Muscle-specific kinase (MuSK) is a receptor tyrosine kinase essential for neuromuscular junction (NMJ) formation and maintenance, yet its regulation remains poorly understood. Crystallographic studies of wild-type MuSK revealed an autoinhibited conformation with tyrosines in the activation loop (A-loop) anchored within the catalytic cleft to stabilize the closed, inactive conformation. We showed previously that additional phosphorylation of an A-loop serine may "prime" MuSK for activation to sensitize it to ligand(s) in certain settings. Here, we employed crystallography, biochemical assays, and hydrogen-deuterium exchange and mass spectrometry (HDX-MS) to test this hypothesis. We found that introducing a phosphomimetic S752D mutation disrupts autoinhibitory A-loop interactions to increase ATP-binding affinity and catalytic turnover. Using HDX-MS, we further observed that the S752D mutation increases A-loop structural flexibility to relieve autoinhibition. The S752D mutation also stabilizes the juxtamembrane (JM) NPXY motif region, a docking site for the adaptor Dok7, possibly priming MuSK for downstream signaling. Together, these findings reveal dynamic transitions that underlie relief of MuSK autoinhibition and provide a mechanistic framework for understanding MuSK activation at the NMJ.
Despite the prominence of laboratory rats in behavioral neuroscience and complex trait genetics, a significant gap exists in the functional rat genomics database that explains the regulation of gene expression at the genome level. To address this, we analyzed genome-wide Hi-C data from the frontal cortex of ten strains in the Hybrid Rat Diversity Panel. While originally generated to improve the rat genome assembly, these data provided a unique opportunity to characterize the regulatory landscape of the adult rat brain. We identified an average of 5,899 ± 1,997 (STD) loops per sample and integrated these with over 3 million curated CTCF binding sites, which serve as architectural anchors for chromatin loops. Our multi-stage filtering workflow identified 15,085 unique, high-confidence regulatory interactions throughout the genome. As a validation of our approach, we observed that genes with the highest loop counts, including Foxo1, Gja1 , and Spry2 , were significantly enriched for developmental processes, reflecting the role of 3D genome structure in maintaining adult neuronal identity. The resulting resource addresses a critical deficiency in rat functional genomics and provides a foundation for dissecting the genetic architecture of complex traits.
Effective management of infected wounds demands biocompatible and breathable dressings capable of real-time monitoring and autonomous therapeutic regulation, which conventional passive dressings fail to provide. Here, we present a permeable liquid metal (LM)-based fibrous patch (LMFP) that integrates closed-loop temperature-humidity monitoring with on-demand photothermal antibacterial therapy. The LM mechanoelectrical dual-bond interconnects, achieved through graphene oxide (GO) and its oxygen-containing groups, establish a stable and conductive LM-sensor coupling that ensures precise temperature-humidity sensing under humid, dynamic wound environments. Comprehensive in vitro and in vivo evaluations demonstrate its validity of wound monitoring, accelerated healing, and intelligent closed-loop wound management. This work introduces a next-generation intelligent wound patch integrating breathable architecture, adaptive sensing, and responsive therapy, offering substantial advantages for enhanced infection control and tissue regeneration in sensitive and vulnerable skin environments.
Vibrio parahaemolyticus (V. parahaemolyticus), a significant pathogen in aquatic products, necessitates rapid and precise detection to prevent foodborne disease outbreaks. Early detection of bacterial pathogens is crucial for effective prevention and control. In this study, we developed a field-deployable, "sample-in, answer-out" workflow that integrates efficient DNA extraction with visual fluorescence loop-mediated isothermal amplification (LAMP) for the on-site detection of V. parahaemolyticus. This extraction protocol enables efficient one-pot DNA release within 10 min, eliminating the need for additional purification steps. In addition, coupled with an optimized fluorescence LAMP assay targeting V. parahaemolyticus, the entire process-from sample to result-can be completed within approximately 50 min. The optimized assay exhibited high sensitivity, detecting as low as 1 copy/µL of plasmid DNA and 10 CFU per reaction from pure bacterial culture and spiked seawater samples. This extraction-amplification integrated approach demonstrates robust performance and high practicality for on-site early warning and pathogen monitoring in aquaculture safety settings.
Congestion is the dominant driver of hospitalization in acute heart failure and relief of congestion remains a central therapeutic target. Loop diuretics are first-line therapy for decongestion, yet their dosing and escalation in routine practice often rely on subjective bedside assessment, variable urine output thresholds, weight change, and delayed laboratory trends approaches that incompletely capture the primary pharmacodynamic goal of diuretic therapy: natriuresis. Neurohormonal activation and renal sodium avidity in heart failure can lead to poor diuretic response despite apparently adequate diuretic dosing, and persistent congestion is consistently associated with adverse outcomes. In this context, early measurement of spot urinary electrolytes especially urinary sodium, and potentially urinary chloride and urinary creatinine offers an objective, rapid method to quantify natriuretic response and identify inadequate decongestion early enough to adjust therapy. This review summarizes the pathophysiologic basis linking renal sodium handling to congestion, critiques traditional metrics used to titrate diuretics, synthesizes the clinical evidence supporting spot urinary sodium-based assessment (observational cohorts and emerging randomized/protocolized strategies), and outlines pragmatic implementation considerations, including confounders such as chronic kidney disease, concomitant SGLT2 inhibitors, and timing of sampling. While natriuresis-guided strategies reliably improve natriuresis and process-of-care metrics, definitive evidence for improved hard outcomes remains evolving, underscoring the need for standardized protocols and larger outcomes trials.
Elevated DNA replication stress is a common feature of cancer cells, rendering them dependent on ATR/Chk1 signaling, which controls the DNA replication stress checkpoint, for survival. Although the activation of ATR/Chk1 signaling is well-established, how this signal is maintained until replication stress is fully resolved is less understood. Here, we investigated the roles of RAD9-HUS1-RAD1 interacting nuclear orphan 1 (RHNO1) in cancer progression and its involvement in maintaining ATR/Chk1 signaling during the DNA replication stress response. We demonstrate that depletion of RHNO1 significantly inhibits cancer cell proliferation in vitro and tumor growth in vivo . Mechanistically, we show that, while RHNO1 is dispensable for initial ATR/Chk1 signal activation, it is upregulated and stabilized following replication stress. RHNO1 is required to sustain ATR/Chk1 signaling, prevent premature checkpoint collapse, and suppress genomic instability. Under basal conditions, RHNO1 is rapidly degraded by the proteasome, however, RHNO1 is phosphorylated and stabilized following DNA replication stress. Stabilization of RHNO1 is mediated by ATR/Chk1 signaling which promotes RHNO1 localization to stressed replication forks marked by phosphorylated RPA32. Together, our data reveals a novel positive feedback loop wherein ATR/Chk1 signaling activation stabilizes RHNO1, which, in turn, is required to sustain the signal and thus the replication stress response. This work identifies RHNO1 as a key component in the cellular replication stress response and highlights its potential as a therapeutic target for tumor cells reliant on ATR/Chk1 signaling.
Two-photon lithography fabricates three-dimensional structures with 100-nanometer resolution; yet its industrial adoption is hindered by poor reproducibility and the need for complex manual tuning processes. While post-fabrication metrology, such as scanning electron microscopy, characterizes final morphologies, it cannot prevent manufacturing errors. Here we show a two-photon lithography platform powered by a single-cavity dual-comb laser that addresses this limitation through real-time correction. During fabrication, one laser comb, after frequency conversion, performs two-photon printing, while the dual-comb system simultaneously performs in-situ phase measurements across the full work field at 360 hertz. By feeding the phase profiles into a dynamic model, the platform automatically modulates printing parameters to correct height errors. We demonstrate this capability through a continuous 14-hour fabrication process to manufacture different millimeter-scale diffractive optical elements with less than 100-nm absolute errors. The resulting devices exhibit superior signal-to-noise ratios, process repeatability, and focus quality. This closed-loop dual-comb platform offers a cost-effective, scalable solution for high-precision, high-yield nanomanufacturing.
Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer, which is difficult to diagnose and treat due to lack of biomarkers, high metastasis and recurrence. Ribosomal protein S6 kinase (RSK2) is frequently overexpressed and hyperactivated, thereby regulating proliferation and metastasis in many solid cancers including breast cancer. However, the underlying mechanisms of RSK2 in TNBC metastasis are poorly understood. Here, we explored the role of RSK2 in migration and invasion of TNBC. Our findings revealed that overexpression of RSK2 promoted Epithelial-mesenchymal transition (EMT) to induce migration and invasion of TNBC cells MDA-MB-231 and 4T1, whereas RSK2 knockdown decreased them. Mechanically, our results showed that RSK2 positively regulated the MEK/ERK pathway and enhanced GALNT5 mRNA expression and stability by RNA-seq analysis and experimental validation. Furthermore, knockdown of GALNT5 attenuated the MEK/ERK pathway. Finally, the in vivo results found that RSK2 promoted growth and EMT of TNBC. Collectively, our findings indicated that the MEK/ERK/RSK2 positive feedback loop promoted EMT to induce migration and invasion of TNBC via enhancing GALNT5 mRNA stability, and it was suggested that RSK2 may provide a novel therapeutic target for TNBC patients.
Ventricular preexcitation causes subclinical LV dysfunction despite preserved LVEF. Pressure-strain loop (PSL) analysis, a novel method, sensitively quantifies myocardial work to assess such functional alterations. This study evaluated global and segmental myocardial work alterations by PSL in these patients.MethodsSeventy-seven patients with ventricular preexcitation (stratified into right-AP [n = 33], left-AP [n = 16], and septal-AP [n = 28] groups) and 25 controls underwent speckle-tracking echocardiography. Global longitudinal strain (GLS) and mechanical dyssynchrony indices-the standard deviation of time-to-peak strain (PSD) and maximal temporal difference (ΔT)-were quantified. PSL analysis derived myocardial work parameters (work efficiency [WE], constructive work [CW], wasted work [WW]) at global, basal-apex, and segmental levels, with focused assessment of six basal LV segments. Septal AP and right AP demonstrated significantly increased global and basal-middle ventricular WW along with reduced WE compared to controls (all P < 0.01). Additionally, the GLS in right AP was mildly less negative than in controls (P < 0.05). PSD and ΔT were significantly elevated in the basal-middle ventricular levels of septal and right AP compared to controls (P < 0.05). Furthermore, PSD exhibited a moderate negative correlation with GWE (r = -0.59, P < 0.001). Multivariate linear regression analysis identified PSD as an independent factor associated with increased GWW (β = 1.71, P = 0.03), decreased GWE (β = -0.136, P < 0.001), and reduced GCW (β = -7.93, P < 0.001). PSL analysis effectively identifies subclinical left ventricular dysfunction in ventricular preexcitation patients with preserved LVEF, particularly pronounced in right-sided and septal accessory pathway subgroups. Ventricular mechanical dyssynchrony emerges as a key mechanistic contributor to these functional impairments, highlighting its pivotal role in early-stage electromechanical uncoupling.
Leaf senescence is a highly regulated biological process that marks the final stage of leaf development. The initiation and progression of this process are precisely controlled by complex regulatory networks involving numerous endogenous factors. Here, we demonstrate that ONAC005 functions as a negative regulator of the onset of leaf senescence. ONAC005 expression gradually declines during both natural (NS) and dark-induced (DIS) senescence. onac005 mutants generated by T-DNA insertion- and CRISPR/Cas9-mediated mutagenesis exhibited precocious leaf yellowing under NS and DIS conditions, while ONAC005-overexpressing plants retained leaf greenness much longer than the wild type. ONAC005 overexpression downregulates the expression of senescence-associated genes (SAGs) and chlorophyll degradation genes (CDGs), whereas onac005 mutation upregulates their expression. ONAC005 reduces the response to abscisic acid (ABA), thereby delaying ABA-induced senescence. Through in vivo and in vitro DNA-binding assays, we demonstrated that ONAC005 directly binds to the promoters of OsNAP and OsNAP-regulated CDGs, leading to transcriptional repression. Furthermore, we identified specific DNA motifs that ONAC005 preferentially recognizes. Taken together, these results suggest that ONAC005 acts as a repressor of leaf senescence by repressing the OsNAP-mediated senescence signaling pathway.
Road salt contamination is an increasing environmental concern in cold-climate cities, where repeated applications lead to long-term chloride accumulation in soils and waterways. Salt-tolerant vegetation (i.e., halophytes) can help intercept this chloride, but the resulting salt-laden biomass presents a secondary management challenge. This study evaluates the feasibility of recovering chloride from the biomass of the grasses Panicum virgatum and Sporobolus michauxianus using two water-based leaching approaches. Passive rinsing removed up to 70% of chloride at high water volumes, while active leaching achieved near complete (≈100%) recovery with fourfold less water. Based on plant densities and tissue chloride concentrations measured at a Canadian field site, preliminary estimates indicate that halophyte stands could recover 4-21 g Cl-/m2 annually, comparable to the salt applied during a single winter road treatment. To our knowledge, this study is the first to demonstrate a scalable approach to reclaim road salt from phytoremediation biomass. Scaling considerations-including biomass collection and transport, volume reduction, dewatering, pre-treatment, water reuse, and brine concentration-suggest that the system could be integrated into existing vegetation management infrastructure. Life-cycle assessment and cost-benefit analysis are significant next steps. Implementing circular biomass-based salt recovery could reduce salt procurement, improve resilience to supply disruptions, and support more sustainable winter maintenance practices.
Low anterior resection syndrome (LARS) is a common functional problem after sphincter-preserving rectal cancer surgery and includes urgency, frequent bowel movements, clustering, and fecal incontinence. Diverting ileostomy may further disrupt the intestinal environment and alter the gut microbiota, potentially worsening bowel dysfunction after ileostomy closure. However, evidence remains limited on whether bowel stimulation with probiotics before ileostomy closure can improve postoperative bowel function and reduce LARS severity. This study aims to evaluate the safety, feasibility, and efficacy of probiotic bowel stimulation through the distal limb of a diverting ileostomy before ileostomy closure in patients with rectal cancer. This single-center randomized controlled trial will be conducted at Keimyung University Dongsan Medical Center, Republic of Korea. Eligible participants are adults aged 18-80 years with clinical stage II or III rectal adenocarcinoma who completed neoadjuvant chemoradiotherapy and underwent laparoscopic or robotic low anterior resection with total mesorectal excision and diverting ileostomy, and who are scheduled for elective ileostomy closure. Participants will be randomly assigned in a 1:1 ratio to receive either 250 mL of normal saline with 4 g of Lacidofil or 250 mL of normal saline alone via the distal limb of the ileostomy once daily for 2 weeks before closure. The primary outcome is the LARS score 3 months after ileostomy closure. Secondary outcomes include postoperative complications, bowel recovery, stool habits, laboratory findings, and length of hospital stay. Analyses will primarily follow the intention-to-treat principle. The study was approved by the Institutional Review Board of Keimyung University Dongsan Medical Center (DSMC-2024-03-016) and registered with the Clinical Research Information Service (KCT0011052). Recruitment is planned to begin in March 2026 and is expected to continue through March 2029. At the time of manuscript submission, the study is in the pre-enrollment stage, with no participants recruited and no data analysis performed. Results are expected to be published in 2029. This trial will provide prospective evidence on whether probiotic bowel stimulation before ileostomy closure is a safe and effective strategy for improving postoperative bowel function and alleviating LARS in patients undergoing rectal cancer surgery with diverting ileostomy.
A balanced immune response is required to limit the microbes without causing damage to the host. The forkhead box O (FoxO)-mediated immunity plays a pivotal role in maintaining microbiota homeostasis by regulating the expression of antimicrobial effectors in non-infected arthropods. However, the mechanism by which FoxO activity is appropriately coordinated remains unclear. In this study, we elucidated a feedback loop that coordinates FoxO-mediated antibacterial response using shrimp as a model. In this feedback loop, the commensal hemolymph microbiota maintains basal activation of FoxO, which determines the expression of antimicrobial effectors, platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF)-related factor 1 (Pvf1) and PDGF/VEGF-related receptor 4 (Pvr4). This ligand-receptor system enhances phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt) activity, limiting the excessive activation of FoxO and expression of antimicrobial effectors. This feedback loop is essential for maintaining the equilibrium of the microbiota, and its strength increases following a pathogenic infection, reducing the incidence of infection-induced mortality and tissue damage. This study revealed a microbiota-initiated feedback loop that balances FoxO-mediated antibacterial immunity for the establishment of microbiota homeostasis, and provides new insights into the functional diversification of PDGF/VEGF signaling.
Simulation plays a crucial role in assessing autonomous driving systems, where the generation of realistic multi-agent behaviors is a key aspect. In multi-agent simulation, the primary challenges include behavioral multimodality and closed-loop distributional shifts. In this study, we formulate a unified mixture model (UniMM) framework revisit mixture models for generating multimodal agent behaviors, which can cover the mainstream methods including regression-based continuous mixture models and discrete NTP GPT-like discrete models. Furthermore, we introduce a closed-loop sample generation approach tailored for mixture models to mitigate distributional shifts. Within the UniMM framework, we recognize critical configurations from both the model and data perspectives. We conduct a systematic examination of various model configurations, and comprehensively characterize their effects including positive component matching, continuous regression, prediction horizon, and the number of components. Moreover, our investigation into the data configuration highlights the pivotal role of closed-loop samples in achieving realistic simulations. To extend the benefits of closed-loop samples across a broader range of mixture models, we further introduce a temporal disentanglement-and-alignment mechanism to address the shortcut learning and off-policy learning issues. Leveraging insights from our exploration, the distinct variants proposed within the UniMM framework, including discrete, anchor-free, and anchor-based models, all achieve state-of-the-art performance on the WOSAC benchmark.
CRISPR-Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA at specific sites and has been adapted as a powerful tool for genome manipulation. Cas9 recognizes its target through multiple conformational transitions coordinated between the Cas9 ribonucleoprotein and the DNA duplex. Such transitions, and consequently Cas9 targeting specificity, are expected to be significantly influenced by the collective duplex physical properties referred to as DNA shape. To advance our currently limited understanding of the interplay between DNA shape and Cas9 target interrogation, we solved two cryo-EM structures of SpyCas9 bound to a cognate target embedded in a relaxed 95-base-pair DNA double-stranded minicircle. The Cas9-bound DNA segment engages in similar interactions involved in PAM-binding and R-loop initiation as those observed in Cas9-bound linear DNA. However, R-loop is limited to less than three base-pairs, thus interfering with Cas9 cleavage. The minicircle DNA, which is fully resolved, retains its global shape. As Cas9 locally unwinds the protospacer, the closed-ring topology constrains the movement of the paired PAM-distal DNA duplex, thus interfering with R-loop propagation. These data provide detailed insight into the interplay between DNA shape and Cas9 structure and function, and may shed light on genome-editing and manipulation in environments with varied DNA topologies.
All-atom simulations of RNA using molecular dynamics have the promise of modeling conformational preferences, folding thermodynamics, conformational change kinetics, and binding affinities of small molecule therapeutics. These simulations rely on a force field, a set of equations and parameters that model the potential energy as a function of conformation using classical mechanics. One popular force field for RNA is Amber OL3, with the most recent iteration derived in 1999 and with subsequent updates to backbone dihedral parameters. The Amber force field, while frequently used, is known to have limitations; for example, it does not properly stabilize native structures against alternative structures. Here, we provide a new approach to fitting the non-bonded parameters for the force field, specifically atom-centered point charges for electrostatics and the Lennard-Jones parameters. The parameters are fit to quantum mechanics (QM) interaction energies calculated with symmetry-adapted perturbation theory (SAPT), including embedded point charges to represent the electrostatic field from solvent and adjacent nucleotides. In this pilot study with a limited set of fitting data, we use the Amber ff99 equations and atom types unchanged. With the revised parameters, we observe improvement in the stability of native structures relative to alternative structures. Native tetraloop conformations, which unfold with the Amber OL3 force field, are stable on the microsecond timescale with our new force field parameters. We also see improvement in the conformational preferences of tetramers. Crucially, A-form helices are still well-modeled, but we observe additional flexibility in an internal loop that is not consistent with NMR data. Overall, we provide evidence that this new approach to fitting RNA force field parameters to SAPT interaction energies with native-structure context represented as embedded point charges is promising. It offers a flexible solution for revising the equations in future work or for extension to other molecules that interact with RNA, such as proteins and small molecules. We call this new set of force field parameters Amber RNA.ROC26.
Neuroinflammation plays a critical role in the pathogenesis of neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis. This review explores the underlying mechanisms of neuroinflammation, with a focus on the roles of microglia, astrocytes, peripheral immune cell infiltration, and cytokine release. The complex interplay between oxidative stress and chronic inflammation accelerates neurodegeneration, contributing to disease progression. Microglial activation, initially protective, becomes harmful when sustained over time, driving chronic inflammation. Similarly, astrocytes once considered passive are now recognized for their active involvement in both inflammatory and neuroprotective responses, depending on their activation state. Prolonged activation of microglia and astrocytes compromises blood-brain barrier integrity, facilitating immune cell infiltration and amplifying inflammatory cascades, thereby worsening neuronal damage. We also detail the molecular mediators of the vicious cycle linking oxidative stress and chronic inflammation, including redox-sensitive transcription factors and mitochondrial dysfunction. Furthermore, the reviewed mechanisms highlight a pathogenic feedback loop, wherein oxidative stress and neuroinflammation fuel each other through NF-κB and Nrf2 pathways, suggesting that dual-target therapeutic strategies may be most effective. This review synthesizes evidence to propose that the failure to resolve neuroinflammation, driven by a self-reinforcing loop between oxidative stress and glial dysfunction, represents a critical, targetable node common to PD, AD, and MS.
Conformationally responsive DNA nanoswitches have previously been developed and validated for a variety of biosensing applications including detection of DNA, microRNA, and viral RNA/DNA. Here we develop new methodology for enhancing the sensitivity of DNA-based sensing by recycling a fixed number of targets for repeated reuse. We achieved target-dependent enzymatic ligation of looped nanoswitches and showed that subsequent removal of target does not affect the ligated loop. Through cyclic annealing, ligation, and target removal, we can linearly control signal amplification up to hundreds of cycles. This method adds an important new capability for low abundance targets without the need for target amplification.
Vitamin K epoxide reductase (VKORC1) catalyzes the reduction of vitamin K epoxide to quinone and hydroquinone, a cofactor essential for the activation of clotting factors. Mutations in VKORC1 are known to confer resistance to vitamin K antagonists (VKAs) such as warfarin in both humans and rodents, yet the structural mechanisms underlying these resistances remain poorly understood. Using AlphaFold3-predicted structures, molecular docking and molecular dynamics simulations, we investigated the effects of four major resistance-causing mutations - W59G and L128S in Mus musculus, L120Q and Y139C in Rattus norvegicus - on VKORC1 structure and warfarin binding. Three mutations (W59G, L120Q and L128S) disrupted a hydrophobic cluster linking the ER luminal loop to transmembrane helices TM3-TM4, inducing and ER loop drift and altering helices orientation. These structural changes reduced warfarin affinity compared to the wild-type enzyme. In contrast, the Y139C mutation caused minimal perturbation and preserved wild-type binding energy, suggesting that it may confer resistance through a different mechanism. Collectively, six hydrophobic residues (V54, W59, L120, I123, L124, L128) were identified as a conserved structural pattern critical for VKORC1 stability and inhibitor sensitivity. This integrative structural study provides a mechanistic framework for understanding VKA resistance in VKORC1. It highlights a hydrophobic core essential for enzyme integrity and drug binding, offering a molecular basis for monitoring emerging mutations and for designing next-generation anticoagulants active against resistant VKORC1 variants.