Motile Cilia Research Articles

Fisiologi Mukosiliar Bronkus

One of the defense systems possessed by the respiratory tract is mucociliary transport. In order for this system to work properly, coordination of 3 components is required, namely: the motile cila, the periciliar layer (PCL), and the mucus layer. The mucus layer will catch foreign particles that enter the respiratory tract. The motile cilia will push the mucus to keep moving towards the proximal airway until it finally reaches the oropharynx and is swallowed. Cilia movement is certainly influenced by PCL which is a medium for cilia to move. In some lung diseases, this component can be impaired so that the mucociliary transport process does not work effectively.

Sperm long non-coding RNAs as markers for ram fertility.

It is critical in sheep farming to accurately estimate ram fertility for maintaining reproductive effectiveness and for production profitability. However, there is currently a lack of reliable biomarkers to estimate semen quality and ram fertility, which is hindering advances in animal science and technology. The objective of this study was to uncover long non-coding RNAs (lncRNAs) in sperm from rams with distinct fertility phenotypes. Mature rams were allocated into two groups: high and low fertility (HF; n = 31; 94.5 ± 2.8%, LF; n = 25; 83.1 ± 5.73%; P = 0.028) according to the pregnancy rates sired by the rams (average pregnancy rate; 89.4 ± 7.2%). Total RNAs were isolated from sperm of the highest- and lowest-fertility rams (n = 4, pregnancy rate; 99.2 ± 1.6%, and 73.6 ± 4.4%, respectively) followed by next-generation sequencing of the transcripts. We uncovered 11,209 lncRNAs from the sperm of rams with HF and LF. In comparison to each other, there were 93 differentially expressed (DE) lncRNAs in sperm from the two distinct fertility phenotypes. Of these, 141 mRNAs were upregulated and 134 were downregulated between HF and LF, respectively. Genes commonly enriched for 9 + 2 motile cilium and sperm flagellum were ABHD2, AK1, CABS1, ROPN1, SEPTIN2, SLIRP, and TEKT3. Moreover, CABS1, CCDC39, CFAP97D1, ROPN1, SLIRP, TEKT3, and TTC12 were commonly enriched in flagellated sperm motility and sperm motility. Differentially expressed mRNAs were enriched in the top 16 KEGG pathways. Targets of the differentially expressed lncRNAs elucidate functions in cis and trans manner using the genetic context of the lncRNA locus, and lncRNA sequences revealed 471 mRNAs targets of 10 lncRNAs. This study illustrates the existence of potential lncRNA biomarkers that can be implemented in analyzing the quality of ram sperm and determining the sperm fertility and is used in breeding soundness exams for precision livestock farming to ensure food security on a global scale.

Loss of Katnal2 leads to ependymal ciliary hyperfunction and autism-related phenotypes in mice.

Autism spectrum disorders (ASD) frequently accompany macrocephaly, which often involves hydrocephalic enlargement of brain ventricles. Katnal2 is a microtubule-regulatory protein strongly linked to ASD, but it remains unclear whether Katnal2 knockout (KO) in mice leads to microtubule- and ASD-related molecular, synaptic, brain, and behavioral phenotypes. We found that Katnal2-KO mice display ASD-like social communication deficits and age-dependent progressive ventricular enlargements. The latter involves increased length and beating frequency of motile cilia on ependymal cells lining ventricles. Katnal2-KO hippocampal neurons surrounded by enlarged lateral ventricles show progressive synaptic deficits that correlate with ASD-like transcriptomic changes involving synaptic gene down-regulation. Importantly, early postnatal Katnal2 re-expression prevents ciliary, ventricular, and behavioral phenotypes in Katnal2-KO adults, suggesting a causal relationship and a potential treatment. Therefore, Katnal2 negatively regulates ependymal ciliary function and its deletion in mice leads to ependymal ciliary hyperfunction and hydrocephalus accompanying ASD-related behavioral, synaptic, and transcriptomic changes.

Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare, genetic disease characterized by dysfunctional motile cilia and abnormal mucociliary clearance, resulting in chronic sino-oto-pulmonary disease, neonatal respiratory distress, subfertility, and organ laterality defects. Over the past 2 decades, research and international collaborations have led to an improved understanding of disease prevalence, classic and variable phenotypes, novel diagnostics, genotype-phenotype correlations, long term morbidity, and innovative therapeutics. However, PCD is often underrecognized in clinical settings and the recent analyses of genetic databases suggest that only a fraction of these patients are being accurately diagnosed. Knowledge of significant advancements, from pathophysiology to the expanded range of clinical manifestations, will have important clinical impacts. These may include increasing disease recognition, improving diagnostic testing and management, and establishing an adequate pool of affected patients to enroll in upcoming clinical therapeutic trials. The objective of this state-of-the-art review is for readers to gain a greater understanding of the clinical spectrum of motile ciliopathies, cutting-edge diagnostic practices, emerging genotype-phenotype associations, and currently accepted management of people with PCD.

The methylome of motile cilia.

Cilia are highly complex motile, sensory, and secretory organelles that contain perhaps 1000 or more distinct protein components, many of which are subject to various posttranslational modifications such as phosphorylation, N-terminal acetylation, and proteolytic processing. Another common modification is the addition of one or more methyl groups to the side chains of arginine and lysine residues. These tunable additions delocalize the side-chain charge, decrease hydrogen bond capacity, and increase both bulk and hydrophobicity. Methylation is usually mediated by S-adenosylmethionine (SAM)-dependent methyltransferases and reversed by demethylases. Previous studies have identified several ciliary proteins that are subject to methylation including axonemal dynein heavy chains that are modified by a cytosolic methyltransferase. Here, we have performed an extensive proteomic analysis of multiple independently derived cilia samples to assess the potential for SAM metabolism and the extent of methylation in these organelles. We find that cilia contain all the enzymes needed for generation of the SAM methyl donor and recycling of the S-adenosylhomocysteine and tetrahydrofolate byproducts. In addition, we find that at least 155 distinct ciliary proteins are methylated, in some cases at multiple sites. These data provide a comprehensive resource for studying the consequences of methyl marks on ciliary biology.

Different recovery patterns of the surviving bivalve Mytilus galloprovincialis based on transcriptome profiling exposed to spherical or fibrous polyethylene microplastics

Microplastics (MPs) are pervasive pollutants exuded from anthropogenic activities and ingested by animals in different ecosystems. This transcriptomic profiling study aimed to explore the impact of polyethylene MPs on Mytilus galloprovincialis, an ecologically significant bivalve species. The toxicity of two MPs types was found to result in increased cellular stress when exposed up to 14 days. Moreover, recovery mechanisms were also observed in progress. Mussels exhibited different gene expression patterns and molecular regulation in response to cellular reactive oxygen species (ROS) stress. The transcriptome analysis demonstrated a notable hindrance in cilia movement as MPs ingested through gills. Subsequent entry resulted in a significant disruption in the cytoskeletal organization, cellular projection, and cilia beat frequency. On day 4 (D4), signal transduction and activation of apoptosis evidenced the signs of toxic consequences. Mussels exposed to spherical MPs shown significant recovery on day 14 (D14), characterized by the upregulation of anti-apoptotic genes and antioxidant genes. The expression of P53 and BCL2 genes was pivotal in controlling the apoptotic process and promoting cell survival. Mussels exposed to fibrous MPs displayed a delayed cell survival effect. However, the elevated physiological stress due to fibrous MPs resulted in energy transfer by compensatory regulation of metabolic processes to expedite cellular recovery. These observations highlighted the intricate and varied reaction of cell survival mechanisms in mussels to recover toxicity. This study provides critical evidence of the ecotoxicological impacts of two different MPs and emphasizes the environmental risks they pose to aquatic ecosystems. Our conclusion highlights the detrimental effects of MPs on M. galloprovincialis and the need for more stringent regulations to protect marine ecosystems.

Sea Anemone-Inspired Slippery Liquid-Infused Porous Surface (SLIPS) with Bionic Cilia for Responsive 4D Antifouling.

The formation process of biofouling is actually a 4D process with both spatial and temporal dimensions. However, most traditional antifouling coatings, including slippery liquid-infused porous surface (SLIPS), are limited to performing antifouling process in the 2D coating plane. Herein, inspired by the defensive behavior of sea anemones' wielding toxic tentacles, a "4D SLIPS" (FSLIPS) is constructed with biomimetic cilia via a magnetic field self-assembly method for antifouling. The bionic cilia move in 3D space driven by an external magnetic field, thereby preventing the attachment of microorganisms. The FSLIPS releases the gaseous antifoulant (nitric oxide) at 1D time in response to light, thereby achieving a controllable biocide effect on microorganisms. The FSLIPS regulates the movement of cilia via the external magnetic field, and controls the release of NO overtime via the light response, so as to adjust the antifouling modes on demand during the day or night. The light/magnetic response mechanism endow the FSLIPS with the ability to adjust the antifouling effect in the 4D dimension of 1D time and 3D space, effectively realizing the intelligence, multi-dimensionality and precision of the antifouling process.

In vitro platform to model the function of ionocytes in the human airway epithelium

BackgroundPulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium.MethodshiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties.ResultsMature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells.ConclusionOur results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.

Theoretical analysis of Ellis fluid flow in two layers due to metachronal propulsion subject to heat and mass transfer: Application in biological function

This study uses a metaphor to describe the motion of cilia with heat and mass transfer, and it highlights the importance of research for understanding biological function. This research article depicts analysis of double-layer of cilia induced flow for non-Newtonian (Ellis fluid) with additional effects of thermal and concentration fields. The Ellis fluid model is considering as mucus present in Human bronchial airways. Respiratory tract and bronchial airways consisted of ciliated epithelium which is further divided into two layers, Airway Ciliary Layer (ACL) and Peri Ciliary liquid Layer (PCL). Due to which double-layer model has been constructed in this investigation. Mathematical modeling of double-layer flow problem has been simplified using long-wavelength and small-Reynolds number approximation. Consequent governing equations with boundary conditions gives an exact/accurate result for velocity field, temperature distribution and concentration distribution in double-layers. The influence of emerging parameters on velocity profile, temperature distribution, concentration field was illuminated by graphs and discussed comprehensively. Contour plots have also been plotted to visualize flow pattern in both layers. Evidenced graphs show that velocity profile and temperature profile are the highest in inner layer of fluid (PCL) and concentration profile is the highest at outer layers of fluid (ACL).

Estimates of primary ciliary dyskinesia prevalence: a scoping review.

Primary ciliary dyskinesia (PCD) is a rare multisystem genetic disease caused by dysfunctional motile cilia. Despite PCD being the second most common inherited airway disease after cystic fibrosis, PCD continues to be under-recognised globally owing to nonspecific clinical features and the lack of a gold standard diagnostic test. Commonly repeated prevalence estimates range from one in 10 000 to one in 20 000, based on regional epidemiological studies with known limitations. The purpose of this scoping review was to appraise the PCD literature, to determine the best available global PCD prevalence estimate and to inform the reader about the potential unmet health service needs in PCD. The primary objective of the present study was to systematically review the literature about PCD prevalence estimates. A scoping review was conducted following the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for scoping reviews (PRISMA-ScR) methodology. Included studies estimated PCD prevalence and used cohort, clinical or genomic data. Case reports, conference abstracts, review articles, animal studies or non-English articles were excluded. A literature review identified 3484 unique abstracts; 34 underwent full-text review and eight met the inclusion/exclusion criteria. Seven articles were based on epidemiological studies of specific geographical regions and provided prevalence estimates that ranged from approximately one to 44.1 in 100 000. Only one study estimated global prevalence, using two large genomic databases, and calculated it to be ∼13.2 in 100 000 (based on pathogenic variants in 29 disease-causing genes). A population-based genomic approach for estimating global prevalence has found that PCD is much more prevalent than previously cited in the literature. This highlights the potential unmet health service needs of people living with PCD.

Planar Cell Polarity in the Multiciliated Epithelial Lining of the Mouse Eustachian Tube.

Planar cell polarity (PCP) signaling, essential for uniform alignment and directional beating of motile cilia, has been investigated in multiciliated epithelia. As a complex structure connecting the middle ear to the nasopharynx, the eustachian tube (ET) is important in the onset of ear-nose-throat diseases. However, PCP signaling, including the orientation that is important for ciliary motility and clearance function in the ET, has not been studied. We evaluated PCP in the ET epithelium. Morphometric examination of the mouse ET. We performed electron microscopy to assess ciliary polarity in the mouse ET, along with immunohistochemical analysis of PCP protein localization in the ET epithelium. We discovered PCP in the ET epithelium. Motile cilia were aligned in the same direction in individual and neighboring cells; this alignment manifested as ciliary polarity in multiciliated cells. Additionally, PCP proteins were asymmetrically localized between adjacent cells in the plane of the ET. The multiciliated ET epithelium exhibits polarization, suggesting novel structural features that may be critical for ET function. NA Laryngoscope, 134:3795-3801, 2024.

Combined approaches, including long-read sequencing, address the diagnostic challenge of HYDIN in primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD), a disorder of the motile cilia, is now recognised as an underdiagnosed cause of bronchiectasis. Accurate PCD diagnosis comprises clinical assessment, analysis of cilia and the identification of biallelic variants in one of 50 known PCD-related genes, including HYDIN. HYDIN-related PCD is underdiagnosed due to the presence of a pseudogene, HYDIN2, with 98% sequence homology to HYDIN. This presents a significant challenge for Short-Read Next Generation Sequencing (SR-NGS) and analysis, and many diagnostic PCD gene panels do not include HYDIN. We have used a combined approach of SR-NGS with bioinformatic masking of HYDIN2, and state-of-the-art long-read Nanopore sequencing (LR_NGS), together with analysis of respiratory cilia including transmission electron microscopy and immunofluorescence to address the underdiagnosis of HYDIN as a cause of PCD. Bioinformatic masking of HYDIN2 after SR-NGS facilitated the detection of biallelic HYDIN variants in 15 of 437 families, but compromised the detection of copy number variants. Supplementing testing with LR-NGS detected HYDIN deletions in 2 families, where SR-NGS had detected a single heterozygous HYDIN variant. LR-NGS was also able to confirm true homozygosity in 2 families when parental testing was not possible. Utilising a combined genomic diagnostic approach, biallelic HYDIN variants were detected in 17 families from 242 genetically confirmed PCD cases, comprising 7% of our PCD cohort. This represents the largest reported HYDIN cohort to date and highlights previous underdiagnosis of HYDIN-associated PCD. Moreover this provides further evidence for the utility of LR-NGS in diagnostic testing, particularly for regions of high genomic complexity.

Thermal dynamics assessment for multi-phase flow analysis with motile cilia and electric double layer effects: Application of Levenberg–Marquardt backpropagation NNs

Ciliary movement holds substantial importance in the realm of biomedical research. Disorders arising from impaired ciliary motion, such as primary ciliary dyskinesia and specific respiratory ailments, underscore the critical need to investigate both the normal functioning and dysfunction of cilia. Such research is fundamental to the development of effective treatments and therapies. This work explores the application of Levenberg–Marquardt backpropagation neural networks (LMBP-NNs) for simulating the flow of a non-Newtonian Jeffery fluid in peristaltic motion. Inspired by synchronous ciliary movement, microfluidic systems mimicking cilia functionality have been developed. These artificial cilia can generate controlled fluid flow patterns, enabling various applications such as drug delivery, mixing, and particle sorting in lab-on-a-chip devices. The present study focuses on measuring thermal and mass transfer in beating cilia during multiphase peristaltic pumping of Jeffery fluid in two- and three-dimensional channels using transverse magnetohydrodynamics in a porous medium. The governing equations for the non-Newtonian Jeffery fluid in both the fluid and dusty phases are formulated, and exact solutions are calculated for fluid phase velocity, particulate phase velocity, temperature, concentration, and pressure gradient. The physical behavior is investigated by graphing dimensionless parameters for velocity, temperature, and concentration profiles in both solid and liquid phases. The trapping phenomena is depicted by showing streamlines of various dimensionless characteristics such as porosity velocity and volume percentage. The LMBP-NNs approach is then used to train, test, and verify the neural network models using the reference datasets. The accuracy of the LMBP-NN is assessed using statistical data such as mean square error, curve fitting graphs, regression plots, and error histograms. The study also investigates flow model limits for momentum, temperature, and concentration profiles using visual representations. This study illustrates the computational capability of LMBP-NNs in modeling the peristaltic flow of Jeffery fluid and gives useful insights into fluid flow behavior in microfluidic devices containing artificial cilia. The findings help to better understand and optimize these systems for a variety of fluid manipulation and transportation applications.

Metachronal Motion of Biological and Artificial Cilia.

Cilia are slender, hair-like cell protrusions that are present ubiquitously in the natural world. They perform essential functions, such as generating fluid flow, propulsion, and feeding, in organisms ranging from protozoa to the human body. The coordinated beating of cilia, which results in wavelike motions known as metachrony, has fascinated researchers for decades for its role in functions such as flow generation and mucus transport. Inspired by nature, researchers have explored diverse materials for the fabrication of artificial cilia and developed several methods to mimic the metachronal motion observed in their biological counterparts. In this review, we will introduce the different types of metachronal motion generated by both biological and artificial cilia, the latter including pneumatically, photonically, electrically, and magnetically driven artificial cilia. Furthermore, we review the possible applications of metachronal motion by artificial cilia, focusing on flow generation, transport of mucus, particles, and droplets, and microrobotic locomotion. The overall aim of this review is to offer a comprehensive overview of the metachronal motions exhibited by diverse artificial cilia and the corresponding practical implementations. Additionally, we identify the potential future directions within this field. These insights present an exciting opportunity for further advancements in this domain.

Advancing Primary Ciliary Dyskinesia Diagnosis through High-Speed Video Microscopy Analysis.

Primary ciliary dyskinesia (PCD) is an inherited disorder that impairs motile cilia, essential for respiratory health, with a reported prevalence of 1 in 16,309 within Hispanic populations. Despite 70% of Puerto Rican patients having the RSPH4A [c.921+3_921+6del (intronic)] founder mutation, the characterization of the ciliary dysfunction remains unidentified due to the unavailability of advanced diagnostic modalities like High-Speed Video Microscopy Analysis (HSVA). Our study implemented HSVA for the first time on the island as a tool to better diagnose and characterize the RSPH4A [c.921+3_921+6del (intronic)] founder mutation in Puerto Rican patients. By applying HSVA, we analyzed the ciliary beat frequency (CBF) and pattern (CBP) in native Puerto Rican patients with PCD. Our results showed decreased CBF and a rotational CBP linked to the RSPH4A founder mutation in Puerto Ricans, presenting a novel diagnostic marker that could be implemented as an axillary test into the PCD diagnosis algorithm in Puerto Rico. The integration of HSVA technology in Puerto Rico substantially enhances the PCD evaluation and diagnosis framework, facilitating prompt detection and early intervention for improved disease management. This initiative, demonstrating the potential of HSVA as an adjunctive test within the PCD diagnostic algorithm, could serve as a blueprint for analogous developments throughout Latin America.

Abstract 5622: Uncovering the transcriptional regulation of TPPP3

Abstract Tubulin Polymerization Promoting Protein 3 (TPPP3) is a protein coding gene primarily associated with microtubule bundling. However more recent research is beginning to reveal more diverse roles it may play in the cell, including B-catenin signaling and endothelial injury. TPPP3 has also been associated with increased cancer growth and proliferation as well epithelial to mesenchymal transition. However, it is unknown how TPPP3 is regulated. We used high grade serous ovarian cancer (HGSOC) as a model to uncover the regulation of TPPP3 as it is highly expressed in many HGSOC cases. To start, we downloaded ~500 HGSOC RNA-seq datasets from The Cancer Genome Atlas (TCGA) and, using python, created a co-expression network to identify a subset of genes exhibiting expression patterns correlated with TPPP3 across all datasets, resulting in the identification of 222 genes. Dynein arm assembly, cilia movement and assembly, and microtubule movement and structure were among the top GO-terms after gene ontology analysis. The genes associated with these processes are typically upregulated in the fallopian tube tissue which aligns with the fact that a majority of HGSOCs originate in the fallopian tube. Fork-head box J1 (FOXJ1) is a transcription factor associated with ciliogenesis and was among the top 222 genes from the co-expression network. It has not been confirmed to transcriptionally regulate TPPP3, but their expression patterns line up across many tissue types. In addition, the TPPP3 promoter contains an RFX2 binding site. RFX2 is DNA binding protein that FOXJ1 uses as a scaffold to bind to proximal promoters due to it binding at to DNA at a distal location. As a result of this evidence, we hypothesize that FOXJ1 transcriptionally regulates TPPP3 via RFX2. To test this hypothesis, we inhibited FOXJ1 and identified TPPP3 downregulation. We are further investigating the regulatory function of how TPPP3 is regulated. Citation Format: James W. Lord, Dmitry Leshchiner, Sachi Horibata. Uncovering the transcriptional regulation of TPPP3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5622.

Peristaltic mechanism of Ellis fluid with viscous dissipation and thermal radiation induced by cilia wave

Bioheat transfer analysis in tissue has attracted the attention of numerous researchers due to its widespread potential applications in the medical field, mainly in thermotherapy and the human thermoregulation system. Also, temperature regulation of the human body primarily occurs through bioheat transfer. Due to the widespread biomedical applications of bio-heat transfer, we aim to investigate the movement of biofluid and bioheat in human organs with the influences of thermal radiation and ciliary waves. The mathematical model for Ellis fluid flow through a tube includes the metachronal wave of cilia motion and convective conditions. The governing equations are created based on mass, momentum conservation, and energy. The current problem is displayed and exact solutions are managed under long wavelength [Formula: see text] and low Reynolds number [Formula: see text] approximations. An analytical approach is employed to derive expressions for longitudinal velocity, temperature, pressure gradient, and stream function as a function of the parameters of the problem. The physical behavior of the peristaltic motion of the Ellis fluid is explained in detail and illustrated graphically for various parameter values. The results of the current study provide potential information for advancement in the biomedical industry, particularly in the development of biomedical devices and processes.

Gene dosage of independent dynein arm motor preassembly factors influences cilia assembly in Chlamydomonas reinhardtii.

Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene DNAAF4. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii, a ciliated unicellular alga, the DNAAF4 ortholog is called PF23. The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (pf23-4) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.

Covid-19 in cystic fibrosis patients compared to the general population: Severity and virus-host cell interactions

BackgroundPeople with cystic fibrosis (pwCF) are considered at risk of developing severe forms of respiratory viral infections. We studied the consequences of COVID-19 and virus-host cell interactions in CF vs. non-CF individuals. MethodsWe enrolled CF and non-CF individuals, with /without COVID-like symptoms, who underwent nasopharyngeal swab for detection of SARS-CoV-2. Gene expression was evaluated by RNA sequencing on the same nasopharyngeal swabs. Criteria for COVID-19 severity were hospitalization and requirement or increased need of oxygen therapy. ResultsThe study included 171 patients (65 pwCF and 106 non-CF individuals). Among them, 10 pwCF (15.4 %) and 43 people without CF (40.6 %) tested positive at RT-PCR. Symptomatic infections were observed in 8 pwCF (with 2 requiring hospitalization) and in 11 individuals without CF (6 requiring hospitalization). Host transcriptomic analysis revealed that genes involved in protein translation, particularly ribosomal components, were downregulated in CF samples irrespective of SARS-CoV-2 status. In SARS-CoV-2 negative individuals, we found a significant difference in genes involved with motile cilia expression and function, which were upregulated in CF samples. Pathway enrichment analysis indicated that interferon signaling in response to SARS-CoV-2 infection was upregulated in both pwCF and non-CF subjects. ConclusionsCOVID-19 does not seem to be more severe in CF, possibly due to factors intrinsic to this population: the lower expression of ribosomal genes may downregulate the protein translation machinery, thus creating an unfavorable environment for viral replication.

Skewed X-chromosome inactivation drives the proportion of DNAAF6-defective airway motile cilia and variable expressivity in primary ciliary dyskinesia

BackgroundPrimary ciliary dyskinesia (PCD) is a rare airway disorder caused by defective motile cilia. Only male patients have been reported with pathogenic mutations in X-linked DNAAF6, which result in the...