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Dysfunction of motile cilia can impair mucociliary clearance in the airway and result in primary ciliary dyskinesia (PCD). We previously showed that mutations in central pair apparatus (CPA) genes perturb ciliary motility and result in PCD in mouse models. However, little is known about how epithelial cell types in the ciliary microenvironment of the upper airway respond to defects in ciliary motility and mucociliary clearance. Here, we have used single-cell RNA sequencing to investigate responses in tracheal epithelial cells from mice with mutations in CPA genes Cfap221/ Pcdp1, Cfap54, and Spef2. Expected cell types were identified, along with an unidentified cell type not expressing markers of typical airway cells. Deuterosomal cells were found to exist in two states that differ largely in expression of genes involved in differentiation into ciliated cells. Functional enrichment analysis of differentially expressed genes (DEGs) revealed important cellular functions and molecular pathways for each cell type that are altered in mutant mice. Overlapping DEGs shed light on general responses to cilia dysfunction, while unique DEGs indicate that some responses may be specific to the individual mutation and ciliary defect., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Neurofibromatosis type 2 (NF2) is a rare disorder that causes vestibular schwannomas (VS), meningiomas and ependymomas. To date, there is no FDA approved drug-based treatment for NF2. We have previously identified that BET inhibition can selectively reduce growth of the NF2-null schwannoma and Schwann cells in vitro and tumorigenesis in vivo and, separately, reported that inhibition of Focal Adhesion Kinase 1 (FAK1) via crizotinib has antiproliferative effects in NF2-null Schwann cells. The current study was aimed at determining whether combined BET and FAK inhibition can synergize and to identify the mechanisms of action. A panel of normal and NF2-null Schwann and schwannoma cell lines were used to characterize the effects of combined BET and FAK inhibition in vitro and in vivo using pharmacological and genetic approaches. The mechanism of action was explored by chromatin immunoprecipitation, ChIP-PCR, western blotting, and functional approaches. We find that combined BET and FAK inhibition are synergistic and inhibit the proliferation of NF2-null schwannoma and Schwann cell lines in vitro and in vivo, by arresting cells in the G1/S and G2/M phases of the cell cycle. Further, we identify the mechanism of action through the downregulation of FAK1 transcription by BET inhibition, which potentiates inhibition of FAK by 100-fold. Our findings suggest that combined targeting of BET and FAK1 may offer a potential therapeutic option for the treatment of NF2-related schwannomas., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Lower respiratory tract infection (LRTI) including pneumonia, bronchitis, and bronchiolitis is the sixth leading cause of mortality around the world and leading cause of death in children under 5 years. Systemic immune response to viral infection is well characterized. However, there is little data regarding the immune response at the upper respiratory tract mucosa. The upper respiratory mucosa is the site of viral entry, initial replication and the first barrier against respiratory infections. Lower respiratory tract samples can be challenging to obtain and require more invasive procedures. However, nasal wash (NW) samples from the upper respiratory tract can be obtained with minimal discomfort to the patient., Method: In a pilot study, we developed a protocol using NW samples obtained from hospitalized children with LRTI that enables single cell RNA sequencing (scRNA-seq) after the NW sample is methanol-fixed., Results: We found no significant changes in scRNA-seq qualitative and quantitative parameters between methanol-fixed and fresh NW samples., Conclusions: We present a novel protocol to enable scRNA-seq in NW samples from children admitted with LRTI. With the inherent challenges associated with clinical samples, the protocol described allows for processing flexibility as well as multicenter collaboration., Impact: There are no significant differences in scRNA-seq qualitative and quantitative parameters between methanol fixed and fresh Pediatric Nasal wash samples. The study demonstrates the effectiveness of methanol fixation process on preserving respiratory samples for single cell sequencing. This enables Pediatric Nasal wash specimen for single cell RNA sequencing in pediatric patients with respiratory tract infection and allows processing flexibility and multicenter collaboration., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Normal differentiation and induced reprogramming require the activation of target cell programs and silencing of donor cell programs. In reprogramming, the same factors are often used to reprogram many different donor cell types. As most developmental repressors, such as RE1-silencing transcription factor (REST) and Groucho (also known as TLE), are considered lineage-specific repressors, it remains unclear how identical combinations of transcription factors can silence so many different donor programs. Distinct lineage repressors would have to be induced in different donor cell types. Here, by studying the reprogramming of mouse fibroblasts to neurons, we found that the pan neuron-specific transcription factor Myt1-like (Myt1l) exerts its pro-neuronal function by direct repression of many different somatic lineage programs except the neuronal program. The repressive function of Myt1l is mediated via recruitment of a complex containing Sin3b by binding to a previously uncharacterized N-terminal domain. In agreement with its repressive function, the genomic binding sites of Myt1l are similar in neurons and fibroblasts and are preferentially in an open chromatin configuration. The Notch signalling pathway is repressed by Myt1l through silencing of several members, including Hes1. Acute knockdown of Myt1l in the developing mouse brain mimicked a Notch gain-of-function phenotype, suggesting that Myt1l allows newborn neurons to escape Notch activation during normal development. Depletion of Myt1l in primary postmitotic neurons de-repressed non-neuronal programs and impaired neuronal gene expression and function, indicating that many somatic lineage programs are actively and persistently repressed by Myt1l to maintain neuronal identity. It is now tempting to speculate that similar 'many-but-one' lineage repressors exist for other cell fates; such repressors, in combination with lineage-specific activators, would be prime candidates for use in reprogramming additional cell types.