Gram-negative bacteria pose a threat to global healthcare mainly because their outer membrane (OM) provides an intrinsic barrier to many antimicrobials. Key to this barrier function is the asymmetric structure of the OM, with phospholipids constituting the inner leaflet and lipopolysaccharides, the outer leaflet. Although the mechanism of phospholipid transport between the inner membrane (IM) and OM remains poorly understood, recent studies implicate TamB, YhdP, and YdbH as functionally redundant proteins mediating this process in Escherichia coli. Accordingly, the collective loss of these three paralogs is lethal, and any one of them is sufficient for growth. YdbH is anchored to the IM, and its periplasmic repeating β-sheet groove domain interacts with the OM lipoprotein YnbE via β-strand augmentation to form an intermembrane bridge. Additionally, YnbE multimerizes, and the periplasmic protein YdbL is proposed to modulate YnbE multimerization to facilitate its stacking on the C-terminus of YdbH. Here, we demonstrate that excess YdbL specifically inhibits the function of the YdbH-YnbE complex since overexpression of ydbL causes lethality in the ΔyhdP ΔtamB double mutant, but the presence of both ydbH and ynbE in trans abrogates this lethality. We resolve high-resolution structural data for YdbL and ascertain its interaction site with the YnbE C-terminal α-helix, with residues mediating this interface highly conserved and critical for YdbL function. Finally, we show that YdbL is protected from degradation by the protease DegP when complexed with YnbE. Overall, our data support a model in which YdbL ensures proper YdbH-YnbE intermembrane bridge formation by directly interacting with YnbE. The mechanism underlying phospholipid transport between the inner and outer membranes of gram-negative bacteria remains enigmatic. Bacterial bridge-like protein systems such as the YdbH-YnbE complex resemble proteins found at membrane contact sites between eukaryotic organelles. These proteins are proposed to mediate intermembrane phospholipid transport, which is essential for growth of the outer membrane (OM). Here, we define the role of YdbL, a periplasmic protein that specifically modulates the YdbH-YnbE system. YdbL directly interacts with YnbE and controls the formation of the YdbH-YnbE complex. Additionally, we reveal that YdbL is selectively degraded by the periplasmic protease DegP. We propose a regulatory model that connects the YdbH-YnbE complex assembly and controls the levels of YdbL, providing new insight into OM homeostasis in gram-negative bacteria.
The rising prevalence of polymyxin resistance in multidrug-resistant Klebsiella pneumoniae presents a critical situation with limited therapeutic options. Genomic sequencing of 15 clinical polymyxin-resistant Klebsiella pneumoniae with multidrug resistance revealed MgrB inactivation, predominantly disrupted by insertion sequences in IS1, IS4, IS5 family, was the leading cause of polymyxin resistance. Comparative transcriptomics of WT, ΔmgrB, and ΔmgrBΔphoP were performed to elucidate the MgrB-PhoPQ regulatory network. This study revealed systemwide analysis of the regulatory network, and identified species-specific PhoPQ regulon in Klebsiella pneumoniae. Beyond the classical MgrB-PhoPQ-ArnBCADTEF pathway, we identified a previously unannotated PhoPQ-regulated gene, 144bp-LN739_RS09850, encoding a homolog of Ecr from Enterobacter cloacae. The protein has been reported to confer colistin heteroresistance, and the underlying mechanism hasn't been functionally validated. This study revealed that overexpression of Ecr homologs decreased colistin susceptibility in both Klebsiella pneumoniae and Enterobacter cloacae, but this phenotype was abolished upon phoP deletion, confirming PhoP's essential role. Consistent with this dependency, comparative transcriptomics of Ecr-overexpressing K. pneumoniae versus control revealed significant up-regulation of mgrB, phoPQ, arnBCADTE, and pmrD. Bacterial two-hybrid assays further demonstrated direct Ecr-PhoQ interaction. EMSA confirmed that PhoP directly binds to ecr promoter in vitro, and β-galactosidase reporter assay demonstrated that PhoP enhanced ecr promoter activity, indicating that PhoP regulates ecr expression by directly controlling its transcription. Collectively, these findings suggest that PhoP may directly activate the transcription of Ecr, Ecr feedback activates the PhoPQ system via interaction with PhoQ, leading to induction of the arn operon and consequent polymyxin resistance.
Colorectal cancer (CRC) is a leading cause of cancer death worldwide, mainly due to cancer cell proliferation and migration. Although chondroitin sulfate (CS) is involved in cancer progression, its regulatory mechanisms remain unclear. To investigate the role and mechanism of chondroitin polymerizing factor 2 (CHPF2) and CS in CRC progression, as well as to evaluate the therapeutic potential of ponicidin. The correlation between CHPF2 and prognosis was analyzed in clinical samples. Mechanistically, ponicidin was found to target CHPF2, suppress CS synthesis, and consequently block the Wnt/β-catenin pathway. Its anti-tumor efficacy was validated in cellular, organoid, and animal models. We identified CHPF2, a key enzyme in CS synthesis, as a critical driver of CRC. CHPF2 is significantly overexpressed in CRC tissues, and its high expression correlates with advanced disease stage and poor patient prognosis. Functionally, CHPF2 drives tumor cell proliferation, migration, and survival by enhancing CS production. Mechanistically, CS promotes the activation of the Wnt/β-catenin signaling pathway and epithelial-mesenchymal transition (EMT). This effect is associated with CS-dependent modifications of Wnt1; however, further investigation is required to determine whether Wnt1 is directly modified by CS chains or indirectly affected via CS-modified proteoglycans. Furthermore, the natural diterpenoid ponicidin derived from Rabdosia rubescens directly targets CHPF2 and inhibits its enzymatic activity, thereby reducing CS biosynthesis and subsequently blocking the Wnt/β-catenin signaling pathway. The anti-tumor efficacy of ponicidin was validated in cellular models, patient-derived organoids, and primary CRC models, demonstrating its potent inhibitory effects on tumor growth and metastasis. Our study reveals the oncogenic role of the CHPF2/CS axis in CRC, establishes CHPF2 as a novel therapeutic target, and provides compelling preclinical evidence supporting ponicidin as a promising CHPF2-targeted agent for CRC treatment.
To investigate the factors influencing exercise intention among the people after stroke by developing a comprehensive causal model. This study is the first to examine the factors influencing exercise intention among people after stroke using the Health Action Process Approach (HAPA) theory and structural equation modeling. Data were collected from 299 people after stroke. Perceived benefits and barriers to exercise were evaluated with the Exercise Benefits/Barriers Scale (EBBS). Exercise self-efficacy was assessed using the Exercise self-efficacy scale. Exercise intention was assessed using the Exercise Intention Scale. People after stroke's EBBS score was (112.54 ± 13.67); exercise self-efficacy score was (41.68 ± 10.95); and exercise intention score was (14.41 ± 3.48). The total score of EBBS was positively correlated with the total score of exercise self-efficacy and the total score of exercise intention (r = 0.623, 0.681, both P < 0.05), and the total score of exercise self-efficacy was positively correlated with the total score of exercise intention (r = 0.646, P < 0.05). Structural equation modeling showed several causal pathways. Perceived barriers to exercise had an indirect effect on exercise intentions through exercise self-efficacy (β = -0.183, 95% CI -0.286 to -0.101, P < 0.05). Perceived benefits to exercise not only directly affected exercise intentions (β = 0.246, 95% CI 0.073 to 0.424, P < 0.05), but also indirectly affected exercise intentions through exercise self-efficacy (β = 0.152, 95% CI 0.084 to 0.231, P < 0.05). Additionally, perceived benefits and perceived barriers to exercise negatively influenced each other (β = -0.681, P < 0.05). The perceived benefits and barriers to exercise, exercise intention and exercise self-efficacy level of people after stroke need to be improved. Among them, the influence of exercise self-efficacy on exercise intention is the most significant. Consequently, attention and active measures should be directed toward improving exercise self-efficacy in this population, as this would increase their exercise intention and reduce the risk of relapse.
Recent multicenter studies aim to define the effects of mothers' blood glucose levels during pregnancy on mammary gland function and breast milk composition. Gene expression measured in cells in milk can serve as a liquid biopsy to evaluate the molecular biology of the mammary gland. Critical to this aim is reproducible and high-quality extraction of RNA from milk and harmonized collection protocols from across centers. To address this, we performed a study to optimize milk RNA quality metrics where samples are collected at multiple centers and shipped to a central laboratory for processing and analysis. Lactating mothers provided breast milk following informed consent. The treatments of the samples were as follows: (1) 200 µL of fresh, never frozen milk used as control (FRESH); (2) 1.7 or 5 mL of milk frozen and thawed on ice before adding TRIzol (FRZ); (3) 200 µL of milk frozen and thawed after adding TRIzol (FRZ 200); and (4) 200 µL of milk with 20 µL of RNA preservative added, frozen and thawed after adding TRIzol (FRZ + INH). In all scenarios, RNA was extracted using TRIzol followed by purification with a Qiagen RNeasy Mini kit. For the FRZ 200 and FRZ 200 + INH samples, TRIzol was directly added at the start of thawing, before extraction. Outcomes included RNA concentration, RNA purity (260/280 ratio), RNA fragmentation (DV 200), RNA integrity number (RIN), quantification via Qubit fluorescence-based assays, and visualization of RNA size on an Agilent TapeStation. A RIN cut-off value ≥ 7 indicated acceptable quality for transcriptomics studies. RNA quality metrics were modeled as continuous outcomes using linear mixed-effects regression models. For FRESH milk (n = 15) the estimated marginal mean (EMM) RIN was 7.48 (SE 0.29). For FRZ 200, (n = 22), the EMM RIN was 6.94 (SE 0.26). Samples of FRZ 200 + INH (n = 21) had an EMM RIN of 7.81 (SE 0.26). However, FRZ (n = 19) samples demonstrated markedly reduced RNA integrity with an EMM RIN of 1.92 (SE 0.26). RIN was not significantly different in FRESH versus FRZ 200 or FRZ 200 + INH milk. FRZ 200 + INH samples showed a statistically significant improvement in RIN compared to FRZ 200 (p = 0.0038). RNA quantities were sufficient for sequencing across all treatments. The addition of TRIzol directly to a 200 µL aliquot of milk at the start of thawing provided the highest integrity of extracted milk RNA, as measured by RIN. Adding RNase inhibitor at the time of sample collection, prior to freezing, also enhanced RNA integrity. We have developed a method to optimize the integrity of RNA from frozen human milk samples. This is a crucial methods improvement for multi-center studies where freezing of milk samples is often required prior to RNA extraction and analysis. These results can inform reproducible research protocols for evaluating the use of breastmilk as a liquid biopsy for mammary gland function.
Hybridization and adaptive introgression are increasingly recognized as important components of how natural selection shapes plant speciation and ecological diversification, yet their roles in tropical clades remain poorly understood. We investigated these processes in Pandanus Parkinson (Pandanaceae), a palaeotropical tree genus of high ecological significance and the tenth-most diverse tree genus worldwide. Although morphological evidence suggested hybridization across the Indian Ocean, its genomic basis remained untested. Using 398 total samples (331 nuclear ones for Pandanus), we assessed hybridization and adaptive phenotypes by: (i) testing for broad and morphology-specific transoceanic gene flow through genome-wide conflict, gene conflict, and admixture analyses from Angiosperms353 data; (ii) evaluating whether gene flow occurred via long-distance dispersal or through a bridge clade; and (iii) testing directly and indirectly for adaptive introgression. We found strong, statistically significant evidence of bidirectional transoceanic gene flow, with nuclear clade Ba acting as a critical genetic and biogeographical bridge between Asian and Afro-Malagasy lineages. Contrary to predictions, the striking morphological similarity between geographically disjunct "swamp" lineages reflects convergent evolution rather than shared introgressive history, likely shaped by distinct macroclimatic regimes. Support for adaptive introgression in the bridge clade rested on a consilience of independent evidence: inferred chloroplast capture, differential purifying selection on plastome clades, correlated distribution of a novel foliar water-storage trait, and ecological niche overlap, all coinciding with Miocene forest contraction, Tethys Sea closure, and increasing climatic seasonality. These results highlight hybridization's central role in shaping tropical tree diversity and facilitating ecological adaptation under environmental change.
The human microbiome, a dynamic endocrine organ, exerts profound systemic influence through the production of bioactive metabolites. While the microbiome-gut-brain axis is well-established, the direct conduit between the gut microbiota and the reproductive system, the Microbiome-Gut-Gonad Axis, remains an emerging paradigm. This review explored cutting-edge evidence to construct a comprehensive model of the Microbiome-Gut-Gonad axis, focusing on the mechanistic roles of specific microbial metabolites in both physiological reproductive function and the pathogenesis of endocrine disorders. We move beyond mere correlation to elucidate how gut-derived molecules, such as short-chain fatty acids (SCFAs), secondary bile acids, and indole derivatives, directly and indirectly modulate the hypothalamic-pituitary-gonadal (HPG) axis by modulating the production of neuropeptides and hormones (Gonadotropin-releasing hormone (GnRH)) that regulate reproductive functions and also steroidogenesis and gametogenesis. We examine novel mechanisms including: the epigenetic regulation of steroidogenic enzymes by butyrate; the modulation of enterohepatic circulation of estrogens by β-glucuronidase-producing bacteria; and the role of tryptophan metabolites as ligands for aryl hydrocarbon receptor (AhR) in ovarian and testicular function. Furthermore, we critically appraise the disruptive potential of dysbiosis-driven metabolite shifts in PCOS, endometriosis, and male infertility, highlighting microbial metabolite signatures as promising exploratory biomarkers that require standardized, multi-center clinical validation before diagnostic use. At present, these signatures should be considered candidate biomarkers only, because external validation cohorts, assay reproducibility, and clinically meaningful estimates of sensitivity, specificity, predictive values, and clinical utility have not yet been established. Therapeutically, we evaluate innovative interventions, including precision probiotics, postbiotics, and dietary strategies targeting specific bacterial guilds, but these approaches remain investigational because current human evidence is still limited and heterogeneous. Finally, by integrating microbial endocrinology into reproductive medicine, this review establishes a new framework for understanding the etiology of reproductive endocrine disorders and paves the way for microbiome-targeted therapeutic avenues. Importantly, the evidence base is tiered: mechanistic statements in this review are drawn primarily from in vitro and animal studies, human disease links are described separately as observational evidence, and interventional claims are limited to early clinical studies and randomized trial summaries.
Ischemia-reperfusion injury (IRI) greatly impairs lung transplantation (LTx) outcomes, with no effective treatments. Although existing studies have confirmed that cell death and inflammation responses are critical in LTx-IRI, the specific cell death profiles of various parenchymal and inflammatory cells remain to be elucidated. Using human single-cell RNA sequencing data from LTx-IRI, we identified activation of genes related to cell death and inflammation pathways. We examined the effects and mechanisms of a RIPK3 inhibitor, GSK'872, on IRI with a rat LTx model. GSK'872, added to lung preservation solution, injected to recipients, or in combination, reduced alveolar hemorrhage, perivascular edema, neutrophil infiltration and suppressed necroptosis, pyroptosis, and inflammation in lung tissues. GSK'872 decreased MLKL phosphorylation in type 2 alveolar epithelial cells and macrophages, and RIPK3 phosphorylation in neutrophils. GSK'872 induced apoptosis in RAW264.7 macrophages via RIPK1 and caspase 3 cleavage in a cold ischemia/warm reperfusion (CI/R) cell culture model. GSK'872 inhibited lipopolysaccharide (LPS)-stimulated neutrophil extracellular traps (NETs) formation with suppressed necroptosis and pyroptosis. GSK'872 did not rescue BEAS-2B lung epithelial cells from CI/R-induced cell death. However, conditioned medium from GSK'872-treated macrophages (after CI/R) or neutrophils (challenged by LPS) reduced CI/R-induced decrease in BEAS-2B cell viability. GSK'872 alleviates LTx-IRI by inhibiting necroptosis and pyroptosis in macrophages and neutrophils directly, and protects lung epithelial cells via blocking soluble mediators indirectly. Administration of GSK'872 to lung preservation solution and/or injection to recipients may be new treatment options for IRI in LTx.
Global increases in the intensity and frequency of elevated temperatures is threatening ecosystem stability and crop yield. Understanding plant thermomorphogenesis is critical for developing climate-resilient crops, yet the underlying mechanisms remain to be clarified. Here, we identify the BEL1-LIKE HOMEODOMAIN transcription factor BLH1 as a critical negative regulator of thermomorphogenesis that modulates the key BRASSINAZOLE-RESISTANT 1 (BZR1)-PHYTOCHROME INTERACTING FACTOR 4 (PIF4) thermomorphogenic regulatory module. Overexpression of BLH1 or its homologs confers high-temperature (HT) insensitivity, whereas blh higher-order mutants exhibit HT hypersensitivity. BLH1 expression is directly repressed by BZR1 and is down-regulated by HT. We further demonstrate that BLH1 directly binds to the PIF4 promoter to repress its transcription and concurrently interacts with the PIF4 protein to inhibit its activity. Overexpression of BLH1 rescues the elongated hypocotyl phenotype in bzr1-1D or PIF4 overexpression plants. Our findings define a BZR1-BLH1-PIF4 regulatory axis that modulates the BZR1-PIF4-auxin-BR-BZR1 positive feedback loop, ensuring a balanced thermomorphogenic response to HT.
The miniaturization and integration of electronic/photonic devices demand precise control over light at the micro-scale. However, achieving tailored optical anisotropy through intrinsic material design, rather than external components, remains a significant challenge. Herein, we report a general and programmable strategy for the growth of one-dimensional organic crystals with precisely tunable asymmetric architectures via a spatially defined temperature gradient. By leveraging the competitive, facet-dependent growth kinetics under a thermal bias, continuous and precise control over the structural asymmetry is achieved in single crystals, with a tunable morphological anisotropy ranging from 9% to 81%. The resulting asymmetric crystals exhibit a pronounced direction-dependent optical response, yielding a photoluminescence intensity contrast ratio as high as 113.3, which scales directly with the degree of structural asymmetry. This work establishes a material-based platform for applying direction-dependent photonic properties directly into crystal morphology, paving the way for advanced organic photonic materials with built-in anisotropy.
The aberrant activation of the NOTCH1 signaling pathway underlies the aggressive malignancy and poor prognosis of T-cell acute lymphoblastic leukemia (T-ALL). T-ALL cell lines (Jurkat and Molt4) were treated with chiglitazar to evaluate viability, proliferation, apoptosis, and cell cycle. RNA-seq, qRT-PCR, and Western blotting were used to examine NOTCH1 signaling. Mechanistic assays included luciferase reporter, DNA affinity precipitation, co-immunoprecipitation, and ChIP. In vivo, cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models were generated by intravenous engraftment of leukemic cells into sublethally irradiated mice, followed by treatment with chiglitazar alone or combined with venetoclax. Therapeutic efficacy was assessed by survival, flow cytometric tumor burden, and histopathology (HE and IHC). We report that therapeutic activation of peroxisome proliferator-activated receptor α (PPARα) significantly represses the leukemogenesis of T-ALL in vitro and in vivo by blocking the NOTCH1 signaling pathway. Mechanistically, PPARα directly binds to the promoter region of the NOTCH1 gene and inhibits its transcriptional activity. Furthermore, PPARα interacts with signal transducer and activator of transcription 3 (STAT3) and attenuates the transcriptional activation effect of STAT3 on the NOTCH1 gene promoter. Importantly, we also found that therapeutic activation of PPARα using chiglitazar synergizes with venetoclax to suppress T-ALL progression in PDX models. We conclude that targeting PPARα to suppress T-ALL progression by blocking the NOTCH1 pathway represents a potential novel therapeutic strategy for the treatment of T-ALL.
Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system associated with alterations in gut commensals, including Akkermansia muciniphila (A. muciniphila). However, its role in MS remains unclear. Here, we report elevated serum lipopolysaccharide (LPS) and anti-LPS IgG levels in patients with relapsing-remitting MS (RRMS), indicating compromised gut barrier integrity. Notably, RRMS patients also exhibited increased serum anti-A. muciniphila IgA and enhanced A. muciniphila-induced Th17 responses in peripheral blood mononuclear cells (PBMCs). Using experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we found that A. muciniphila colonization worsened EAE severity, with increased infiltration of GM-CSF+CD4+ and IL-17 A+CD4+ T cells in spinal cord. Mechanistically, A. muciniphila colonization enhanced tryptophan metabolism and elevated levels of aryl hydrocarbon receptor (AhR) agonists, including indole derivatives, during EAE. Although A. muciniphila does not directly metabolize tryptophan, it promotes expansion of tryptophan-utilizing bacterium Alistipes onderdonkii (A. onderdonkii) through mucin degradation. We further demonstrate that A. onderdonkii utilizes mucin-derived metabolites, including galactose and N-acetylneuraminic acid (NANA). Importantly, dietary tryptophan restriction significantly attenuated EAE severity. Collectively, these findings reveal a cross-feeding mechanism in which A. muciniphila supports growth of A. onderdonkii, thereby enhancing microbial tryptophan metabolism and production of AhR agonists that drive Th17-mediated neuroinflammation.
Bacterial meningitis caused by Streptococcus pneumoniae is a lethal central nervous system infection, yet conventional intravenous vancomycin struggles to cross the blood-brain barrier effectively. Interestingly, the natural pathology of this pathogen originates from nasopharyngeal colonization, disseminates into systemic bacteremia, and ultimately breaches the meninges. Inspired by this sequential invasion, we hypothesized that administering vancomycin directly at the exact starting point via a nasal spray could achieve a simultaneous "three-in-one" eradication of all infection stages. To realize this goal and overcome the bottleneck of nasal delivery, we developed a vancomycin nasal spray using hydroxypropyl methylcellulose as a viscosity modifier. By systematically tuning the formulation viscosity, we achieved a synchronous optimization of the macroscopic spray morphology and microscopic droplet behavior. This aerodynamic balance minimized premature droplet impaction at the anterior nasal valve and prevented excessive gravitational settling in the main nasal meatus. Quantitative analysis in a 3D-printed human nasal cast demonstrated that the optimized formulation F4 maximized target site coverage, achieving a total nasal meatus deposition of 2491.7 μg and a peak olfactory deposition fraction of 5.06%. The optimized spray increased cerebrospinal fluid bioavailability by 2.93-fold and drastically reduced peripheral renal exposure by 74.93% compared to intravenous injection. In a pneumococcal infection rat model, the intranasal therapy demonstrated superior multidimensional bactericidal efficacy, clearing 89.81% of the local nasopharyngeal colonies, 97.11% of the systemic bacteremia, and 93.83% of the intracerebral bacterial load. This robust pathogen clearance was accompanied by the prompt resolution of localized neuroinflammation, systemic procalcitonin levels, and circulating leukocyte abnormalities. Ultimately, this aerodynamically engineered formulation provides an anatomically inspired and highly effective intervention paradigm for managing complex central nervous system infections.
Two-dimensional (2D) materials exhibit a wide range of electronic properties that make them promising candidates for next-generation nanoelectronic devices. Accurate prediction of their quantum transport behavior is therefore of both fundamental and technological importance. While the Non-Equilibrium Green's Function (NEGF) formalism coupled with Density Functional Theory (DFT) provides reliable insights, its high computational cost limits applications to large-scale or high-throughput studies. Here we present DeePTB-NEGF, a framework that combines a deep learning-based tight-binding Hamiltonian derived directly from first-principles calculations (DeePTB) with efficient quantum transport simulations implemented in the DPNEGF package. We validate the method on five prototypical 2D materials (graphene, hexagonal boron nitride (h-BN), [Formula: see text], [Formula: see text], and black phosphorus) demonstrating excellent agreement with conventional DFT-NEGF for band structures and transmission spectra. Beyond single-material benchmarks, we showcase the framework's versatility by exploring strain engineering (uniaxial strain on graphene and biaxial strain on [Formula: see text]), substitution doping in [Formula: see text], and current-voltage characteristics of a graphene field-effect transistor (FET). A scaling analysis reveals that DeePTB-NEGF can simulate systems with hundreds of atoms in minutes, achieving speed-ups of over [Formula: see text] compared to DFT-NEGF for heterostructures such as graphene/h-BN/graphene. These results establish DeePTB-NEGF as a powerful tool for autonomous, high-throughput design of quantum transport in microscopic heterostructures, enabling rapid prototyping of next-generation 2D devices.
The RNA interference (RNAi) pathway regulates gene expression and viral defense and has been harnessed in therapeutic solutions to inhibit otherwise undruggable proteins by preventing translation. Dicer initiates RNAi by generating cleaved RNA products that bind to a target mRNA to promote gene silencing. Mutations to the Dicer catalytic domain cause DICER1 syndrome, which increases the risk of cancers, including early childhood variants. However, the catalytic mechanism remains poorly defined due to the lack of structural data for Dicer bound to a substrate (or substrate mimic) in the presence of divalent ions known to be critical for nuclease activity. This study uses molecular dynamics (MD) simulations to uncover the first atomic level structure of the wild-type Dicer-RNA complex, including the binding pattern of two catalytically essential Mg2+ ions. Subsequently, quantum mechanics/molecular mechanics (QM/MM) techniques are used to elucidate the Dicer catalytic mechanism for phosphodiester bond cleavage. Among three fully characterized pathways, our data suggest catalysis is only feasible when both active site Mg2+ ions are directly coordinated to the RNA substrate and a hydroxide ion nucleophile is bound to an active site Mg2+ ion. Phosphodiester bond hydrolysis proceeds through a two-step mechanism involving a phosphorane intermediate that is stabilized by direct Mg2+-substrate coordination and a hydrogen bond with K1806. This newly identified mechanism is consistent with experimental kinetic data, the impact of mutating the corresponding lysine in mouse Dicer, and the active site architectures and proposed mechanisms for other related nucleases. Directed by the enhanced understanding of wild-type Dicer function, MD simulations subsequently show that six known DICER1 syndrome-causing mutants likely impede catalysis by inducing unique active site disruptions that inhibit Mg2+-ion coordination to the substrate. By furthering our knowledge of the structure and catalytic mechanism of wild-type and mutant Dicer, this work unveils the molecular underpinnings of DICER1 syndrome and opens the door for the development of enhanced RNAi-based therapeutics and biotechnologies.
Precisely tailoring the macroscopic morphology of covalent organic frameworks (COFs) fundamentally drives their physicochemical properties. However, the robust and highly directional nature of covalent bonds makes such control at the single-crystal level a formidable challenge. Resolving this bottleneck, we establish a synergistic Brønsted and Lewis dual-acid catalytic strategy to dictate the controlled axial growth and morphological evolution of large (≥50 μm) three-dimensional(3D) COFs single crystals (the XNU-375-X; X = 1-6, a, p, EtOH) featuring a dia topology. Modulating the concentration of dysprosium trifluoromethanesulfonate (Dy(OTf)3), acting as the Lewis acid, drastically suppresses the twinning rate. Consequently, this targeted regulation drives a continuous morphological transition from octahedral to tetragonal bipyramidal geometries. Single-crystal X-ray diffraction (SCXRD) explicitly confirms the microscopic structural consistency throughout this macroscopic evolution. Crucially, during guest solvent removal and exchange, these crystallographic analyses directly capture a rare structural flexibility and dynamic "breathing" effect, evidenced by a massive 46% volume variation. Density functional theory (DFT) calculations elucidate the underlying growth kinetics. Conditional on the specific exposed facets ({100} versus {001}), Dy3+ exhibits differential adsorption behaviors that effectively passivate lateral free amine sites. To the extent that these sites govern horizontal proliferation, this selective binding simultaneously promotes ordered c-axis stacking and intrinsic self-correction. Ultimately, this work bridges the gap in the precision morphological tailoring of 3D COFs single crystals, providing a robust platform for the anisotropic growth and targeted synthesis of complex porous architectures.
Adolescent screen exposure is increasing, yet clinically interpretable thresholds for cognitive risk are unclear. This study examined associations between daily screen time and cognitive screening performance and derived a screen-time cutoff associated with cognitive impairment. We conducted an observational cross-sectional study (March-April 2022) at a private junior high school in Indonesia during online learning. Students completed digital questionnaires reporting educational and recreational screen time and a directly reported overall estimate; a computed overall (educational + recreational) was generated to assess reporting consistency. Cognitive function was assessed using the MoCA-Ina, with < 24 as the primary impairment threshold based on recent psychometric evidence favoring lower cutoffs for improved classification accuracy. Sixty-seven adolescents were included (34 girls, 50.7%; 33 boys, 49.3%), with median age 13.0 years (12.0-16.0) and median MoCA-Ina 25.0 (19.0-31.0). MoCA-Ina did not differ by sex (girls 25.0 [19.0-31.0] vs. boys 26.0 [20.0-30.0]; p = 0.244). Recreational screen time correlated inversely with MoCA-Ina (ρ = -0.446, p < 0.001), as did overall screen time (ρ = -0.360, p = 0.003), whereas educational screen time was not associated (ρ = -0.061, p = 0.624). In adjusted regression, overall screen time remained negatively associated with MoCA-Ina (β = -0.24 per hour/day; 95% CI - 0.41 to - 0.07; p = 0.007), while age was positively associated (β = 0.96; 95% CI 0.07 to 1.85; p = 0.034). All variance inflation factors were below 2.5, indicating no substantial multicollinearity. ROC analysis showed fair discrimination (AUC 0.66; optimism-corrected AUC after bootstrap internal validation [1,000 resamples]: 0.63) with an optimal cutoff > 8.97 h/day (sensitivity 83.3%, specificity 48.8%, PPV 47.6%, NPV 84.0%); risk of impairment was higher above the cutoff (RR 2.98; 95% CI 1.15-7.72; p = 0.010; OR 4.77; 95% CI 1.40-16.31). High daily screen exposure was associated with poorer cognitive screening performance. The > 8.97-hour/day threshold represents a preliminary, hypothesis-generating cutoff that may help identify adolescents at elevated likelihood of cognitive impairment, pending external validation in larger, more diverse samples. 071/K-LKJ/ETIK/II/2022.
Youth violence is a significant public health concern, for which a number of preventive approaches have demonstrated promise in reducing the overall harm and associated effects. Yet relatively few of those approaches have explicitly addressed the role of structural racism and discrimination, which is a potential root cause of youth violence. This paper introduces a special issue of Prevention Science, "Structural Approaches to Youth Violence Prevention: Addressing Racism and Discrimination," which includes a collection of original papers from multiple disciplines that outline promising approaches for advancing prevention science in this important and timely direction. The overarching goal of the special issue is to provide insights related to youth violence prevention by directly addressing racism and discrimination at the structural level. The papers in this issue include systematic reviews, proposed approaches to policy and practice, intervention development and process, and evaluation research. After identifying common themes across the papers, we conclude with some future directions for research on structural interventions for youth violence prevention.
Radiotherapy remains a cornerstone for non‑small cell lung cancer (NSCLC). However, its efficacy is frequently limited by radioresistance, a phenomenon closely associated with cancer stem-like cells (CSCs). Although our previous work demonstrated that epicatechin (EC) sensitizes NSCLC cells to ionizing radiation (IR), whether this effect involves modulation of CSCs and the underlying mechanisms remain to be elucidated. A radioresistant NSCLC cell line (A549RR) was established via fractionated irradiation and employed in both in vitro assays and murine xenograft models. To assess its radiosensitizing potential, EC was administered prior to IR exposure. Cell viability and colony formation capacity were measured to evaluate radiosensitivity. Putative targets of EC were predicted using network pharmacology and validated through molecular docking. The direct interaction between EC and CXCL8 was examined by cellular thermal shift assay (CETSA). The functional relevance of CXCL8 in EC-mediated effects was assessed through both loss-of-function (shRNA-mediated knockdown) and gain-of-function (CXCL8 overexpression) approaches. CSC properties were evaluated by tumor sphere formation assays and immunoblotting for stemness markers. EC significantly enhanced NSCLC radiosensitivity both in vitro and in vivo, concomitant with marked suppression of CSC stemness. CXCL8 was identified as a direct functional target of EC: its expression was substantially upregulated in radioresistant cells but downregulated upon EC treatment. CETSA confirmed a direct interaction between EC and CXCL8 in cells. Functionally, CXCL8 knockdown suppressed cell viability, colony formation capacity, and CSC properties in A549RR cells. Conversely, overexpression of CXCL8 abrogated EC-induced radiosensitization and restored CSC phenotypes in both cellular and xenograft models. EC overcomes NSCLC radioresistance by directly targeting CXCL8, thereby disrupting CSC-like traits.
Inducing localized mineralized lesions offers a promising drug-free strategy for tumor suppression, yet calcium-phosphate systems are limited by slow crystallization, high ion requirements, and poor organelle specificity. Here, we develop a silicene-derived nanoplatform that serves as an "inorganic silicic acid reservoir", enabling controlled, organelle-specific biosilicification for cancer therapy. Silicene nanosheets are sequentially engineered with tannic acid and PEI-anchored triphenylphosphonium (TPTS), conferring high colloidal stability, efficient endosomal escape, and selective mitochondrial targeting. Within the oxidative mitochondrial milieu, TPTS undergoes programmed hydrolysis to release Si(OH)4, which condenses in situ to form silica directly on mitochondrial membranes. The resulting confined mineral deposits disrupt membrane potential, impede metabolite trafficking, and precipitate a catastrophic energetic collapse that drives apoptosis. This platform delivers two major advances: (1) Intracellular mineralization redefinition-precursors shift from intrinsic labile physiological ions to exogenous bio-orthogonal nano-reservoir, enabling sustained, site-specific silicic acid release; (2) High therapeutic potency - organelle-level precise therapy surpasses conventional high-dose-dependent cellular-scale mineralization, achieving 81.79% tumor inhibition in ectopic models and 65.81% even in the more challenging orthotopic TNBC models, without inducing systemic toxicity. Together, these results establish a generalizable paradigm for spatially programmed mineralization therapy and position silicene as a versatile foundation for next-generation organelle-targeted cancer interventions.