Syndromic ciliopathy: a taiwanese single-center study.

BACKGROUND: Syndromic ciliopathies are a group of congenital disorders characterized by broad clinical and genetic overlap, including obesity, visual problems, skeletal anomalies, mental retardation, and renal diseases. The hallmark of the pathophysiology among these disorders is defective ciliary functions or formation. Many different genes have been implicated in the pathogenesis of these diseases, but some patients still remain unclear about their genotypes. METHODS: The aim of this study was to identify the genetic causes in patients with syndromic ciliopathy. Patients suspected of or meeting clinical diagnostic criteria for any type of syndromic ciliopathy were recruited at a single diagnostic medical center in Southern Taiwan. Whole exome sequencing (WES) was employed to identify their genotypes and elucidate the mutation spectrum in Taiwanese patients with syndromic ciliopathy. Clinical information was collected at the time of patient enrollment. RESULTS: A total of 14 cases were molecularly diagnosed with syndromic ciliopathy. Among these cases, 10 had Bardet-Biedl syndrome (BBS), comprising eight BBS2 patients and two BBS7 patients. Additionally, two cases were diagnosed with Alström syndrome, one with Oral-facial-digital syndrome type 14, and another with Joubert syndrome type 10. A total of 4 novel variants were identified. A recurrent splice site mutation, BBS2: c.534 + 1G > T, was present in all eight BBS2 patients, suggesting a founder effect. One BBS2 patient with homozygous c.534 + 1G > T mutations carried a third ciliopathic allele, TTC21B: c.264_267dupTAGA, a nonsense mutation resulting in a premature stop codon and protein truncation. CONCLUSIONS: Whole exome sequencing (WES) assists in identifying molecular pathogenic variants in ciliopathic patients, as well as the genetic hotspot mutations in specific populations. It should be considered as the first-line genetic testing for heterogeneous disorders characterized by the involvement of multiple genes and diverse clinical manifestations.

A neurodevelopmental disorder associated with a loss-of-function missense mutation in RAB35.

Rab35 (Ras-associated binding protein) is a small GTPase that regulates endosomal membrane trafficking and functions in cell polarity, cytokinesis, and growth factor signaling. Altered Rab35 function contributes to progression of glioblastoma, defects in primary cilia formation, and altered cytokinesis. Here, we report a pediatric patient with global developmental delay, hydrocephalus, a Dandy-Walker malformation, axial hypotonia with peripheral hypertonia, visual problems, and conductive hearing impairment. Exome sequencing identified a homozygous missense variant in the GTPase fold of RAB35 (c.80G>A; p.R27H) as the most likely candidate. Functional analysis of the R27H-Rab35 variant protein revealed enhanced interaction with its guanine-nucleotide exchange factor, DENND1A and decreased interaction with a known effector, MICAL1, indicating that the protein is in an inactive conformation. Cellular expression of the variant drives the activation of Arf6, a small GTPase under negative regulatory control of Rab35. Importantly, variant expression leads to delayed cytokinesis and altered length, number, and Arl13b composition of primary cilia, known factors in neurodevelopmental disease. Our findings provide evidence of altered Rab35 function as a causative factor of a neurodevelopmental disorder.

Practical guide for the diagnosis and management of primary ciliary dyskinesia.

OBJECTIVE: Primary ciliary dyskinesia (PCD) is a relatively rare genetic disorder that affects approximately 1 in 20,000 people. Approximately 50 genes are currently known to cause PCD. In light of differences in causative genes and the medical system in Japan compared with other countries, a practical guide was needed for the diagnosis and management of Japanese PCD patients. METHODS: An ad hoc academic committee was organized under the Japanese Rhinologic Society to produce a practical guide, with participation by committee members from several academic societies in Japan. The practical guide including diagnostic criteria for PCD was approved by the Japanese Rhinologic Society, Japanese Society of Otolaryngology-Head and Neck Surgery, Japanese Respiratory Society, and Japanese Society of Pediatric Pulmonology. RESULTS: The diagnostic criteria for PCD consist of six clinical features, six laboratory findings, differential diagnosis, and genetic testing. The diagnosis of PCD is categorized as definite, probable, or possible PCD based on a combination of the four items above. Diagnosis of definite PCD requires exclusion of cystic fibrosis and primary immunodeficiency, at least one of the six clinical features, and a positive result for at least one of the following: (1) Class 1 defect on electron microscopy of cilia, (2) pathogenic or likely pathogenic variants in a PCD-related gene, or (3) impairment of ciliary motility that can be repaired by correcting the causative gene variants in iPS cells established from the patient's peripheral blood cells. CONCLUSION: This practical guide provides clinicians with useful information for the diagnosis and management of PCD in Japan.

Genetic variants in primary cilia-related genes associated with the prognosis of first-line chemotherapy in colorectal cancer.

BACKGROUND: Primary cilia are antenna-like organelles that conduct physical and chemical signals, which affect cell proliferation, migration, and differentiation. Some researchers have reported the correlation between primary cilia-related genes and prognosis of colorectal cancer (CRC). METHODS: The association between single nucleotide polymorphisms (SNPs) of primary cilia-related genes and outcome after the first-line chemotherapy was explored by the Cox regression model. Expression qualitative trait locus (eQTL) analysis was performed to explore the impact of SNPs on gene expression. Tumor Immune Estimation Resource and TISIDB databases were used for investigating the relevance between ODF2L and tumor infiltration immune cells and immunomodulators. RESULTS: We identified that rs4288473 C allele of ODF2L had poor progression-free survival (PFS) and overall survival (OS) of CRC patients in the additive model (adjusted HRPFS = 1.39, 95% CI = 1.14-1.70, p = 1.36 × 10-3 , and adjusted HROS = 1.31, 95% CI = 1.03-1.65, p = 2.62 × 10-2 ). The stratified analysis indicated that rs4288573 CC/CT genotype was involved with poor prognosis in the irinotecan-treated subgroup (PPFS = 1.03 × 10-2 , POS = 3.29 × 10-3 ). Besides, ODF2L mRNA expression level was notably up-regrated in CRC tissues. The C allele of rs4288573 was notably related to higher ODF2L mRNA expression levels based on eQTL analysis. Functionally, knockdown of ODF2L inhibited cell proliferation and decrease the chemoresistance of HCT-116 and DLD-1 cells to irinotecan. CONCLUSION: Our study indicates that rs4288573 in ODF2L is a potential predictor of the chemotherapy prognosis of CRC.

Lianhua Qingke Preserves Mucociliary Clearance in Rat with Acute Exacerbation of Chronic Obstructive Pulmonary Disease by Maintaining Ciliated Cells Proportion and Protecting Structural Integrity and Beat Function of Cilia.

Purpose: Acute Exacerbation of Chronic Obstructive Pulmonary Disease (AECOPD) is a sudden worsening of symptoms in patients with Chronic Obstructive Pulmonary Disease (COPD), such as cough, increased sputum volume, and sputum purulence. COPD and AECOPD are characterized by damage to cilia and increased mucus secretion. Mucociliary clearance (MCC) functions as part of the primary innate system of the lung to remove harmful particles and pathogens together with airway mucus and is therefore crucial for patients with COPD. Methods: AECOPD was induced by cigarette smoke exposure (80 cigarettes/day, 5 days/week for 12 weeks) and lipopolysaccharide (LPS) instillation (200 µg, on days 1, 14, and 84). Rats administered Lianhua Qingke (LHQK) (0.367, 0.732, and 1.465 g/kg/d) or Eucalyptol, Limonene, and Pinene Enteric Soft Capsules (ELP, 0.3 g/kg/d) intragastrically. Pulmonary pathology, Muc5ac+ goblet cell and ß-tubulin IV+ ciliated cells, and mRNA levels of forkhead box J1 (Foxj1) and multiciliate differentiation and DNA synthesis associated cell cycle protein (MCIDAS) were assessed by hematoxylin and eosin staining, immunofluorescence staining, and RT-qPCR, respectively. Ciliary morphology and ultrastructure were examined through scanning electron microscopy and transmission electron microscopy. Ciliary beat frequency (CBF) was recorded using a high-speed camera. Results: Compared to the model group, LHQK treatment groups showed a reduction in inflammatory cell infiltration, significantly reduced goblet cell and increased ciliated cell proportion. LHQK significantly upregulated mRNA levels of MCIDAS and Foxj1, indicating promoted ciliated cell differentiation. LHQK protected ciliary structure and maintained ciliary function via increasing the ciliary length and density, reducing ciliary ultrastructure damage, and ameliorating random ciliary oscillations, consequently enhancing CBF. Conclusion: LHQK enhances the MCC capability of ciliated cells in rat with AECOPD by preserving the structural integrity and beating function of cilia, indicating its therapeutic potential on promoting sputum expulsion in patients with AECOPD.

Nasal polyps show decreased mucociliary transport despite vigorous ciliary beating.

OBJECTIVE: Mucociliary transport function in the airway mucosa is essential for maintaining a clean mucosal surface. This function is impaired in upper and lower airway diseases. Nasal polyps are a noticeable pathological feature that develop in some of the patients with chronic rhinosinusitis. Like ordinary nasal mucosae, nasal polyps have a ciliated pseudostratified epithelium with vigorous ciliary beating. We measured ex vivo Mucociliary Transport Velocity (MCTV) and Ciliary Beat Frequency (CBF) and explored the expressions of Planar Cell Polarity (PCP) proteins in nasal polyps in comparison with turbinate mucosae. METHODS: Inferior turbinates and nasal polyps were surgically collected from patients with chronic rhinosinusitis. Ex vivo MCTV and CBF were measured using a high-speed digital imaging system. Expressions of PCP proteins were explored by fluorescence immunohistochemistry and quantitative RT-PCR. RESULTS: The MCTV of nasal polyps was significantly lower than that of the turbinates (7.43 ±â€¯2.01 vs. 14.56 ±â€¯2.09 µm/s; p = 0.0361), whereas CBF did not differ between the two tissues. The MCTV vector was pointed to the posteroinferior direction in all turbinates with an average inclination angle of 41.0 degrees. Immunohistochemical expressions of Dishevelled-1, Dishevelled-3, Frizzled3, Frizzled6, Prickle2 and Vangl2 were lower in the nasal polyps than in the turbinates. Confocal laser scanning microscopy showed that Frizzled3 was localized along the cell junction on the apical surface. The expression levels of mRNAs for Dishevelled-1, Dishevelled-3 and Frizzled3 in the nasal polyps were also decreased in comparison with the turbinates. CONCLUSION: These results indicate that muco ciliary transport in nasal polyps is impaired although vigorous ciliary beating is maintained, and that the impairment may be caused by a decrease in Dishevelled/Frizzled proteins and resultant PCP disarrangement. LEVEL OF EVIDENCE: Level 3.

Ultrastructural cilia defects in multi-ciliated uterine glandular epithelial cells from women with reproductive failure.

In brief: The causes of subfertility and recurrent pregnancy loss are often unclear. This study shows that endometrial gland cilia from women with subfertility have ultrastructural defects. Abstract: Endometrial glands secrete products into the endometrium and are necessary for embryo implantation and successful pregnancy. However, structural and functional abnormalities in endometrial gland cilia from women with reproductive failure remain poorly understood. This was a cross-sectional study where endometrial biopsies were collected at days 19-23 of the menstrual cycle from women with unexplained recurrent pregnancy loss (n = 15), unexplained subfertility (n = 11) or from egg donor control participants (n = 10). Endometrial gland cilia ultrastructure was imaged by transmission electron microscopy and cilia defects assessed by an electron-microscopist from a national primary ciliary dyskinesia diagnostic centre. Endometrial glands were isolated, and the cilia beat frequency recorded by high speed video. Subfertile women have proportionately lower ultrastructurally normal cilia (P < 0.05); higher frequency of absent dynamin arms (P < 0.01) or inner arm defects (P < 0.01) and lower cilia beat frequency (P < 0.05). The mechanisms underlying these obversions have yet to be determined. Recent studies have identified cilia related gene expression changes associated with reproductive failure and this study adds to the growing body of literature revealing structural and functional changes. The observation that cilia defects occurred at a higher frequency in endometrial glands of subfertile women raises the question of its mechanistic role in implantation.

Effects of Sesquiterpene Lactones on Primary Cilia Formation (Ciliogenesis).

Sesquiterpene lactones (SLs), plant-derived metabolites with broad spectra of biological effects, including anti-tumor and anti-inflammatory, hold promise for drug development. Primary cilia, organelles extending from cell surfaces, are crucial for sensing and transducing extracellular signals essential for cell differentiation and proliferation. Their life cycle is linked to the cell cycle, as cilia assemble in non-dividing cells of G0/G1 phases and disassemble before entering mitosis. Abnormalities in both primary cilia (non-motile cilia) and motile cilia structure or function are associated with developmental disorders (ciliopathies), heart disease, and cancer. However, the impact of SLs on primary cilia remains unknown. This study evaluated the effects of selected SLs (grosheimin, costunolide, and three cyclocostunolides) on primary cilia biogenesis and stability in human retinal pigment epithelial (RPE) cells. Confocal fluorescence microscopy was employed to analyze the effects on primary cilia formation (ciliogenesis), primary cilia length, and stability. The effects on cell proliferation were evaluated by flow cytometry. All SLs disrupted primary cilia formation in the early stages of ciliogenesis, irrespective of starvation conditions or cytochalasin-D treatment, with no effect on cilia length or cell cycle progression. Interestingly, grosheimin stabilized and promoted primary cilia formation under cilia homeostasis and elongation treatment conditions. Thus, SLs have potential as novel drugs for ciliopathies and tumor treatment.

Certain heterozygous variants in the kinase domain of the serine/threonine kinase NEK8 can cause an autosomal dominant form of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) resulting from pathogenic variants in PKD1 and PKD2 is the most common form of PKD, but other genetic causes tied to primary cilia function have been identified. Biallelic pathogenic variants in the serine/threonine kinase NEK8 cause a syndromic ciliopathy with extra-kidney manifestations. Here we identify NEK8 as a disease gene for ADPKD in 12 families. Clinical evaluation was combined with functional studies using fibroblasts and tubuloids from affected individuals. Nek8 knockout mouse kidney epithelial (IMCD3) cells transfected with wild type or variant NEK8 were further used to study ciliogenesis, ciliary trafficking, kinase function, and DNA damage responses. Twenty-one affected monoallelic individuals uniformly exhibited cystic kidney disease (mostly neonatal) without consistent extra-kidney manifestations. Recurrent de novo mutations of the NEK8 missense variant p.Arg45Trp, including mosaicism, were seen in ten families. Missense variants elsewhere within the kinase domain (p.Ile150Met and p.Lys157Gln) were also identified. Functional studies demonstrated normal localization of the NEK8 protein to the proximal cilium and no consistent cilia formation defects in patient-derived cells. NEK8-wild type protein and all variant forms of the protein expressed in Nek8 knockout IMCD3 cells were localized to cilia and supported ciliogenesis. However, Nek8 knockout IMCD3 cells expressing NEK8-p.Arg45Trp and NEK8-p.Lys157Gln showed significantly decreased polycystin-2 but normal ANKS6 localization in cilia. Moreover, p.Arg45Trp NEK8 exhibited reduced kinase activity in vitro. In patient derived tubuloids and IMCD3 cells expressing NEK8-p.Arg45Trp, DNA damage signaling was increased compared to healthy passage-matched controls. Thus, we propose a dominant-negative effect for specific heterozygous missense variants in the NEK8 kinase domain as a new cause of PKD.

Primary cilia: Structure, dynamics, and roles in cancer cells and tumor microenvironment.

Despite the initiation of tumor arises from tumorigenic transformation signaling in cancer cells, cancer cell survival, invasion, and metastasis also require a dynamic and reciprocal association with extracellular signaling from tumor microenvironment (TME). Primary cilia are the antenna-like structure that mediate signaling sensation and transduction in different tissues and cells. Recent studies have started to uncover that the heterogeneous ciliation in cancer cells and cells from the TME in tumor growth impels asymmetric paracellular signaling in the TME, indicating the essential functions of primary cilia in homeostasis maintenance of both cancer cells and the TME. In this review, we discussed recent advances in the structure and assembly of primary cilia, and the role of primary cilia in tumor and TME formation, as well as the therapeutic potentials that target ciliary dynamics and signaling from the cells in different tumors and the TME.

Repurposing small molecules for nephronophthisis and related renal ciliopathies.

Nephronophthisis is an autosomal recessive tubulointerstitial nephropathy, belonging to the ciliopathy disorders, characterized by fibrosis and/or cysts. It is the most common genetic cause of kidney failure in children and young adults. Clinically and genetically heterogeneous, it is caused by variants in ciliary genes, resulting in either an isolated kidney disease or syndromic forms in association with other manifestations of ciliopathy disorders. No curative treatment is currently available. Over the past 2 decades, advances in understanding disease mechanisms have identified several dysregulated signaling pathways, some shared with other cystic kidney diseases. Notably, molecules previously developed to target these pathways have shown promising beneficial effects in orthologous mouse models. In addition to these knowledge-based repurposing approaches, unbiased "in cellulo" phenotypic screens of "repurposing" libraries identified small molecules able to rescue the ciliogenesis defects observed in nephronophthisis conditions. Those compounds appeared to act on relevant pathways and, when tested, showed beneficial nephronophthisis-associated kidney and/or extrarenal defects in mice. In this review, we have summarized those studies that highlight the drug repurposing strategies in the context of a rare disorders, such as nephronophthisis-related ciliopathies, with broad genetic heterogeneity and systemic manifestations but with shared disease mechanisms.

Primary Cilia Are Frequently Present in Small Cell Lung Carcinomas but Not in Non-Small Cell Lung Carcinomas or Lung Carcinoids.

Most human malignant neoplasms show loss of primary cilia (PC). However, PC are known to be retained and involved in tumorigenesis in some types of neoplasms. The PC status in lung carcinomas remains largely uninvestigated. In this study, we comprehensively assessed the PC status in lung carcinomas. A total of 492 lung carcinomas, consisting of adenocarcinomas (ACs) (n = 319), squamous cell carcinomas (SCCs) (n = 152), and small cell lung carcinomas (SCLCs) (n = 21), were examined by immunohistochemical analysis using an antibody against ARL13B, a marker of PC. The PC-positive rate was markedly higher in SCLCs (81.0%) than in ACs (1.6%) and SCCs (7.9%). We subsequently performed analyses to characterize the PC-positive lung carcinomas further. PC-positive lung carcinomas were more numerous and had longer PC than normal cells. The presence of PC in these cells was not associated with the phase of the cell cycle. We also found that the PC were retained even in metastases from PC-positive lung carcinomas. Furthermore, the hedgehog signaling pathway was activated in PC-positive lung carcinomas. Because ARL13B immunohistochemistry of lung carcinoids (n = 10) also showed a statistically significantly lower rate (10.0%) of PC positivity than SCLCs, we searched for a gene(s) that might be upregulated in PC-positive SCLCs compared with lung carcinoids, but not in PC-negative carcinomas. This search, and further cell culture experiments, identified HYLS1 as a gene possessing the ability to regulate ciliogenesis in PC-positive lung carcinomas. In conclusion, our findings indicate that PC are frequently present in SCLCs but not in non-SCLCs (ACs and SCCs) or lung carcinoids, and their PC exhibit various specific pathobiological characteristics. This suggests an important link between lung carcinogenesis and PC.

Patent hepatic ciliated foregut remnant resulting in an umbilicobiliary sinus tract, with gallbladder agenesis, in an 8-wk-old male French Bulldog.

Hepatic ciliated foregut remnants or cysts are congenital abnormalities resulting from retention of embryonic ciliated foregut within the liver. These structures are rarely reported in the human medical literature and have not been reported in the veterinary literature previously, to our knowledge. We describe here a case of an 8-wk-old male French Bulldog with a congenital patent hepatic ciliated foregut remnant resulting in an umbilicobiliary sinus tract. The dog also had concurrent gallbladder agenesis. The patient had yellow fluid discharging from the umbilicus, mimicking a patent urachus. Surgical exploration, removal, and histology provided a conclusive diagnosis of a hepatic foregut remnant and therapeutic resolution of the clinical signs. The histologic appearance of a hepatic foregut remnant is classical, namely a duct composed of 4 layers: an inner ciliated epithelial lining, loose connective tissue, smooth muscle, and a fibrous capsule.

Ciliated Cells in Renal Calyx mucosa: Metaplastic Change or Developmental Abnormality? Immunohistochemical Study of one Case and Review of the Literature.

Ciliated epithelial cells have been rarely observed in urothelium lined mucosa. Only extremely rare reports in the literature have described this phenomenon and no cases have been described in other sites than the male urethra. Herein, we illustrate the finding of ciliated pseudostratified columnar cells in the renal calyx mucosa adjacent to an area of urothelial invasive carcinoma in an 82 year-old man with previous history of nephrolithiasis. The ciliated cells covered a linear extension of 0.5 cm: they were positive for keratin 7 and keratin 8/18 and negative for keratin 20. Alcian blue staining was positive in some vacuoles in the apical cytoplasm of the same cells whereas PAS (Periodic Acid-Schiff) staining was negative. GATA3 resulted negative in ciliated cells except for a layer in the basal portion of the epithelium, just above the basal membrane. The actual prevalence of ciliated epithelia in the urinary tract is not well documented and the current knowledge on the subject is limited to electron scanning microscopy studies. The significance of this phenomenon remains unknown: it could be either a developmental abnormality or more probably a metaplastic change. Associated urolithiasis, which has been described in both a previous report and in the present one, could hypothetically represent a possible trigger for this unusual cell change. However, this hypothesis needs to be confirmed through further investigation.

Alterations of the Primary Cilia Gene SPAG17 and SOX9 Locus Noncoding RNAs Identified by RNA-Sequencing Analysis in Patients With Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is characterized by immune activation, vasculopathy, and unresolving fibrosis in the skin, lungs, and other organs. We performed RNA-sequencing analysis on skin biopsy samples and peripheral blood mononuclear cells (PBMCs) from SSc patients and unaffected controls to better understand the pathogenesis of SSc. METHODS: We analyzed these data 1) to test for case/control differences and 2) to identify genes whose expression levels correlate with SSc severity as measured by local skin score, modified Rodnan skin thickness score (MRSS), forced vital capacity (FVC), or diffusing capacity for carbon monoxide (DLco). RESULTS: We found that PBMCs from SSc patients showed a strong type I interferon signature. This signal was found to be replicated in the skin, with additional signals for increased extracellular matrix (ECM) genes, classical complement pathway activation, and the presence of B cells. Notably, we observed a marked decrease in the expression of SPAG17, a cilia component, in SSc skin. We identified genes that correlated with the MRSS, DLco, and FVC in SSc PBMCs and skin using weighted gene coexpression network analysis. These genes were largely distinct from the case/control differentially expressed genes. In PBMCs, type I interferon signatures negatively correlated with the DLco. In SSc skin, ECM gene expression positively correlated with the MRSS. Network analysis of SSc skin genes that correlated with clinical features identified the noncoding RNAs SOX9-AS1 and ROCR, both near the SOX9 locus, as highly connected, "hub-like" genes in the network. CONCLUSION: These results identify noncoding RNAs and SPAG17 as novel factors potentially implicated in the pathogenesis of SSc.

Motile Ciliary Disorders of the Nasal Epithelium in Adults With Bronchiectasis.

BACKGROUND: Motile ciliary disorder (MCD) has been implicated in chronic inflammatory airway diseases such as asthma and COPD. RESEARCH QUESTION: What are the characteristics of MCD of the nasal epithelium and its association with disease severity and inflammatory endotypes in adults with bronchiectasis? STUDY DESIGN AND METHODS: In this observational study, we recruited 167 patients with bronchiectasis and 39 healthy control participants who underwent brushing of the nasal epithelium. A subgroup of patients underwent bronchoscopy for bronchial epithelium sampling (n = 13), elective surgery for bronchial epithelium biopsy (n = 18), and blood sampling for next-generation sequencing (n = 37). We characterized systemic and airway inflammatory endotypes in bronchiectasis. We conducted immunofluorescence assays to profile ultrastructural (dynein axonemal heavy chain 5 [DNAH5], dynein intermediate chain 1 [DNAI1], radial spoke head protein 9 [RSPH9]) and ciliogenesis marker expression (Ezrin). RESULTS: MCD was present in 89.8% of patients with bronchiectasis, 67.6% showed secondary MCD, and 16.2% showed primary plus secondary MCD. Compared with healthy control participants, patients with bronchiectasis yielded abnormal staining patterns of DNAH5, DNAI1, and RSPH9 (but not Ezrin) that were more prominent in moderate to severe bronchiectasis. MCD pattern scores largely were consistent between upper and lower airways and between large-to-medium and small airways in bronchiectasis. Coexisting nasal diseases and asthma did not confound nasal ciliary ultrastructural marker expression significantly. The propensity of MCD was unaffected by the airway or systemic inflammatory endotypes. MCD, particularly an ultrastructural abnormality, was notable in patients with mild bronchiectasis who showed blood or sputum eosinophilia. INTERPRETATION: Nasal ciliary markers profiling provides complimentary information to clinical endotyping of bronchiectasis.

Ciliated Hepatic Foregut Cysts: Not as Rare as Previously Believed.

Background. Ciliated hepatic foregut cysts (CHFCs) are uncommon cystic lesions within the liver. CHFCs can undergo a malignant transformation to form a primary squamous cell carcinoma of the liver. The true incidence and natural history of CHFCs is unknown and the risk of malignant transformation is unclear. We present a single centre's experience of CFHC management. Methods. A retrospective review of a departmental database identified all patients with CHFCs over a 4 year time period. Patients with CHFCs confirmed on histological assessment or suspected on radiological imaging were included in this study. Clinical information regarding patient demographics, symptomatic presentation, surgical management and histopathological features were noted. The radiological characteristics of CHFCs were recorded and the malignant transformation rate was calculated. Results. 15 patients with CHFC were identified (7 histologically confirmed and 8 radiologically suspected cases). All patients were asymptomatic and the CHFCs were incidental findings. No CHFC developed an interval change in cyst features or underwent a malignant transformation during follow up. MRI serves as the most sensitive modality to diagnose CHFC. Conclusions. CHFCs may be more prevalent than previously reported. Definitive management should encompass a patient centred discussion regarding the merits of long term follow up with serial imaging versus resection on an individual basis once CHFC is diagnosed.

Activation of Hedgehog signaling by the oncogenic RELA fusion reveals a primary cilia-dependent vulnerability in supratentorial ependymoma.

BACKGROUND: Supratentorial RELA fusion (ST-RELA) ependymomas (EPNs) are resistant tumors without an approved chemotherapeutic treatment. Unfortunately, the molecular mechanisms that lead to chemoresistance traits of ST-RELA remain elusive. The aim of this study was to assess RELA fusion-dependent signaling modules, specifically the role of the Hedgehog (Hh) pathway as a novel targetable vulnerability in ST-RELA. METHODS: Gene expression was analyzed in EPN from patient cohorts, by microarray, RNA-seq, qRT-PCR, and scRNA-seq. Inhibitors against Smoothened (SMO) (Sonidegib) and Aurora kinase A (AURKA) (Alisertib) were evaluated. Protein expression, primary cilia formation, and drug effects were assessed by immunoblot, immunofluorescence, and immunohistochemistry. RESULTS: Hh components were selectively overexpressed in EPNs induced by the RELA fusion. Single-cell analysis showed that the Hh signature was primarily confined to undifferentiated, stem-like cell subpopulations. Sonidegib exhibited potent growth-inhibitory effects on ST-RELA cells, suggesting a key role in active Hh signaling; importantly, the effect of Sonidegib was reversed by primary cilia loss. We, thus, tested the effect of AURKA inhibition by Alisertib, to induce cilia stabilization/reassembly. Strikingly, Alisertib rescued ciliogenesis and synergized with Sonidegib in killing ST-RELA cells. Using a xenograft model, we show that cilia loss is a mechanism for acquiring resistance to the inhibitory effect of Sonidegib. However, Alisertib fails to rescue cilia and highlights the need for other strategies to promote cilia reassembly, for treating ST-RELA tumors. CONCLUSION: Our study reveals a crucial role for the Hh pathway in ST-RELA tumor growth, and suggests that rescue of primary cilia represents a vulnerability of the ST-RELA EPNs.

Non-contact optical in-vivo sensing of cilia motion by analyzing speckle patterns.

Cilia motion is an indicator of pathological-ciliary function, however current diagnosis relies on biopsies. In this paper, we propose an innovative approach for sensing cilia motility. We present an endoscopic configuration for measuring the motion frequency of cilia in the nasal cavity. The technique is based on temporal tracking of the reflected spatial distribution of defocused speckle patterns while illuminating the cilia with a laser. The setup splits the optical signal into two channels; One imaging channel is for the visualization of the physician and another is, defocusing channel, to capture the speckles. We present in-vivo measurements from healthy subjects undergoing endoscopic examination. We found an average motion frequency of around 7.3 Hz and 9.8 Hz in the antero-posterior nasal mucus (an area rich in cilia), which matches the normal cilia range of 7-16 Hz. Quantitative and precise measurements of cilia vibration will optimize the diagnosis and treatment of pathological-ciliary function. This method is simple, minimally invasive, inexpensive, and promising to distinguish between normal and ciliary dysfunction.

Evaluation of ciliary functions and ciliary beat frequency via cell culture method in patients with primary ciliary dyskinesia.

BACKGROUND: Cell culture increases both diagnostic specificity and sensitivity of primary ciliary dyskinesia (PCD) and the most important reason to use cell culture for definitive diagnosis in PCD is to exclude secondary ciliary defects. Here we aimed to evaluate the cilia functions and cilia ultrastructural abnormalities after ciliogenesis of cell culture in patients with definitive diagnosis of PCD. We also aimed to compare high speed videomicroscopy (HSVM) results of patients before and after ciliogenesis and to compare them with electron microscopy, genetic and immunofluorescence results in patients with positive diagnosis of PCD. METHODS: This study was conducted as a cross-sectional study in patients with PCD. HSVM, transmission electron microscopy (TEM) and immunofluorescence staining results of the nasal biopsy samples taken from patients with the definitive diagnosis of PCD were evaluated and HSVM findings before and after cell culture were described. RESULTS: Ciliogenesis and regrowth in the cell culture occurred in the nasal biopsy sample of eight patients with PCD. The mean age of the patients was 15.5±4.2 years (8.5-18 years). Mean beat frequency was found to be 7.54±1.01 hz (6.53-9.45 hz) before cell culture, and 7.36±0.86 hz (6.02-7.99 hz) after cell culture in the nasal biopsy of patients. There was no significant difference in the beat frequency of PCD patients before and after cell culture. Ciliary function analysis showed the similar beating pattern before and after cell culture in patients with PCD. CONCLUSIONS: This study showed us that there was no difference between cilia beat frequency and beat pattern before and after cell culture in patients with definitive diagnosis of PCD and repeated HSVM would be a useful diagnostic approach in patients who have no possibility to reach other diagnostic methods.