Biomolecules

Background: Subepithelial connective tissue grafting remains a reference treatment for predictable root coverage. Although short- and medium-term outcomes of tunnel-based procedures are well documented, evidence regarding stability beyond 10 years remains limited. This study evaluated the long-term clinical performance of a minimally invasive tunnel technique combined with subepithelial connective tissue grafting (SCTG) under routine clinical conditions. Methods: This retrospective longitudinal cohort study included 74 patients (57 women and 17 men) contributing 710 gingival recession sites treated between 2009 and 2025. All sites were treated with a tunnel approach and SCTG, with enamel matrix derivative (EMD) used in selected cases. The mean follow-up was 6.0 for 4.0 years, with a maximum observation period of 16 years. The primary outcome was recession depth reduction. Secondary outcomes included complete root coverage (CRC), mean root coverage, and long-term marginal stability. Clinically relevant relapse was defined as a 1 mm increase in recession after initial healing. Results: Mean recession reduction was 2.72 mm. Complete root coverage was achieved at 83.4% of treated sites. At the final available follow-up, no treated site showed a clinically relevant relapse of 1 mm after initial healing, and no site deteriorated beyond its baseline recession level. Treatment effects were observed across anterior and posterior regions. Conclusions: Within the limitations of a retrospective cohort design, tunnel surgery combined with SCTG was associated with high root-coverage predictability and durable marginal soft-tissue stability for observation periods extending to 16 years. These real-world data support phenotype-enhancing, minimally invasive soft-tissue augmentation as a durable therapeutic strategy for localized and multiple gingival recessions. Keywords: gingival recession; tunnel technique; subepithelial connective tissue graft; root coverage; periodontal plastic surgery; long-term stability

Traumatic optic neuropathy (TON) occurs due to direct or indirect injury to the optic nerve and is a significant cause of visual disability. So far, there is no effective treatment. The lack of understanding of the cellular mechanisms by which trauma induces inflammation and damage in retinal neurons is a critical knowledge gap in developing effective therapies. We have studied the role of the arginase 1 (A1) enzyme in this pathology. We have found previously that treatment with a long-acting form of human recombinant A1, pegylated A1 (PEG-A1) after optic nerve crush limits activation of retinal microglia and macrophages (M{Phi}) and reduces inflammation, thereby decreasing injury and protecting visual function. Here we report on studies designed to demonstrate the therapeutic efficacy of PEG-A1 in mouse models of direct and indirect TON and to elucidate the underlying mechanisms. We used ONC to model direct TON and sonication-induced trauma to the supraorbital rim to model indirect TON (SI-TON). At different times after injury, mice were treated with PEG-A1 which was delivered systemically by i.p. injection or locally by intravitreal injection. In order to assess the role of A1-induced activation of the ornithine/polyamine pathway in the protective effects of PEG-A1, some mice were treated with the ornithine decarboxylase (ODC) inhibitor, difluoromethylornithine (DFMO) immediately after the PEG-A1 treatment. Retinal function was determined by OptoMotry and electroretinography. Retinal injury and microglia/M{Phi} activation were assessed by immunofluorescence imaging. Expression of inflammatory cytokines was determined by Western blotting and quantitative RT PCR. Liquid chromatography mass spectrometry was used to analyze changes in arginase/ODC pathway metabolites. Results showed that PEG-A1 treatment improved neuronal survival and visual function whether delivered systemically or intravitreally. This neuroprotection was associated with decreased microglia/M{Phi} activation, decreased inflammatory cytokine expression, and increased formation of L-ornithine and putrescine. Furthermore, DFMO treatment blocked these effects, indicating that PEG-A1 limits retinal injury and preserves vision after ocular injury by activating ODC. ODC processes the arginase product L-ornithine to form polyamines which are known to promote reparative functions. Thus, PEG-A1 therapy offers a new strategy to limit trauma-induced vision loss and promote repair after TON.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in older adults. It is characterised by early retinal pigment epithelium (RPE) dysfunction followed by progressive photoreceptor degeneration. Cigarette smoking is a major environmental risk factor for AMD, and hydroquinone (HQ), a redox-active cigarette smoke component, induces oxidative stress and apoptosis in RPE cells. To analyse how RPE stress contributes to photoreceptor degeneration, we employed a retinal co-culture model composed of human induced pluripotent stem cell-derived RPE (iPSC-RPE) cells in conjunction with porcine neuroretina explants. Exposure to HQ induced oxidative stress in iPSC-RPE cells as well as retinal photoreceptors (RPR), resulting in apoptosis, executed at least in part by caspase activation. Concomitantly, HQ caused endoplasmic reticulum (ER) stress (ERAD) in RPR followed by their degeneration, evidenced by reduced outer nuclear layer (ONL) rows and shortened RPR outer segments (OS). Based on earlier results, which suggest a perturbation of proteostasis due to HQ, we tested whether ML240, a bona fide inhibitor of valosin-containing protein (VCP), would influence the degree of degenerative activities. ML240 did not prevent HQ-induced apoptosis in iPSC-RPE cells. However, it significantly preserved photoreceptor integrity, retaining OS length and cone density in HQ-stressed co-cultures. Proteomic analysis suggested that ML240 reshapes stress response patterns of the HQ-exposed neuroretina, as evidenced by a reduction in ERAD-associated markers, increased levels of antioxidant response proteins, and the preservation of cytochrome c enrichment in photoreceptor inner segments, which indicates improved mitochondrial integrity consistent with the observed preservation of photoreceptor structure. Together, these findings establish the iPSC-RPE/neuroretina co-culture as a platform to analyse pathophysiological features of AMD, dissect cell type-specific retinal responses to environmental stress and test neuroprotective pharmacological approaches to protect photoreceptors in oxidative stress-associated retinal degeneration.

Preeclampsia is a pregnancy complication characterized by hypertension, proteinuria, and end-organ dysfunction. Abnormal placentation leading to reduced placental perfusion may contribute to its development. Previous studies demonstrated that the activation of the apelin receptor (APJ) system has hypotensive, renoprotective, and antioxidant effects in preeclamptic rat models. Apelin and elabela (ELA) can stimulate the proliferation of trophoblast cells, suggesting a role in embryonic development. However, the mechanisms underlying the actions of apelin or ELA in trophoblast cells are not well understood, particularly in hypoxic settings. The immortalized HTR-8/SVneo trophoblastic cells were treated with cobalt chloride (CoCl2) at 0.2 mM for 24 hours to mimic hypoxic conditions. RT-qPCR, ELISA or Western blotting was used to measure mRNA or protein levels of apelin, elabela, and the components of IL-6 signaling in cell lysates or conditioned media. The exposure to CoCl2 increased total apelin and elabela content approximately 2-fold in the conditioned media but did not affect APJ levels. CoCl2 upregulated proinflammatory cytokine concentrations: soluble fms-like tyrosine kinase 1 (sFlt-1), soluble gp130 (sgp130), interleukin-6 (IL-6), and sIL-6 receptor (IL-s6R). Both apelin and elabela downregulated IL-6 mRNA but had no effect on sFlt-1 mRNA. Apelin attenuated sgp130, while ELA decreased the membrane form of IL-s6R. Apelin also decreased the pSTAT3/STAT3 ratio. CoCl2-induced hypoxia upregulated the pro-inflammatory milieu in HTR-8/SVneo cells. Local activation of this peptidergic system may be a compensatory response of the trophoblast cells to hypoxia as exogenous apelin and elabela treatment ameliorated the hypoxia-induced pro-inflammatory milieu.

Abstract Aim: To test whether neighbouring-tooth restorations affect a focal tooths periodontal status (within-mouth spillover), and whether accounting for them alters the established association at the restored surface. Materials and methods: Prospective tooth-surface data from the Study of Health in Pomerania (SHIP): SHIP-START (N=2,715/1,982/1,397, follow-ups 1-3) and SHIP-TREND (N=2,123). Own and neighbouring-tooth restoration were defined at baseline; probing depth (PD), clinical attachment level (CAL), bleeding on probing (BOP) and deep pockets (PD >=4 mm) at follow-up. A neighbourhood-exposure mixed model with generalized propensity-score adjustment estimated both effects, with two corroborating estimators. Results: Own restoration was associated with worse periodontal status at that surface (crown PD exp(beta) up to 1.10). Neighbouring-tooth restorations raised focal-tooth PD (spillover exp(beta) 1.03-1.05) and deep-pocket risk (risk ratio up to 1.25); CAL and BOP showed none consistently. It attenuated with tooth-lag, nearing the null by two to three teeth in most estimators. Ignoring interference modestly inflated the direct effect; an opposing-tooth negative control showed none; untreated caries reproduced it. Conclusions: Dental restorations exert a within-mouth spillover on neighbouring-tooth PD: their periodontal footprint extends beyond the restored tooth. The direct association persists, modestly attenuated, after accounting for the neighbourhood.

Objectives: Collaboration among health care professionals and the services they provide can be strengthen by interprofessional education (IPE). IPE can be implemented at the undergraduate level. Accordingly, the objective of the present study was to evaluate senior students' understanding of the terms interdisciplinarity and multidisciplinarity within the context of IPE. Methods: A retrospective cross-sectional study design was used. Students understanding of interdisciplinarity and multidisciplinarity was assessed through an assessment question completed by three consecutive cohorts of senior undergraduate dental students at the UBC Faculty of Dentistry between 2021/22 and 2023/24 (N = 177). Responses had a maximum of 100 words and were categorized into one of four predetermined themes: concordant knowledge (when both definitions were correct), discordant rhetoric (when both definitions were incorrect), switched ideas (when the definitions were reversed), and altered discourse (when the concepts of discipline and specialty were conflated). Descriptive and inferential statistical analyses were performed using SPSS Version 31. Results: Of the 177 students enrolled, 164 provided responses to the question on multidisciplinarity and interdisciplinarity: 60 students in 2021/22, 51 students in 2022/23, and 53 students in 2023/24; the mean age was 25 years and 88 were female. Of the four predetermined themes, 45.7% of responses reflected concordant knowledge, 15.9% discordant rhetoric, 18.3% switched ideas, and 20.1% altered discourse. The logistic regression analysis showed age associated with a higher probability of providing the correct definitions (adjusted OR = 1.39; 95% CI: 1.06 - 1.83; p = 0.017). Conclusions: Knowledge of interdisciplinary and multidisciplinary appeared to be retained by the majority of students. However, dental education programs, alongside other health professional training programs, should continue to incorporate interprofessional education through both didactic and experiential learning opportunities to equip students with the skills necessary to provide collaborative care in future practice.

Amyloid fibrils are highly ordered protein aggregates characterized by a conserved cross-{beta}-sheet architecture despite originating from structurally diverse precursor proteins. Growing evidence suggests that interactions between different amyloidogenic proteins can modulate aggregation pathways through heterologous cross-seeding; however, the influence of seed polymorphism on the structure and biological properties of cross-seeded fibrils remains poorly understood. Here, we investigated the cross-seeding of native human insulin by two structurally distinct polymorphs of hen egg-white lysozyme (HEWL): flexible fibrils (FFs) and rigid fibrils (RFs). Native insulin remained stable under physiological conditions and underwent spontaneous fibrillation only under acidic conditions. In contrast, both HEWL polymorphs efficiently induced insulin aggregation at physiological pH, bypassing the nucleation barrier. Thioflavin T fluorescence, circular dichroism spectroscopy, and transmission electron microscopy revealed that lysozyme FFs templated the formation of insulin flexible fibrils (IFFs), whereas lysozyme RFs produced insulin rigid fibrils (IRFs), demonstrating that the structural characteristics of the parental HEWL polymorphs were propagated during heterologous cross-seeding. The toxicity of the resulting insulin fibrils was evaluated in SH-SY5Y neuronal cells and CCF-STTG1 astrocytes. IFFs exhibited minimal cytotoxicity and only subtle morphological alterations, whereas IRFs caused modest reductions in cell viability accompanied by more pronounced cellular damage. These findings demonstrate that the structural polymorphism of HEWL fibrils governs both the architecture and biological activity of cross-seeded insulin fibrils, highlighting amyloid polymorphism as an important determinant of heterologous amyloid propagation and a potential design principle for engineering functional amyloid-based biomaterials and protein delivery platforms.

Cell confluency is a major determinant of cell-cell communication, protein interactions, access to nutrients, and signalling dynamics, thereby significantly impacting biological outcomes. Lung cancer cells like A549 are widely used as screening models for scientific studies wherein their growth in vitro progress from non-confluent to confluent growth. In this study, we investigated the transcriptomic adaptations associated with the transition of A549 cells from baseline non-confluent to confluent growth. Comparative transcriptomic analysis between confluent and cells at baseline identified 815 upregulated and 671 downregulated transcripts. Pathway enrichment analysis of deregulated transcripts in confluent cells revealed enhanced cholesterol and sterol biosynthetic pathways, along with suppression of chromosomal segregation and mitotic pathways. At confluency, an increased expression of glucose transporters (SLC2, SLC60, and SL37 families) and glycolytic pathways, and a decrease in amino acid transporters (SLC1, SLC7, SLC38, and SLC36) and amino acid metabolic pathways is observed. A reduced one-carbon metabolic signature (SHMT2, DHFR, and MTHFD2) and enhanced fatty acid precursor synthesis (HMGCLL1, ALDH6A1, and AASS) were also observed at confluency. 1H NMR profiling of culture media revealed higher glucose and glutamine utilisation with lactate accumulation during culture maturation. Collectively, the data suggest transcriptome-level rewiring in A549 cells with preferential biosynthesis of lipids and sterols at confluency and underscore the importance of considering culture maturity in cancer biology, metabolism, and therapeutic studies.

Objective. A deep learning (DL) model was used to convert smartphone videos of a complete arch implant cast into 3D scans. The aim of current study was to determine if a custom scan body (SB) with geometric features and coating would outperform regular PEEK stock SB in this DL scenario. The DL-derived scan outcomes were compared with those obtained from a conventional splinted open-tray impression and from photogrammetry. Materials and Methods. A maxillary edentulous model with six implants and multi-unit abutment analogs was scanned using four protocols: conventional splinted open-tray impression (CO), photogrammetry (PG; Icam4D), DL using stock SBs (DLS) and DL using custom SBs (DLC). Each protocol was repeated for 10 times. The DL scans were produced from smartphone videos with a high-fidelity, multi-view 3D construction AI model (Neuralangelo). The custom designed SB incorporated geometric features and was fabricated via 3D printing followed by a spray coating. Accuracy (trueness and precision) was assessed using three measurements: Root Mean Square (RMS), linear deviation, and angular deviation. Results. DLC outperformed DLS in both trueness and precision regarding RMS and linear measurements (p<0.001). CO and PG demonstrated the highest RMS and linear trueness, with no significant difference between them (RMS: p=0.93; linear: p=0.663). PG achieved the best precision across RMS, linear and angular measurements. Conclusion. The optimised SB significantly improves the accuracy of DL-based approach for full-arch implant scan comparing to regular PEEK stock scan bodies. While early stage, neural surface reconstruction has potential as a viable option for full-arch implant rehabilitation.

Background: Serial glial fibrillary acidic protein (GFAP) trajectories have become an important framework for contextualizing evolving secondary-injury pathophysiology after moderate-to-severe traumatic brain injury (msTBI). However, total GFAP pools release and clearance signals that may be less useful for longitudinal bedside decisions than a proteoform-resolved assay. We compared total GFAP with neoGFAP, defined here as calpain-generated GFAP proteoforms intended to index active astroglial proteolysis during the subacute phase. Methods: We analyzed 651 serial serum samples from 95 msTBI patients from a previously described single-site cohort. Total GFAP and neoGFAP were measured on the same MSD platform from 6 to 240 hours after injury. Early (6 to 72 h) and late (96 to 240 h) windows, data-derived tertiles, and serial trajectory summaries were calculated directly from serial samples. Models were benchmarked against age plus admission post-resuscitation Glasgow Coma Scale (GCS) and the admission IMPACT extended risk score using five-fold stratified cross-validation. Outcomes were unfavorable outcome (GOSE 1 to 4), less-than-good recovery (GOSE 1 to 6), Disability Rating Scale (DRS) [&ge;]15, mortality, and neuroimaging worsening at 6 months. Results: The cohort contributed 95 serial biomarker profiles, with 90 participants evaluable for 6-month GOSE and 89 for DRS. Unfavorable outcome occurred in 57/90 (63.3%), and less-than-good recovery in 79/90 (87.8%). For unfavorable outcome, IMPACT plus early neoGFAP reached AUROC 0.85 versus 0.84 for IMPACT plus early total GFAP and 0.81 for IMPACT alone. For less-than-good recovery, IMPACT plus late neoGFAP achieved AUROC 0.90 versus 0.84 for late total GFAP and 0.82 for IMPACT alone. Secondary analyses for DRS, mortality, and neuroimaging worsening showed smaller differences. Conclusions: In this retrospective analysis, neoGFAP provided clearer incremental value than total GFAP for recovery-oriented monitoring, especially when late-window reassessment of patients who remained at risk for less-than-good recovery was required. Results support prospective testing of neoGFAP as a pathophysiology-informed adjunct to serial bedside decision making, repeat-assessment thresholds, and recovery stratification.

The binding affinities of a chimeric analog of a riboflavin derivative linked to biotin, (6- (7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl 5-((3aS,4S,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), referred to as C6-Rf-biotin-tag, to the riboflavin binding retain or streptavidin are in the M range, 1.29 {+/-} 0.277 and 3.00 {+/-} 0.459, respectively. These values suggest that C6-Rf-biotin-tag has potential applications in diagnostic assay and labelling target flavin binding proteins. The C6-Rf-biotin-tag which was characterized with respect to physical and biochemical properties retains UV/Vis spectroscopic and fluorescence behavior similar to riboflavin.

Plant-derived small molecules possess highly diverse physicochemical properties, and the computational design of their protein recognition elements depends not only on the global structural quality of candidate backbones, but also on whether the local binding pocket, ligand-contact pattern, and predefined recognition conformation can be consistently retained after sequence design and structural back-prediction. To explore pocket-design strategies for different types of natural-product small molecules, this study selected capsaicin, (4R)-limonene, and quercetin as model ligands, representing a flexible amphipathic molecule, a compact hydrophobic monoterpene, and a rigid polyphenolic flavonoid scaffold, respectively, and covering the dimensions of pungent sensory flavor, volatile aroma, and flavonoid functional constituents. A ligand- physicochemical-property-guided computational design and multi-stage prioritization framework was established for candidate protein binders. The results showed that candidates with favorable initial global structural scores did not necessarily form reasonable local small-molecule binding pockets, indicating that evaluation of the local ligand environment is essential for candidate prioritization. After screening, 31 partial- pocket candidate backbones for capsaicin, 75 buried hydrophobic-pocket candidate backbones for (4R)-limonene, and 56 pocket-qualified candidate backbones for quercetin were obtained. Further sequence design and structural back-prediction analyses indicated that a subset of candidates could maintain the original pocket geometry and major ligand-contact patterns after sequence realization. Overall, these results suggest that the physicochemical properties of different plant-derived small molecules substantially influence the efficiency of de novo protein pocket formation, with compact hydrophobic ligands being more compatible with buried hydrophobic- pocket strategies, whereas flexible or multipolar ligands require a more refined balance between hydrophobic burial and polar exposure. This study provides a pre- experimental computational prioritization framework for natural-product small- molecule-recognizing proteins and offers candidate resources for subsequent protein expression, in vitro binding validation, active-constituent enrichment, and development of small-molecule biorecognition tools. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/743643v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@8fe6c2org.highwire.dtl.DTLVardef@176cef2org.highwire.dtl.DTLVardef@10c8201org.highwire.dtl.DTLVardef@2b28cf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Objectives: Dentists prescribe approximately one in ten antibiotics worldwide, yet antimicrobial stewardship (AMS) remains underemphasized in dental education. Large language models (LLMs) may support AMS training, but their proficiency and clinical reasoning in this context remain unclear. We evaluated GPT-4o's accuracy and clinical reasoning on dental antibiotic prescribing questions, stratified by question difficulty. Methods: We assembled 125 multiple-choice questions on dental antibiotic prescribing from eight peer-reviewed studies (2017-2023). GPT-4o answered each question and generated a clinical justification. Accuracy was assessed against source-study answer keys and examined across difficulty quartiles. Justifications were evaluated using an adapted 12-axis human-evaluation framework assessing scientific consensus, extent and likelihood of harm, inappropriate and missing content, bias, and both correct and incorrect comprehension, retrieval, and reasoning. Prophylaxis-specific questions were analysed separately. Results: GPT-4o correctly answered 72% of questions. Accuracy remained relatively stable across difficulty quartiles (78%, 78%, 65%, 70%). Experts rated 95.4% of justifications positively across the 12 axes. Comprehension, retrieval, and reasoning each exceeded 96.2% positive ratings. Missing content was the main weakness (7.8%), and 7.1% of justifications showed a moderate-to-severe potential for harm. Performance on prophylaxis-specific questions (98.1%) exceeded non-prophylaxis questions (93.0%). Conclusions: GPT-4o demonstrated moderate-to-high proficiency and clinically defensible reasoning in dental antibiotic prescribing questions. However, residual risks indicate that it is not suitable for unsupervised clinical use but shows potential as a supervised AMS educational tool.

Purpose: Oxidative stress is thought to contribute to the development of age-related cataracts (ARCs), but the mechanisms by which oxidative damage leads to the opacification of the lens remain unclear. Previous studies suggest that oxidative stress can disrupt lens proteostasis. Therefore, it was hypothesized that ARCs arise from proteomic changes driven by an age-associated decline in oxidative stress defenses that interact with the lens proteostatic state. To test this hypothesis, proteomic analyses of lenses exposed to oxidative stress were performed to examine oxidative and proteostatic stress responses in vivo. Methods: Cataract formation was induced by injecting hydrogen peroxide into the aqueous humor of adult zebrafish. nrf2fh318/fh318 zebrafish were used to model the reduced oxidative stress protection observed in aged human lenses, while cryaba-/- zebrafish were used to model impaired lens proteostasis. Resulting opacities in WT, cryaba-/-, nrf2fh318/fh318, and cryaba-/-; nrf2fh318/fh318 lenses were quantified and proteomic changes in the cortex were analyzed using data independent acquisition Parallel Accumulation Serial Fragmentation mass spectrometry. Results: Hydrogen peroxide treatment induced the formation of cortical cataracts. Proteomic results showed that, dependent on genotype and day, oxidative stress activates the unfolded and mitochondrial unfolded protein responses. Additional changes were also observed in energy metabolism, Ca2+ homeostasis, protein degradation, and cytoskeletal and extracellular matrix remodeling pathways. Conclusions: Treated zebrafish lenses successfully model ARC and mass spectrometry proteomics identified the unfolded and mitochondrial unfolded protein responses as potential therapeutic targets for ARC.

Objective White smoke inhalation injury (WSI) causes severe acute lung damage with no specific therapy currently available. Sphingolipid metabolism is implicated in pulmonary inflammation, but its transcriptional regulatory landscape in WSI remains unexplored. This study aimed to identify key sphingolipid metabolism related genes and evaluate their regulatory roles and therapeutic potential in WSI. Methods We established a rat model of WSI and performed integrated bulk RNA sequencing, weighted gene coexpression network analysis (WGCNA), and single-cell RNA sequencing (scRNAseq) to screen for differentially expressed sphingolipid metabolism-related genes (DESRGs). Protein-protein interaction (PPI) network with four centrality algorithms was used to prioritize hub genes. In silico gene knockout and molecular docking were conducted to assess regulatory functions and identify potential drug candidates. Results We identified 22 DESRGs that were predominantly enriched in DNA replication and cell cycle pathways rather than canonical sphingolipid metabolic processes. PPI consensus prioritized three hub genes--Top2a, Ttk, and Ccna2--with Top2a exhibiting the highest expression in epithelial cells and significant downregulation after smoke exposure. ScRNAseq revealed immune cell infiltration and epithelial differentiation trajectories. Virtual knockout showed that Top2a depletion affected the largest transcriptomic fraction (~0.4%) and was enriched in lysosome biogenesis, innate immunity, phagocytosis, and lipid catabolism. Molecular docking identified thalidomide as a high affinity ligand for Top2a (Vina score: -8.5 kcal/mol). Conclusion Our multiomics integrative framework identifies Top2a as a central regulatory hub linking sphingolipid associated inflammation to epithelial responses in WSI, and nominates thalidomide as a potential drug repurposing candidate. These findings provide prioritized targets for future translational investigation.

BackgroundObservational comparisons of former smokers who use non-combusted nicotine products with former smokers who quit without them require that two quantities be measured precisely: which product is being used, and how long ago cigarette smoking stopped. Neither quantity is recorded by the National Health Insurance Service (NHIS) screening instrument used in a recent Korean cohort study of post-cessation e-cigarette use and lung cancer risk. We characterized both quantities in a contemporaneous, nationally representative survey of the same population. MethodsWe analyzed the public-release microdata of the Korea National Health and Nutrition Examination Survey (KNHANES), 2018 to 2023, restricted to adults aged 19 years and older. Former smokers were identified by smoking status, and cessation duration was taken from the item recording months since the last cigarette. Former smokers currently using a heated tobacco product (HTP) or an e-cigarette (EC) were compared with former smokers using neither. KNHANES 2018 asked a generic e-cigarette question and, separately, a checklist naming HTP brands, allowing the two product classes to be separated. Distributions were compared with rank-based methods, the age-duration relationship with Theil-Sen regression, and residual imbalance by restricting the comparison group to respondents age-matched to within two years. ResultsThe 2018 analytic sample comprised 1,348 former smokers, of whom 43 currently used HTP or EC and 1,305 used neither. Among the product-using former smokers, 58% reported HTP use without e-cigarette use, 21% reported both, and 21% reported e-cigarette use without HTP use; 79% reported any HTP use. Median cessation duration was 0.7 years (IQR 0.25 to 1.5) among product users and 12.0 years (IQR 5.0 to 20.0) among those using neither (Kolmogorov- Smirnov D = 0.76, P < 0.001), with the product user having quit more recently in 92% of cross-group pairs. The separation persisted within the short-term (<5 year) stratum (D = 0.34, P < 0.001; 73% of pairs) and after age matching, where the residual gap was 9.3 years. Cessation duration rose with age among those using no product (Theil-Sen slope +0.30 years per year) but was flat among product users (-0.01). Restricting to the screening-eligible stratum used in the cohorts high-risk analysis did not attenuate the imbalance: among those aged 50 to 80, median cessation among no-product quitters rose to 15.5 years (n = 858), and adding a 20 pack-year criterion left 421 no-product quitters with a median of 11.0 years against three HTP/EC users who had quit 0.25, 1.0 and 2.0 years earlier, despite closely matched cumulative exposure (mean 37.6 vs 37.7 pack-years). The overall contrast reproduced in every wave from 2018 to 2023, with an age-matched residual of 9 to 11 years. ConclusionsIn a nationally representative survey of the same population and the same calendar year as the NHIS screening cohort analyzed by Kim et al., Korean former smokers using non-combusted nicotine products differed from other former smokers in two respects that bear directly on how such comparisons should be read. First, they were predominantly HTP users: 79% reported any HTP use, and only 21% reported e-cigarette use without HTP use. Second, they had stopped smoking approximately a decade more recently, a difference that survived stratification at five years and exact age matching. Neither quantity is recorded in the NHIS screening instrument. Cohort estimates comparing post-cessation product users with other quitters should therefore be interpreted with caution if they do not precisely characterize product composition and to time since cessation, and future studies should measure both directly.

Lysophosphatidic acid (LPA) is widely recognized as an extracellular lipid mediator; however, the functional significance of intracellularly produced LPA remains poorly understood. Here, we investigated the regulatory mechanism and functional role of a LPA-producing lysophospholipase D GDE4, also known as GDPD1, in prostate cancer cells. GDE4 expression is induced under ER stress conditions in a PERK-dependent manner and requires the transcription factor ATF3. Disruption of GDE4 expression resulted in altered intracellular levels of LPA and LPA precursor lysophosphatidylethanolamine, accompanied by reduced cell proliferation. RNA sequencing and subsequent validation identified a set of genes downregulated in GDE4-depleted cells. Pharmacological inhibition experiments indicated that peroxisome proliferator-activated receptor and {gamma} (PPAR and PPAR{gamma}) signaling pathways contribute to the regulation of these GDE4-dependent genes. Collectively, our findings suggest that GDE4-dependent lipid remodeling is associated with PPAR/{gamma}-mediated transcriptional regulation under ER stress conditions. These results provide a potential framework for understanding the link between intracellular lipid metabolism and stress-responsive gene regulation.

Human iPSC-derived brain organoids are revolutionizing tools to study layers biology, synergize disease modeling, and accelerate therapeutic discoveries that overcome obstacles in monolayer cell culture or animal models. The neurovascular unit including vasculature and microglia is critical for brain development, maintenance of synaptic plasticity and neural activity, and the high metabolic demands of long-term culture. We present a methodology to incorporate these important components during organoid generation and discuss potential approach, aiming consistent production of vascularized organoids for longitudinal study. We also demonstrate that this vascularized organoid is a versatile platform to model brain cancer and traumatic brain injury.

Multimodal large language models (MLLMs) are increasingly applied to image-based clinical reasoning, yet their diagnostic reliability in periodontal image interpretation, and the underlying source of their errors, remain poorly characterized. This study evaluated six architecturally distinct MLLMs (Claude Sonnet 4.5, GPT-5.0, Gemini 2.5, GLM-4.6, Sonar, and Grok 4.1) using 50 image-based multiple-choice questions drawn from the American Academy of Periodontology In-Service Examination, spanning clinical photographs, histopathology, radiographs, cardiac rhythm strips, and anatomical illustrations. A sequential two-phase experimental design was used: in Phase 1, each model independently described each image, selected an answer, and provided a supporting citation; in Phase 2, applied only to questions answered incorrectly, models were given an expert-validated visual description and asked to re-answer, allowing diagnostic improvement through visual correction to be measured directly. Expert ground truth for image content was established by a board-certified periodontist and independently validated by a second board-certified periodontist. Model outputs were classified using a dual-process error taxonomy adapted from Norman's model of diagnostic reasoning, distinguishing perceptual errors, arising from inaccurate visual feature extraction, from cognitive errors, arising from flawed reasoning despite accurate perception, with cognitive errors further subdivided into correctable and persistent subtypes, and additional categories capturing compound perceptual-cognitive failures and compensatory reasoning that overcame inaccurate perception. Diagnostic accuracy and error type distribution varied significantly across models and image modality. Correcting inaccurate visual descriptions in Phase 2 improved diagnostic accuracy for a subset of previously incorrect responses, indicating that a meaningful share of errors originated at the level of visual perception rather than clinical reasoning; conversely, a distinct subset of errors persisted despite accurate corrected visual input, indicating reasoning-level failures independent of perceptual accuracy. Some models also reached correct answers despite generating inaccurate image descriptions, reflecting compensatory reasoning resilient to perceptual error. These findings show that aggregate accuracy scores conflate mechanistically distinct failure modes, and that perceptual and cognitive errors carry different implications for how MLLMs might be safely deployed or improved for diagnostic image interpretation. The expert-guided visual correction framework introduced here provides a generalizable, mechanism-based approach to benchmarking multimodal AI diagnostic performance that extends beyond periodontics to other visually driven diagnostic domains in medicine. As MLLMs become increasingly accessible to clinicians, residents, and dental educators, distinguishing perceptual from cognitive failure is essential for guiding responsible clinical use, targeting model refinement, and informing AI-augmented dental education and competency assessment.

Background: Pulmonary arterial hypertension (PAH) is a serious disease with poor prognosis, especially in infants or preterm babies and there is still no optimal treatment for this disease. Noradrenalin (NE) is a vasoactive mediator which is released by sympathetic ganglion. According to previous studies, NE/1-adrenoreceptors is not only in regulating normal physiologic responses, but also in the pathogenesis of PAH. However, the mechanisms of NE in PAH are not fully understood. Methods: Human PASMC (PASMC) was used in this study. Cell viability assay and Wound healing assay were used to evaluate the proliferation and migration of PASMC. Immunoprecipitation and western blots analysis were used to investigate the mechanisms which involved in NE-induced PASMC proliferation. Results: We investigated that NE could induce human PASMC proliferation and migration. Furthermore, we first find that endothelin 1 (ET-1) signaling pathway plays an important role in NE-induced PASMC proliferation. ET1 is a critical molecular which is known for regulating cell growth and migration. We investigated that NE could increase NE-1 secretion, further enhancing ET-1 bind to its receptors. For further clarifying the downstream signals in NE/ET-1 induced PASMC proliferation, we detected the phosphorylation and expression levels of ERK and JNK. Conclusions: By combining the results from ours and previous studies, we believed that JNK/c-jun pathway may play an important role in NE-induced PASMC proliferation. Key Words: Noradrenaline; Pulmonary Arterial Hypertension; Pulmonary Artery Smooth Muscle Cells; Endothelin-1; JNK/c-Jun Signaling.