Despite newly introduced treatment options, the 5-year survival rate for non-small cell lung cancer (NSCLC) remains below 20%, indicating a remaining need for new treatment strategies. Benzamil (BZ) has demonstrated inhibitory effects on brain tumors and osteosarcoma; however, its effects on NSCLC cells remain unknown. In this study, we investigated the potential anti-cancer effects of BZ in a panel of NSCLC cell lines. Compared to cells with EGFR L858R/T790M double mutations (H1975), BZ induced greater cytotoxicity in EGFR wild-type (A549, H1299, H292) and EGFR ex19del (PC9) cells. Mechanistically, BZ treatment was associated with inhibition of Akt, ERK1/2, and Stat3 signaling and increased mitochondrial damage. It was also associated with reduced Drp1 and Fis1 expression. Moreover, BZ induced release of cytochrome c, Smac/Diablo, and HtrA2/Omi from mitochondria. Additionally, BZ enhanced osimertinib-induced cytotoxicity in EGFR-mutant cells, and it suppressed H1975-derived cancer spheres. These findings support a proposed model in which BZ-induced mitochondrial damage is accompanied by the release of cytochrome c, Smac/Diablo, and HtrA2/Omi. These changes were accompanied by caspase activation and cell line-dependent alterations in XIAP expression, consistent with their potential contribution to apoptosis in NSCLC cells.

Hypomagnesemia secondary to cisplatin-induced nephrotoxicity is caused by a tubular defect that results in renal loss of magnesium. The objective was to evaluate the effect of amiloride in patients with cisplatin-induced hypomagnesemia. To this end, we included nine cancer patients who were evaluated sequentially in three stages: without corrective treatment, with magnesium supplementation, and with combined treatment of magnesium plus amiloride. Magnesium supplementation increased serum magnesium levels compared with no treatment (1.22 ± 0.07 vs. 1.01 ± 0.13 mg/dl; p < 0.01), without reducing urinary magnesium excretion. The addition of amiloride produced a further increase in serum magnesium levels (1.38 ± 0.17 mg/dl; p <0.05) and was associated with a significant decrease in magnesium excretion and fractional magnesium excretion (p <0.01). No episodes of hyperkalemia were observed. Amiloride reduced hypermagnesuria and increased serum magnesium levels in patients with cisplatin-induced nephrotoxicity.

The endothelial surface glycocalyx (ESG) is a dense, carbohydrate-rich mesh of glycoproteins, proteoglycans, and glycosaminoglycans (GAGs) that coats the surface of endothelial cells (ECs). Mechanical cues, such as shear stress, at the ESG can be rapidly translated into biochemical signals. An impairment in this mechanosignaling pathway is linked to cardiovascular complications, such as diabetes, stroke, sepsis, and atherosclerosis, due to the decrease in the potent vasodilator, nitric oxide (NO). ESG plays a role in this pathway; however, the receptor-level pathways remain obscure. We present an atomic force microscopy (AFM) force-to-signal assay that delivers real-time, pico- to nano-newton, receptor-targeted mechanical inputs to a single live EC while simultaneously recording NO production via fluorescence. We apply controlled pulling forces to ESG components on mouse brain ECs to investigate which ESG elements are relevant to mechanosensing and which transient receptor potential (TRP) ion channels support Ca[2+]-dependent NO production. Our key findings are the following: (1) heparan sulfate (HS) and glypican-1 act as proximal mechano-sensors that trigger rapid NO signaling. (2) Pharmacological perturbation with SKF96365 markedly suppressed the mechanically evoked NO response, whereas a smaller but significant response persisted in the presence of amiloride. Together, our findings highlight glypican-1 engagement and the potential involvement of TRP-channel-associated Ca[2+] entry in the ESG-mediated mechanosignaling pathway. Additionally, by directly coupling a molecule-specific mechanical input to a quantitative fluorescent readout, this technique provides a direct, single-cell map of endothelial mechanotransduction from receptor to downstream signal, enabling exploration of relevant targets for cardiovascular pathologies associated with glycocalyx dysfunction.

Niobium pentoxide (Nb2O5) is a promising photocatalyst for environmental remediation and solar hydrogen production; however, the role of synthesis parameters in governing its phase composition and charge-carrier dynamics remains insufficiently understood. Herein, we systematically investigate the effect of synthesis pH (2, 4, and 6) on the structural, morphological, electronic, and photocatalytic properties of Nb2O5 nanoparticles prepared via two hydrothermal routes. pH variation in Route 1 promotes a phase transition from pseudohexagonal TT-Nb2O5 (pH 2) to hydrated Nb2O5 nH2O (pH 4-6), accompanied by pronounced morphological evolution from nanoneedles to lamellar structures. In contrast, Route 2 preserves the TT-Nb2O5 phase regardless of post-treatment pH, highlighting its structural stability. In organic pollutant degradation, all samples were active toward Rhodamine B and amiloride, where mechanistic studies revealed a hole-dominated oxidation pathway with secondary involvement of superoxide radicals. Hydrated Nb2O5·nH2O phases exhibited strong adsorption capacity, whereas TT-Nb2O5 showed higher intrinsic photocatalytic efficiency. Stability tests confirmed sustained activity over four cycles. TT-Nb2O5 materials also exhibited superior charge-transfer properties and were active in H2 evolution, achieving up to 4104.5 μmol g[-1] after 8 h (Nb2O5-AP2). Optimization of Pt loading revealed an optimal cocatalyst content of 0.5 wt %, while long-term assays confirmed stable H2 production over 24 h of operation. Photoluminescence results indicated that recombination suppression contributes to performance enhancement but does not solely govern activity. These findings demonstrate that synthesis pH governs phase formation, surface chemistry, and charge-carrier dynamics in Nb2O5, providing fundamental insights into the rational design of niobium-based photocatalysts tailored for integrated environmental and energy applications.

Background/Objectives: Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in cardiovascular disease, particularly through the transfer of regulatory microRNAs. However, direct evidence of cytosolic microRNA delivery by mesenchymal stem cell (MSC)-derived EVs into cardiomyocytes remains limited. Many prior studies have inferred transfer without fully excluding endogenous microRNA induction or surface-bound artifacts. The objective of this study was to determine whether MSC-derived EV microRNA undergoes functional cytoplasmic delivery into cardiomyocytes and to identify the endocytic pathways involved. Methods: To eliminate background from endogenous microRNA expression, MSC-derived EVs were loaded with the exogenous microRNA cel-miR-39-3p. H9C2 cardiomyocytes were treated with increasing vesicle doses, and intracellular microRNA levels were quantified using real-time polymerase chain reactions. Cytosolic accessibility was assessed indirectly using a cholesterol-modified antagomir targeting cel-miR-39-3p. Pharmacologic inhibitors of macropinocytosis (5-(N-ethyl-N-isopropyl) amiloride (EIPA)) and clathrin-mediated endocytosis (chlorpromazine and ES9-17) were applied to delineate uptake pathways, with transferrin assays confirming clathrin inhibition. Results: EV treatment produced a dose-dependent increase in intracellular cel-miR-39-3p levels. Antagomir administration reduced intracellular microRNA levels by 89%, providing indirect functional evidence that transferred cel-miR-39-3p reached an antagomir-accessible cytosolic compartment. At the highest preparation dose, EIPA and ES9-17 reduced cell-associated cel-miR-39-3p by 71% and 36%, respectively, consistent with contributions from EIPA-sensitive and clathrin-associated uptake processes. Notably, chlorpromazine increased microRNA accumulation despite blocking transferrin uptake, suggesting compensatory or membrane curvature-dependent effects. Conclusions: These findings provide strong functional evidence that exogenous cel-miR-39-3p associated with MSC EV preparations reaches an antagomir-accessible cytoplasmic compartment in H9C2 cardiomyocytes and implicate EIPA-sensitive uptake consistent with macropinocytosis and clathrin-associated uptake. Additional genetic, localization, and pulse-chase studies are required to establish the precise intracellular trafficking mechanisms. This work strengthens the mechanistic foundation for EV-based therapies and informs the rational development of cell-free strategies for cardiovascular disease.

Urinary plasminogen has been implicated in the progression of proteinuric kidney diseases, yet its direct role in mediating injury along the nephron remains incompletely understood. Experimental models commonly used to study plasminogen-mediated damage are limited by incomplete phenotypic fidelity or interspecies differences, hindering translational relevance. Here, we used kidney organoids generated from H9 human embryonic stem cells to investigate the effects of plasminogen exposure and to evaluate urokinase-type plasminogen activator (uPA) inhibition with amiloride as a therapeutic strategy. Kidney organoids were exposed to plasminogen in the presence or absence of amiloride, and uPA activity was quantified using a fluorescence-based assay. Compartment-specific injury was assessed by confocal microscopy, automated image analysis, and AI-assisted nuclear segmentation, while targeted transcriptomic changes were evaluated using bulk quantitative PCR. Plasminogen exposure induced glomerular injury, characterized by reduced podocalyxin, nephrin, podocin, and WT1 expression, decreased glomerular cell density, and increased apoptosis. Proximal tubules exhibited reduced LTL and megalin expression, with increased KIM-1, whereas distal tubules were relatively preserved. Amiloride effectively inhibited uPA activity and partially restored podocyte and proximal tubular marker expression, but did not prevent apoptosis, indicating incomplete protection against plasminogen-induced injury. These findings support preferential plasminogen-induced injury along the glomerular-proximal tubular axis and highlight the utility of human kidney organoids for modeling this process, while underscoring the need for further validation and broader molecular profiling to strengthen their application in mechanistic studies and therapeutic discovery.

Mineralocorticoid receptor antagonists (MRAs), aldosterone synthase inhibitors (ASIs), and epithelial sodium channel (ENaC) blockade target different levels of the aldosterone-related sodium-retention pathway in resistant hypertension (RH), but the comparative efficacy of individual agents remains uncertain. We conducted a focused drug-level network meta-analysis of randomized controlled trials (RCTs) comparing pathway-directed therapies in adults with RH. PubMed, Scopus, Embase, Cochrane CENTRAL, Cochrane Reviews, and ClinicalTrials.gov were searched from inception to July 20, 2026. The primary outcome was change in office systolic blood pressure (BP). Secondary outcomes included office diastolic BP and 24-hour ambulatory systolic and diastolic BP. Ten RCTs including 2865 participants were analyzed. In the primary drug-level analysis, amiloride, spironolactone, baxdrostat, eplerenone, and lorundrostat significantly reduced office systolic BP compared with placebo, with mean differences (MDs) of -14.08, -10.78, -9.09, -8.14, and -6.80 mmHg, respectively. Osilodrostat showed a statistically uncertain effect, with an MD of -2.61 mmHg. At the class level, both MRAs and ASIs showed clinically meaningful reductions in office systolic BP. Secondary outcomes were generally consistent with the primary analysis, although fewer trials contributed to these networks. Sensitivity analyses did not materially change the primary findings. These results support the aldosterone-related sodium-retention pathway as an important therapeutic target in RH. MRAs remain the established reference add-on therapy, whereas newer ASIs and amiloride may represent pathway-based alternatives for selected patients. Trial Registration: INPLASY202670099.

Chronic airway diseases such as cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) pose substantial clinical challenges. Here, we explore the p.C97W variant in WAP four-disulfide core domain protein 2 (WFDC2), proposed as a new genetic origin of respiratory distress, especially among Koreans. Whole-exome and whole-genome sequencing (WES/WGS) are performed on 64 patients from 62 families presenting with severe bronchiectasis and chronic rhinosinusitis. Pathogenic variants are found in 19.4% of families, including a novel homozygous WFDC2 missense variant (c.291 C > G, p.Cys97Trp) in five unrelated families. WFDC2 is expressed in lung epithelial cells, and the p.C97W variant impairs WFDC2 protein folding, secretion, and function. Wfdc2 p.C147W knock-in mice exhibit respiratory failure due to the hyperactive epithelial sodium channel (ENaC) linked to increased PRSS8 activity and recapitulate human disease. Treatment with amiloride, an ENaC inhibitor, improves survival and respiratory function in these mice. In conclusion, the p.C97W variant in WFDC2 is a critical genetic factor in severe chronic airway disease that shares clinical features with CF and PCD. Given its implications for diagnosis and treatment, genetic testing for WFDC2 mutations in individuals with CF- or PCD-like symptoms is recommended.

Acid Sensing Ion Channels (ASICs) are widely expressed in the nervous system and involved in an increasing number of functions such as nociception, mechanoreception, and taste transduction, to name a few. In the CNS, ASICs are involved in synaptic plasticity, learning/memory, and in acidosis-mediated neuronal injury. In retina, ASICs are expressed in rodent, rabbit and monkey retina and pharmacological and gene knockdown studies have indicated changes in the earliest phases of electroretinogram that point to effects on phototransduction. This finding led us to investigate the electrophysiological/pharmacological properties of ASICs in cultured primate rod and cone cells. Patch-clamp recordings showed transient ASIC currents with a threshold pH of ~ 7.0 and pH0.5 of 6.15 ± 0.03 and 6.33 ± 0.03 for cultured rod and cone cells, respectively. The currents were almost completely blocked by amiloride and highly sensitive to PcTx-1. Our results for the first time demonstrate the expression of functional ASICs on primate photoreceptors and suggest that ASIC currents in these cells are mediated predominantly by homomeric ASIC1a channels.

In recent years, bovine enterovirus (BEV) has been increasingly associated with diarrhea in cattle in China, posing new challenges for disease control in the cattle industry. However, the mechanisms underlying BEV pathogenesis and virulence remain poorly understood. Infectious cDNA clones provide a powerful tool for dissecting viral replication and pathogenic mechanisms. In this study, we generated a full-length infectious cDNA clone of the BEV-F isolate HB19-1. Three overlapping fragments spanning the complete viral genome were amplified by RT-PCR and assembled downstream of a cytomegalovirus (CMV) promoter placed immediately upstream of the 5' untranslated region (5'UTR). To establish a reporter virus system, the green fluorescent protein (GFP) gene was inserted between the 5'UTR and the N terminus of VP4, followed by a 2A cleavage sequence (IKTAG) at the C terminus of GFP. The recombinant rHB19-GFP virus was successfully rescued. Growth curve analysis demonstrated that rHB19-GFP exhibited slower replication kinetics at early time points relative to the parental HB19-1 virus, with no significant difference in their peak viral titers. This GFP-expressing reporter virus enables convenient monitoring of BEV replication and provides a useful platform for antiviral screening. Using this system, we found that 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) inhibited BEV replication, suggesting its potential as an antiviral candidate. Overall, the rHB19-GFP infectious clone developed here offers a practical tool for studying BEV biology and for identifying antiviral compounds against BEV.

While under physiological conditions, extracellular pH in the brain is reasonably constant, several mechanisms induce transient and localized pH fluctuations. Multiple pathologies involve prolonged changes in pH. Therefore, detecting and monitoring pH changes is crucial for the proper functioning of the nervous system. Acid-Sensing Ion Channels (ASICs) are Na+-permeable ion channels, predominantly expressed in the nervous tissue and activated by protons. Thus, ASICs act as pH sensors, leading to neuronal excitation when the pH drops. ASICs belong to the epithelial sodium channel/degenerin (ENaC/DEG) family of amiloride-sensitive transmembrane ion channel proteins. There are at least eight different ASIC subunits encoded by five genes. Functional ASICs assembled in the plasma membrane are homo- or heteromeric trimers. Trimers containing ASIC1a are of particular interest as, in addition to sodium ions, they also conduct calcium ions and thus can regulate multiple cellular processes. ASICs are involved in numerous physiological functions and pathological states. Biophysical properties of ASICs, their evolutionary origins, regulation and pharmacology, their roles in the peripheral nervous system and in the regulation of synaptic transmission have been recently reviewed in many details. Here we shall focus on brain ASICs in the context of the following issues. (i) The role of ASICs in the homeostasis of extracellular рН in the brain; (ii) interactions of ASICs with major neurotransmitters and (iii) ASICs as emerging key factors and drug targets in brain pathology with focus on stroke, neurological consequences of neonatal hyperbilirubinemia, and Alzheimer's disease.

A recent randomised trial demonstrated the non-inferiority of amiloride versus spironolactone in resistant hypertension, with exploratory analyses suggesting heterogeneity in treatment response based on baseline aldosterone-renin ratio (ARR). We evaluated associations between baseline renin-aldosterone status and blood pressure-lowering responses to each agent. This post hoc subgroup analysis used data from the abovementioned trial. Among 114 patients, 113 had available ARR data and were included and stratified by median ARR into two groups. Subgroup analyses were also performed according to baseline plasma renin activity (PRA) levels. The primary endpoint was the change in home systolic blood pressure (SBP) from baseline to 12 weeks. Patients with higher ARR were older and had higher baseline SBP than those with lower ARR. Multivariate regression analysis showed that female sex, higher nighttime SBP, higher ARR, and lower PRA were associated with a greater reduction in home SBP in response to treatment. In treatment-specific analyses, the home SBP-lowering response to spironolactone was pronounced in patients with higher ARR (-18.8 ± 11.6 vs -9.8 ± 8.4 mmHg, vs. lower ARR) and lower PRA (-19.1 ± 11.8 vs. -10.1 ± 8.4 mmHg, vs. higher PRA), yielding significant ARR-by-treatment (p = 0.046) and PRA-by-treatment (p = 0.027) interactions. In contrast, the home SBP-lowering response to amiloride was not significantly different according to baseline ARR or PRA. These exploratory findings suggest that renin-aldosterone profiling may help identify biological heterogeneity in treatment and warrant prospective evaluation as a candidate tool for treatment selection in resistant hypertension. This post-hoc analysis of a randomized trial in resistant hypertension evaluated. whether renin-aldosterone status predicts differential BP responses to spironolactone versus. amiloride. Women, higher nighttime SBP, higher aldosterone-renin ratio (ARR), and lower. plasma renin activity (PRA) were associated with greater BP reduction. Spironolactone showed greater efficacy in patients with high ARR or low PRA, with significant treatment-by-ARR (p = 0.046) and treatment-by-PRA (p = 0.027) interactions. Amiloride demonstrated consistent efficacy regardless of renin-aldosterone profile. No significant between-drug difference was efficacy regardless of renin-aldosterone profile. No significant between-drug difference was observed within individual strata.