Journal of Dental Research

Background Alveolar bone assessment in periodontal practice relies on radiography and clinical probing, both of which have well-documented limitations in precision. Intraoral high-frequency ultrasonography (US) offers a radiation-free alternative with potential for sub-millimeter resolution, the validity and precision for detecting minute osseous changes have not been established. The purpose of this study was to evaluate the concurrent validity and measurement precision of intraoral US for detecting alveolar bone-level changes in patients undergoing crown lengthening and osseous surgery, thereby enabling its translation to monitor osseous changes in patients with periodontitis. Methods Ten patients (28 tooth sites) undergoing crown lengthening or osseous surgery at a USC Advanced Grad Perio clinic were enrolled in this prospective observational study. Distance from the cementoenamel junction (CEJ) to the Alveolar bone crest (ABC) was measured at pre- and post-operative time points using a 40 MHz handheld intraoral US transducer and, intraoperatively, by standardized clinical photography. Agreement was assessed by Pearson correlation and Bland-Altman analysis. Measurement precision was quantified using the standard error of measurement (SEM) and minimum detectable change (MDC). Results Preoperative agreement between methods was excellent (r = 0.977; Bland-Altman bias = -0.009 mm; 95% limits of agreement [LoA]: +-0.40 mm). Post-operative correlation remained strong (r = 0.912; bias = 0.123 mm; LoA: -0.85 to +1.10 mm). Both methods detected statistically significant post-surgical increases in the ABC-to-CEJ distance (p < 0.001), as anticipated. US demonstrated substantially superior precision: preoperative SEM 0.058 mm with US versus 0.128 mm clinically, yielding MDC values of 0.160 mm (US) versus 0.354 mm (clinical), providing a 2.2-fold precision advantage. Conclusions Intraoral US demonstrated strong concurrent validity with clinical photography and a reproducible precision advantage in detecting alveolar bone-level changes in patients with periodontitis. These findings support its clinical utility as a radiation-free, high-sensitivity bone monitoring tool. Larger longitudinal studies with CBCT validation are warranted.

Relapse of aligned mandibular anterior teeth and the progressive collapse of the mandibular anterior arch are historically striking problems for orthodontists. The etiology of this collapse, and the cause of mesial molar drift, are unknown. However, light continuous (quasi-continuous) intra-oral pressures and forces applied to the mandibular dentition have been implicated. To explore this further, we use three-dimensional finite element analysis to investigate the influence of these intra-oral loads (tongue pressure, lip-cheek pressure, and interdental force) on mandibular arch collapse and mesial molar drift. Dentitions of three-dimensional finite element mandibular models were subjected to a wide range of simulated tongue pressures, lip-cheek pressures, and transseptal fiber-mediated interdental forces reported in the literature. Resulting crown displacement measurements from these isolated loads were made along with measurements resulting from simultaneous combined application of literature-defined mean tongue pressure, lip-cheek pressure, and interdental force. Our results indicate that tongue pressure alone results in generalized arch expansion and tooth spacing while lip-cheek pressure and interdental force result in generalized arch collapse, anterior crowding, and mesial molar displacement. Simultaneous application of tongue pressure, lip-cheek pressure, and interdental force mean values, as would occur in vivo, results in incisor crowding, intercanine width reduction, and mesial molar displacement. Our results suggest mandibular anterior arch collapse (incisor crowding / intercanine width reduction), and mesial molar displacement result from simultaneous application of tongue pressure, lip-cheek pressure, and interdental force.

This cross-sectional study evaluated the association of tooth loss and oral health-related quality of life (OHRQoL) with sexual function in adult women attending a primary dental care service. Methodology: Ninety-nine sexually active women aged 19-66 years were consecutively recruited from a primary dental care service between January and October 2023. Tooth loss was quantified by standardized oral examination. OHRQoL was assessed using the Oral Health Impact Profile-14 (OHIP-14), and sexual function was assessed using the Female Sexual Function Index (FSFI). Sexual dysfunction was defined as FSFI <=26.5. Spearman rank correlation was used for bivariate analyses. Multivariable logistic regression was used to evaluate factors associated with sexual dysfunction, including number of missing teeth, OHIP-14 score, age, and relationship status. Results: Tooth loss was present in 83.8% of participants, with a median of 4 missing teeth (interquartile range [IQR], 1-10). Sexual dysfunction was identified in 62.6% of women. FSFI scores were negatively correlated with number of missing teeth (rho = -0.407; p < 0.001), OHIP-14 score (rho = -0.279; p = 0.005), and age (rho = -0.334; p < 0.001). In multivariable logistic regression, OHIP-14 score was independently associated with sexual dysfunction (OR = 1.05; 95% CI, 1.01-1.10; p = 0.015), whereas number of missing teeth was not independently associated after adjustment. Conclusion: Worse OHRQoL was independently associated with sexual dysfunction, whereas tooth loss was associated with lower FSFI scores only in bivariate analysis. These findings are compatible with the hypothesis that the impact of tooth loss on sexual function may be partly explained by oral health-related quality of life, but longitudinal studies are required to test causal and mediational pathways. Keywords: tooth loss; oral health; quality of life; sexual dysfunction, physiological; women; cross-sectional studies

Background: Accurately identifying patients who will require opioids after third molar extraction could improve pain management while supporting opioid stewardship. This study evaluated surgeon accuracy in predicting supplemental opioid use following treatment with ibuprofen and acetaminophen. Methods: Patients (N=85) undergoing third molar extraction were treated with a standardized analgesic regimen of ibuprofen+acetaminophen, with supplemental opioid if needed. Four surgeons independently reviewed preoperative radiographs, assessed surgical difficulty using the Pederson scale, and rated the likelihood of supplemental opioid use on a 5-point Likert scale. Inter-rater reliability was assessed using intraclass correlation coefficients (ICC). The relationship between surgeon ratings and postoperative opioid use was evaluated using logistic regression and receiver operating characteristic (ROC) analysis. Results: Seventeen patients used supplemental opioid analgesics. Inter-rater reliability among surgeons was moderate (ICC3=0.606, 95%CI: 0.505-0.700), while reliability of the average rating across surgeons was good (ICC3k = 0.860, 95% CI: 0.804-0.903). Median surgeon rating was not associated with postoperative opioid use (OR: 0.800, 95% CI: 0.414-1.51, p=0.496) and demonstrated poor discrimination (AUC: 0.551, 95% CI: 0.392-0.710). Surgeon ratings were positively associated with Pederson score (beta=0.073, 95%CI: 0.050-0.096; p<0.001). Conclusions: Surgeons demonstrated moderate agreement, but these assessments did not accurately identify patients who ultimately required supplemental opioids. Surgeon judgments appeared to be influenced by anticipated surgical difficulty. Practical Implications: Clinicians should follow current recommendations against routine "just-in-case" opioid prescribing after third molar extraction. Future studies should focus on identifying clinical and biological predictors of inadequate analgesic response to NSAIDs to support individualized pain management strategies.

We conducted a multi-ancestry genome-wide association study (GWAS) of developmental defects of enamel (DDE) in the primary dentition among 6,061 U.S. preschool-aged children (3--5 years). We investigated four DDE phenotypes (demarcated opacities, diffuse opacities, hypoplastic defects, and a combined DDE trait) leveraging main-effect models, joint gene-sex interaction testing (2df), and sex-stratified analyses. SNP-based heritability for the combined DDE trait was estimated at 20%, with concordance analyses robustly supporting a genetic etiology. We identified 39 unique genome-wide significant loci (P<5 x 10-8;), with five surpassing a study-wide Bonferroni-corrected statistical significance criterion (P<1.25 x 10-9), including Y RNA and ALDH1A1. The main-effect GWAS identified 20 loci, including HBS1L and MYB, genes regulating hematopoiesis with plausible roles in amelogenesis. Joint test and sex-stratified analyses revealed 19 additional loci, including ALDH1A1, TENM2, and DLGAP2, demonstrating sex-specific heterogeneity. Nineteen loci exhibited sex-specific differences after Bonferroni correction (P<2 x 10-3), including genes involved in retinoic acid signaling (ALDH1A1), odontogenesis (TENM2), and neurodevelopment (DLGAP2, CDH10). Pathway enrichment highlighted ectodermal and synapse organization networks, suggesting shared etiological mechanisms between DDE and systemic conditions like neurofibromatosis and autism spectrum disorder. Notably, no locus generalized in an external GWAS of permanent dentition DDE, underscoring fundamental biological differences in the genetic architectures governing primary versus permanent enamel formation. Crucially, a comprehensive cross-trait pleiotropy lookup against early childhood caries (ECC) revealed no shared genetic architecture, supporting the notion that the established clinical and epidemiological association between DDE and ECC is likely driven by structural defects increasing caries lesion susceptibility rather than genetic pleiotropy. By integrating gene-sex interaction testing, this study offers novel insights into the complex, sexually dimorphic genetic etiology of DDE and augments the biological evidence base that can support the development of precision pediatric dentistry.

This study compared and evaluated two widely used deep learning-based artificial intelligence (AI) models, U-Net++ and YOLOv11, for quantifying tooth and root exposure on intraoral camera images of the mandibular anterior lingual region. Intraoral images of the mandibular anterior lingual region were collected from 291 patients (mean age, 52.8 years) at a university hospital dental clinic with institutional review board approval (YUMC IRB 2021-07-019-002). A total of 266 eligible images (mean, 5.50 teeth per image; 3.70 teeth with root exposure) were annotated. YOLOv11 and U-Net++ were fine-tuned using five-fold cross-validation with data augmentation. Model performance was evaluated on a held-out test set of 40 images using Dice coefficient, Intersection over Union (IoU), accuracy, mean Average Precision at an IoU threshold of 0.5 (mAP50), Lins concordance correlation coefficient (CCC), and intraclass correlation coefficient (ICC). Confidence intervals were estimated using 10,000 bootstrap iterations. For tooth segmentation, U-Net++ demonstrated superior performance, with high accuracy (0.981), Dice coefficient (0.971), and IoU (0.944). In contrast, for root segmentation, YOLOv11 outperformed U-Net++, achieving higher Dice (0.860 vs. 0.746) and IoU (0.762 vs. 0.631). Notably, YOLOv11 showed stronger agreement with the ground truth for quantifying the exposed root ratio (ERR) (CCC, 0.973; ICC, 0.975). These findings suggest that accurate detection of root exposure is important for assessing periodontal tissue loss and that YOLOv11 is a promising model for root exposure quantification in intraoral images. YOLOv11-based quantification of root exposure may serve as a useful adjunctive AI tool for screening and monitoring periodontal conditions and may support individualized treatment planning.

Background/Objectives Dental anxiety remains a significant psychological barrier to oral healthcare, particularly for invasive surgical procedures such as tooth extraction. Aromatherapy using Lavandula angustifolia (lavender) has been proposed as a non-pharmacological adjunct to manage acute preoperative anxiety. This clinical trial evaluated the efficacy of inhaled lavender essential oil on anxiety severity in patients awaiting elective single-tooth extraction. Methods An assessor-blinded randomized controlled clinical trial with limited participant blinding (inherent to the recognizable scent of the intervention), registered with the Iranian Registry of Clinical Trials (IRCT20190920044824N1), was conducted among 60 patients requiring non-surgical tooth extraction. Participants were randomly assigned to an intervention group (n=30), receiving two drops of pure lavender essential oil on a sterile gauze for 20 minutes of inhalation, or a control group (n=30), receiving sweet almond oil as an olfactory placebo. The primary outcome was the severity of somatic anxiety symptoms, measured pre- and post-intervention using the Beck Anxiety Inventory (BAI). Because a significant baseline-by-treatment interaction was detected, a General Linear Model (GLM) retaining this interaction term was used to estimate adjusted group means at the grand-mean baseline, with bootstrap 95% confidence intervals (10,000 resamples). Two pre-specified checks of robustness were performed: (i) an unadjusted, baseline-naive Mann-Whitney U comparison of raw post-intervention scores, and (ii) a sensitivity analysis re-fitting the adjusted model after excluding two baseline outliers identified by inspection of model residuals. Results There were no significant baseline differences between the intervention and control groups regarding sex distribution (p=0.100), mean age (p=0.479), or pre-intervention BAI scores (Mann-Whitney p=0.215). A significant baseline-by-treatment interaction was detected (p<0.001). In the full sample (n=60), the baseline-adjusted GLM estimated a lower post-intervention BAI mean in the intervention group (22.38) than the control group (23.09), an adjusted difference of -0.71 (bootstrap 95% CI: -1.41 to -0.06; p=0.028). However, this signal was not robust: after excluding two patients identified as outliers in the model residuals (n=58), the adjusted difference attenuated to -0.51 and was no longer statistically significant (bootstrap 95% CI: -1.11 to 0.10; p=0.098). The unadjusted comparison of raw post-intervention scores was likewise not significant in the full sample (Mann-Whitney p=0.754). Conclusions A baseline-adjusted analysis of this trial produced an initial signal suggesting a small anxiolytic effect of inhaled lavender, but this signal was not stable under a pre-specified outlier-exclusion sensitivity analysis and was not supported by the unadjusted comparison of raw scores. Taken together, the totality of evidence from this trial is insufficient to conclude that inhaled lavender aromatherapy produces a reliable reduction in acute preoperative dental anxiety. Beyond its clinical findings, this trial offers a concrete, quantified illustration of how baseline imbalance and a small number of influential observations can generate a fragile, model-dependent treatment signal in a modestly sized randomized trial, a methodological caution directly relevant to the design and analysis of future small RCTs in dental and complementary medicine research.

Orofacial clefts (OFCs), including cleft lip (CL), cleft palate (CP), and cleft lip with cleft palate (CLP), are among the most common craniofacial malformations in humans, with a birth prevalence of approximately 1 in 1,000 live births globally. Non-syndromic forms of OFC are predominantly genetic, with significant variability in prevalence across populations. Understanding the genetic underpinnings of OFCs remains a key public health priority, given the substantial medical and societal burden of these conditions. Recent genome-wide association studies (GWAS) have implicated numerous genetic loci, but challenges remain due to genetic heterogeneity and complex gene-environment interactions. This study aimed to identify sex-specific genetic risk factors for cleft lip with or without cleft palate (CL/P) through a meta-analysis of whole genome sequencing (WGS) data from 1,922 case-parent trios across eight diverse cohorts. Our approach revealed four SNPs in three distinct regions that showed genome-wide significant sex-specific effects. However, despite each of these SNPs passing standard quality control filters, follow-up analyses showed that these signals most likely were technical artifacts caused by sequencing errors, in particular mis-mapped reads due to sequence similarities with the sex chromosomes. These findings highlight the necessity for careful scrutiny when studying differences between the sexes in genetic association studies.

This protocol describes a standardized and reproducible minimally invasive approach for establishing mouse models of bisphosphonate-related osteonecrosis of the jaw (BRONJ) and osteoradionecrosis of the jaw (ORNJ). The method combines a unified low-trauma oral surgical procedure with disease-specific injury induction strategies to generate robust and clinically relevant models of jaw osteonecrosis. For BRONJ, systemic zoledronic acid administration is coupled with mandibular first molar extraction using tape-assisted mouth opening and customized bent micro-forceps, minimizing soft tissue damage and reducing procedural variability. For ORNJ, a customized lead-shielding platform enables precise, noninvasive mandible-targeted irradiation, producing reproducible bone injury while limiting off-target radiation exposure. Together, these complementary models provide a consistent and minimally invasive framework for investigating jaw osteonecrosis arising from distinct etiologies. The protocol supports comprehensive downstream analyses, including micro-computed tomography, histology, and immunofluorescence, and facilitates mechanistic studies of disease pathogenesis, bone regeneration, and therapeutic intervention.

Periodontitis, a chronic inflammatory disease affecting approximately 40% of U.S. adults aged 30 years and older, is characterized by dysbiosis of the dental plaque microbiome. However, although scaling and root planing (SRP) is the cornerstone of periodontal treatment, its effects on the taxonomic composition and functional potential of the dental plaque microbiome remain incompletely understood. In this study, we used whole-metagenome shotgun sequencing to characterize taxonomic composition and functional potential in dental plaque microbiomes collected from 39 patients with Stage II or III generalized periodontitis before and 3-4 months after SRP. Consistent with clinical improvement, periodontal therapy significantly reduced bleeding on probing and plaque index. Whole-metagenome shotgun sequencing identified 3.18 million non-redundant genes and 12,353 microbial species across 78 samples, revealing increased gene and species richness after treatment, along with a significant restructuring of microbial community. Established periodontal pathogens, including Porphyromonas gingivalis and Tannerella forsythia, as well as the emerging pathogen Escherichia coli, decreased following treatment, whereas health-associated early colonizers, including multiple Actinomyces species and Streptococcus cristatus, increased. Functional annotation using the Carbohydrate-Active Enzymes (CAZy) database identified treatment-associated differences in several carbohydrate-active enzymes, including multiple glycosyltransferases, indicating remodeling of the predicted functional potential of the dental plaque microbiome. These findings demonstrate that successful SRP promotes coordinated taxonomic and predicted functional remodeling of the dental plaque microbiome and highlight the value of shotgun metagenomic sequencing for characterizing both taxonomic and functional recovery following periodontal therapy.

Background: Children in Central Africa face compounded vulnerabilities, yet no comprehensive review has synthesized evidence on ECC prevalence or risk factors. This study aims to determine the prevalence and identify risk factors associated with early childhood caries (ECC) in the Central Africa Sub-region. Methods: This systematic review and meta-analysis was registered with the International Prospective Register of Systematic Reviews (PROSPERO) under the ID CRD420251019424. A comprehensive literature search was conducted across PubMed, African Journals Online, Scopus, Web of Science, CINAHL, and ProQuest as well as through Google Scholar and other grey literature sources, with no language restrictions. Eligible studies were limited to cross-sectional, cohort, case-control, and clinical trials that reported baseline data on the prevalence and risk factors of ECC in the Central Africa sub-region among children under the age of six. There was no restriction on publication date. Exclusion criteria included letters to the editor, commentaries, studies without accessible full texts, systematic reviews, reviews lacking original data, studies with overlapping data already included, and non-peer-reviewed sources such as books. Study quality was assessed using the Joanna Briggs Institute Critical Appraisal Tool. A random-effects meta-analysis was conducted using Review Manager 5.4.1, and heterogeneity was assessed using the I statistic. Results: Five studies met the eligibility criteria, encompassing 640 participants. Three of the studies were from Cameroon, and one from the Democratic Republic of Congo and the Central African Republic, respectively. One study that did not report the prevalence of ECC and another non-peer-reviewed publication were excluded from the meta-analysis. The prevalence of ECC in the region ranged between 29.4% and 80% with a pooled prevalence of 57% (95% CI: 22 to 92%; I = 98%). Identified risk factors for ECC were related to oral hygiene behaviours, diet and feeding practices, parental/caregiver factors, and access to dental care. Conclusions: ECC is highly prevalent in Central Africa, driven by preventable factors. Urgent region-specific policies and programs are needed to improve preventive services and address data gaps across this under-resourced subregion.

Background Growing evidence suggests that the gut microbiome influences nasal and ocular allergic inflammation through gut-mucosal immune interactions. Yet, its association with Allergic rhinitis (AR) and allergic eye symptoms (AES) remains incompletely understood in large population-based cohorts. Objective To examine associations between the gut microbiome and self-reported AR and AES in Finnish adults. Methods Shallow metagenomic sequencing was performed on stool samples from a population-based cohort (FINRISK02; n = 7,231). Microbial taxonomic and functional profiles were compared between individuals with AR (n = 1,950), AES (n = 1,554), combined allergies (AR and/or AES; n = 2,305), and controls without reported symptoms (n = 3,175). Results Allergic groups exhibited lower microbial richness and phylogenetic diversity than controls. Shared microbial and functional signatures were observed across AR and AES, consistent with their high co-occurrence (N = 1,199). Compared with controls, allergic groups showed enrichment of 17 bacterial species, predominantly from the Clostridia class, including taxa previously associated with asthma, chronic obstructive pulmonary disease, and atopic dermatitis. Allergic individuals also exhibited enrichment of pathways related to mucosal carbohydrate processing, shikimate metabolism, histidine turnover, and broader amino acid metabolism. Concurrent enrichment of histidine biosynthesis and degradation suggested altered microbial histidine metabolism. Conclusions Adult allergic symptoms are associated with gut microbiome taxonomic and functional alterations linked to mucosal barrier function and immune-related metabolism, supporting a shared gut-mucosal immune axis across allergic phenotypes.

Background: Short-chain fatty acids (SCFAs) are microbial metabolites with immunoregulatory properties. Human milk contains SCFAs which have been proposed as potential modulators of infant immune development. We aimed to examine associations between human milk SCFA concentrations and infant allergic disease outcomes in a high-risk cohort of infants of atopic mothers. Methods: SCFAs were measured by targeted liquid chromatography-mass spectrometry in human milk samples collected at 3 and 6 months postpartum from atopic mothers enrolled in the Infant Fish Oil Supplementation (IFOS) Study (n=147). Associations between milk SCFA concentrations and early childhood allergic disease outcomes (atopic dermatitis, food allergy, allergic rhinitis, and allergen sensitisation at 1 and 2-3 years) were examined using logistic regression. Results: Human milk SCFA concentrations were broadly stable between 3 and 6 months postpartum, except for acetate which was significantly elevated at 6 months. No significant associations were observed between human milk SCFA concentrations and any allergic disease outcome after correction for multiple comparisons (all p>0.05). Conclusions: Human milk SCFA concentrations are not associated with allergic disease outcomes up to 4 years of age. These findings suggest that oral SCFA exposure via human milk is insufficient to reduce infant allergy risk, and that gut SCFA production may be a more relevant target for future allergy prevention research.

Background: The hyperplasia and dysplasia stage (pre-cancer) offers a viable opportunity to reduce the incidence and mortality of oral cancer through early prevention. Smartphone-based Artificial Intelligence (AI) enabled screening of potentially malignant oral lesions offers a scalable solution for this in resource-constrained settings. However, developing accurate and explainable AI segmentation models require high-quality, pixel-level annotated data. This process that is prohibitively expensive, time-consuming, and prone to inter-observer subjectivity among clinical experts. Methods: We designed and empirically validated a Deep Learning-driven Human-in-the-Loop (HITL) framework to pixel-annotate a dataset of 3026 clinical oral images. Using an iterative pseudo-labeling pipeline, we evaluated the model's learning dynamics and performance evolution across five training cycles. We conducted controlled experiments to quantify the networks tolerance to intermediate level of label noise (unreviewed pseudo-labels) to resolve clinical subjectivity using pixel-wise Cohen's Kappa and the STAPLE consensus algorithm. Results: Iterative self-training produced sustained improvements in lesion detection and spatial localization. However, controlled experiments revealed that including even a modest fraction ({approx}10%) of unreviewed pseudo-labels led to a three-to-four-fold increase in training convergence instability and induced a conservative prediction bias that negatively impacted model recall. When measured against multi-expert ground truth, the model's performance converged with the inter-rater reliability ceiling ({kappa} {approx} 0.65), indicating that its predictions fell within the envelope of human agreement. Conclusions: Our findings emphasize that a final expert-driven quality assurance step remains absolutely essential to mitigate training instability, confirmation bias, and clinically unacceptable drops in recall caused by label noise. Overall, this work provides a scalable, empirically validated blueprint for building domain-specific medical imaging datasets in low-resource global health settings, where the dual challenges of annotation cost and inter-observer variability are most acute.

The COVID-19 pandemic has imposed a reevaluation of safety protocols across various sectors, including the arts. This study addresses a critical gap in understanding SARS-CoV-2 persistence on materials commonly associated with musical instruments and scores, such as alloys, varnishes, reeds, and paper. While previous research has explored viral survival on various surfaces, limited data exists for materials specific to musical contexts. In this work, we investigate the efficacy of quarantine as a non-destructive method for inactivating SARS-CoV-2 on 16 materials, including brass, silver plating, ABS plastic, ebonite, and various varnishes and paper types. Results revealed significant variability in viral persistence across materials. Non-porous surfaces like metals and ABS plastic cleared infectivity within 3 days, while porous materials such as reeds and music scores required up to 7 days. Gold-plated brass and certain varnishes showed intermediate persistence, with infectivity clearing after 4 days. These findings are in agreement with prior studies indicating that SARS-CoV-2 survival is highly dependent on surface composition, with porous and organic-coated materials retaining viable virus longer due to reduced environmental stress. Our results highlight the feasibility of stratified quarantine protocols based on material type, offering practical guidelines for musicians and institutions and provides critical insights for mitigating SARS-CoV-2 transmission risks in musical settings.

Purpose: Animal models of myopia typically induce monocular refractive shifts via form deprivation (FD) or lens-induced myopia (LIM), modeling susceptibility to myopia, but with potentially limited applicability to childhood myopia. Here we describe a novel, genetically diverse mouse model of naturally occurring refractive error (NORE) with three distinct refractive phenotypes: hyperopic, myopic, and intermediate. Methods: C57BL/6J mice were mated to 129S2/SvPasCrl mice to create F1 or F2 offspring. Refractive errors in male and female F1 (N=21) and F2 (N=101) mice were assessed on postnatal days (P) 28 and 42 using photorefractometry. In a subset of mice (N=30 - 40), corneal radius of curvature, axial ocular dimensions, retinal and visual function were assessed. Results: F2 mice were classified as NORE with either hyperopic (RE [&ge;] 0 diopters (D) at P28 and P42), myopic (RE<0D at P28 and P42) or intermediate (RE<0D at P28 and RE [&ge;] 0D at P42) refractions based on individual trajectories. All ocular parameters changed with age, with significantly slower growth in axial length and vitreous chamber depth in the intermediate versus myopic mice (p<0.05). Lens thickness was smaller in the myopic group at P28. Differences in refraction were not attributed to variances in retinal function or dopamine signaling. Conclusions: NORE mice represent a novel, genetically diverse wild-type mouse model that, unlike traditional models, does not require interventions such as FD or LIM to induce myopia. NORE mice provide a valuable tool for future investigations of genetic and environmental mechanisms and targeted therapeutic strategies for refractive errors.

Reporters are widely used in Drosophila genetics to visualize gene expression and cell lineages. However, uncharacterized limitations in specific reporter lines can lead to data misinterpretation. Here, we identify a consistent, driver-independent tdTomato signal in the adult proventriculus from the widely used lexAop-tdTomato.nls reporter line. This signal was observed across multiple lexA driver combinations and was directly detectable in lexAop-tdTomato.nls responder-alone adult proventriculi lacking any lexA driver and without antibody staining. In contrast, no comparable native red fluorescence was detected in larval proventriculi under the same no-antibody imaging condition. Mouse and rabbit anti-RFP immunostaining further supported the presence of proventriculus-associated tdTomato/RFP antigen in adult responder-alone animals. In larval responder-alone proventriculi, antibody-amplified staining was antibody-source-dependent: a detectable signal was observed only with rabbit anti-RFP, whereas mouse and rat anti-RFP produced no reliable detectable signal under the same staining condition. A driver-matched comparison using lexAop-RFP.nls did not reproduce the proventricular signal, arguing against detectable ectopic activity of the tested lexA driver in this tissue. However, because lexAop-tdTomato.nls and lexAop-RFP.nls differ in reporter/transgene architecture and possibly genomic insertion context, the underlying cause cannot be assigned specifically to the lexAop sequence. Our findings highlight the necessity of including driver-negative and no-antibody controls when using this reporter line in adult Drosophila proventriculus and gut studies.

Background: Single cell RNA sequencing provides a wealth of information to explore the complexities of the tumour microenvironment, but crucially the spatial topology of the tumour is lost and studying cellular interactions is limited. Spatial transcriptomics aims to address this however the technique remains cost prohibitive for the generation of data from meaningfully-sized clinical cohorts. In contrast, spatial proteomic profiling with multiplex immunofluorescence, preserves spatial interactions, is relatively cost accessible, and is scalable for large clinical cohorts to address powerful translational questions. Whilst multiplex approaches have advanced in recent years, we note that cancer-associated fibroblasts (CAFs) have been explored in less detail, potentially due to difficulties associated with CAF heterogeneity and the diversity of markers used to define them. Methods: We designed, optimised, and validated a multiplex immunofluorescence panel that combines four frequently used CAF markers; alpha smooth muscle actin (aSMA), fibroblast activation protein (FAP), podoplanin (PDPN) and platelet-derived growth factor receptor alpha (PDGFRa) with CD8 and pan-cytokeratin. Here we share our methodology and the practical considerations taken to inform the final panel design. We also highlight the benefits of robust optimisation experiments.

Lung transplantation (LT) is the ultimate treatment option for patients suffering from end stage cystic fibrosis (CF). Most LT-patients, colonized pre-LT by Pseudomonas aeruginosa witness colonization of their non-CF allograft within a few days or weeks post-LT, thereby compromising graft and life expectancy. How P. aeruginosa isolates adapted for years to the specific CF lung environment efficiently colonize and survive in the non-CF allograft environment remains unclear. To address this question, we collected sequential isolates from CF LT-recipients and non-CF LT-recipients and performed phenotypic and genetic analyses of pairs of early and late isolates from LT-patients. We found evidence for mutations compatible with a switch from biofilm to planktonic lifestyle as well as loss of mucoid phenotypes. Hypermutators, characteristic of chronic CF-adapted isolates, were also found in four LT-patients. Their persistence in the non-CF allograft environment suggests a continuous seeding from the sinuses. Our results suggest that in CF LT-recipients efficient colonisation by P. aeruginosa of the allograft implies both adaptation and continuous seeding from the sinuses to the lower respiratory tract.

Background Diagnostic interpretation of kidney allograft biopsies using the Banff classification remains variable, but the determinants of this variability are not fully defined. We performed a global, fully digital multi-reader study to identify the principal drivers of disagreement in Banff-based assessment. Methods Thirty six kidney transplant biopsies were independently scored by 67 renal pathologists on a standardized digital platform. Readers assessed Banff lesions on hematoxylin and eosin, periodic acid Schiff, and Jones' silver stains; final diagnostic categories were assigned using prespecified Banff-based decision rules. Interobserver agreement was quantified with Gwet's agreement coefficient (AC) statistics. Determinants of diagnostic agreement were evaluated) using pairwise mixed-effects logistic regression, and reader similarity was examined by principal component analysis (PCA) with post hoc molecular annotation. Results Agreement for final diagnostic categories was moderate (Gwet's AC1, 0.55; 95% CI, 0.47 - 0.63). Lesion-level agreement varied substantially, with lowest agreement for selected threshold-dependent inflammatory or semi-quantitative lesions, including interstitial inflammation in areas of IFTA, peritubular capillaritis and arteriolar hyalinosis. Diagnostic concordance differed markedly across biopsies, indicating strong case-level heterogeneity. In pairwise models, differences in active inflammatory and vascular lesion scoring were the strongest correlates of diagnostic disagreement; reader experience and geography contributed minimally. Principal component analysis showed reader variation was organized along two dominant axes: a rejection-calling threshold axis linked mainly to tubulointerstitial inflammatory injury, and a T cell-mediated (TCMR/TI) and antibody-mediated/microvascular (AMR/MVI) inflammation-oriented phenotypic classification axis. Conclusion Interobserver variation in Banff-based kidney transplant biopsy assessment is structured rather than random and driven mainly by how readers threshold and integrate key inflammatory lesion compartments rather than experience or geographic location.