ACE-2 Blockade & TMPRSS2 Inhibition Mitigate SARS-CoV-2 Severity Following Cigarette Smoke Exposure in Airway Epithelial Cells In Vitro.

Cigarette smoking is associated with COVID-19 prevalence and severity, but the mechanistic basis for how smoking alters SARS-CoV-2 pathogenesis is unknown. A potential explanation is that smoking alters the expression of the SARS-CoV-2 cellular receptor and point of entry, angiotensin converting enzyme-2 (ACE-2), and its cofactors including transmembrane protease serine 2 (TMPRSS2). We investigated the impact of cigarette smoking on the expression of ACE-2, TMPRSS2, and other known cofactors of SARS-CoV-2 infection and the resultant effects on infection severity in vitro. Cigarette smoke extract (CSE) exposure increased ACE-2 and TMPRSS2 mRNA expression compared to air control in ferret airway cells, Calu-3 cells, and primary human bronchial epithelial (HBE) cells derived from normal and COPD donors. CSE-exposed ferret airway cells inoculated with SARS-CoV-2 had a significantly higher intracellular viral load versus vehicle-exposed cells. Likewise, CSE-exposure increased both SARS-CoV-2 intracellular viral load and viral replication in both normal and COPD HBE cells over vehicle control. Apoptosis was increased in CSE-exposed, SARS-CoV-2-infected HBE cells. Knockdown of ACE-2 via an antisense oligonucleotide (ASO) reduced SARS-CoV-2 viral load and infection in CSE-exposed ferret airway cells that was augmented by co-administration of camostat mesylate to block TMPRSS2 activity. Smoking increases SARS-CoV-2 infection via upregulation of ACE2 and TMPRSS2.

Quantitative Measurement of Rate of Targeted Protein Degradation.

Targeted protein degradation (TPD) is a therapeutic approach that leverages the cell's natural machinery to degrade targets instead of inhibiting them. This is accomplished by using mono- or bifunctional small molecules designed to induce the proximity of target proteins and E3 ubiquitin ligases, leading to ubiquitination and subsequent proteasome-dependent degradation of the target. One of the most significant attributes of the TPD approach is its proposed catalytic mechanism of action, which permits substoichiometric exposure to achieve the desired pharmacological effects. However, apart from one in vitro study, studies supporting the catalytic mechanism of degraders are largely inferred based on potency. A more comprehensive understanding of the degrader catalytic mechanism of action can help aspects of compound development. To address this knowledge gap, we developed a workflow for the quantitative measurement of the catalytic rate of degraders in cells. Comparing a selective and promiscuous BTK degrader, we demonstrate that both compounds function as efficient catalysts of BTK degradation, with the promiscuous degrader exhibiting faster rates due to its ability to induce more favorable ternary complexes. By leveraging computational modeling, we show that the catalytic rate is highly dynamic as the target is depleted from cells. Further investigation of the promiscuous kinase degrader revealed that the catalytic rate is a better predictor of optimal degrader activity toward a specific target compared to degradation magnitude alone. In summary, we present a versatile method for mapping the catalytic activity of any degrader for TPD in cells.

Preoperative vs Postoperative Opioid Prescriptions and Prolonged Opioid Refills Among US Youths.

Importance: High-risk practices, including dispensing an opioid prescription before surgery when not recommended, remain poorly characterized among US youths and may contribute to new persistent opioid use. Objective: To characterize changes in preoperative, postoperative, and refill opioid prescriptions up to 180 days after surgery. Design, Setting, and Participants: This retrospective cohort study was performed using national claims data to determine opioid prescribing practices among a cohort of opioid-naive youths aged 11 to 20 years undergoing 22 inpatient and outpatient surgical procedures between 2015 and 2020. Statistical analysis was performed from June 2023 to April 2024. Main Outcomes and Measures: The primary outcome was the percentage of initial opioid prescriptions filled up to 14 days prior to vs 7 days after a procedure. Secondary outcomes included the likelihood of a refill up to 180 days after surgery, including refills at 91 to 180 days, as a proxy for new persistent opioid use, and the opioid quantity dispensed in the initial and refill prescriptions in morphine milligram equivalents (MME). Exposures included patient and prescriber characteristics. Multivariable logistic regression models were used to estimate the association between prescription timing and prolonged refills. Results: Among 100 026 opioid-naive youths (median [IQR] age, 16.0 [14.0-18.0] years) undergoing a surgical procedure, 46 951 (46.9%) filled an initial prescription, of which 7587 (16.2%) were dispensed 1 to 14 days before surgery. The mean quantity dispensed was 227 (95% CI, 225-229) MME; 6467 youths (13.8%) filled a second prescription (mean MME, 239 [95% CI, 231-246]) up to 30 days after surgery, and 1216 (3.0%) refilled a prescription 91 to 180 days after surgery. Preoperative prescriptions, increasing age, and procedures not typically associated with severe pain were most strongly associated with new persistent opioid use. Conclusions and Relevance: In this retrospective study of youths undergoing surgical procedures, of which, many are typically not painful enough to require opioid use, opioid dispensing declined, but approximately 1 in 6 prescriptions were filled before surgery, and 1 in 33 adolescents filled prescriptions 91 to 180 days after surgery, consistent with new persistent opioid use. These findings should be addressed by policymakers and communicated by professional societies to clinicians who prescribe opioids.

Single-cell analysis reveals transcriptional dynamics in healthy primary parathyroid tissue.

Studies on human parathyroids are generally limited to hyperfunctioning glands owing to the difficulty in obtaining normal human tissue. We therefore obtained non-human primate (NHP) parathyroids to provide a suitable alternative for sequencing that would bear a close semblance to human organs. Single-cell RNA expression analysis of parathyroids from four healthy adult M. mulatta reveals a continuous trajectory of epithelial cell states. Pseudotime analysis based on transcriptomic signatures suggests a progression from GCM2 hi progenitors to mature parathyroid hormone (PTH)-expressing epithelial cells with increasing core mitochondrial transcript abundance along pseudotime. We sequenced, as a comparator, four histologically characterized hyperfunctioning human parathyroids with varying oxyphil and chief cell abundance and leveraged advanced computational techniques to highlight similarities and differences from non-human primate parathyroid expression dynamics. Predicted cell-cell communication analysis reveals abundant endothelial cell interactions in the parathyroid cell microenvironment in both human and NHP parathyroid glands. We show abundant RARRES2 transcripts in both human adenoma and normal primate parathyroid cells and use coimmunostaining to reveal high levels of RARRES2 protein (also known as chemerin) in PTH-expressing cells, which could indicate that RARRES2 plays an unrecognized role in parathyroid endocrine function. The data obtained are the first single-cell RNA transcriptome to characterize nondiseased parathyroid cell signatures and to show a transcriptomic progression of cell states within normal parathyroid glands, which can be used to better understand parathyroid cell biology.

COVID-19 mRNA Vaccination Responses in Individuals With Sickle Cell Disease: ASH RC Sickle Cell Research Network Study.

Children and adults with sickle cell disease (SCD) have increases in morbidity and mortality with COVID-19 infections. The ASH Research Collaborative Sickle Cell Disease Research Network performed a prospective COVID-19 vaccine study to assess antibody responses and analyze whether mRNA vaccination precipitated any adverse effects unique to individuals with SCD. Forty-one participants received two doses of the Pfizer-BioNTech vaccine and provided baseline blood samples prior to vaccination and 2 months after the initial vaccination for analysis of IgG reactivity against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Six month IgG reactivity against the viral RBD was also available in 37 patients. Post-vaccination reactogenicity was common and similar to the general population. There were no fevers that required inpatient admission. Vaso-occlusive pain within 2-3 days of 1st or 2nd vaccination was reported by 5 (12%) participants including 4 (10%) who sought medical care. Twenty-seven participants (66%) were seropositive at baseline, and all 14 (34%) initially seronegative participants converted to seropositive post vaccination. Overall, mRNA vaccination had a good risk benefit-profile in individuals with sickle cell disease.This mRNA vaccine study also marks the first evaluation of vaccine safety and antibody response in very young children with sickle cell disease. NCT05139992.

Switching to e-cigarettes as harm reduction among individuals with chronic disease who currently smoke: Results of a pilot randomized controlled trial.

INTRODUCTION: E-cigarettes (ECs) may be an effective harm reduction strategy for individuals with conditions like chronic obstructive pulmonary disease (COPD), asthma, coronary artery disease (CAD), and peripheral arterial disease (PAD) who smoke combustible cigarettes (CCs). Our aim was to examine how individuals with chronic conditions transition from CCs to ECs and its impact on health outcomes. METHODS: In a pilot randomized controlled trial (RCT), patients with COPD, asthma, CAD/PAD who currently smoke CCs and have not used nicotine replacement therapy (NRT) or ECs in the past 14 days were randomized to receive ECs or combination NRT with behavioral counselling. Disease symptoms, acceptability/satisfaction (TSQM-9) and feasibility, and cigarettes per day (CPD), and/or EC use were collected at baseline, 3-, and 6-months. Descriptive statistics and a linear regression were conducted to explore changes in CPD and chronic condition-specific assessments (CAT, SAQ-7, ACT) that assess COPD, asthma, and CAD/PAD symptom change. RESULTS: At 3-months, the EC group (n=63, mean CPD=9±11) reduced their CPD by 54% vs. 60% in the NRT group (n=58, mean CPD=7±6), p=0.56. At 6-months, 17.5% had switched completely to ECs while 23% quit smoking in the NRT arm. CAT scores showed a significant 6-point reduction in the EC arm (p=0.03). Participants scored an average of 69±27 for EC effectiveness, 87±23 for convenience, and 75±27 for overall satisfaction. CONCLUSIONS: This pilot study suggests that ECs may be a safer alternative for chronic condition patients using CCs and warrants further research on expected smoking cessation/reduction among individuals who use ECs. IMPLICATIONS: The findings from this pilot RCT hold significant implications with chronic conditions such as COPD, asthma, CAD and PAD who smoke CCs. The observed reduction in cigarettes per day and improvement in respiratory symptoms suggest that switching to ECs appears feasible and acceptable among those with chronic diseases. These results suggest that ECs may offer an alternative for individuals struggling to quit CC smoking through existing pharmacotherapies. This study supports further exploration of switching to ECs as a harm reduction strategy among CC users who have been unsuccessful at quitting by other means.

Infarct density defined by ADC threshold is associated with long-term functional outcome after endovascular thrombectomy.

OBJECTIVES: Endovascular thrombectomy (EVT) dramatically improves clinical outcomes, but the reduction in final infarct volume only accounts for 10-15% of the treatment benefit. We aimed to develop a novel MRI-ADC-based metric that quantify the degree of tissue injury to test the hypothesis that it outperforms infarct volume in predicting long-term outcome. MATERIALS AND METHODS: A single-center cohort consisted of consecutive acute stroke patients with anterior circulation large vessel occlusion, successful recanalization via EVT (mTICI ≥2b), and MRI of the brain between 12 hours and 7 days post-EVT. Imaging was processed via RAPID software. Final infarct volume was based on the traditional ADC <620 threshold. Logistic regression quantified the association of lesion volumes and good outcome (90-day modified Rankin Scale ≤2) at a range of lower ADC thresholds (<570, <520, and <470). Infarct density was calculated as the percentage of the final infarct volume below the ADC threshold with the greatest effect size. Univariate and multivariate logistic regression quantified the association between imaging/clinical metrics and functional outcome. RESULTS: 120 patients underwent MRI after successful EVT. Lesion volume based on the ADC threshold <470 had the strongest association with good outcome (OR: 0.81 per 10mL; 95% CI: 0.66-0.99). In a multivariate model, infarct density (<470/<620 * 100) was independently associated with good outcome (aOR 0.68 per 10%; 95% CI: 0.49-0.95), but final infarct volume was not (aOR 0.98 per 10mL; 95% CI: 0.85-1.14). CONCLUSIONS: Infarct density after EVT is more strongly associated with long-term clinical outcome than infarct volume.

Wood ducks and hooded mergansers as interspecific brood parasites: An evaluation of parasitic egg survival.

Conspecific and interspecific brood parasitism are alternate reproductive strategies more pervasive in waterfowl than in any other group of birds. While previous research has measured costs incurred by nest hosts incubating parasitized clutches, few studies have focused on the relative success of parasites. Here, we evaluated the success of wood duck (Aix sponsa) and hooded merganser (Lophodytes cucullatus) eggs laid parasitically in Louisiana and Mississippi. We monitored nest boxes, assigned eggs in each nest as host or parasitic, and determined the number of eggs that hatched and failed. Across all study areas (1994-1999 and 2020-2023), we monitored 1750 wood duck and 377 hooded merganser nests; ~13% of wood duck and ~24% of hooded merganser nests were interspecifically parasitized. We modeled egg survival of 2925 host and 691 parasitic eggs from 197 successful nests (≥1 hatched egg, regardless of species). Wood duck eggs laid in hooded merganser nests had lower survival [0.293, CI = 95% credible intervals (after, CI) = 0.176, 0.439] than hooded merganser eggs (0.762, CI = 0.704, 0.810) laid in wood duck nests. Clutch size negatively influenced parasitic wood duck egg survival (ß = -.24, CI = -0.39, -0.10) but had a slight positive influence on parasitic hooded merganser eggs (ß = .08, CI = 0.04, 0.12). Our results revealed that hooded merganser eggs experience higher success when laid parasitically in wood duck nests, whereas wood duck eggs experience lower success when laid parasitically in hooded merganser nests. Our results reveal new complexity in waterfowl interspecific brood parasitism, where the success of parasitic eggs is species-, host-, and context-specific.

The effects of buprenorphine and morphine during pregnancy: Impact of exposure length on maternal brain, behavior, and offspring neurodevelopment.

The escalating incidence of opioid-related issues among pregnant women in the United States underscores the critical necessity to understand the effects of opioid use and Medication for Opioid Use Disorders (MOUDs) during pregnancy. This research employed a translational rodent model to examine the impact of gestational exposure to buprenorphine (BUP) or morphine on maternal behaviors and offspring well-being. Female rats received BUP or morphine before conception, representing established use, with exposure continuing until postnatal day 2 or discontinued on gestational day 19 to mimic treatment cessation before birth. Maternal behaviors - including care, pup retrieval, and preference - as well as hunting behaviors and brain neurotransmitter levels were assessed. Offspring were evaluated for mortality, weight, length, milk bands, surface righting latency, withdrawal symptoms, and brain neurotransmitter levels. Our results reveal that regardless of exposure length (i.e., continued or discontinued), BUP resulted in reduced maternal care in contrast to morphine-exposed and control dams. Opioid exposure altered brain monoamine levels in the dams and offspring, and was associated with increased neonatal mortality, reduced offspring weight, and elevated withdrawal symptoms compared to controls. These findings underscore BUP's potential disruption of maternal care, contributing to increased pup mortality and altered neurodevelopmental outcomes in the offspring. This study calls for more comprehensive research into prenatal BUP exposure effects on the maternal brain and infant development with the aim to mitigate adverse outcomes in humans exposed to opioids during pregnancy.

Direct-Acting Oral Anticoagulants and Antiseizure Medications for Atrial Fibrillation and Epilepsy and Risk of Thromboembolic Events.

Importance: Direct-acting oral anticoagulants (DOACs) are commonly prescribed with antiseizure medications (ASMs) due to concurrency of and the association between atrial fibrillation (AF) and epilepsy. However, enzyme-inducing (EI) ASMs may reduce absorption and accelerate metabolism of DOACs, potentially lowering DOAC levels and elevating thromboembolism risk. Objective: To assess the rates of thromboembolic and major bleeding events in adults with AF and epilepsy dispensed DOACs and EI ASMs vs DOACs with non-EI ASMs. Design, Setting, and Participants: This active-comparator, new-user cohort study included US health care data from the Clinformatics Data Mart database from October 2010 to September 2021 for a nationally representative population of adults with AF and epilepsy. Exposure: Evaluations included episodes of contiguous coadministration of DOACs for AF with EI ASMs (exposed) or non-EI ASMs (referent) for epilepsy. Main Outcomes and Measures: Thromboembolic events (primary outcome) and major bleeding events (secondary outcome) were identified based on a series of validated, diagnosis-based coding algorithms. Data-adaptive, high-dimensional propensity score matching was used to control for observed confounders and proxies for unobserved confounders. Adjusted hazard ratios (AHRs) were estimated using Cox proportional hazards regression models with robust variance estimators to account for clustering within matched pairs. Results: This study included 14 078 episodes (median age, 74 [IQR, 67-81]; 52.4% female) and 14 158 episodes (median age, 74 [IQR, 67-81]; 52.4% female) of incident DOAC and ASM use that met eligibility criteria for assessment of thromboembolic and major bleeding outcomes, respectively. Incidence was 88.5 per 1000 person-years for thromboembolic events and 68.3 per 1000 person-years for bleeding events. Compared with use of non-EI ASMs, use of EI ASMs with DOACs was not associated with a difference in risk of thromboembolic events (AHR, 1.10; 95% CI, 0.82-1.46) but was associated with a reduction in risk of major bleeding events (AHR, 0.63; 95% CI, 0.44-0.89). Conclusions and Relevance: In this cohort study, EI ASMs were not associated with alteration in DOAC efficacy. Further research is needed on the reduction in bleeding risk associated with EI ASMs, as this may suggest that pharmacokinetic interactions are associated with lowering DOAC levels without negating therapeutic effects.

Ophthalmic findings in the KIF1A-associated neurological disorder (KAND).

PURPOSE: To define the ophthalmic manifestations in KIF1A-associated neurological disorder (KAND), a rare, progressive neurodegenerative disorder caused by pathogenic variants in the KIFA1 gene. DESIGN: Cross-sectional study. METHODS: Clinical ophthalmic examination and multimodal imaging were performed for 24 participants enrolled in the KIF1AOutcome measures, Assessments, Longitudinal And endpoints (KOALA) Study. Visual evoked potentials (VEP) were performed on select participants. RESULTS: The average central visual acuity in pediatric participants was 20/43 (logMAR 0.329, range 0.0-1.0), and 20/119 (logMAR 0.773, range 0.471-1.351) in adults. Ninety-five percent of participants examined had some degree of optic nerve atrophy detected by clinical examination and/or optical coherence tomography (OCT). Almost forty percent had strabismus. Color vision, visual fields and stereopsis were impaired in most participants who were able to participate in testing. VEP showed varying degrees of signal slowing and diffuseness. CONCLUSIONS: Optic nerve atrophy is the primary ocular finding in individuals with KAND and is present at higher prevalence than previously reported. The degree of the atrophy is likely dependent on the severity of the pathogenic variant and possibly the age of the patient. Adults had worse vision on average than children, suggesting possible decline in vision with age. Strabismus in this cohort was common. Visual evoked potentials showed findings consistent with optic neuropathy and visual dysfunction even in the absence of obvious structural changes on OCT. Families should be counseled regarding visual impairment in KAND patients, so as to obtain appropriate support and assistance to maximize safety, functionality, and learning.

Molecular insights into the low complexity secreted venom of Calliactis polypus.

Sea anemones are venomous animals that rely on their venom for prey capture, defence against predators and intraspecific competition. Currently, comprehensive molecular and evolutionary analyses of the toxin repertoire for sea anemones are limited by a lack of proteomic data for most species. In this study, proteo-transcriptomic analyses were used to expand our knowledge of the proteinaceous components of sea anemone venom by determining the secreted venom proteome of Calliactis polypus. Electro-mechanical stimulation was used to obtain the secreted venom of C. polypus. We identified a low complexity proteome that was dominated by toxins with similarity to known neurotoxins, as well as six novel toxin candidates. The novel putative toxin candidates were found to be taxonomically restricted to species from the superfamily Metridioidea. Furthermore, the secreted venom of C. polypus had only three putative toxins in common with the venom of acontia from the same species, and little similarity with the secreted venom of closely-related species. Overall, this demonstrates that regionalised and lineage-specific variability in toxin abundance is common among sea anemone species. Moreover, the limited complexity of the toxin repertoire found in C. polypus supports the idea that peptide neurotoxins make up the dominant toxin arsenal found in the venom of sea anemones.

METAPHOR: Metabolic evaluation through phasor-based hyperspectral imaging and organelle recognition for mouse blastocysts and oocytes.

Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading to repeated transfer cycles. To reduce time-to-pregnancy and stress for patients, there is a need for a diagnostic tool to better select embryos and oocytes based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging and Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination and AI to deliver the metabolic profile of embryos and oocytes based on intrinsic autofluorescence signals. We used it to classify i) mouse blastocysts cultured under standard conditions or with depletion of selected metabolites (glucose, pyruvate, lactate); and ii) oocytes from young and old mouse females, or in vitro-aged oocytes. The imaging process was safe for blastocysts and oocytes. The METAPHOR classification of control vs. metabolites-depleted embryos reached an area under the ROC curve (AUC) of 93.7%, compared to 51% achieved for human grading using brightfield imaging. The binary classification of young vs. old/in vitro-aged oocytes and their blastulation prediction using METAPHOR reached an AUC of 96.2% and 82.2%, respectively. Finally, organelle recognition and segmentation based on the flavin adenine dinucleotide signal revealed that quantification of mitochondria size and distribution can be used as a biomarker to classify oocytes and embryos. The performance and safety of the method highlight the accuracy of noninvasive metabolic imaging as a complementary approach to evaluate oocytes and embryos based on their physiology.

RSV Vaccination in the adult pulmonary patient.

TOPIC IMPORTANCE: Since its discovery in 1957, respiratory syncytial virus (RSV) has been widely recognized as a common and deadly pathogen. While early studies focused on the impact of RSV on the health of children, more recent data show that RSV imposes a significant burden on individuals aged 70 years or greater. RSV also substantially harms the health of individuals with cardiopulmonary diseases. REVIEW FINDINGS: Early efforts to develop an RSV vaccine were hampered by toxicity due to antibody-enhanced viral pneumonia and a lack of efficacy in vaccines that targeted the post-fusion configuration of the F fusion protein, which is crucial to the pathogenesis of RSV-mediated injury. A newer wave of vaccines has targeted a stabilized prefusion F protein, generating effective neutralizing antibodies and reducing the burden of mild and severe RSV lower respiratory tract injury. This review focuses on the burden of RSV in patients with pulmonary diseases, highlights the tumultuous path from the early days of RSV vaccine development to the modern era, and offers insights into key gaps in knowledge that must be addressed in order to adequately protect the vulnerable population of patients with severe pulmonary diseases.

Responses of wheat (Triticum aestivum L.) constitutively expressing four different monolignol biosynthetic genes to Fusarium head blight caused by Fusarium graminearum.

The Fusarium head blight (FHB) pathogen Fusarium graminearum produces the trichothecene mycotoxin deoxynivalenol (DON) and reduces wheat yield and grain quality. Spring wheat (Triticum aestivum L.) genotype CB037 was transformed with constitutive expression (CE) constructs containing sorghum (Sorghum bicolor L. (Moench)) genes encoding monolignol biosynthetic enzymes, caffeoyl-Coenzyme A (CoA) 3-O-methyltransferase (SbCCoAOMT), 4-coumarate-CoA ligase (Sb4CL), or coumaroyl shikimate 3-hydroxylase (SbC3'H), or monolignol pathway transcriptional activator, SbMyb60. Spring wheats were screened for Type I (resistance to initial infection, using spray inoculations) and Type II (resistance to spread within the spike, using single floret inoculations) resistances in the field (spray) and greenhouse (spray and single floret). Following field inoculations, disease index, percent Fusarium damaged kernels (FDK), and DON measurements of CE plants were similar to or greater than CB037. For greenhouse inoculations, the area under the disease progress curve (AUDPC) and FDK were determined. Following screens, focus was placed on two each, SbC3'H and SbCCoAOMT CE lines because of trends towards decreased AUDPC and FDK observed following single floret inoculations. These four lines were as susceptible as CB037 following spray inoculations. However, single floret inoculations showed that these CE lines had significantly reduced AUDPC (P<0.01) and FDK (P≤0.02) compared with CB037, indicating improved Type II resistance. None of these CE lines had increased acid detergent lignin, as compared with CB037, indicating that lignin concentration may not be a major factor in FHB resistance. The SbC3'H and SbCCoAOMT CE lines are valuable for investigating phenylpropanoid-based resistance to FHB.

Pulmonary Fibrosis Ferret Model Demonstrates Sustained Fibrosis, Restrictive Physiology, and Aberrant Repair.

Rationale: The role of MUC5B mucin expression in IPF pathogenesis is unknown. Bleomycin-exposed rodent models do not exhibit sustained fibrosis or airway remodeling. Unlike mice, ferrets have human-like distribution of MUC5B expressing cell types and natively express the risk-conferring variant that induces high MUC5B expression in humans. We hypothesized that ferrets would consequently exhibit aberrant repair to propagate fibrosis similar to human IPF. Methods: Bleomycin (5U/kg) or saline-control was micro-sprayed intratracheally then wild-type ferrets were evaluated through 22 wks. Clinical phenotype was assessed with lung function. Fibrosis was assessed with µCT imaging and comparative histology with Ashcroft scoring. Airway remodeling was assessed with histology and quantitative immunofluorescence. Results: Bleomycin ferrets exhibited sustained restrictive physiology including decreased inspiratory capacity, decreased compliance, and shifted Pressure-Volume loops through 22 wks. Volumetric µCT analysis revealed increased opacification of the lung bleomycin-ferrets. Histology showed extensive fibrotic injury that matured over time and MUC5B-positive cystic structures in the distal lung suggestive of honeycombing. Bleomycin ferrets had increased proportion of small airways that were double-positive for CCSP and alpha-tubulin compared to controls, indicating an aberrant 'proximalization' repair phenotype. Notably, this aberrant repair was associated with extent of fibrotic injury at the airway level. Conclusions: Bleomycin-exposed ferrets exhibit sustained fibrosis through 22 wks and have pathologic features of IPF not found in rodents. Ferrets exhibited proximalization of the distal airways and other pathologic features characteristic of human IPF. MUC5B expression through native cell types may play a key role in promoting airway remodeling and lung injury in IPF.

Within-host influenza viral diversity in the pediatric population as a function of age, vaccine, and health status.

Seasonal influenza virus predominantly evolves through antigenic drift, marked by the accumulation of mutations at antigenic sites. Because of antigenic drift, influenza vaccines are frequently updated, though their efficacy may still be limited due to strain mismatches. Despite the high levels of viral diversity observed across populations, most human studies reveal limited intrahost diversity, leaving the origin of population-level viral diversity unclear. Previous studies show host characteristics, such as immunity, might affect within-host viral evolution. Here we investigate influenza A viral diversity in children aged between 6 months and 18 years. Influenza virus evolution in children is less well characterized than in adults, yet may be associated with higher levels of viral diversity given the lower level of pre-existing immunity and longer durations of infection in children. We obtained influenza isolates from banked influenza A-positive nasopharyngeal swabs collected at the Children's Hospital of Philadelphia during the 2017-18 influenza season. Using next-generation sequencing, we evaluated the population of influenza viruses present in each sample. We characterized within-host viral diversity using the number and frequency of intrahost single-nucleotide variants (iSNVs) detected in each sample. We related viral diversity to clinical metadata, including subjects' age, vaccination status, and comorbid conditions, as well as sample metadata such as virus strain and cycle threshold. Consistent with previous studies, most samples contained low levels of diversity with no clear association between the subjects' age, vaccine status, or health status. Further, there was no enrichment of iSNVs near known antigenic sites. Taken together, these findings are consistent with previous observations that the majority of intrahost influenza virus infection is characterized by low viral diversity without evidence of diversifying selection.