3T3 cell line and primary astrocyte culture. Then Wistar rats were divided into four groups: I- Control, II- type 2 diabetes mellitus (T2DM), III- T2DM + Met and IV- T2DM + E. uniflora. To induce T2DM, groups II, III and IV received a high fat diet (HFD) for 3 weeks followed by a single intraperitoneal (i.p.) dose of streptozotocin (STZ, 35mg/kg). Animals of group I received normal diet and vehicle (i.p.). Group III received Met (250mg/kg) and group IV received E. uniflora (200mg/kg) intragastric pathway, once a day, throughout all experimental protocol. Animals from the groups I and II received water in the same volume. Results showed that cell viability was not affected. In-vivo, E. uniflora and Met prevented the change in serum levels of glucose, cholesterol, LDL, triglyceride and interleukin-6. Furthermore, extract and Met improved oxidative stress markers and antioxidant enzyme activity in the brain (cerebral cortex, hippocampus, striatum). Furthermore, extract enhanced the downstream insulin signaling pathway, including insulin receptor substrate 1, forkhead box protein O-3a, as well as activated the nuclear factor erythroid 2-related factor 2 in the cerebral cortex. This study indicates that E. uniflora extract may have potential in preventing complications associated with T2DM; nevertheless, additional studies are required to confirm these effects and establish their clinical significance.