Cilia play essential roles in normal human development and health; cilia dysfunction results in diseases such as primary ciliary dyskinesia (PCD). Despite their importance, the native structure of ...human cilia is unknown, and structural defects in the cilia of patients are often undetectable or remain elusive because of heterogeneity. Here we develop an approach that enables visualization of human (patient) cilia at high-resolution using cryo-electron tomography of samples obtained noninvasively by nasal scrape biopsy. We present the native 3D structures of normal and PCD-causing RSPH1-mutant human respiratory cilia in unprecedented detail; this allows comparisons of cilia structure across evolutionarily distant species and reveals the previously unknown primary defect and the heterogeneous secondary defects in RSPH1-mutant cilia. Our data provide evidence for structural and functional heterogeneity in radial spokes, suggest a mechanism for the milder RSPH1 PCD phenotype and demonstrate that cryo-electron tomography can be applied to human disease by directly imaging patient samples.
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia, but the genetic cause is not defined for all patients with PCD.
To identify disease-causing ...mutations in novel genes, we performed exome sequencing, follow-up characterization, mutation scanning, and genotype-phenotype studies in patients with PCD.
Whole-exome sequencing was performed using NimbleGen capture and Illumina HiSeq sequencing. Sanger-based sequencing was used for mutation scanning, validation, and segregation analysis.
We performed exome sequencing on an affected sib-pair with normal ultrastructure in more than 85% of cilia. A homozygous splice-site mutation was detected in RSPH1 in both siblings; parents were carriers. Screening RSPH1 in 413 unrelated probands, including 325 with PCD and 88 with idiopathic bronchiectasis, revealed biallelic loss-of-function mutations in nine additional probands. Five affected siblings of probands in RSPH1 families harbored the familial mutations. The 16 individuals with RSPH1 mutations had some features of PCD; however, nasal nitric oxide levels were higher than in patients with PCD with other gene mutations (98.3 vs. 20.7 nl/min; P < 0.0003). Additionally, individuals with RSPH1 mutations had a lower prevalence (8 of 16) of neonatal respiratory distress, and later onset of daily wet cough than typical for PCD, and better lung function (FEV1), compared with 75 age- and sex-matched PCD cases (73.0 vs. 61.8, FEV1 % predicted; P = 0.043). Cilia from individuals with RSPH1 mutations had normal beat frequency (6.1 ± Hz at 25°C), but an abnormal, circular beat pattern.
The milder clinical disease and higher nasal nitric oxide in individuals with biallelic mutations in RSPH1 provides evidence of a unique genotype-phenotype relationship in PCD, and suggests that mutations in RSPH1 may be associated with residual ciliary function.
Primary ciliary dyskinesia (PCD) is a rare lung disease caused by mutations that impair the function of motile cilia, resulting in chronic upper and lower respiratory disease, reduced fertility, and ...a high prevalence of situs abnormalities. The disease is genetically and phenotypically heterogeneous, with causative mutations in > 50 genes identified, and clinical phenotypes ranging from mild to severe. Absence of
(
), a component of the outer dynein arm docking complex, results in a failure to assemble outer dynein arms (ODAs), mostly immotile cilia, and a typical PCD phenotype. We identified a female (now 34 years old) with an unusually mild clinical phenotype who has a homozygous non-canonical splice mutation (c.1502+5G>A) in
. To investigate the mechanism for the unusual phenotype, we performed molecular and functional studies of cultured nasal epithelial cells. We demonstrate that this splice mutation results in the expression of a truncated protein that is attached to the axoneme, indicating that the mutant protein retains partial function. This allows for the assembly of some ODAs and a significant level of ciliary activity that may result in the atypically mild clinical phenotype. The results also suggest that partial restoration of ciliary function by therapeutic agents could lead to significant improvement of disease symptoms.
Highly asymmetric yttria stabilized zirconia (YSZ) hollow fibre membranes were fabricated by a modified phase inversion and sintering technique using a mixture of NMP and water as internal coagulant. ...The obtained YSZ hollow fibres possess a thin dense outer skin layer of thickness 3–5
μm and a thicker porous sub-layer with a surface porosity of 28.8%. The crystal phase of the YSZ hollow fibres retains the same cubic fluorite structure as the original YSZ powders after the spinning and sintering process. The thin dense skin layer is integrated perfectly with the porous layer and is especially suitable for use as an electrolyte film in micro-tubular solid oxide fuel cells (MT-SOFCs).
Cationic cell-penetrating peptides (CPPs), such as transactivator of transcription (TAT) peptide, have been proposed as effective drug carriers to improve intracellular delivery of biological ...macromolecules. Amphibian skin-derived Kunitz-type trypsin inhibitors (KTIs), short counterparts of KTIs from plant sources, were found to possess potent serine protease inhibitory activity. However, poor transmembrane permeability of these molecules has largely hindered the study of the full spectrum of their biological actions. As a result, this study aimed to extend the biological activities of amphibian KTIs by their conjugation to cationic CPPs. Herein, a novel peptide (kunitzin-OV2) and its phenylalanine-substituted analogue F9-kunitzin-OV2 (F9-KOV2) were evaluated for inhibition of trypsin/chymotrypsin and showed weak antibacterial activity against Escherichia coli (E. coli). As expected, the conjugation to TAT peptide did not increase membrane lysis compared with the original kunitzin-OV2, but effectively assisted this complex to enter cells. TAT-kunitzin-OV2 (TAT-KOV2) exhibited a 32-fold increase in antibacterial activity and an enhanced bactericidal rate against E. coli. In addition, the conjugation enabled the parent peptides to exhibit antiproliferative activity against cancer cells. Interestingly, TAT-F9-kunitzin-OV2 (TAT-F9-KOV2) showed stronger antiproliferative activity against human breast cancer (MCF-7) and human glioblastoma (U251MG) cell lines, which TAT-KOV2 did not possess. Moreover, TAT-F9-KOV2 showed a 20–25-fold increase in antiproliferative capacity against human lung cancer (H157, H460) cell lines compared with TAT-KOV2. Therefore, the conjugation of CPPs effectively solves the problem of cell penetration that short KTIs lack and provides evidence for new potential applications for their subsequent development as new antibacterial and anticancer agents.
Studies of primary ciliary dyskinesia (PCD) have been hampered by the lack of a suitable animal model because disruption of essential ciliary genes in mice results in a high incidence of lethal ...hydrocephalus. To develop a viable mouse model for long-term studies of PCD, we have generated a transgenic mouse line in which two conserved exons of the mouse intermediate dynein chain gene, Dnaic1, are flanked by loxP sites (Dnaic1(flox/flox)). Dnaic1 is the murine homolog of human DNAI1, which is mutated in approximately 10% of human PCD cases. These mice have been crossed with mice expressing a tamoxifen-inducible Cre recombinase (CreER). Treatment of adult Dnaic1(flox/flox)/CreER(+/-) mice with tamoxifen results in an almost complete deletion of Dnaic1 with no evidence of hydrocephalus. Treated animals have reduced levels of full-length Dnaic1 mRNA, and electron micrographs of cilia demonstrate a loss of outer dynein arm structures. In treated Dnaic1(flox/flox)/CreER(+/-) animals, mucociliary clearance (MCC) was reduced over time. After approximately 3 months, no MCC was observed in the nasopharynx, whereas in the trachea, MCC was observed for up to 6 months, likely reflecting a difference in the turnover of ciliated cells in these tissues. All treated animals developed severe rhinosinusitis, demonstrating the importance of MCC to the health of the upper airways. However, no evidence of lung disease was observed up to 11 months after Dnaic1 deletion, suggesting that other mechanisms are able to compensate for the lack of MCC in the lower airways of mice. This model will be useful for the study of the pathogenesis and treatment of PCD.
Genetic defects in motile cilia cause primary ciliary dyskinesia (PCD), a rare disease with no specific therapeutics. Individuals with PCD often have impaired fertility and laterality defects and ...universally suffer from upper and lower airway diseases. Chronic rhinosinusitis is a universal feature of PCD, and mucus accumulation and subsequent infections of the sinonasal cavity cause significant morbidity in individuals with PCD. Despite this, there are no approved treatments that specifically target mucus.
The goals of this study were to determine whether computed tomography (CT) imaging could be used to quantify mucus accumulation and whether the use of a mucolytic agent to reduce disulfide cross-links present in mucins would improve the effectiveness of nasal lavage at removing mucus in a murine model of PCD.
Adult mice with a deletion of the axonemal dynein Dnaic1 were imaged using CT scanning to characterize mucus accumulation. The animals were then treated by nasal lavage with saline, with/without the disulfide-reducing agent tris(2-carboxyethyl)phosphine. Post-treatment CT scans were used to quantify improvement in the sinonasal cavity.
Mucus accumulation in the nasal cavity was readily quantified by CT. Compared to sham-treated control animals, nasal lavage with/without a mucolytic agent resulted in a significant reduction of accumulated mucus (
< 0.01). Treatment with the mucolytic agent showed a greater reduction of accumulated mucus than treatment with saline alone.
The results suggest that inclusion of a mucolytic agent may increase the effectiveness of nasal lavage at reducing mucus burden in PCD.
We reported a 32-year-old male with a sellar solitary fibrous tumor who presented with headache and affliction in the left eye. Serum biochemical examination revealed hypoglycemia. The tumor was ...assumed to be a nonfunctional pituitary adenoma preoperatively. A subtotal resection of the tumor was performed. Immunohistochemically, atypical solitary fibrous tumor was established. The residual tumor had no progression or distant metastasis at a 44-month follow-up after gamma-knife stereoradiotherapy.
Cilia are essential to many diverse cellular processes. Although many major axonemal components have been identified and studied, how they interact to form a functional axoneme is not completely ...understood. To further our understanding of the protein composition of human airway cilia, we performed a semiquantitative analysis of ciliary axonemes using label-free LC/MSE, which identified over 400 proteins with high confidence. Tubulins were the most abundant proteins identified, with evidence of 20 different isoforms obtained. Twelve different isoforms of axonemal dynein heavy chain were also identified. Absolute quantification of the nontubulin components demonstrated a greater than 75-fold range of protein abundance (RSPH9;1850 fmol vs CCDC103;24 fmol), adding another level of complexity to axonemal structure. Of the identified proteins, ∼70% are known axonemal proteins. In addition, many previously uncharacterized proteins were identified. Unexpectedly, several of these, including ERICH3, C1orf87, and CCDC181, were present at high relative abundance in the cilia. RT-PCR analysis and immunoblotting confirmed cilia-specific expression for eight uncharacterized proteins, and fluorescence microscopy demonstrated unique axonemal localizations. These studies have provided the first quantitative analysis of the ciliary proteome and have identified and characterized several previously unknown proteins as major constituents of human airway cilia.
Respiratory infections are a major cause of morbidity and mortality in the elderly. Previous reports have suggested that mucociliary clearance (MCC) is impaired in older individuals, but the cause is ...unclear. To unravel the mechanisms responsible for the age-associated decline in MCC, we investigated the MCC system in young (3 mo) and old (2 yr) C57BL/6 mice. We found that old mice had significantly reduced MCC function in both the upper and lower airways compared with young mice. Measurement of bioelectric properties of isolated tracheal and bronchial tissue revealed a significant decrease in Cl(-) secretion, suggesting that the older mice may have a reduced ability to maintain a sufficiently hydrated airway surface for efficient MCC. Ciliary beat frequency was also observed to be reduced in the older animals; however, this reduction was small relative to the reduction in MCC. Interestingly, the level of the major secreted mucin, Muc5b, was found to be reduced in both bronchioalveolar lavage and isolated tracheal tissue. Our previous studies of Muc5b(-/-) mice have demonstrated that Muc5b is essential for normal MCC in the mouse. Furthermore, examination of Muc5b(+/-) and wild-type animals revealed that heterozygous animals, which secrete ∼50% of the wild-type level of Muc5b, also demonstrate a markedly reduced level of MCC, confirming the importance of Muc5b levels to MCC. These results demonstrate that aged mice exhibit a decrease in MCC and suggest that a reduced level of secretion of both Cl(-) and Muc5b may be responsible.
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