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Background To aid in the diagnosis of Primary Ciliary Dyskinesia (PCD) and to evaluate the respiratory epithelium in respiratory disease, normal age-related reference ranges are needed for ciliary beat frequency (CBF), beat pattern and ultrastructure. Our aim was to establish reference ranges for healthy Chinese children. Methods Ciliated epithelial samples were obtained from 135 healthy Chinese children aged below 18 years by brushing the inferior nasal turbinate. CBF and beat pattern were analysed from high speed video recordings. Epithelial integrity and ciliary ultrastructure were assessed using transmission electronic microscopy. Results The mean CBF from 135 children studied was 10.1 Hz (95% CI 9.8 to 10.4). Approximately 20% (ranged 18.0–24.2%) of ciliated epithelial edges were found to have areas of dyskinetically beating cilia. Normal beat pattern was observed in ciliated epithelium from all subjects. We did not find any effect of exposure to second hand smoke on CBF in our subjects. Microtubular defects were found in 9.3% of all of the cilia counted in these children, while other ciliary ultrastructural defects were found in less than 3%. Conclusions We established the reference range for CBF, beat pattern and ultrastructure in healthy Chinese children. Using similar methodology, we found a lower overall mean CBF than previously obtained European values. This study highlights the need to establish normative data for ciliary function in different populations.

Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of two central singlets and nine outer doublet microtubules. Cryo-electron microscopy-based studies have revealed a complex network inside the lumen of both tubules composed of microtubule-inner proteins (MIPs). However, the functions of most MIPs remain unknown. Here, we present single-particle cryo-EM-based analyses of the Tetrahymena thermophila native doublet microtubule and identify 42 MIPs. These data shed light on the evolutionarily conserved and diversified roles of MIPs. In addition, we identified MIPs potentially responsible for the assembly and stability of the doublet outer junction. Knockout of the evolutionarily conserved outer junction component CFAP77 moderately diminishes Tetrahymena swimming speed and beat frequency, indicating the important role of CFAP77 and outer junction stability in cilia beating generation and/or regulation. Using cryo-EM, the authors identified 42 MIPs, including outer junction protein CFAP77 and outer dense fibers, in native doublet microtubules of Tetrahymena thermophila . Knockout of CFAP77 reduced ciliary beat frequency and led to outer junction damage.

We present numerical and analytical predictions of mucociliary clearance based on the continuum description of a viscoelastic mucus film, where momentum transfer from the beating cilia is represented via a Navier-slip boundary condition introduced by Bottier et al. (PLoS Comput. Biol., vol. 13, issue 7, 2017a, e1005552). Mucus viscoelasticity is represented via the Oldroyd-B model, where the relaxation time and the viscosity ratio have been fitted to experimental data for the storage and loss moduli of different types of real mucus, ranging from healthy to diseased conditions. We solve numerically the fully nonlinear governing equations for inertialess flow, and develop analytical solutions via asymptotic expansion in two limits: (i) weak viscoelasticity, i.e. low Deborah number; (ii) low cilia beat amplitude (CBA). All our approaches predict a drop in the mucus flow rate in relation to the Newtonian reference value, as the cilia beat frequency is increased. This relative drop increases with decreasing CBA and slip length. In diseased conditions, e.g. mucus properties characteristic of cystic fibrosis, the drop reaches 30 % in the physiological frequency range. In the case of healthy mucus, no significant drop is observed, even at very high frequency. This contrasts with the deterioration of microorganism propulsion predicted by the low-amplitude theory of Lauga (Phys. Fluids, vol. 19, issue 8, 2007, 083104), and is due to the larger beat amplitude and slip length associated with mucociliary clearance. In the physiological range of the cilia beat frequency, the low-amplitude prediction is accurate for both healthy and diseased conditions. Finally, we find that shear-thinning, modelled via a multi-mode Giesekus model, does not significantly alter our weakly viscoelastic and low-amplitude predictions based on the Oldroyd-B model.

Chronic obstructive pulmonary disease (COPD) is characterised by airflow limitation and infective exacerbations, however, in-vitro model systems for the study of host-pathogen interaction at the individual level are lacking. Here, we describe the establishment of nasopharyngeal and bronchial organoids from healthy individuals and COPD that recapitulate disease at the individual level. In contrast to healthy organoids, goblet cell hyperplasia and reduced ciliary beat frequency were observed in COPD organoids, hallmark features of the disease. Single-cell transcriptomics uncovered evidence for altered cellular differentiation trajectories in COPD organoids. SARS-CoV-2 infection of COPD organoids revealed more productive replication in bronchi, the key site of infection in severe COVID-19. Viral and bacterial exposure of organoids induced greater pro-inflammatory responses in COPD organoids. In summary, we present an organoid model that recapitulates the in vivo physiological lung microenvironment at the individual level and is amenable to the study of host-pathogen interaction and emerging infectious disease. Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory respiratory disease characterized by airflow limitation and infective exacerbations. Here, Chan et al. report the generation of nasopharyngeal and bronchial COPD organoids derived from adult stem cells and employ them in the study of host-pathogen interactions, including SARS-CoV-2 and Pseudomonas aeruginosa.

Background Rhinoviruses (RV) are the major cause of common colds in healthy individuals and are associated with acute exacerbations in patients with chronic lung diseases. Yet, no vaccines or effective treatment against RV are available. This study investigated the effect of Euphorbium compositum SN (ECSN6), a multicomponent, multitarget medication made from natural ingredients, on the mucosal barrier network during RV infection. Methods Mucociliary-differentiated airway epithelial cell cultures were infected with RV or sham, and treated with 20% ECSN6 or placebo twice daily. Barrier integrity was assessed by measuring transepithelial resistance (TER), permeability to inulin, and expression and localization of intercellular junctions proteins (IJ). Ciliary beat frequency (CBF), expression of pro-inflammatory cytokines, antiviral interferons and mucins, and viral load were also measured. C57BL/6 mice were infected intranasally with RV or sham and treated with 40% ECSN6 or placebo twice daily. Inflammation of sinunasal mucosa, localization of E-cadherin, viral load and mucin gene expression were determined. Results ECSN6-treated, uninfected cell cultures showed small, but significant increase in TER over placebo, which was associated with enhanced localization of E-cadherin and ZO-1 to IJ. In RV-infected cultures, treatment with ECSN6, but not placebo prevented RV-induced (1) reduction in TER, (2) dissociation of E-cadherin and ZO-1 from the IJ, (3) mucin expression, and (4) CBF attenuation. ECSN6 also decreased RV-stimulated expression of pro-inflammatory cytokines and permeability to inulin. Although ECSN6 significantly increased the expression of some antiviral type I and type III interferons, it did not alter viral load. In vivo, ECSN6 reduced RV-A1-induced moderate inflammation of nasal mucosa, beneficially affected RV-A1-induced cytokine responses and Muc5ac mRNA expression and prevented RV-caused dissociation of E-cadherin from the IJ of nasal mucosa without an effect on viral clearance. Conclusions ECSN6 prevents RV-induced airway mucosal barrier dysfunction and improves the immunological and mucociliary barrier function. ECSN6 may maintain integrity of barrier function by promoting localization of tight and adherence junction proteins to the IJ. This in turn may lead to the observed decrease in RV-induced pro-inflammatory responses in vitro. By improving the innate defenses of the airway mucosal barrier network, ECSN6 may alleviate respiratory symptoms caused by RV infections.

Electronic cigarettes (e-cigs) have been introduced as a safer alternative to traditional combustible cigarettes and have been growing in popularity. E-cig e-liquids all contain the carrier compounds, vegetable glycerin (VG), propylene glycol (PG), and nicotine, together with different flavors, but the effects of inhalation of these compounds on the airway are not well understood. This study investigates the effects of e-cig exposure on primary human airway epithelial cells grown in air–liquid interface (ALI) cultures, specifically focusing on mucociliary clearance, the lung’s primary host defense mechanism whereby pathogens and particles trapped by mucus are cleared by unidirectional beating by ciliated cells. We developed a microcontroller-based exposure system to reproducibly examine cellular and molecular changes in ALI cultures from e-cig exposure. Here we show heterogeneous, donor-dependent effects of different e-cig flavors on airway epithelial cells. Examining the effects of the unflavored carrier compounds common to all e-cigs, we found that ALI airway cultures exposed to PG:VG (30:70 ratio) with 5% nicotine unflavored e-cigs show a reduction in ciliary beat frequency. Moreover, using transmission electron microscopy, we identified defects in ciliary ultrastructure induced by unflavored e-cigs. Phosphoproteomic analysis uncovered changes in phosphorylation of proteins involved in cadherin and actin binding and the Rho GTPase signaling pathway, which are all involved in cytoskeletal remodeling that may influence ciliary structure and function. Altogether, our findings suggest that exposure to all e-cigs reduces mucociliary clearance.

A multiplexed fiber laser sensing system for cell temperature is proposed. To the best of the authors' knowledge, this is the first multilongitudinal mode (MLM) optical fiber laser sensor array designed for cell temperature sensing. A two-channel cell temperature sensing system with high sensitivity and real-time sensing capability is achieved. The temperature change of human hepatoellular carcinomas (HepG2) cells under the influence of exogenous chemical aflatoxin B1 (AFB1) can be monitored in real time. A fiber laser cavity consists of a pair of fiber Bragg gratings (FBGs) with matched central wavelengths and a piece of erbium-doped fiber (EDF). The static FBG is utilized for design of fiber laser cavity and laser modes selection. In comparison, the sensing FBG is used for cell temperature sensing. The sensing FBG has a length of 10 mm and a diameter of . Beat frequency signals (BFS) are generated by MLM lasers after optical-to-electrical conversion at a photodetector. Frequency change of a BFS is closely related to the reflected wavelength change of the sensing FBG. Through frequency division multiplexing, two fiber laser cavities are designed in the sensing system for two-channel temperature sensing. Frequency shift of a BFS that represents temperature change of cells can be automatically recorded in seconds. A two-channel cell temperature sensing system is designed with high sensitivities of 101.62 and , respectively. The temperature change of HepG2 cells under the influence of exogenous chemical AFB1 is monitored in real time. The proposed system has the advantages of simple structure, high sensitivity, and two-channel sensing capability. Our study provides a simple and effective method to design a fiber laser sensor system without complex demodulation techniques and expensive optical components.

The airways of the human respiratory system are covered by a protective layer, which is known as airway surface liquid (ASL). This layer consists of two relatively distinct sub-layers; a mucus layer (ML), and a periciliary liquid layer (PCL). In addition, the airways are lined with a dense mat of hair-like structures, called cilia, which beat back and forth in a co-ordinated manner and mainly propel the mucus layer. Such interaction between the cilia and mucus is called ‘muco-ciliary clearance’ (MCC) which is essential to clear the respiratory airways from the inhaled toxic particles deposited on the mucus. The complex nature of lung clearance mechanisms limit the ability to conduct experiments to investigate micro-scale physiological phenomena. As such, modelling techniques are commonly implemented to investigate the effects of biological parameters on the lung muco-ciliary clearance. In the present work, modelling techniques of cilia-ASL interactions – including continuum cilia modelling and discrete cilia modelling – are reviewed and the numerical procedures and level of complexity related to each technique are explained. This is followed by a detailed analysis of the airway surface liquid modelling approaches. In addition, findings of numerical investigations related to the effects of various parameters such as ciliary beat frequency (CBF), mucus rheology, metachronal waves of cilia, surface tension at the PCL-mucus interface, ciliary length, ciliary density, and airway surface liquid depth on the bronchial and tracheal ASL transport are reviewed. This review also explains how these biological parameters can alter the internal power required to perform ciliary beating. Lastly, the main limitations of current numerical works are discussed and significant research directions are brought forward that may be considered in future models to better understand this complex human biological system and its vital clearance mechanism.

Background Silica nanoparticles (SiNPs) have numerous beneficial properties and are extensively used in cosmetics and food industries as anti-caking, densifying and hydrophobic agents. However, the increasing exposure levels experienced by the general population and the ability of SiNPs to penetrate cells and tissues have raised concerns about possible toxic effects of this material. Although SiNPs are known to affect the function of the airway epithelium, the molecular targets of these particles remain largely unknown. Given that SiNPs interact with the plasma membrane of epithelial cells we hypothesized that they may affect the function of Transient Receptor Potential Vanilloid 4 (TRPV4), a cation-permeable channel that regulates epithelial barrier function. The main aims of this study were to evaluate the effects of SiNPs on the activation of TRPV4 and to determine whether these alter the positive modulatory action of this channel on the ciliary beat frequency in airway epithelial cells. Results Using fluorometric measurements of intracellular Ca 2+ concentration ([Ca 2+ ] i ) we found that SiNPs inhibit activation of TRPV4 by the synthetic agonist GSK1016790A in cultured human airway epithelial cells 16HBE and in primary cultured mouse tracheobronchial epithelial cells. Inhibition of TRPV4 by SiNPs was confirmed in intracellular Ca 2+ imaging and whole-cell patch-clamp experiments performed in HEK293T cells over-expressing this channel. In addition to these effects, SiNPs were found to induce a significant increase in basal [Ca 2+ ] i , but in a TRPV4-independent manner. SiNPs enhanced the activation of the capsaicin receptor TRPV1, demonstrating that these particles have a specific inhibitory action on TRPV4 activation. Finally, we found that SiNPs abrogate the increase in ciliary beat frequency induced by TRPV4 activation in mouse airway epithelial cells. Conclusions Our results show that SiNPs inhibit TRPV4 activation, and that this effect may impair the positive modulatory action of the stimulation of this channel on the ciliary function in airway epithelial cells. These findings unveil the cation channel TRPV4 as a primary molecular target of SiNPs.

Vitamin D supplementation has been suggested to enhance immunity during respiratory infection season. We tested the effect of active vitamin D (calcitriol) supplementation on key airway innate immune mechanisms in vitro. Primary human airway epithelial cells (hAECs) grown at the air liquid interface were supplemented with 10-7 M calcitriol for 24 hours (or a time course) and their antimicrobial airway surface liquid (ASL) was tested for pH, viscoscity, and antibacterial and antiviral properties. We also tested hAEC ciliary beat frequency (CBF). Next, we assessed alterations to hAEC gene expression using RNA sequencing, and based on results, we measured neutrophil migration across hAECs. Calcitriol supplementation enhanced ASL bacterial killing of Staphylococcus aureus (p = 0.02) but did not enhance its antiviral activity against 229E-CoV. It had no effect on ASL pH or viscosity at three timepoints. Lastly, it did not affect hAEC CBF or neutrophil migration, although there was a trend of enhanced migration in the presence of a neutrophil chemokine (p = 0.09). Supplementation significantly altered hAEC gene expression, primarily of AMP-related genes including CAMP and TREM1. While vitamin D supplementation did not have effects on many airway innate immune mechanisms, it may provide a useful tool to resolve respiratory bacterial infections.