Search Results (1,023)

Neurogranin (Ng) is a postsynaptic calmodulin-binding protein highly enriched in forebrain neurons and widely implicated in synaptic plasticity. However, whether Ng contributes more broadly to neuronal network maturation and cellular homeostasis remains unclear. Here, we examined the consequences of silencing or restoring Ng to adult physiological levels in primary hippocampal neurons. Ng expression promoted dendritic expansion, increased synaptic number, and shifted the axon initial segment toward the soma, consistent with structural adaptations to enhanced connectivity. Calcium (Ca²⁺) imaging revealed a marked increase in spontaneous neuronal activity and network synchronization, which was confirmed by electrophysiological recordings showing enhanced burst firing and spike synchrony. At the molecular level, Ng altered Ca²⁺/calmodulin (CaM) signaling by increasing total CaM levels, reducing Ca²⁺/CaM-dependent protein kinase II (CaMKII) abundance while increasing its relative autophosphorylation, and downscaling specific ionotropic glutamate receptors. Despite elevated network activity, Ng expression enhanced neuronal metabolic competence and viability, reduced cellular stress signaling and induced modest caspase-3 activation without engagement of apoptotic pathways. Together, these results indicate that Ng promotes neuronal maturation and coordinated network activity while engaging compensatory mechanisms that preserve excitatory balance and neuronal resilience. Our findings identify Ng as a molecular integrator linking Ca²⁺/CaM signaling with the structural and functional maturation of neuronal networks. Full article

Erythropoietin-producing hepatocellular receptor A2 (EphA2) has emerged as a key mediator that promotes tumor malignant progression. EphA2 overexpression and its non-canonical signaling lead to oncogenic transformation, metabolic reprogramming, resistance to treatments, and metastasis. Therefore, strategies targeting EphA2 have been evaluated in clinical trials. However, the clinical effects were not sufficient. An anti-EphA2 monoclonal antibody (mAb), Ea₂Mab-7 (mouse IgG₁, κ), demonstrated high affinity and specificity among Eph receptors. In this study, we produced recombinant class-switched Ea₂Mab-7 variants, including Ea₂Mab-7-mG_2a (mouse IgG_2a) and Ea₂Mab-7-hG₁ (human IgG₁). Both Ea₂Mab-7-mG_2a and Ea₂Mab-7-hG₁ recognized human triple-negative breast cancer MDA-MB-231, pancreatic cancer MIA PaCa-2, and colorectal cancer HCT-15 in flow cytometry. Furthermore, both Ea₂Mab-7-mG_2a and Ea₂Mab-7-hG₁ exerted significant antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against these tumors. In mouse xenograft models of breast, pancreatic, and colorectal cancers, both mAbs demonstrated antitumor activity. These results indicate the potential of Ea₂Mab-7 variants for the treatment of EphA2-positive cancers. Full article

Tumor initiation and metastatic progression are driven by context-dependent genetic alterations that disrupt tumor suppressor pathways, metabolic homeostasis, and signaling networks. However, the initial drivers that transform normal cells into malignant ones and their context dependency remain elusive. To address this, we aimed to systematically identify and characterize these drivers across cancer types, species, and microenvironments. We constructed customized clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout (KO) libraries targeting high-frequency mutated and downregulated genes associated with liver hepatocellular carcinoma (LIHC) and breast carcinoma (BRCA) and conducted parallel functional screens in non-cancerous mouse and human fibroblast cell lines under two-dimensional (2D), three-dimensional (3D), and in vivo conditions. Strikingly, TP53 and NF1 emerged as pan-context drivers consistently enriched across immortalization, tumorigenesis, and metastasis in both LIHC and BRCA settings, while most other identified drivers were largely species-, tissue-, and microenvironment-specific with limited cross-model overlap. Despite this heterogeneity, all drivers converge on core pathways including epigenetic regulation, metabolic reprogramming, and growth factor signaling. Unlike prior studies on established cancer cells, this work defines the genetic barriers restricting the malignant transformation of primary normal cells, offering a new framework for early cancer evolution. Full article

Stroke, a leading cause of mortality and long-term disability, induces complex cascades of oxidative stress and neuroinflammation that exacerbate brain injury. The peroxiredoxin (Prx; Prdx) family, composed of six thiol-dependent antioxidant enzymes (Prx1–6), plays a pivotal role in regulating redox homeostasis and immune responses in the brain. This review synthesizes current knowledge on the isoform-specific roles of Prxs in both ischemic and hemorrhagic stroke, highlighting their dual and context-dependent functions. Intracellular Prxs generally protect neurons and maintain blood–brain barrier (BBB) integrity, while extracellular Prxs—particularly Prx1 and Prx2—act as damage-associated molecular patterns (DAMPs), activating toll-like receptor signaling and amplifying inflammation. Isoforms such as Prx3 and Prx4 exhibit mitochondrial and endothelial protective effects, respectively, whereas Prx6 demonstrates complex roles influenced by its acidic, Ca²⁺-independent, phospholipase A2 (aiPLA2) activity and cellular localization. We also discuss emerging tools for studying Prx biology and explore the translational potential of Prxs as biomarkers and therapeutic targets. Ultimately, a nuanced understanding of Prx dynamics offers new avenues for stroke diagnosis, prognosis, and treatment. Full article

A thin-film surface plasmon resonance (SPR) sensor is presented using a prism-coupled Kretschmann configuration and an optimized multilayer architecture incorporating black phosphorus (BP) as an ultrathin overlayer. The response is modeled at 633 nm under TM polarization using the transfer-matrix method. Low-concentration sensing conditions in the 1–5 ng/mL range are represented through small effective-refractive-index perturbations of the aqueous sensing medium, providing a preliminary optical framework for evaluating refractive-index response in biosensing-related scenarios. The coupling prism, Au film thickness, and Si₃N₄ spacer thickness are optimized to control resonance depth, linewidth, and angular shift. The optimized CaF₂/Au/Si₃N₄/BP configuration exhibits systematic condition-dependent displacement of the SPR minimum and an evanescent-field distribution that remains strongly localized at the sensing interface while extending into the sensing medium, enabling refractive-index interrogation. High angular sensitivity is obtained at low levels, reaching 517.62°/RIU at 2 ng/mL and 482.82°/RIU at 1 ng/mL, with quality factors above 120 RIU⁻¹ in the same regime. Composite indicators (figure of merit and contrast signal factor) peak at intermediate levels, whereas resonance broadening at higher levels reduces the quality factor and increases the inferred limit of detection, evidencing a sensitivity–resolution trade-off. Benchmarking against reported SPR platforms indicates that BP-assisted interface engineering provides a competitive low-level operating window within a preliminary refractive-index-sensing framework that is relevant to future biosensor design. These results motivate further experimental validation, including BP stabilization, surface biofunctionalization, and practical implementation under liquid-phase sensing conditions. Full article

Accurate prediction of vibration responses in hydraulic structures during flood discharge is essential for ensuring safe and stable operation. This study develops a hybrid deep learning model that combines Convolutional Neural Networks (CNN), Bidirectional Long Short-Term Memory (BiLSTM), and a Channel Attention (CA) mechanism, optimized through Bayesian Optimization (BO), to predict dam gantry crane beam displacements. Time-lagged Pearson correlation and Maximum Information Coefficient (MIC) are applied to select the informative input features. The CNN-BiLSTM-CA model captures both spatial patterns and temporal dependencies in vibration signals. BO tunes model hyperparameters, while Partial Dependence (PD) analysis provides insight into how these parameters affect prediction accuracy. The model is validated using vibration data from an arch dam in Southwest China during flood discharge. Results show that CNN parameters have a greater impact on prediction accuracy than BiLSTM parameters, underscoring the importance of spatial feature extraction. Ablation studies confirm each component’s contribution. Compared with existing methods, the proposed model achieves superior accuracy with a Root Mean Square Error (RMSE) of 5.49, Mean Absolute Error (MAE) of 4.34, and correlation coefficient (R) of 99.42%. This framework provides a reliable and interpretable tool for predicting structural vibrations in hydraulic engineering under complex discharge conditions. Full article

Screening for stomatal opening enhancers and their application via foliar spraying represents a feasible strategy to increase CO₂ assimilation flux by augmenting stomatal aperture, thereby enhancing photosynthesis and promoting plant growth. However, the lack of relevant research on forage crops has significantly limited the implementation of this strategy in forage production. In this study, using vetch (Vicia sativa) and alfalfa (Medicago sativa) as experimental materials, we first established a stable observation system tailored for evaluating stomatal opening regulation in forages: incubating abaxial epidermal peels in a solution containing 0.5% KCl (pH 6.0) under light conditions for 4 h resulted in stably opened stomata. Utilizing this system, we systematically screened the regulatory effects of 14 stomatal opening modulators, including signaling molecules, phytohormones, and amino acids. The results indicated that stomatal opening in both vetch and alfalfa exhibited pronounced concentration-dependent and species-specific responses to the modulators. Supplementation with appropriate concentrations of EGTA, GA₃, MT, His, and Pro significantly promoted stomatal opening in vetch, with increases ranging from 21% to 35%. In contrast, appropriate concentrations of Ca²⁺, H₂O₂, MJ, His, Glu, Met, Arg, and Ala effectively enhanced stomatal opening in alfalfa, with increases of 8% to 34%. To further validate the reliability of the screening system, we selected Met, which showed no regulatory effect on vetch stomata but enhanced opening in alfalfa, for foliar application validation. The results demonstrated that Met treatment had no significant effect on stomatal aperture in vetch but significantly increased it in alfalfa, consistent with the initial screening results. This consistency further confirmed the reliability of our established screening system for identifying stomatal opening enhancers in forages. Correspondingly, foliar Met application did not affect vetch growth but significantly promoted alfalfa growth, increasing biomass by 18%. In conclusion, this study established a stable screening system for stomatal opening enhancers specifically for vetch and alfalfa and successfully identified several species-specific enhancers using this system. Foliar application of these species-specific enhancers effectively increased stomatal aperture and promoted growth in target forage species, demonstrating promising potential for enhancing forage yield. Full article

Neuron-targeted therapies for Alzheimer’s disease (AD) have shown limited efficacy, highlighting the need to explore glial-based mechanisms of neuroprotection. Here, we show that astrocyte mitochondrial uncoupling via viral overexpression of uncoupling protein 4 (UCP4) restores neuronal circuits and ion channel function in aged 3xTG AD mice with overt symptoms. Spontaneous local field potential recordings revealed a partial recovery of hippocampal and subicular sharp wave ripple oscillations, electrophysiological signatures of neuronal circuits known to be altered in AD. Combined whole-cell patch-clamp electrophysiology with two-photon Ca²⁺ imaging further demonstrated that UCP4 modulates activity-dependent Ca²⁺ influx, A-type potassium channel function, and enhances glial cell line-derived neurotrophic factor (GDNF) signaling. These findings identify astrocytic mitochondrial uncoupling as a potent mechanism enhancing neuronal resilience and restoring circuit function in symptomatic AD brains. Full article

Cytotoxic CD8 T lymphocytes are crucial in antiviral immune responses. However, their recruitment to infection sites renders them at risk of viral infection, which could affect their effector activity. CD8 T lymphocytes express RIG-I, which detects cytosolic viral RNA and subsequently induces antiviral gene expression. We investigated how Influenza A virus infection and synthetic triphosphorylated double-stranded RNA, a specific RIG-I ligand, influence TCR-dependent effector responses in primary human CD8 T cells. Cells were isolated from healthy donors and either infected with the reassortant virus RG-PR8-Brazil78 (H1N1) or transfected with the synthetic RNA. Proliferation, degranulation, and cytokine production upon anti-CD3/CD28 stimulation were assessed using flow cytometry and intracellular cytokine staining. Type I IFN production and downstream signaling were measured using IFN-I reporter assay and Western blotting. CRISPR/Cas9 gene editing was employed to knock out RIG-I and STAT2 to evaluate their roles in antiviral responses. Influenza A virus infection of CD8 T cells stimulated RIG-I and activated downstream pathways, including TBK1 and NF-κB, resulting in type-I interferon secretion. Transfection of cytotoxic CD8 T lymphocytes with synthetic RIG-I ligands not only stimulated these pathways but also enhanced the proliferation of CD8 T cells in vitro and protected them from influenza A virus infection. In line with a positive effect on CD8 effector function, both influenza A virus infection and RIG-I ligand transfection enhanced CD8 T cell degranulation and cytokine secretion. Conversely, activation of CD8 T lymphocytes via CD3/CD28 crosslinking increased their susceptibility to influenza A virus infection. We demonstrated that RIG-I stimulation by virus infection or RIG-I ligand transfection promotes intrinsic antiviral pathways and enhances CD8 T-cell effector functions and proliferation. This suggests that RIG-I agonists could enhance and prolong the effector function of cytotoxic CD8 T lymphocytes in immunotherapy. Full article

In patients, extraintestinal manifestations of inflammatory bowel disease (IBD) are attenuated ventricular contractile function and arrhythmia. To determine the mechanism of IBD-induced changes in ventricular function, we used a mouse model of dextran sodium sulfate (3.5% weight/volume; 7 days)-induced colitis. Changes in cardiac function were quantified in isolated ventricular myocytes (VM) by cell shortening, imaging of [Ca²⁺]_i, reactive oxygen species (ROS), and t-tubular density. During colitis, VMs exhibited attenuated cell-shortening and altered Ca²⁺-handling properties. A prolonged Ca²⁺ transient (CaT) rise time correlated with an increased coefficient of variation in the subcellular Ca²⁺ release and an attenuated t-tubular density. T-tubular loss was accompanied by increased ROS production, calpain-2 (CAPN2) expression, junctophilin-2 (JPH-2) cleavage, and autophagy. Inhibition of Angiotensin-converting enzyme during colitis (Perindopril: 3 mg/kg/day) prevented the increase in CAPN2, ROS production, autophagy, and t-tubular remodeling. It failed, however, to restore full length JPH-2. We conclude that, during IBD, the angiotensin II (AngII)-induced loss of t-tubular integrity and altered cellular Ca²⁺ handling can be prevented by suppression of the AngII-dependent increase in CAPN2 and autophagy and thus suppression of AngII signaling might benefit IBD patients with cardiac manifestations of the disease. Full article

The diamondback moth (Plutella xylostella) severely threatens global oilseed rape (Brassica napus L.) production. This study demonstrates that CRISPR/Cas9-mediated knockout of two homologous BnaFAH, involved in tyrosine degradation, confers enhanced Brassica napus resistance to Plutella xylostella under a 2-day short-day (SD2) photoperiod. Multi-omics analyses revealed that this resistance is associated with a coordinated response: BnaFAH deficiency triggers reactive oxygen species (ROS) accumulation, which is closely associated with activating the jasmonic acid (JA) biosynthetic and signaling pathways. This led to significant upregulation of key JA biosynthetic genes and accumulation of JA, its precursors (OPDA, OPC-4, and OPC-6), and bioactive conjugates (JA-Ile and JA-Phe). Pharmacological analyses support the central role of JA, as exogenous application of methyl jasmonate (MeJA) enhanced insect resistance, whereas the JA biosynthesis inhibitor DIECA suppressed resistance. Scavenging ROS with sodium selenite prevented both JA pathway upregulation and insect resistance, suggesting that ROS may act upstream to activate the JA biosynthetic and signaling pathways. These findings support a previously unrecognized “photoperiod-dependent ROS-JA” defense module, revealing how metabolic perturbation under specific environmental cues can be co-opted to enhance plant immunity, offering new targets for breeding resistant rapeseed varieties. Full article

Background/Objectives: Primary dysmenorrhea is a common gynecological disorder characterized by painful uterine contractions. Danggui Buxue Decoction (DBD) is used to treat menstrual irregularities, but its mechanism in primary dysmenorrhea remains unclear. This study investigated the efficacy of DBD against dysmenorrhea and its calcium signaling-related mechanism. Methods: DBD components were analyzed by UPLC–Orbitrap MS. Isolated uterine muscle strips precontracted with oxytocin (OT, 50 ng/mL) or KCl (60 mM) were used to assess the effects of DBD and its active compounds (Quercetin, Formononetin, Ononin, Ferulic acid, Senkyunolide I, Calycosin, Ligustilide, Calycosin-7-O-β-D-glucoside). Ca²⁺-dependent experiments, intracellular calcium release assays, and inhibitor treatments (Nifedipine, 2-APB) were performed to evaluate the involvement of L-type calcium channels and the IP₃R pathway. A primary dysmenorrhea model induced by estradiol benzoate and oxytocin was used to assess the analgesic effects, histopathology, inflammatory factors, and IP₃/Ca²⁺-related proteins and genes following DBD and Quercetin treatment. Results: A total of 161 compounds were identified in DBD. DBD and its eight active constituents relaxed OT (50 ng/mL) or KCl (60 mM)-induced uterine contractions, with Quercetin, Calycosin, and Ligustilide showing particularly prominent relaxant activity. These three compounds suppressed extracellular calcium influx and intracellular calcium release through the blockade of L-type calcium channels and IP₃R. In vivo, DBD and Quercetin alleviated pain, reduced inflammation, and decreased uterine Ca²⁺ and IP₃ levels in dysmenorrhea mice. Conclusions: DBD and its active component Quercetin promote uterine relaxation by lowering Ca²⁺ levels, which is achieved through suppression of L-type calcium channels and the IP₃/Ca²⁺ pathway. This contributes to their therapeutic action against primary dysmenorrhea. Full article

Chronic hypoxia is a hallmark of aging and retinal diseases such as age-related macular degeneration (AMD), yet the molecular mechanisms that enable retinal pigment epithelium (RPE) cells to survive under sustained low-oxygen conditions remain poorly understood. To address this, we conducted transcriptomic profiling and a genome-wide CRISPR-Cas9 loss-of-function screen in ARPE-19 cells exposed to chronic hypoxia (1% and 5% O₂), mimicking the retinal disease environment. The CRISPR screen identified genes whose loss compromises RPE viability or fitness under hypoxia, while transcriptomic profiling revealed oxygen-dependent shifts in key functional modules. These findings converged on pathways related to mitochondrial function, extracellular matrix remodeling, vascular signaling, and cell cycle regulation, identifying unique functional nodes specific to RPE cells. These core processes are also implicated in retinal diseases, such as AMD. Together, these complementary approaches provide an integrated view of the molecular networks driving RPE adaptation to hypoxic stress and highlight novel gene candidates that may serve as therapeutic targets in retinal disease. Full article

CDKL5 Deficiency Disorder (CDD) is a severe neurodevelopmental encephalopathy characterized by early disruptions of synaptic maturation and network stability, leading to persistent motor, cognitive, and behavioral impairments. Given the role of the endocannabinoid system in synaptic development, neuroinflammation, and neuronal resilience, we investigated whether the sustained enhancement of endogenous 2-arachidonoylglycerol (2-AG) signaling via monoacylglycerol lipase (MAGL) inhibition could mitigate key pathological features in adult Cdkl5 knockout (KO) mice. Using an intermittent 6-week treatment, the MAGL inhibitor JZL184 robustly increased plasma 2-AG levels, reduced MAGL protein levels, and activated CB1-AKT signaling without evidence of receptor desensitization. Despite this clear pharmacodynamic efficacy, behavioral effects were domain-specific: neither dose ameliorated core behavioral deficits, although the higher dose selectively reduced stereotypic jumping and modestly improved cue-dependent associative memory. At the cellular level, JZL184 induced biologically meaningful effects, partially restoring dendritic spine maturation in the primary somatosensory cortex and increasing neuronal survival in the vulnerable CA1 hippocampal region. In contrast, microglial responses were dose-dependent and divergent, with the lower dose exerting anti-inflammatory effects, while the higher dose increased cortical microglial density and Allograft Inflammatory Factor-1 (AIF-1) expression, suggesting engagement of compensatory or off-target mechanisms. Overall, these findings show that MAGL inhibition activates neuroprotective pathways and ameliorates select structural deficits in adult Cdkl5 KO mice, but is insufficient to produce broad behavioral recovery, highlighting the domain-specific effects of selective 2-AG enhancement via MAGL inhibition and the need for developmentally informed or multimodal therapeutic strategies in CDD. Full article

Intervertebral disc degeneration is characterized by inflammation, extracellular matrix breakdown, and neurovascular ingrowth, processes that contribute to discogenic, chronic back pain. The transient receptor potential canonical 6 (TRPC6) channel is a calcium-permeable ion channel implicated in inflammation and pain signaling in multiple tissues; however, its functional role in human disc cells remain unknown. Here, we investigated the expression, activation, and downstream consequences of TRPC6 activation using Hyp9, a pharmacological activator of TRPC6. TRPC6 transcripts were consistently detected across all donors examined (n = 17). Functional TRPC6 activation induced a rapid, dose-dependent calcium (Ca²⁺) influx across 0.5–100 µM Hyp9. TRPC6 activation did not reduce metabolic activity or increase cytotoxicity at concentrations commonly used for in vitro TRPC6 activation. Mechanistically, TRPC6 activation induced mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways, as demonstrated by increased phosphorylation of p38 and extracellular signal-regulated kinase (ERK), degradation of the inhibitor of κB-alpha (IκB-α), and increased nuclear translocation of the NF-κB p65 subunit. Downstream of these early signaling events, TRPC6 activation elicited a robust inflammatory and catabolic response with upregulation of IL-6, IL-8, COX-2, MMP-1, MMP-3, NGF, and VEGF, with corresponding increases in protein secretion. These findings identify TRPC6 as an important signaling node linking calcium influx to inflammatory, catabolic, and neuro- and angiogenesis-associated pathways in disc cells, highlighting TRPC6 as a potential therapeutic target in degenerative disc disease. Full article