Uncovering strain- and age-dependent innate immune responses to SARS-CoV-2 infection in air-liquid-interface cultured nasal epithelia.

Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, with the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly infected and uninfected bystander cells and heightened in Alpha-infection. Alpha highly infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control cells, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2.

Changes in State Laws on Suicide Prevention Training for School Staff, 2002-2022.

OBJECTIVES: Youth suicide is an urgent public health problem. Gatekeeper training aims to prevent suicide by training people to identify warning signs and make referrals to appropriate services. Many states in the United States have enacted gatekeeper training laws (GTLs) to train school staff in suicide prevention. The objectives of this study were to describe the development of a dataset on GTLs and use the dataset to summarize trends in uptake of GTLs from 2002 through 2022 as well as differences in characteristics (eg, frequency and duration of training) of GTLs. METHODS: We used publicly available legal databases from all 50 states and the District of Columbia to conduct a policy surveillance assessment of GTLs. We cross-checked data with the American Foundation for Suicide Prevention's 2022 Suicide Prevention in Schools (K-12) issue brief and used Westlaw Edge to conduct a sensitivity analysis. We included the following data in the full dataset: type of laws (encouraged, mandatory, or conditional mandatory), date passed, effective date, frequency of training, and length of training. RESULTS: In 2022, 49 states and the District of Columbia had GTLs, 31 of which were mandatory laws. In 2002, only 6 states had such laws, and none were mandatory. CONCLUSION: The growing proliferation of laws on suicide prevention training for school staff warrants evaluation of the laws' effectiveness. Our policy surveillance data may be used to better understand the role of these laws in a school-based approach to youth suicide prevention.

Acceptability of COVID-19 booster vaccine in malaysia: a cross-sectional study.

Despite the high efficacy and safety demonstrated in clinical trials, COVID-19 booster vaccination rates in Malaysia remain below 50% among the general public. This study explores the factors influencing public acceptance of the COVID-19 booster vaccine among the Malaysian population. The questionnaire included variables on sociodemographics, knowledge, and the Health Belief Model (HBM) constructs. Based on the Chi-squared test of contingencies, a t-test and multivariate logistic regression analysis on 411 collected responses, the findings revealed that older participants, individuals of Chinese ethnicity, and those with higher education levels and incomes were more willing to accept booster vaccinations. The analysis further identified perceived susceptibility, perceived severity and perceived barriers as significant predictors influencing booster vaccination acceptance rates. Healthcare policymakers may consider targeting interventions to diminish the obstacles associated with booster vaccinations. These intervention strategies include implementing health intervention programmes, such as public health awareness initiatives, to raise awareness of the risks and severity of COVID-19, ultimately encouraging higher uptake of booster vaccines.

A Feasibility Randomized Controlled Trial of Prehabilitation During Neoadjuvant Chemotherapy for Women with Breast Cancer: A Mixed Methods Study.

BACKGROUND: Limited data exist regarding the role of multimodal prehabilitation during neoadjuvant chemotherapy (NACT) for breast cancer. Determining large trial feasibility and identifying signals of prehabilitation benefit are needed. PATIENTS AND METHODS: We conducted a randomized controlled feasibility trial of multimodal prehabilitation versus usual care during NACT among women diagnosed with non-metastatic breast cancer. Intervention participants received an individualized exercise program, dietetic support, and stress management counseling during NACT. The trial assessed feasibility via rates of recruitment, attrition, adherence, and study-related adverse events. Physical fitness (Six Minute Walk Test, grip strength, anthropometrics) and patient-reported outcomes were assessed at baseline, after NACT completion, and 6 months after surgery as exploratory outcomes, and analyzed using linear mixed effects models. Qualitative data were collected from a subsample to understand feasibility and acceptability of prehabilitation. RESULTS: A total of 72 participants were enrolled from the 123 eligible patients (recruitment rate of 53%). There was a 13% attrition rate and no intervention-related adverse events. Participants in the prehabilitation group had better 6-min walk distance at the post-chemotherapy timepoint [between group difference of 49.43 m, 95% confidence interval (CI) - 118.1, 19.2] and at the post-surgery timepoint (27.3, 95% CI -96.8, 42.2) compared with the control group. Prehabilitation participants reported better quality of life, less fatigue, and improved physical activity levels compared with usual care participants. Interviews revealed that the intervention had a positive impact on the treatment experience. CONCLUSIONS: This study demonstrated feasibility and improvement in physical and psychosocial outcomes. Larger trials assessing intervention efficacy to confirm indications of prehabilitation benefit are warranted.

Considering the Role of Information and Context in Promoting Health-Related Behavioral Change.

This poster considers the role that information and context may play in health management. We employ a well-known taxonomy of techniques for promoting behavioral change to consider how social media authors describe their recovery from substance use. We harvest discussion posts from subreddits, or discussion forums, about alcohol, cannabis, and opioids, and perform content analysis to identify behavioral change techniques (BCTs) described in the content. We then consider the role that the context of information use plays in these BCTs, as well as how interventions and technologies might be leveraged to better support the recovery process.

Hierarchical assembly of tryptophan zipper peptides into stress-relaxing bioactive hydrogels.

Soft materials in nature are formed through reversible supramolecular assembly of biological polymers into dynamic hierarchical networks. Rational design has led to self-assembling peptides with structural similarities to natural materials. However, recreating the dynamic functional properties inherent to natural systems remains challenging. Here we report the discovery of a short peptide based on the tryptophan zipper (trpzip) motif, that shows multiscale hierarchical ordering that leads to emergent dynamic properties. Trpzip hydrogels are antimicrobial and self-healing, with tunable viscoelasticity and unique yield-stress properties that allow immediate harvest of embedded cells through a flick of the wrist. This characteristic makes Trpzip hydrogels amenable to syringe extrusion, which we demonstrate with examples of cell delivery and bioprinting. Trpzip hydrogels display innate bioactivity, allowing propagation of human intestinal organoids with apical-basal polarization. Considering these extensive attributes, we anticipate the Trpzip motif will prove a versatile building block for supramolecular assembly of soft materials for biotechnology and medicine.

Q1291H-CFTR molecular dynamics simulations and ex vivo theratyping in nasal epithelial models and clinical response to elexacaftor/tezacaftor/ivacaftor in a Q1291H/F508del patient.

Background: Cystic fibrosis (CF) is caused by a wide spectrum of mutations in the CF transmembrane conductance regulator (CFTR) gene, with some leading to non-classical clinical presentations. We present an integrated in vivo, in silico and in vitro investigation of an individual with CF carrying the rare Q1291H-CFTR allele and the common F508del allele. At age 56 years, the participant had obstructive lung disease and bronchiectasis, qualifying for Elexacaftor/Tezacaftor/Ivacaftor (ETI) CFTR modulator treatment due to their F508del allele. Q1291H CFTR incurs a splicing defect, producing both a normally spliced but mutant mRNA isoform and a misspliced isoform with a premature termination codon, causing nonsense mediated decay. The effectiveness of ETI in restoring Q1291H-CFTR is largely unknown. Methods: We collected clinical endpoint measurements, including forced expiratory volume in 1 s percent predicted (FEV1pp) and body mass index (BMI), and examined medical history. In silico simulations of the Q1291H-CFTR were compared to Q1291R, G551D, and wild-type (WT)-CFTR. We quantified relative Q1291H CFTR mRNA isoform abundance in patient-derived nasal epithelial cells. Differentiated pseudostratified airway epithelial cell models at air liquid interface were created and ETI treatment impact on CFTR was assessed by electrophysiology assays and Western blot. Results: The participant ceased ETI treatment after 3 months due to adverse events and no improvement in FEV1pp or BMI. In silico simulations of Q1291H-CFTR identified impairment of ATP binding similar to known gating mutants Q1291R and G551D-CFTR. Q1291H and F508del mRNA transcripts composed 32.91% and 67.09% of total mRNA respectively, indicating 50.94% of Q1291H mRNA was misspliced and degraded. Mature Q1291H-CFTR protein expression was reduced (3.18% ± 0.60% of WT/WT) and remained unchanged with ETI. Baseline CFTR activity was minimal (3.45 ± 0.25 µA/cm2) and not enhanced with ETI (5.73 ± 0.48 µA/cm2), aligning with the individual's clinical evaluation as a non-responder to ETI. Conclusion: The combination of in silico simulations and in vitro theratyping in patient-derived cell models can effectively assess CFTR modulator efficacy for individuals with non-classical CF manifestations or rare CFTR mutations, guiding personalized treatment strategies and optimizing clinical outcomes.

Molecular and Functional Characteristics of Airway Epithelium under Chronic Hypoxia.

Localized and chronic hypoxia of airway mucosa is a common feature of progressive respiratory diseases, including cystic fibrosis (CF). However, the impact of prolonged hypoxia on airway stem cell function and differentiated epithelium is not well elucidated. Acute hypoxia alters the transcription and translation of many genes, including the CF transmembrane conductance regulator (CFTR). CFTR-targeted therapies (modulators) have not been investigated in vitro under chronic hypoxic conditions found in CF airways in vivo. Nasal epithelial cells (hNECs) derived from eight CF and three non-CF participants were expanded and differentiated at the air-liquid interface (26-30 days) at ambient and 2% oxygen tension (hypoxia). Morphology, global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility and ion transport) of basal stem cells and differentiated cultures were assessed. hNECs expanded at chronic hypoxia, demonstrating epithelial cobblestone morphology and a similar proliferation rate to hNECs expanded at normoxia. Hypoxia-inducible proteins and pathways in stem cells and differentiated cultures were identified. Despite the stem cells' plasticity and adaptation to chronic hypoxia, the differentiated epithelium was significantly thinner with reduced barrier integrity. Stem cell lineage commitment shifted to a more secretory epithelial phenotype. Motile cilia abundance, length, beat frequency and coordination were significantly negatively modulated. Chronic hypoxia reduces the activity of epithelial sodium and CFTR ion channels. CFTR modulator drug response was diminished. Our findings shed light on the molecular pathophysiology of hypoxia and its implications in CF. Targeting hypoxia can be a strategy to augment mucosal function and may provide a means to enhance the efficacy of CFTR modulators.

MiRNAs Overexpression and Their Role in Breast Cancer: Implications for Cancer Therapeutics.

MicroRNAs have a plethora of roles in various biological processes in the cells and most human cancers have been shown to be associated with dysregulation of the expression of miRNA genes. MiRNA biogenesis involves two alternative pathways, the canonical pathway which requires the successful cooperation of various proteins forming the miRNA-inducing silencing complex (miRISC), and the non-canonical pathway, such as the mirtrons, simtrons, or agotrons pathway, which bypasses and deviates from specific steps in the canonical pathway. Mature miRNAs are secreted from cells and circulated in the body bound to argonaute 2 (AGO2) and miRISC or transported in vesicles. These miRNAs may regulate their downstream target genes via positive or negative regulation through different molecular mechanisms. This review focuses on the role and mechanisms of miRNAs in different stages of breast cancer progression, including breast cancer stem cell formation, breast cancer initiation, invasion, and metastasis as well as angiogenesis. The design, chemical modifications, and therapeutic applications of synthetic anti-sense miRNA oligonucleotides and RNA mimics are also discussed in detail. The strategies for systemic delivery and local targeted delivery of the antisense miRNAs encompass the use of polymeric and liposomal nanoparticles, inorganic nanoparticles, extracellular vesicles, as well as viral vectors and viruslike particles (VLPs). Although several miRNAs have been identified as good candidates for the design of antisense and other synthetic modified oligonucleotides in targeting breast cancer, further efforts are still needed to study the most optimal delivery method in order to drive the research beyond preclinical studies.

Genome-wide tiled detection of circulating Mycobacterium tuberculosis cell-free DNA using Cas13.

Detection of microbial cell-free DNA (cfDNA) circulating in the bloodstream has emerged as a promising new approach for diagnosing infection. Microbial diagnostics based on cfDNA require assays that can detect rare and highly fragmented pathogen nucleic acids. We now report WATSON (Whole-genome Assay using Tiled Surveillance Of Nucleic acids), a method to detect low amounts of pathogen cfDNA that couples pooled amplification of genomic targets tiled across the genome with pooled CRISPR/Cas13-based detection of these targets. We demonstrate that this strategy of tiling improves cfDNA detection compared to amplification and detection of a single targeted locus. WATSON can detect cfDNA from Mycobacterium tuberculosis in plasma of patients with active pulmonary tuberculosis, a disease that urgently needs accurate, minimally-invasive, field-deployable diagnostics. We thus demonstrate the potential for translating WATSON to a lateral flow platform. WATSON demonstrates the ability to capitalize on the strengths of targeting microbial cfDNA to address the need for point-of-care diagnostic tests for infectious diseases.

Assessment of Pre-Clinical Liver Models Based on Their Ability to Predict the Liver-Tropism of Adeno-Associated Virus Vectors.

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors. Multiple clinical trials have been undertaken for this target in the past 15 years; however, we are still to see market approval of the first liver-targeted adeno-associated virus (AAV)-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically and clinically predictive preclinical models. To this end, this study reports findings of a functional evaluation of 6 AAV vectors in 12 preclinical models of the human liver, with the aim to uncover which combination of models is the most relevant for the identification of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.

High-Throughput Neutralization and Serology Assays Reveal Correlated but Highly Variable Humoral Immune Responses in a Large Population of Individuals Infected with SARS-CoV-2 in the US between March and August 2020.

The ability to measure neutralizing antibodies on large scale can be important for understanding features of the natural history and epidemiology of infection, as well as an aid in determining the efficacy of interventions, particularly in outbreaks such as the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Because of the assay's rapid scalability and high efficiency, serology measurements that quantify the presence rather than function of serum antibodies often serve as proxies of immune protection. Here, we report the development of a high-throughput, automated fluorescence-based neutralization assay using SARS-CoV-2 virus to quantify neutralizing antibody activity in patient specimens. We performed large-scale testing of over 19,000 COVID-19 convalescent plasma (CCP) samples from patients who had been infected with SARS-CoV-2 between March and August 2020 across the United States. The neutralization capacity of the samples was moderately correlated with serological measurements of anti-receptor-binding domain (RBD) IgG levels. The neutralizing antibody levels within these convalescent-phase serum samples were highly variable against the original USA-WA1/2020 strain with almost 10% of individuals who had had PCR-confirmed SARS-CoV-2 infection having no detectable antibodies either by serology or neutralization, and ~1/3 having no or low neutralizing activity. Discordance between neutralization and serology measurements was mainly due to the presence of non-IgG RBD isotypes. Meanwhile, natural infection with the earliest SARS-CoV-2 strain USA-WA1/2020 resulted in weaker neutralization of subsequent B.1.1.7 (alpha) and the B.1.351 (beta) variants, with 88% of samples having no activity against the BA.1 (omicron) variant. IMPORTANCE The ability to directly measure neutralizing antibodies on live SARS-CoV-2 virus in individuals can play an important role in understanding the efficacy of therapeutic interventions or vaccines. In contrast to functional neutralization assays, serological assays only quantify the presence of antibodies as a proxy of immune protection. Here, we have developed a high-throughput, automated neutralization assay for SARS-CoV-2 and measured the neutralizing activity of ~19,000 COVID-19 convalescent plasma (CCP) samples collected across the United States between March and August of 2020. These data were used to support the FDA's interpretation of CCP efficacy in patients with SARS-CoV-2 infection and their issuance of emergency use authorization of CCP in 2020.

Self-induced panniculitis in adolescence: Three cases and review of the literature.

Self-induced trauma is a rare cause of panniculitis in adolescents and may be associated with undiagnosed underlying psychological conditions. The condition often poses a diagnostic challenge as patients usually present with non-specific signs and symptoms, and often a concealed history of self-induced trauma. Here, we present three adolescent patients with self-induced traumatic panniculitis. All three patients were repeatedly admitted with non-specific cutaneous lesions as well as multiple somatic complaints. After extensive evaluation to exclude organic causes, psychiatric assessment eventually revealed underlying psychosomatic disorders as a cause of the self-induced trauma.

S945L-CFTR molecular dynamics, functional characterization and tezacaftor/ivacaftor efficacy in vivo and in vitro in matched pediatric patient-derived cell models.

Cystic Fibrosis (CF) results from over 400 different disease-causing mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. These CFTR mutations lead to numerous defects in CFTR protein function. A novel class of targeted therapies (CFTR modulators) have been developed that can restore defects in CFTR folding and gating. This study aimed to characterize the functional and structural defects of S945L-CFTR and interrogate the efficacy of modulators with two modes of action: gating potentiator [ivacaftor (IVA)] and folding corrector [tezacaftor (TEZ)]. The response to these modulators in vitro in airway differentiated cell models created from a participant with S945L/G542X-CFTR was correlated with in vivo clinical outcomes of that participant at least 12 months pre and post modulator therapy. In this participants' airway cell models, CFTR-mediated chloride transport was assessed via ion transport electrophysiology. Monotherapy with IVA or TEZ increased CFTR activity, albeit not reaching statistical significance. Combination therapy with TEZ/IVA significantly (p = 0.02) increased CFTR activity 1.62-fold above baseline. Assessment of CFTR expression and maturation via western blot validated the presence of mature, fully glycosylated CFTR, which increased 4.1-fold in TEZ/IVA-treated cells. The in vitro S945L-CFTR response to modulator correlated with an improvement in in vivo lung function (ppFEV1) from 77.19 in the 12 months pre TEZ/IVA to 80.79 in the 12 months post TEZ/IVA. The slope of decline in ppFEV1 significantly (p = 0.02) changed in the 24 months post TEZ/IVA, becoming positive. Furthermore, there was a significant improvement in clinical parameters and a fall in sweat chloride from 68 to 28 mmol/L. The mechanism of dysfunction of S945L-CFTR was elucidated by in silico molecular dynamics (MD) simulations. S945L-CFTR caused misfolding of transmembrane helix 8 and disruption of the R domain, a CFTR domain critical to channel gating. This study showed in vitro and in silico that S945L causes both folding and gating defects in CFTR and demonstrated in vitro and in vivo that TEZ/IVA is an efficacious modulator combination to address these defects. As such, we support the utility of patient-derived cell models and MD simulations in predicting and understanding the effect of modulators on CFTR function on an individualized basis.

Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface.

Infection control and aggressive antibiotic therapy play an important role in the management of airway infections in individuals with cystic fibrosis (CF). The responses of airway epithelial cells to pathogens are likely to contribute to the pathobiology of CF lung disease. Primary airway epithelial cells obtained from individuals with CF, cultured and differentiated at air-liquid interface (ALI), effectively mimic the structure and function of the in vivo airway epithelium. With the recent respiratory viral pandemics, ALI cultures were extensively used to model respiratory infections in vitro to facilitate physiologically relevant respiratory research. Immunofluorescence staining and imaging were used as an effective tool to provide a fundamental understanding of host-pathogen interactions and for exploring the therapeutic potential of novel or repurposed drugs. Therefore, we described an optimized quantitative fluorescence microscopy assay for the wholemount staining and imaging of epithelial cell markers to identify distinct cell populations and pathogen-specific targets in ALI cultures of human airway epithelial cells grown on permeable support insert membranes. We present a detailed methodology using a graphical user interface (GUI) package to quantify the detected signals on a tiled whole membrane. Our method provided an imaging strategy of the entire membrane, overcoming the common issue of undersampling and enabling unbiased quantitative analysis.

Molecular Dynamics and Theratyping in Airway and Gut Organoids Reveal R352Q-CFTR Conductance Defect.

A significant challenge to making targeted cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies accessible to all individuals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rare CFTR mutation R352Q, with a potential role contributing to intrapore chloride ion permeation, in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using an ion transport assay and forskolin-induced swelling assay, respectively. CFTR potentiators (VX-770, GLPG1837, and VX-445) and correctors (VX-809, VX-445, with or without VX-661) were tested. Data from R352Q-CFTR were compared with data of 20 participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function that was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics simulations of R352Q-CFTR were carried out, which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach of in vitro patient-derived cell models and in silico molecular dynamics simulations to characterize rare CFTR mutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.

Information Communication Technology as Instrumental Activities of Daily Living for Aging-in-Place in Chinese Older Adults With and Without Cognitive Impairment: The Validation Study of Advanced Instrumental Activities of Daily Living Scale.

Background: The capability in applying information communication technology (ICT) is crucial to the functional independence of older peoples of community living nowadays. The proper assessment of individuals' capability of ICT application is the corner stone for the future development of telemedicine in our aging population. Methods: With the recruitment of 300 participants of different functional and social background in home-living, hostel-living, and care-and-attention home living; and through assessing the ability of individuals in instrumental activities of daily living and cognitive assessments, this study aimed at capturing the content validity and construct validity of the Advanced Instrumental Activities of Daily Living (AIADL scale). In addition, this study assess the ability of older peoples in applying ICT and how the functional and social background affects their independence in aging-in-place. Results: The AIADL scale showed good test-retest reliability and good-to-excellent internal consistency. To determine if items of the AIADL scale measure various aspects of community living, exploratory factor analysis revealed a two-factor structure with "home living and management" and "community living". Validity analysis with the known-groups method showed a high overall accuracy of prediction of individuals' capability of independent living in the community. Conclusions: The AIADL scale is a valid and reliable instrument to assess the ability of older adults in handling ICT as part of their instrumental activities in daily living. The scale can reflect capability of older peoples in applying ICT. This instrument can serve as a reference in measuring readiness of individuals in receiving telemedicine and their ability of aging-in-place.

Air-Liquid-Interface Differentiated Human Nose Epithelium: A Robust Primary Tissue Culture Model of SARS-CoV-2 Infection.

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air-liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.

Molecular dynamics and functional characterization of I37R-CFTR lasso mutation provide insights into channel gating activity.

Characterization of I37R, a mutation located in the lasso motif of the CFTR chloride channel, was conducted by theratyping several CFTR modulators from both potentiator and corrector classes. Intestinal current measurements in rectal biopsies, forskolin-induced swelling (FIS) in intestinal organoids, and short circuit current measurements in organoid-derived monolayers from an individual with I37R/F508del CFTR genotype demonstrated that the I37R-CFTR results in a residual function defect amenable to treatment with potentiators and type III, but not type I, correctors. Molecular dynamics of I37R using an extended model of the phosphorylated, ATP-bound human CFTR identified an altered lasso motif conformation which results in an unfavorable strengthening of the interactions between the lasso motif, the regulatory (R) domain, and the transmembrane domain 2 (TMD2). Structural and functional characterization of the I37R-CFTR mutation increases understanding of CFTR channel regulation and provides a potential pathway to expand drug access to CF patients with ultra-rare genotypes.