Ultra‐potent synthetic opioids are powerful analgesics but also a leading cause of drug overdose deaths. Calcium−/voltage‐gated potassium channels of large conductance (BK) control vital physiological processes, with their accessory β1 (encoded by Kcnmb1 in mice) subunit being predominantly expressed in smooth muscle. Thus, we hypothesize that BK β1 determines the effect of fentanyl on cerebral artery diameter. Here we report that fentanyl (0.001–10 μ m ) activates BK heteromers consisting of slo1 + β1 subunits in CHO cells, but not homomers of slo1. In ex vivo pressurized mouse cerebral arteries, pharmacological inhibition of BK or β1 genetic deletion ( Kcnmb1 −/− ) led to fentanyl‐induced vasoconstriction. Naloxone, an opioid receptor antagonist, ablates the fentanyl response in wild‐type cerebral arteries whereas mild constriction persists in Kcnmb 1 −/− arteries. Therefore, we identify BK β1 as a functional fentanyl target which acts independently of opioid receptors to regulate fentanyl action on cerebral artery diameter.
Glioblastoma resists therapy through interconnected survival programs. Combined XPO1, Bcl‐2, and Mcl‐1 inhibition (Eltanexor, Venetoclax, A1210477; EVA) enhances the efficacy of chemotherapy, yet resistance persists. We hypothesized that PI3K/AKT/mTOR (PAM) signaling drives adaptive escape. TCGA/GTEx analyses and immunohistochemistry indicated PAM pathway activation in glioblastoma, with AKT significantly overexpressed, whereas PIK3CA showed a clear trend towards higher expression, although it did not reach statistical significance. qPCR analysis showed that PAM‐component expression was highest in patient‐derived glioblastoma stem‐like cells (GSCs). In U87, U251, and GSCs, the dual PI3K/mTOR inhibitor Dactolisib combined with EVA and 5‐Azacytidine reduced viability and induced apoptosis more effectively than any of the Rapamycin combinations, and the difference in apoptosis was most pronounced in U87 and GSCs. Dactolisib monotherapy shifted the cell cycle towards G1 with a reduced S‐phase fraction. In CAM xenografts, [ 18 F]FDG‐PET/CT showed decreased tracer uptake, and Caspase‐3 activation with PARP cleavage was detected only under EVA + Dactolisib treatment. Taken together, our results suggest that dual PI3K/mTOR inhibition with Dactolisib disables a critical adaptive survival axis, warranting further preclinical evaluation.
This study reports the heterologous production, purification, and characterization of the cytochrome Cyc2 TAG1 cluster 1 (Cyc2 TAG1_1 ) from Ghiorsea bivora TAG1 in the outer membrane of Escherichia coli BL21. Using a specific gene construct we optimized solubilization and purification protocols to obtain high‐purity enzyme. This enabled biophysical characterization by EPR, measurement of its midpoint redox potential, and the development of an iron‐oxidizing assay with 2,6‐dichlorophenolindophenol (DCPIP) as an artificial final electron acceptor. The study provides the first quantitative kinetic characterization of a Cyc2 homolog in vitro . The absence of comparable apparent K m or V max values with a natural or artificial electron acceptor in the literature underscores the novelty of our findings. Cyc2 TAG1_1 exhibited an apparent K m of 627 ± 226 μ m for iron sulfate, consistent with iron‐rich hydrothermal vent environments where G. bivora thrives, and an apparent V max of 19 ± 5 μ m ·s −1 . Despite using an artificial electron acceptor that does not accurately represent the natural partner or the real energetic system, the enzyme exhibits apparent kinetic parameters that retain biological significance, with a catalytic efficiency within the range expected for functional electron transfer enzymes. Cyc2 TAG1_1 exhibited a redox potential of −130 ± 20 mV vs SHE which is the lowest reported for Cyc2 homologs. The EPR spectrum is consistent with a low‐spin ferric heme with two axial ligands within the protein barrel. These findings provide the first biochemical characterization of Cyc2 TAG1_1 and support its proposed role in iron oxidation.
Tissue engineering offers significant potential to advance the understanding and treatment of cardiac disease by generating in vitro models that recapitulate myocardial physiology in both healthy and diseased states. Specifically, hydrogel‐based systems have been widely employed to engineer 3D cardiac tissues due to their ability to support cell viability and mimic aspects of the extracellular matrix. However, conventional hydrogels are inherently isotropic and electrically insulating, limiting their capacity to reproduce the anisotropic structure and conductive properties of native myocardium. To address these limitations, increasingly sophisticated hydrogel platforms have been developed that incorporate conductive, piezoelectric and/or aligned architectures to enhance electrical coupling, guide tissue organisation and promote cardiomyocyte maturation. These hydrogel‐based systems can also be tuned to recapitulate pathological states. Cardiac fibrosis is characterised by increased tissue stiffness, and researchers have sought to model this process by modulating the mechanical microenvironment to induce fibrotic phenotypes. With recently developed hydrogel platforms, mechanical properties can be controlled with both spatial and temporal precision, enabling systematic investigation of how dynamic and heterogeneous stiffness influences tissue formation, remodelling and disease progression. Although these platforms are not yet fully translational, they provide powerful tools to modulate multiple physiological parameters, thereby advancing our understanding of cardiac development and informing strategies to treat prevalent pathological conditions.
This study describes a rapid, visual detection platform for Streptococcus suis , a significant zoonotic pathogen, by integrating loop‐mediated isothermal amplification (LAMP) with CRISPR–Cas12a and a lateral flow immunochromatographic strip (LFIA) readout. Targeting the conserved HtpsC gene, specific LAMP primers and a PAM‐compliant CrRNA were designed. Following isothermal amplification, the Cas12a/CrRNA complex recognizes the HtpsC amplicon, initiating target‐specific cleavage and subsequent trans‐cleavage of a dual‐labeled ssDNA reporter (FAM‐BIO), enabling visual detection on a LFIA; a design which reduces false positives. The optimized assay detected HtpsC‐positive S. suis strains within 45 min and achieved a visual detection limit of 1.1 × 10 2 CFU/mL under laboratory conditions, with no cross‐reactivity observed against the tested nontarget bacteria. In addition to pure‐culture dilutions, the assay was further evaluated using spiked porcine blood samples and achieved similar detection performance, supporting its preliminary applicability in a complex biological matrix. Overall, this study provides an HtpsC‐targeted visual detection workflow for screening of S. suis and is expected to be further applied to on‐site rapid detection of clinical samples.
