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Abstract Introduction Eosinophilic otitis media is an intractable otitis media and a fairly common middle ear disease. However, the pathogenesis of eosinophilic otitis media is obscure. Objective To observe the pathological and ultrastructural changes of the Eustachian tube mucosal epithelium in rats with eosinophilic otitis media and further explore the pathogenesis of eosinophilic otitis media. Methods Animals were intraperitoneally injected with 2000 mg ovalbumin and 100 mg aluminum hydroxide (alum) on day 0, followed by 100 mg ovalbumin and 100 mg alum injection on days 7 and 14. Next they were topically boosted by daily application of 100 mg ovalbumin solution via nasal drip and intratympanic injection of 0.1 mL ovalbumin (1000 mg/mL) in the right ear (group A, n = 80) and 0.1 mL saline in the left ear as control (group B, n = 80) starting on day 21 and continuing for 14 days. The temporal bones were dissected on the 35th, 38th, 41st and 43rd day separately under anesthesia. Scanning electron microscopy, hematoxylin-eosin and toluidine blue staining were used to observe the pathological and morphological changes of Eustachian tube mucosa stained samples. Moreover, inflammatory cells and cilia were counted. Results The epithelium of the Eustachian tube in group A was swollen and thickened. The cilia were arranged in a disorderly manner and partially detached. Eosinophils infiltrated the submucosal layer of the Eustachian tube, and their number increased significantly compared with that in group B (p< 0.05). Simultaneously, mast cell degranulation was observed in group A. Scanning electron microscopy revealed that the cilia were lodged and gathered along the whole length of Eustachian tube in group A. Ciliated cell density was significantly lower than that in Group B (p< 0.01). Conclusion In the eosinophilic otitis media model, allergy caused significant changes in pathology and morphology of the Eustachian tube mucosa, affecting the normal function of the Eustachian tube which played an important role in the occurrence and development of eosinophilic otitis media.
Resumo Introdução A otite média eosinofílica é uma doença relativamente comum de orelha média; entretanto, sua patogênese é ainda obscura, assim como o tratamento. Objetivo Observar as alterações histopatológicas e ultraestruturais do epitélio da mucosa da tuba auditiva em ratos com otite média eosinofílica e investigar a sua patogênese. Método Os animais foram injetados intraperitonealmente com 2.000 mg de ovalbumina e 100 mg de hidróxido de alumínio (alúmen) no dia 0, seguido por 100 mg de ovalbumina e 100 mg de injeção de alúmen nos dias 7 e 14. Em seguida, receberam um reforço tópico através de uma aplicação diária de 100 mg da solução por gotejamento nasal e injeção intratimpânica de 0,1 mL de ovalbumina (1000 mg/mL) na orelha direita (grupo A, n = 80) e 0,1 mL de solução salina na orelha esquerda como controle (grupo B, n = 80), começou no dia 21 e continuou por 14 dias. Os ossos temporais foram dissecados nos dias 35, 38, 41 e 43 separadamente sob anestesia. Foram usadas microscopia eletrônica de varredura e coloração com hematoxilina-eosina e azul de toluidina para observar as alterações histopatológicas e morfológicas das amostras coradas de mucosa da tuba auditiva. Além disso, células inflamatórias e cílios foram contados. Resultados O epitélio da tuba auditiva no grupo A estava edematoso e espessado. Os cílios estavam dispostos de forma desordenada e parcialmente destacados. Os eosinófilos infiltraram a camada submucosa da tuba auditiva e seu número aumentou significantemente em comparação ao grupo B (p < 0,05). Simultaneamente, degranulação dos mastócitos foi observado no grupo A. A microscopia eletrônica de varredura mostrou que os cílios estavam depositados e reunidos ao longo de todo o comprimento da tuba auditiva no grupo A. A densidade das células ciliadas foi significantemente menor do que no grupo B (p < 0,01). Conclusão No modelo de otite média eosinofílica, a alergia causou alterações significativas à histopatologia e na morfologia da mucosa da tuba auditiva, afetou a função normal dela, o que desempenhou um papel importante na ocorrência e no desenvolvimento da otite média eosinofílica.
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Objective@#To investigate the function of primary cilia in regulating the cellular response to temozolomide (TMZ) and ionizing radiation (IR) in glioblastoma (GBM).@*Methods@#GBM cells were treated with TMZ or X-ray/carbon ion. The primary cilia were examined by immunostaining with Arl13b and γ-tubulin, and the cellular resistance ability was measured by cell viability assay or survival fraction assay. Combining with cilia ablation by IFT88 depletion or chloral hydrate and induction by lithium chloride, the autophagy was measured by acridine orange staining assay. The DNA damage repair ability was estimated by the kinetic curve of γH2AX foci, and the DNA-dependent protein kinase (DNA-PK) activation was detected by immunostaining assay.@*Results@#Primary cilia were frequently preserved in GBM, and the induction of ciliogenesis decreased cell proliferation. TMZ and IR promoted ciliogenesis in dose- and time-dependent manners, and the suppression of ciliogenesis significantly enhanced the cellular sensitivity to TMZ and IR. The inhibition of ciliogenesis elevated the lethal effects of TMZ and IR via the impairment of autophagy and DNA damage repair. The interference of ciliogenesis reduced DNA-PK activation, and the knockdown of DNA-PK led to cilium formation and elongation.@*Conclusion@#Primary cilia play a vital role in regulating the cellular sensitivity to TMZ and IR in GBM cells through mediating autophagy and DNA damage repair.
Subject(s)
Humans , Antineoplastic Agents, Alkylating/therapeutic use , Brain Neoplasms/metabolism , Cell Line, Tumor , DNA/therapeutic use , Glioblastoma/metabolism , Radiation, Ionizing , Temozolomide/therapeutic useABSTRACT
Congenital heart disease(CHD)is one of the most common genetic diseases, mainly refers to the abnormal cardiovascular development caused by various abnormal factors during fetal development.Studies have found that the normal development of cardiovascular functional structure requires accurate positioning of the left-right asymmetry.As an essential link in body material metabolism and signal-transducing mechanism, cilia may participate in the pathogenesis of CHD by affecting the distribution of the left-right asymmetry of human organs and tissues during embryonic development.Therefore, a thorough understanding of the role, molecular mechanism, and related regulatory genes of cilia in CHD can provide accurate diagnosis and treatment for clinical work to obtain a better prognosis.Here we review the effects of cilia on the positioning of the left-right asymmetry during embryo development and its role in the pathogenesis of CHD.
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Chordotonal neurons are responsible for sound sensation in Drosophila. However, little is known about how they respond to sound with high sensitivity. Using genetic labeling, we found one of the Drosophila axonemal dynein heavy chains, CG9492 (DNAH5), was specifically expressed in larval chordotonal neurons and showed a distribution restricted to proximal cilia. While DNAH5 mutation did not affect the cilium morphology or the trafficking of Inactive, a candidate auditory transduction channel, larvae with DNAH5 mutation had reduced startle responses to sound at low and medium intensities. Calcium imaging confirmed that DNAH5 functioned autonomously in chordotonal neurons for larval sound sensation. Furthermore, disrupting DNAH5 resulted in a decrease of spike firing responses to low-level sound in chordotonal neurons. Intriguingly, DNAH5 mutant larvae displayed an altered frequency tuning curve of the auditory organs. All together, our findings support a critical role of DNAH5 in tuning the frequency selectivity and the sound sensitivity of larval auditory neurons.
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Objective:To explore the mechanism of highly expressed primary cilia in tibial growth plate chondrocytes accelerating chondrocytes differentiation in young rats with chronic renal failure (CRF).Methods:Forty male 4-week-old SD rats weighing (98±3) g were randomly divided into control group (intragastric administration with distilled water, n=20) and CRF group (intragastric administration with adenine suspension 150 mg·kg -1·d -1, n=20). All the young rats were sacrificed after continuous gavage for 6 weeks. The length of the growth plate was measured with histological sections. Immunofluorescence (IF) was used to detect the expression rate of primary cilia and the level of β-catenin, the key protein of Wnt/β-catenin signaling pathway in tibial growth plate chondrocytes. Chondrocytes isolated from growth plate in two groups were cultured in vitro to P3 generation, and the expression rate of primary cilia in chondrocytes, the levels of Indian hedgehog (IHH) and glycogen synthase kinase 3β (GSK3β) were detected by IF. Co-immunoprecipitation was used to detect the relationship between IHH and GSK3β. Results:Compared with the control group, the relative length of the growth plate was shorter in histological sections [(0.51±0.11) vs (1.00±0.08), t=16.11, P<0.001], the expression rate of primary cilia was higher [(26.3±5.5)% vs (7.6±1.9)%, t=14.37, P<0.001], and the level of β-catenin increased [(7.1±2.0) scores vs (3.6±1.0) scores, t=7.10, P<0.001] in CRF group. In vitro, the expression rate of primary cilia was higher in CRF group chondrocytes [(31.4±8.2)% vs (12.5±3.1)%, t=9.64, P<0.001] than that in control group. The level of IHH in CRF group increased than that in control group [(1 360±270) vs (310±84), t=16.61, P<0.001]. There was no significant difference in GSK3β level of chondrocytes between the two groups [(850±195) vs (780±140), t=1.30, P=0.200]. There was a direct interaction between IHH and GSK3β in CRF group chondrocytes. Conclusions:The expression levels of primary cilia and related protein IHH increase in tibial growth plate chondrocytes of CRF young rats. The IHH protein plays a direct interaction with GSK3β protein, Wnt/β-catenin signaling pathway antagonist, which leads to the activation of Wnt/β-catenin signaling pathway and final accelerated differentiation of chondrocytes. The rapid differentiation of chondrocytes causes the closing trend of growth plate.
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Bone and teeth are hard tissues. Hard tissue diseases have a serious effect on human survival and quality of life. Primary cilia are protrusions on the surfaces of cells. As antennas, they are distributed on the membrane surfaces of almost all mammalian cell types and participate in the development of organs and the maintenance of homeostasis. Mutations in cilium-related genes result in a variety of developmental and even lethal diseases. Patients with multiple ciliary gene mutations present overt changes in the skeletal system, suggesting that primary cilia are involved in hard tissue development and reconstruction. Furthermore, primary cilia act as sensors of external stimuli and regulate bone homeostasis. Specifically, substances are trafficked through primary cilia by intraflagellar transport, which affects key signaling pathways during hard tissue development. In this review, we summarize the roles of primary cilia in long bone development and remodeling from two perspectives: primary cilia signaling and sensory mechanisms. In addition, the cilium-related diseases of hard tissue and the manifestations of mutant cilia in the skeleton and teeth are described. We believe that all the findings will help with the intervention and treatment of related hard tissue genetic diseases.
Subject(s)
Animals , Humans , Cilia , Homeostasis , Quality of Life , Signal TransductionABSTRACT
Multiple morphological abnormalities of the sperm flagella (MMAF) is a specific type of asthenoteratozoospermia, presenting with multiple morphological anomalies in spermatozoa, such as absent, bent, coiled, short, or irregular caliber flagella. Previous genetic studies revealed pathogenic mutations in genes encoding cilia and flagella-associated proteins (CFAPs; e.g., CFAP43, CFAP44, CFAP65, CFAP69, CFAP70, and CFAP251) responsible for the MMAF phenotype in infertile men from different ethnic groups. However, none of them have been identified in infertile Pakistani males with MMAF. In the current study, two Pakistani families with MMAF patients were recruited. Whole-exome sequencing (WES) of patients and their parents was performed. WES analysis reflected novel biallelic loss-of-function mutations in CFAP43 in both families (Family 1: ENST00000357060.3, p.Arg300Lysfs*22 and p.Thr526Serfs*43 in a compound heterozygous state; Family 2: ENST00000357060.3, p.Thr526Serfs*43 in a homozygous state). Sanger sequencing further confirmed that these mutations were segregated recessively in the families with the MMAF phenotype. Semiquantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was carried out to detect the effect of the mutation on mRNA of the affected gene. Previous research demonstrated that biallelic loss-of-function mutations in CFAP43 accounted for the majority of all CFAP43-mutant MMAF patients. To the best of our knowledge, this is the first study to report CFAP43 biallelic loss-of-function mutations in a Pakistani population with the MMAF phenotype. This study will help researchers and clinicians to understand the genetic etiology of MMAF better.
Subject(s)
Adolescent , Adult , Humans , Male , Middle Aged , Infertility, Male/epidemiology , Loss of Function Mutation/genetics , Microtubule Proteins/genetics , Pakistan/epidemiology , Sperm Tail/physiologyABSTRACT
The primary cilium, a sensory organelle that protrudes from the surface of most eukaryotic cells, receives and transduces various critical signals that are essential for normal development and homeostasis. Structural or functional disruption of primary cilia causes a number of human diseases, including cancer. Primary cilia has cross talks with cell cycle and it may act as a cell cycle checkpoint to suppress cancer development. Moreover, primary cilia has cross-regulation with autophagy, which may affect tumor progression. We then discuss the association of the primary cilia with several oncogenic signaling pathways, including Shh, Wnt, Notch and platelet-derived growth factor receptor (PDGFR). Since these signaling pathways are often over-activated in many types of human cancers, primary cilia are likely to play a role in the tumorigenesis by modulating these pathways. Finally, we summarize current progress on the role of cilia during tumorigenesis and the challenges that the cilia-cancer field faces.
Subject(s)
Humans , Autophagy , Carcinogenesis , Cilia , Homeostasis , Signal TransductionABSTRACT
Primary cilia are non-motile, microtubule-based, antennae-like organelle that protrude out from the cell surfaceand perform sensory function or transduce physiological signals in majority of the vertebrate cells. Cilia areassembled on basal bodies that are transformed centrioles. The assembly-disassembly of primary cilia maypose an additional measure on regulating cell cycle in vertebrate cells. While primary cilia are commonly foundin differentiated or quiescent cells that are not cycling, disassembly of primary cilia may promote re-entry ofthese cells into the mitotic cycle, and support proliferation. Many cancer tissues or cancer-derived cells exhibitloss of primary cilia. However, primary cilia may also promote tumorigenesis in some contexts throughgrowth-promoting signalling. This review will shed light on recent advancements of temporal coordination ofciliary disassembly and cell cycle progression, with a focus on how cilia loss may support tumorigenesis invarious epithelial cancers
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Alstrom syndrome (ALMS) is a rare autosomal recessive disorder involving multiple systems.The main clinical manifestations include nystagmus,hearing loss,obesity,insulin resistance,type 2 diabetes,dilated cardiomyopathy,etc.Primary cilia are key organelles.ALMS is classified as a ciliopathy,mainly related to the mutation of ALMS1 gene which affects cilia function,but the specific mechanism remains unclear.At present,the diagnosis of ALMS mainly relies on clinical manifestations and gene sequencing.There are no specific and effective treatment methods except for symptomatic treatment,but early diagnosis and intervention can delay disease progression and improve patients' quality of life.This article reviews recent advances in the pathogenesis,diagnosis,and treatment of ALMS.
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Biomechanical factors play a crucial role in the steady-state maintenance of articular cartilage. The primary cilium (PC) is a kind of organelle which can sense mechanical and chemical signals at the same time. It is also distributed on the surface of chondrocyte membrane. It is involved in multiple signal transduction pathways as well as in the process of chondrocyte phenotype maintenance and material metabolism. Abnormalities in PC are also associated with a variety of human bone and joint diseases. This paper mainly discusses the mechanism of PC in mechanical microenvironment of chondrocytes and the interaction with other signaling pathways, and explores its relationship with bone and joint diseases, so as to provide some scientific basis for clinical and basic research in orthopedics.
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Cilia are protruding cell structures on the cell surface and are found in almost every type of cell.According to the different structures and quantity of tubulins,cilia can be divided into two categories:motor cilia and sensory cilia.Sensory cilia are also called non-motor cilia and primary cilia,due to the composition and number of tubulins.They are closely related to the development of internal organs and many human physiological activities.Recent studies have demonstrated that cilia are involved in regulating the formation of left and right symmetry of the heart structure,and eventually the heart develops into the left-right asymmetry structures.Since congenital heart diseases(CHD)are characterized by abnormalities in the spatial structure of the heart chamber and outflow tract,cilia may play an important role in the pathogenesis of CHD.Cilia,mainly through ciliary transduction signal pathways,regulate both the formation of left and right asymmetrical structures and the polarity and the migration of cells.Therefore,a clear understanding of the regulation mechanism of ciliary signaling pathway on heart development can provide new therapeutic targets and new ideas for the clinical treatment of CHD and may offer new target genes for prenatal screening of CHD.This article summarizes recent advances in the role of cilia in heart development and CHD pathogenesis and its mechanisms.
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Alstrom syndrome(ALMS)is a rare autosomal recessive disorder involving multiple systems.The main clinical manifestations include nystagmus, hearing loss, obesity, insulin resistance, type 2 diabetes, dilated cardiomyopathy, etc.Primary cilia are key organelles.ALMS is classified as a ciliopathy, mainly related to the mutation of ALMS1 gene which affects cilia function, but the specific mechanism remains unclear.At present, the diagnosis of ALMS mainly relies on clinical manifestations and gene sequencing.There are no specific and effective treatment methods except for symptomatic treatment, but early diagnosis and intervention can delay disease progression and improve patients′ quality of life.This article reviews recent advances in the pathogenesis, diagnosis, and treatment of ALMS.
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@#Primary cilia are organelles present on most mammalian cells that sense environmental changes and transduce signaling, and they are the key coordinators of various signaling pathways during tissue development. This article reviews the progress of research on the distribution of primary cilia in tooth development and the related signaling pathways. A literature review shows that in odontogenesis, primary cilia play an important role in the mutual induction of the epithelium and mesenchyme; during the continuous proliferation and differentiation of cells, the distribution of primary cilia is temporally and spatially dependent. Although the reason for this distribution is still unclear, some experimental evidence indicates that this phenomenon is compatible with the function of cells and tissues in which primary cilia are distributed. Primary cilia are involved in the regulation of two important signaling pathways, Hedgehog and Wnt, in odontogenesis. Genes encoding cilia (such as Kif3a, Evc/Evc2 and Ift) can affect the development of teeth by regulating these two signaling pathways, and there is an interaction between the two signaling pathways. Deletion of related genes (such as Ofd1 and Bbs) can damage the transmission of upstream and downstream signals by damaging the structure or function of cilia, thereby causing various types of dental dysplasia, including small teeth, enamel hypoplasia, missing teeth, or craniofacial deformities.
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Unlike adult mammalian heart, zebrafish heart has a remarkable capacity to regenerate after injury. Previous study has shown Notch signaling activation in the endocardium is essential for regeneration of the myocardium and this activation is mediated by hemodynamic alteration after injury, however, the molecular mechanism has not been fully explored. In this study we demonstrated that blood flow change could be perceived and transmitted in a primary cilia dependent manner to control the hemodynamic responsive klf2 gene expression and subsequent activation of Notch signaling in the endocardium. First we showed that both homologues of human gene KLF2 in zebrafish, klf2a and klf2b, could respond to hemodynamic alteration and both were required for Notch signaling activation and heart regeneration. Further experiments indicated that the upregulation of klf2 gene expression was mediated by endocardial primary cilia. Overall, our findings reveal a novel aspect of mechanical shear stress signal in activating Notch pathway and regulating cardiac regeneration.
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Unlike adult mammalian heart, zebrafish heart has a remarkable capacity to regenerate after injury. Previous study has shown Notch signaling activation in the endocardium is essential for regeneration of the myocardium and this activation is mediated by hemodynamic alteration after injury, however, the molecular mechanism has not been fully explored. In this study we demonstrated that blood flow change could be perceived and transmitted in a primary cilia dependent manner to control the hemodynamic responsive klf2 gene expression and subsequent activation of Notch signaling in the endocardium. First we showed that both homologues of human gene KLF2 in zebrafish, klf2a and klf2b, could respond to hemodynamic alteration and both were required for Notch signaling activation and heart regeneration. Further experiments indicated that the upregulation of klf2 gene expression was mediated by endocardial primary cilia. Overall, our findings reveal a novel aspect of mechanical shear stress signal in activating Notch pathway and regulating cardiac regeneration.
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INTRODUCCIÓN: La discinesia ciliar primaria es un trastorno hereditario autosómico recesivo, que afecta la función de las células ciliadas y se caracteriza por infecciones respiratorias a repetición y afecta tanto al tracto respiratorio superior e inferior, puede asociarse con trastornos de la lateralidad orgánica (síndrome de Kartagener), infertilidad y en algunos casos malformaciones. No existe un tratamiento específico; sin embargo, se tratan las infecciones agudas y se realiza seguimiento de la función pulmonar como en el caso clínico que se presenta a continuación. CASO CLÍNICO: Se trata de una mujer de 28 años, con antecedentes de dextrocardia, sinusitis, otitis, bronquitis y neumonías a repetición, asmática, con rinorrea mucoide crónica, que acudió por cuadro persistente de tos productiva y disnea de moderados esfuerzos. Al examen físico destacó: saturación de 80% con FIO2: 21%, cianosis discreta, ruidos cardiacos audibles en hemitórax derecho con reforzamiento del segundo ruido, estertores difusos y frémito aumentado. En la espirometría se detectó patrón obstructivo restrictivo severo, la tomografía demostró la presencia de sinusitis maxilar y esfenoidal, dextrocardia, bronquiectasias e infiltrados difusos, poliesplenia, hepatomegalia e hígado en herradura. Se diagnosticó de síndrome de Kartagener (por dextrocardia, sinusitis y bronquiectasias). EVOLUCIÓN: Durante la estancia hospitalaria la paciente permaneció sin requerimientos de oxígeno suplementario y afebril. Recibió tratamiento antibiótico, corticoides inhalatorios y salbutamol. Se explicó a la paciente y sus familiares la benignidad de la enfermedad y el requerimiento de controles rigurosos por consulta externa. El diagnóstico definitivo por microscopía electrónica no fue realizado por falta de recursos a nivel local. CONCLUSIÓN: La discinesia ciliar primaria por lo general tiene un curso evolutivo de carácter benigno, al ser una enfermedad poco conocida su diagnóstico es tardío. La discinesia ciliar primaria debe ser considera dentro de los diagnósticos diferenciales de un paciente que presenta infecciones respiratorias a repetición.(au)
BACKGROUND: Primary ciliary dyskinesia is an inherited autosomal recessive disorder, which affects the function of ciliated cells and is characterized by recurrent upper and lower respiratory infections. It may be associated with organic laterality disorders (Kartagener syndrome), infertility and in some cases malformations. There is no specific treatment; however, acute infections management and pulmonary function surveillance is recommended, as presented in the case report. CASE REPORT: 28-year-old woman with a history of dextrocardia, sinusitis, otitis, bronchitis and recurrent pneumonia, asthmatic, with chronic mucoid rhinorrhea and recurrent episodes of productive cough and dyspnea. Physical examination revealed an oxygen saturation of 80% at room air, discrete cyanosis, and audible cardiac sounds in the right hemithorax with reinforcement of the second noise, diffuse rales and increased thrill. Pulmonary function test was positive for a severe obstructive - restrictive pattern, computed tomography revealed the presence of maxillary and sphenoid sinusitis, dextrocardia, bronchiectasis, polysplenia hepatomegaly and horseshoe liver. The diagnosis of Kartagener syndrome was made (due to dextrocardia, sinusitis and bronchiectasis). EVOLUTION: During the hospital stay the patient remained without oxygen requirements, she received antibiotic treatment plus corticosteroids and salbutamol. Patient education was carried out, indicating the benignity of the disease and the requirement of close monitoring. Definitive diagnosis by electron microscopy was not available. CONCLUSION: Primary ciliary dyskinesia usually has a benign course of evolution; being an uncommon disease, diagnosis is usually late. Primary ciliary dyskinesia should be considered within the differential diagnosis of patients with recurrent respiratory infection(au)
Subject(s)
Humans , Female , Adult , Asthma , Sinusitis , Kartagener Syndrome , Ciliary Motility Disorders/diagnostic imaging , Dextrocardia , Dyspnea , Respiratory Function Tests , Respiratory Tract Infections , HistoryABSTRACT
Objective: To investigate the role of BBS protein in ciliary signal transduction by studying the pro- karyotic expression, purification and polyclonal antibody preparation of Chlamydomonas reinhardtii protein BBS4. Methods: Prokaryotic Expression Vector pET-28a(+)-bbs4 and pMAL-c2X-bbs4 were constructed by the cDNA sequence of bbs4 Gene from C. reinhardtii, and then transformed into Escherichia coli BL21(DE3)for protein expression. The fusion protein with maltose binding protein(MBP)and 6×His tag was obtained by inducing expression. The purified fusion protein 6×His-BBS4 were used to immunize New Zealand white rabbits and the antiserum was isolated from the blood collected from the ear vein. The titer of the antiserum was measured by indirect ELISA essay, the specificity of the antibody was tested by Western blotting method and immunofluorescence test. Results: Prokaryotic expression plasmids pET-28a(+)-bbs4 and pMAL-c2X-bbs4 were successfully constructed. The relative molecular weights of 6×His-BBS4 and MBP-BBS4 fusion proteins were 45 kDa and 85 kDa, respectively. The purity of the fusion proteins was more than 85%, and the concentration of the fusion proteins was more than 0.5 mg/ml. The proteins were used for immunization. The titer of the fusion proteins was 51 200. Western blotting showed a high specificity for the detection of C. reinhardtii CC-125. Prokaryotic expression of BBS4 protein of C. reinhardtii and preparation of polyclonal antibody were realized. Conclu- sion: The polyclonal antibody against BBS4 of C. reinhardtii was prepared successfully, which laid a foundation for further study on the role of BBS4 in ciliopathies.
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Objective To investigate the effect of different fluid shear stress (FSS) on the regulation of planar cell polarity (PCP) signaling, and further to explore the relationship among FSS, PCP signaling pathway and ciliogenesis. Methods The hydrodynamic cell model of adjustable FSS was established. qPCR and immunofluorescence were used to detect the mRNA expression of PCP signaling pathway core protein Dvl2 and cilia assembly protein IFT88, cell targeting and co-localization under different FSS. Western blot (WB) was used to detect the protein expression of Dvl2 at 18 h under different FSS. Results The qPCR result showed that compared with 1.5 Pa FSS, under 0.1 Pa FSS, the mRNA expression of Dvl2 was higher at 6 h and 18 h (P＜0.05), significantly higher at 12 h (P＜0.01); the mRNA expression of IFT88 was significantly higher at 18 h (P＜0.01). The WB result showed that compared with 0 h, under 0.1 Pa FSS, the protein expression of Dvl2 was higher at 18 h (P＜0.05), significantly lower under 1.5 Pa FSS (P＜0.01); compared with 1.5 Pa FSS, the protein expression of Dvl2 was higher at 18 h under 0.1 Pa FSS (P＜0.05). The immunofluorescence result showed that the positive expression of Dvl2 increased with the loading time on FSS increasing, and gradually aggregated at a point around the nucleus; the positive expression of IFT88 was gradually transferred from the nucleus to the cytoplasm and aggregated at a point under 0.1 Pa FSS, and gradually decreased and depolymerized under 1.5 Pa FSS. Protein Dvl2 and IFT88 were located in the same position in cells under 0.1 Pa FSS and before 18 h under 1.5 Pa FSS, and colocalization of proteins Dvl2 and IFT88 was not observed after 18 h under 1.5 Pa FSS due to IFT88 depolymerization. Conclusions Laminar FSS played an inhibition on the transduction of PCP signaling pathway and a hindrance on the process of ciliogenesis, while low FSS played a promotion on the transduction. PCP signaling pathway might regulate FSS-induced ciliogenesis by Dvl2.
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OBJECTIVE： To prepare the Peppermint oil moisturizing microemulsion for nasal mucosa and survey its mucosal adhesion and cilia toxicity. METHODS： The polyoxyethylene hydrogenated castor oil was used as emulsifier to prepare the Peppermint oil moisturizing microemulsion for nasal mucosa， and the preparation technology was optimized on the basis of comprehensive score by orthogonal design. The microemulsion was characterized and the menthol content was determined by GC. The mucosal adhesion was evaluated by measuring the transport rate by cilia in vivo， and the cilia toxicity of microemulsion was evaluated by measuring the sustained movement time of cilia in vitro. RESULTS： The optimal preparation technology of self-made microemulsion was to firstly disperse the peppermint oil and the emulsifier， then add anhydrous ethanol， edible glycerin and distilled water， and stir at 1 200 r/min for 2 h. The average contents of menthol in the three batches of the microemulsion were 2.682， 2.507 and 2.496 mg/mL （RSD＝2.89％，n＝3）， respectively. The cilia transport rates in vivo were （0.65±0.01）， （0.78±0.03）and （0.92±0.04） cm/min in high-dose， medium-dose， and low-dose groups of self-made microemulsion （2.561， 0.256， 0.128 mg/mL of menthol） respectively， which were significantly lower than normal saline group and compound menthol nasal droups （P＜0.05）. The cilia movement time in vitro were（206.7±4.9）， （226.0±13.5）， （269.3±12.9）min， which were significantly longer than sodium deoxycholate group （P＜0.05）. CONCLUSIONS： The preparation technology of self-made microemulsion is easy-to-handle and controllable in quality. The prepared microemulsion shows good mucosal adhesion without cilia toxicity．