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Background: The larynx consists of the supraglottis, glottis, and subglottis and each differ in tissue composition, lymphatic drainage, ability to counter infections, and response to injuries. However, the cellular mechanisms driving laryngeal homoeostasis remain largely unexplored. As a result, understanding disease pathogenesis within the larynx including idiopathic subglottic stenosis (iSGS) and intubation-related traumatic stenosis has been challenging. Here, we sought to characterise the cellular processes governing laryngeal health and disease.

Methods: As part of the prospective Canadian Airways Research (CARE) iSGS study, we characterised 122,004 high-quality transcriptomes using single nucleus RNA-sequencing to profile 11 human epiglottis and 17 human subglottis biopsies across three different conditions: control, iSGS, and intubation-related traumatic stenosis to define cell populations and pathways associated with disease. We validated our results using cohort-level bulk transcriptomics using 114 human epiglottis and 121 human subglottis.

Findings: We defined the single-cell taxonomy of the human subglottis and epiglottis using single-nucleus sequencing in both healthy and disease states. Mechanistically, we discovered the presence of unique epithelial and fibroblast progenitor subsets within the control subglottis but not within the anatomically adjacent epiglottis. The uncontrolled proliferation of these cellular subsets exhibited skewed sex hormone signalling and orchestrated a fibro-inflammatory cascade. We leveraged cohort-level bulk transcriptomics to define hallmarks of iSGS associated with disease covariates and introduced the first biomarker associated with recurrent relapse. Longitudinal sampling demonstrated that the subglottic microenvironment in patients with iSGS is changing dynamically with and without therapeutic intervention.

Interpretation: Together, our data refines our understanding of laryngeal biology, nominates candidate compounds for iSGS treatment, and serves as a transformative platform for future clinical investigations to further precision laryngology.

Funding: This study was funded by a grant from the American Laryngology Association (#1082), an Academic Medical Organisation of Southwestern Ontario innovation fund grant (INN21-016), grant support from the Departments of Otolaryngology-Head and Neck Surgery at University of Toronto, Canada and Western University, Canada. ACN was supported by the Wolfe Surgical Research Professorship in the Biology of Head and Neck Cancers Fund. PYFZ was supported by a Vanier Canada Graduate Scholarship and PSI Foundation fellowship.

Background: First described in 1972, idiopathic subglottic stenosis (iSGS) is a serious chronic orphan disease characterised by recurrent scarring of the subglottis. Although the cause is unknown, iSGS is almost exclusively restricted to Caucasian females typically in their fourth to sixth decade. However, given its rare incidence (1:400,000), understanding the clinical trajectory and molecular factors associated with iSGS disease development and prognosis has been difficult. In the current study we sought to unravel the pathogenesis of iSGS at the clinical, transcriptional, and genetic level in a prospective cohort.

Methods: We prospectively enrolled 126 patients with iSGS, 104 controls, and 13 patients with traumatic SGS. Within this cohort, we profiled 114 human epiglottis and 121 human subglottis biopsies across three different conditions: control, iSGS, and intubation-related traumatic stenosis using bulk and single nucleus RNA-sequencing. Whole exome sequencing for germline variants was performed for 70 controls and 75 patients with iSGS.

Findings: Patients with iSGS received a median number of five (range 0-18) surgical dilations at a rate of 1.031 dilations (range: 0.12-6.2) per year. Older age at diagnosis and higher Cotton-Myers grade were associated with increased number of surgical dilations over time. Cohort-level bulk transcriptomics found that iSGS pathology was restricted within the subglottis and did not affect anatomically adjacent epiglottis, opposite to previous hypotheses. We further identified cellular subsets associated with iSGS prognosis and severity. Finally, patients with iSGS exhibit lower testosterone predicted using a polygenic score.

Interpretation: Together, our data refines our understanding of laryngeal biology and provides insights into the clinical trajectory of subglottic stenoses. Future research should explore the role of testosterone in the development of iSGS.

Funding: This study was funded by a grant from the American Laryngology Association (#1082), an Academic Medical Organization of Southwestern Ontario innovation fund grant (INN21-016), grant support from the Departments of Otolaryngology-Head and Neck Surgery at University of Toronto and Western University. ACN was supported by the Wolfe Surgical Research Professorship in the Biology of Head and Neck Cancers Fund. PYFZ was supported by a Vanier Canada Graduate Scholarship and PSI foundation fellowship.

Background: The gut microbiota of infants harbours a higher proportion of antibiotic resistance genes (ARGs) compared to adults, even in infants never exposed to antibiotics. Our study aims to elucidate this phenomenon by analysing how different perinatal factors influence the presence of ARGs, mobile genetic elements (MGEs), and their bacterial hosts in the infant gut.

Methods: We searched MEDLINE and Embase up to April 3rd, 2023, for studies reporting infant cohorts with shotgun metagenomic sequencing of stool samples. The systematic search identified 14 longitudinal infant cohorts from 10 countries across three continents, featuring publicly available sequencing data with corresponding metadata. For subsequent integrative bioinformatic analyses, we used 3981 high-quality metagenomic samples from 1270 infants and 415 mothers.

Findings: We identified distinct trajectories of the resistome and mobilome associated with birth mode, gestational age, antibiotic use, and geographical location. Geographical variation was exemplified by differences between cohorts from Europe, Southern Africa, and Northern America, which showed variation in both diversity and abundance of ARGs. On the other hand, we did not detect a significant impact of breastfeeding on the infants' gut resistome. More than half of detected ARGs co-localised with plasmids in key bacterial hosts, such as Escherichia coli and Enterococcus faecalis. These ARG-associated plasmids were gradually lost during infancy. We also demonstrate that E. coli role as a primary modulator of the infant gut resistome and mobilome is facilitated by its increased abundance and strain diversity compared to adults.

Interpretation: Birth mode, gestational age, antibiotic exposure, and geographical location significantly influence the development of the infant gut resistome and mobilome. A reduction in E. coli relative abundance over time appears as a key factor driving the decrease in both resistome and plasmid relative abundance as infants grow.

Funding: Centre for Advanced Study in Oslo, Norway. Centre for New Antibacterial Strategies through the Tromsø Research Foundation, Norway.

Background: Aceruloplasminemia (ACP) is a rare recessive disease caused by loss of ceruloplasmin activity due to pathogenic variants in the ceruloplasmin (CP) gene. ACP causes iron accumulation in various organs, leading to neurodegeneration, anaemia, and diabetes. Estimating ACP prevalence is challenging, particularly as missense variants are not readily identified as pathogenic.

Methods: Heterozygous missense variants likely to impact function were mapped in gnomAD and representative examples analysed for effects on CP activity. This knowledge was complemented by prediction of destabilizing effects of potentially pathogenic missense variants and integrated with loss-of-function mutations. Global ACP prevalence was predicted and compared with a more traditional method.

Findings: Several as yet uncharacterised missense CP variants of pathogenic interest were identified by structure-function in-silico analysis. A representative subset was functionally validated, together with known ACP missense variants. Insights on the relative importance of copper ions coordinating centres in CP and its substrate specificity were discovered. Overall, a destabilizing effect was predicted for 130 missense CP variants. This information, integrated with known ACP missense and loss-of-function CP variants in gnomAD, allowed an estimation of ACP prevalence of 12.6/10⁶. An alternative analysis based on minor allele frequency ≤0.01 resulted in an ACP prevalence as high as 8/10⁶.

Interpretation: These prevalence estimates for ACP are 20-25-fold higher than previously estimated and underscore the applicability of structure-function based analyses of real-world genetic variability to provide an alternative method for representing the frequency of rare disease variants.

Funding: REACT-EU PON 2014-2021, Kedrion S.p.A.

Background: Oligogenic inheritance has been suggested as a possible mechanism to explain the broad phenotype observed in individuals with differences of sex development (DSD) harbouring NR5A1/SF-1 variants.

Methods: We investigated genetic patterns of possible oligogenicity in a cohort of 30 individuals with NR5A1/SF-1 variants and 46,XY DSD recruited from the international SF1next study, using whole exome sequencing (WES) on family trios whenever available. WES data were analysed using a tailored filtering algorithm designed to identify rare variants in DSD and SF-1-related genes. Identified variants were subsequently tested using the Oligogenic Resource for Variant Analysis (ORVAL) bioinformatics platform for a possible combined pathogenicity with the individual NR5A1/SF-1 variant.

Findings: In 73% (22/30) of the individuals with NR5A1/SF-1 related 46,XY DSD, we identified one to seven additional variants, predominantly in known DSD-related genes, that might contribute to the phenotype. We found identical variants in eight unrelated individuals with DSD in DSD-related genes (e.g., TBCE, FLNB, GLI3 and PDGFRA) and different variants in eight genes frequently associated with DSD (e.g., CDH23, FLNB, GLI2, KAT6B, MYO7A, PKD1, SPRY4 and ZFPM2) in 15 index cases. Our study also identified combinations with NR5A1/SF-1 variants and variants in novel candidate genes.

Interpretation: These findings highlight the complex genetic landscape of DSD associated with NR5A1/SF-1, where in several cases, the use of advanced genetic testing and filtering with specific algorithms and machine learning tools revealed additional genetic hits that may contribute to the phenotype.

Funding: Swiss National Science Foundation and Boveri Foundation Zurich.

Extracellular vesicles (EVs) are lipid-enclosed nanovesicles secreted by diverse cell types that orchestrate intercellular communication through cargo delivery. Their pivotal roles span from supporting the development of normal central nervous system (CNS) to contributing to the pathogenesis of neurological diseases. Particularly noteworthy is their involvement in the propagation of pathogenic proteins, such as those involved in neurodegenerative disorders, and nucleic acids, closely linking them to disease onset and progression. Moreover, EVs have emerged as promising diagnostic biomarkers for neurological disorders and as tools for disease staging, owing to their ability to traverse the blood-brain barrier and their specific, stable, and accessible properties. This review comprehensively explores the realm of CNS-derived EVs found in peripheral blood, encompassing their detection methods, transport mechanisms, and diverse roles in various neurodegenerative diseases. Furthermore, we evaluate the potentials and limitations of EVs in clinical applications and highlight prospective research directions in this rapidly evolving field.

Background: Successful seizure onset zone (SOZ) localisation for epilepsy surgery often relies upon intracranial recordings. Accurate delineation requires anatomical detail yet influences of intracranial electrode density on clinical variables have not been systematically studied.

Methods: In this experimental study we compared SOZ localisation between spontaneously captured seizures on higher-density depth and grid electrode arrays (4-5 mm inter-electrode spacing) vs. lower-density resampled versions of those same seizures (8-10 mm spacing). Since traditional review of channel traces would reveal density conditions, we instead projected seizure activity data as heatmaps on patient brain reconstructions and hid electrode locations. Using a single-blinded randomised crossover design, six attending-level epileptologists viewed these visualisations from ten patients under both higher-density and lower-density conditions (n = 120 observations) and digitally annotated SOZs.

Findings: Inter-rater agreement between epileptologists on annotated margins was moderate (average Cohen's kappa: 0.47) and lower for the lower-density condition (p = 0.021, mixed effects model). Scorer confidence ratings did not differ between higher- and lower-density conditions (p = 0.410). The spatial extents of annotated SOZs for higher-density recordings were 25.4% larger on average (p = 0.011) and always closer to true SOZ extents in computer simulations, relative to lower-density.

Interpretation: Epileptologists using higher-density depth and subdural intracranial EEG recordings had higher inter-rater agreement and identified larger extents of SOZs compared to lower-density recordings. While further studies assessing surgical outcomes in more patients are needed, these results suggest higher densities of electrodes on already-implanted hardware may reveal sub-centimetre extensions and clearer functional contiguity of the SOZ(s) for better appraisals of pathophysiological margins in epilepsy surgery.

Funding: This work was supported by the National Institutes of Health through NINDS grant K23NS110920 and through a UCSF Weill Institute for Neurosciences Pilot Award.

Background: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) has been increasingly reported worldwide, posing a severe challenge to public health; however, the mechanisms driving its emergence and global dissemination remain unclear.

Methods: We analysed CR-hvKp strains derived from canonical hvKp backgrounds, and acquired a carbapenemase-encoding gene. These strains were identified from 485 CRKp isolates in the CRACKLE-2 China cohort, 259 CRKp isolates from a multi-centre study, and 67,631 K. pneumoniae genomes available in GenBank. Clinical isolates harbouring the IncFII_K34 KPC-2 plasmid were selected for genome sequencing, RNA-Seq, conjugation assays, in vivo, ex vivo, and in vitro phenotypic characterisation.

Findings: Analysis of clinical CR-hvKp isolates and the 414 genomes from 24 countries available in GenBank identified an IncFII_K34 KPC-2 plasmid as the prevalent KPC plasmid (detected in 25%, 45/178 of KPC-producing CR-hvKp). Compared with the epidemic IncFII_K2 KPC-2 plasmid, the IncFII_K34 KPC-2 plasmid exhibited a 100- to 1000-fold increase in conjugation frequency (10^-4-10^-5 vs. 10^-7) and an in vitro growth advantage under meropenem challenge-likely due to the overexpression of conjugation-related genes and an increased bla_KPC copy number and expression. CR-hvKp isolates and hvKp transconjugants carrying this plasmid often exhibited reduced mucoviscosity, while retaining hypervirulence in both murine models and human neutrophil assays.

Interpretation: The IncFII_K34 plasmid may be a key factor driving the global dissemination of CR-hvKp, underscoring the urgent need for enhanced molecular surveillance of this emerging pathogen.

Funding: National Natural Science Foundation of China and National Institutes of Health.

Background: LAMP3 encodes a lysosomal membrane protein associated with lamellar bodies and has recently been proposed as a candidate gene for childhood interstitial lung diseases (chILD). Here, we identified two LAMP3 variants in a proband with chILD and performed functional validation of these variants as well as the previously reported variants to demonstrate the role of LAMP3 in pathology.

Methods: LAMP3 variants were identified by exome sequencing. Ex vivo studies included mRNA analysis from nasal brushing and lung tissue and immunohistochemistry from lung biopsy. In vitro functional analyses in the A549 cell line included immunofluorescence staining and expression analysis of LAMP3. Interactions between LAMP3 and the surfactant protein (SP)-B and SP-C were evaluated by co-immunoprecipitation.

Findings: Two heterozygous LAMP3 variants (Y302Qfs∗2 and T268M) were identified in a 15 year old boy with chILD. LAMP3 mRNA revealed that the frameshift variant resulted in nonsense-mediated mRNA decay. Reduced LAMP3 expression was confirmed in the patient's lung tissue. Functional studies of the T268M and the previously reported G288R variant revealed reduced levels of the mutant proteins. In addition, impaired N-glycosylation and protein instability were demonstrated with the T268M variant. Finally, we provided evidence for an interaction between LAMP3 and SP-B and SP-C, revealing a direct link between LAMP3 and surfactant metabolism.

Interpretation: LAMP3 bi-allelic variants leading to LAMP3 dysfunction emerges as a cause of chILD associated with a heterogeneous phenotype that remains to be further defined. The close links between LAMP3 and surfactant metabolism could explain the pathophysiology of this genetic disease.

Funding: No specific funding.