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Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections 1 . Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes 2 . The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures. Detailed atomic models of axonemes from algal flagella and human respiratory cilia, which are hair-like protrusions from cells that enable motility and clear mucus from human airways, could provide insights into how they function.

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages. The generation of primary organoids, from fetal fluid-derived epithelial stem or progenitor cells, offers the possibility of modeling different developing tissues during gestation, even beyond the limits of pregnancy termination.

BackgroundPrimary ciliary dyskinesia (PCD), a genetically heterogeneous condition enriched in some consanguineous populations, results from recessive mutations affecting cilia biogenesis and motility. Currently, diagnosis requires multiple expert tests.MethodsThe diagnostic utility of multigene panel next-generation sequencing (NGS) was evaluated in 161 unrelated families from multiple population ancestries.ResultsMost (82%) families had affected individuals with biallelic or hemizygous (75%) or single (7%) pathogenic causal alleles in known PCD genes. Loss-of-function alleles dominate (73% frameshift, stop-gain, splice site), most (58%) being homozygous, even in non-consanguineous families. Although 57% (88) of the total 155 diagnostic disease variants were novel, recurrent mutations and mutated genes were detected. These differed markedly between white European (52% of families carry DNAH5 or DNAH11 mutations), Arab (42% of families carry CCDC39 or CCDC40 mutations) and South Asian (single LRRC6 or CCDC103 mutations carried in 36% of families) patients, revealing a striking genetic stratification according to population of origin in PCD. Genetics facilitated successful diagnosis of 81% of families with normal or inconclusive ultrastructure and 67% missing prior ultrastructure results.ConclusionsThis study shows the added value of high-throughput targeted NGS in expediting PCD diagnosis. Therefore, there is potential significant patient benefit in wider and/or earlier implementation of genetic screening.

The diagnosis of primary ciliary dyskinesia (PCD) requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI) aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns. We retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD n  111) was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture. Ciliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced. The change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia.

Background Primary ciliary dyskinesia can result from a number of different ciliary defects that adversely affect ciliary function resulting markedly reduced or absent mucociliary clearance. Improvement in diagnostic testing is an area of current research. During diagnostic evaluation of PCD we observed ciliated conical protrusions from part of the apical surface of ciliated cells in those diagnosed with PCD. The aim of this study was to investigate if this abnormality was specific to PCD. Methods Epithelial edges from 67 consecutively diagnosed PCD patients, 67 patients consecutively referred for PCD diagnostic testing in whom PCD was excluded, 22 with asthma and 18 with Cystic Fibrosis (CF) were studied retrospectively in a blinded manner using light microscopy. Results Forty six out of 67 patients with PCD had ciliated conical epithelial protrusions, whereas none were seen in patients where PCD was excluded, or in patients with asthma or CF. The sensitivity, specificity, positive predictive value and negative predictive value for the presence of the ciliated conical protrusions to predict a diagnosis of PCD were 76.5, 100, 100 and 77% respectively. Conclusions Characteristic ciliated conical protrusions from ciliated epithelial cells maybe a useful pointer to the diagnosis of PCD. However, their absence does not exclude the diagnosis of PCD.

Air–liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling, infection or inflammation confounding PCD diagnostic results. ALI culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI culture method adopted from April 2018 across three collaborating PCD diagnostic sites, including current University Hospital Southampton COVID-19 risk mitigation measures, and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI culture and 199 (82.9%) were ciliated. Fifty-four of 83 (63.9%) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen; 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated, scanning electron microscopy demonstrated excellent ciliation, and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary, our ALI culture protocol provides high ciliation rates across three centres, minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful, facilitating PCD research.

Background. The role played by pneumolysin and autolysin in pneumococcal meningitis is poorly understood. Methods. A rat model was used to investigate the disease, in which surgical implantation of a cisternal catheter allowed bacterial instillation and cerebrospinal fluid (CSF) sampling. Results. CSF infection of rats with wild-type pneumococci caused meningitis within 26 h, whereas isogenic mutants that do not express pneumolysin (ΔPly) or autolysin (LytA−) caused very mild or no disease. Wild-type infections resulted in pneumococci in the CSF and cortical homogenates, but a minority of the rats infected with ΔPly or LytA− had bacteria in these locations at 26 h. Leukocyte numbers in the CSF were similar after infection with all pneumococci; however, neutrophils and monocytes predominated after wild-type infection, whereas lymphocytes and atypical lymphocytes predominated after infection with the mutants. Wild-type pneumococci caused disruption to the ependyma, but this was not observed in rats infected withΔPly or LytA−. Cells surrounding the ventricles in wild type-infected animals expressed caspase 3, and astrocytes had hypertrophy; both findings were absent in rats infected with the mutants. Conclusions. This study provides strong in vivo evidence that pneumolysin and autolysin play crucial roles in the pathogenesis of pneumococcal meningitis.

Pneumolysin (PLN) is an intracellular toxin of Streptococcus pneumoniae that has been implicated as a major virulence factor in infections caused by this pathogen. Conserved bacterial motifs are recognized by the immune system by pattern recognition receptors among which the family of Toll-like receptors (TLRs) prominently features. The primary objective of the present study was to determine the role of TLR2 and TLR4 in lung inflammation induced by intrapulmonary delivery of PLN. First, we confirmed that purified PLN activates cells via TLR4 (not via TLR2) in vitro, using human embryonic kidney cells transfected with either TLR2 or TLR4. Intranasal administration of PLN induced an inflammatory response in the pulmonary compartment of mice in vivo, as reflected by influx of neutrophils, release of proinflammatory cytokines and chemokines, and a rise in total protein concentrations in bronchoalveolar lavage fluid. These PLN-induced responses were dependent in part, not only on TLR4, but also on TLR2, as indicated by studies using TLR deficient mice. These data suggest that although purified PLN is recognized by TLR4 in vitro, PLN elicits lung inflammation in vivo by mechanisms that may involve multiple TLRs.

Telomeric 3' overhangs can fold into a four-stranded DNA structure termed G-quadruplex (G4), a formation which inhibits telomerase. As telomerase activation is crucial for telomere maintenance in most cancer cells, several classes of G4 ligands have been designed to directly disrupt telomeric structure. We exposed brain tumor cells to the G4 ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) and investigated proliferation, cell cycle dynamics, telomere length, telomerase activity and activated c-Myc levels. Although all cell lines tested were sensitive to RHPS4, PFSK-1 central nervous system primitive neuroectodermal cells, DAOY medulloblastoma cells and U87 glioblastoma cells exhibited up to 30-fold increased sensitivity compared to KNS42 glioblastoma, C6 glioma and Res196 ependymoma cells. An increased proportion of S-phase cells were observed in medulloblastoma and high grade glioma cells whilst CNS PNET cells showed an increased proportion of G1-phase cells. RHPS4-induced phenotypes were concomitant with telomerase inhibition, manifested in a telomere length-independent manner and not associated with activated c-Myc levels. However, anti-proliferative effects were also observed in normal neural/endothelial cells in vitro and ex vivo. This study warrants in vivo validation of RHPS4 and alternative G4 ligands as potential anti-cancer agents for brain tumors but highlights the consideration of dose-limiting tissue toxicities.

BackgroundThe inflamed bronchial mucosal surface is a profoundly hypoxic environment. Neutrophilic airway inflammation and neutrophil-derived proteases have been linked to disease progression in conditions such as COPD and cystic fibrosis, but the effects of hypoxia on potentially harmful neutrophil functional responses such as degranulation are unknown.Methods and resultsFollowing exposure to hypoxia (0.8% oxygen, 3 kPa for 4 h), neutrophils stimulated with inflammatory agonists (granulocyte-macrophage colony stimulating factor or platelet-activating factor and formylated peptide) displayed a markedly augmented (twofold to sixfold) release of azurophilic (neutrophil elastase, myeloperoxidase), specific (lactoferrin) and gelatinase (matrix metalloproteinase-9) granule contents. Neutrophil supernatants derived under hypoxic but not normoxic conditions induced extensive airway epithelial cell detachment and death, which was prevented by coincubation with the antiprotease α-1 antitrypsin; both normoxic and hypoxic supernatants impaired ciliary function. Surprisingly, the hypoxic upregulation of neutrophil degranulation was not dependent on hypoxia-inducible factor (HIF), nor was it fully reversed by inhibition of phospholipase C signalling. Hypoxia augmented the resting and cytokine-stimulated phosphorylation of AKT, and inhibition of phosphoinositide 3-kinase (PI3K)γ (but not other PI3K isoforms) prevented the hypoxic upregulation of neutrophil elastase release.ConclusionHypoxia augments neutrophil degranulation and confers enhanced potential for damage to respiratory airway epithelial cells in a HIF-independent but PI3Kγ-dependent fashion.