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We evaluated the diagnostic performance of newly developed microfluidic microplate-based fluorescent ELISA for anti-SARS-CoV-2 antibody detection: the Veri-Q opti COVID-19 IgG and IgM ELISAs (hereafter, "Opti IgG/M"; MiCo BioMed, Gyeonggi-do, Republic of Korea), in comparison with conventional ELISAs. A total of 270 serum samples were analyzed, among which 90 samples were serially obtained from 25 COVID-19 patients. Another 180 samples were collected from 180 SARS-CoV-2-negative individuals. As comparative assays, we used SCoV-2 Detect IgG/M ELISA (hereafter, "InBios IgG/M"; InBios, Seattle, WA, USA) and Veri-Q COVID-19 IgG/IgM ELISA (hereafter, "Veri-Q IgG/M"; MiCo BioMed). Compared with conventional ELISAs, the Opti IgG yielded 97.1-100.0% positive percent agreement, 95.2-98.0% negative percent agreement, 96.3-97.8% total percent agreement, and kappa values of 0.90-0.94. Between the Opti IgM and the InBios IgM, the values were 93.7%, 96.6%, 95.9%, and 0.89, respectively. For the Opti IgG, sensitivities for the samples collected from 0-7, 8-14, 15-21, and ≥ 22 days after symptom onset were 40.0, 58.3, 94.1, and 100.0%, respectively. The values for the Opti IgM were 30.0, 54.2, 88.2, and 80%, respectively. The diagnostic specificities of the Opti IgG and IgM were 99.4 and 97.2%, respectively. The microfluidic microplate-based fluorescent ELISAs showed comparable diagnostic performance to conventional ELISAs for detecting anti-SARS-CoV-2 antibodies. With the combination of high throughput, a simplified workflow, and the ability to analyze reduced volumes, this new technology has great potential for improving SARS-CoV-2 serologic testing.
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Anticorpos Antivirais , Teste Sorológico para COVID-19 , COVID-19 , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G , Imunoglobulina M , SARS-CoV-2 , Humanos , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Imunoglobulina G/sangue , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , COVID-19/diagnóstico , COVID-19/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Teste Sorológico para COVID-19/métodos , Sensibilidade e Especificidade , Microfluídica/métodos , Microfluídica/instrumentação , Pessoa de Meia-Idade , Feminino , Masculino , IdosoRESUMO
BACKGROUND: Assessment of measurable residual disease (MRD) is an essential prognostic tool for B-lymphoblastic leukaemia (B-ALL). In this study, we evaluated the utility of next-generation sequencing (NGS)-based MRD assessment in real-world clinical practice. METHOD: The study included 93 paediatric patients with B-ALL treated at our institution between January 2017 and June 2022. Clonality for IGH or IGK rearrangements was identified in most bone marrow samples (91/93, 97.8%) obtained at diagnosis. RESULTS: In 421 monitoring samples, concordance was 74.8% between NGS and multiparameter flow cytometry and 70.7% between NGS and reverse transcription-PCR. Elevated quantities of clones of IGH alone (P < 0.001; hazard ratio [HR], 22.2; 95% confidence interval [CI], 7.1-69.1), IGK alone (P = 0.011; HR, 5.8; 95% CI, 1.5-22.5), and IGH or IGK (P < 0.001; HR, 7.2; 95% CI, 2.6-20.0) were associated with an increased risk of relapse. Detection of new clone(s) in NGS was also associated with inferior relapse-free survival (P < 0.001; HR, 18.1; 95% CI, 3.0-108.6). Multivariable analysis confirmed age at diagnosis, BCR::ABL1-like mutation, TCF3::PBX1 mutation, and increased quantity of IGH or IGK clones during monitoring as unfavourable factors. CONCLUSION: In conclusion, this study highlights the usefulness of NGS-based MRD as a routine assessment tool for prognostication of paediatric patients with B-ALL.
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Most patients diagnosed with clear cell renal cell carcinoma (ccRCC) are also detected with small and organ-confined tumors, and the majority of these are classified as clinical tumor stage 1a (cT1a). A considerable proportion of patients with cT1 RCC shows tumor upstaging to pathological stage 3a (pT3a), and these patients have worse oncological outcomes. The role of circulating tumor DNA (ctDNA) in RCC has been limited to monitoring treatment response and resistance. Therefore, the present study aimed to evaluate the potential of ctDNA in predicting pT3a upstaging in cT1a ccRCC. We sequenced plasma samples preoperatively collected from 48 patients who had undergone partial nephrectomy for cT1a ccRCC using data from a prospective cohort RCC. The ctDNA were profiled and compared with clinicopathological ccRCC features to predict pT3a upstaging. Associations between ctDNA, tumor complexity, and pT3a upstaging were evaluated. Tumor complexity was assessed using the anatomical classification system. Univariate analysis used chi-squared and Student's t-tests; multivariate analysis considered significant factors from univariate analyses. Of the 48 patients with cT1a ccRCC, 12 (25%) were upstaged to pT3a, with ctDNA detected in 10 (20.8%), predominantly in patients with renal sinus fat invasion (SFI; n = 8). Among the pT3a group, ctDNA was detected in 75%, contrasting with only 2.8% in patients with pT1a (1/36). Detection of ctDNA was the only significant preoperative predictor of pT3a upstaging, especially in SFI. This study is the first to suggest ctDNA as a preoperative predictor of pT3a RCC upstaging from cT1a based on preoperative radiological images.
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Carcinoma de Células Renais , DNA Tumoral Circulante , Neoplasias Renais , Estadiamento de Neoplasias , Nefrectomia , Humanos , Carcinoma de Células Renais/cirurgia , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/sangue , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Nefrectomia/métodos , Feminino , Masculino , Neoplasias Renais/cirurgia , Neoplasias Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/sangue , Pessoa de Meia-Idade , Idoso , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Estudos Prospectivos , Adulto , Idoso de 80 Anos ou maisRESUMO
PURPOSE: We designed and evaluated the clinical performance of a plasma circulating tumor DNA (ctDNA) panel of 112 genes in various subtypes of lymphoma. MATERIALS AND METHODS: Targeted deep sequencing with an error-corrected algorithm was performed in ctDNA from plasma samples that were collected before treatment in 42 lymphoma patients. Blood buffy coat was utilized as a germline control. We evaluated the targeted gene panel using mutation detection concordance on the plasma samples with matched tissue samples analyzed the mutation profiles of the ctDNA. RESULTS: Next-generation sequencing analysis using matched tissue samples was available for 18 of the 42 patients. At least one mutation was detected in the majority of matched tissue biopsy samples (88.9%) and plasma samples (83.3%). A considerable number of mutations (40.4%) that were detected in the tissue samples were also found in the matched plasma samples. Majority of patients (21/42) were diffuse large B cell lymphoma patients. The overall detection rate of ctDNA in patients was 85.7% (36/42). The frequently mutated genes included PIM1, TET2, BCL2, KMT2D, KLHL6, HIST1H1E, and IRF8. A cutoff concentration (4,506 pg/mL) of ctDNA provided 88.9% sensitivity and 82.1% specificity to predict ctDNA mutation detection. The ctDNA concentration correlated with elevated lactate dehydrogenase level and the disease stage. CONCLUSION: Our design panel can detect many actionable gene mutations, including those at low frequency. Therefore, liquid biopsy can be applied clinically in the evaluation of lymphoma patients, especially in aggressive lymphoma patients.
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DNA Tumoral Circulante , Linfoma , Humanos , DNA Tumoral Circulante/genética , Biópsia Líquida , Mutação , Biomarcadores Tumorais/genética , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
BACKGROUND: Hereditary hemolytic anemia (HHA) refers to a heterogeneous group of genetic disorders that share one common feature: destruction of circulating red blood cells (RBCs). The destruction of RBCs may be due to membranopathies, enzymopathies, or hemoglobinopathies. Because these are genetic disorders, incorporation of next-generation sequencing (NGS) has facilitated the diagnostic process of HHA. METHOD: Genetic data from 29 patients with suspected hereditary anemia in a tertiary hospital were retrospectively reviewed to evaluate the efficacy of NGS on hereditary anemia diagnosis. Targeted NGS was performed with custom probes for 497 genes associated with hematologic disorders. After genomic DNA was extracted from peripheral blood, prepared libraries were hybridized with capture probes and sequenced using NextSeq 550Dx (Illumina, San Diego, CA, USA). RESULT: Among the 29 patients, ANK1 variants were detected in five, four of which were pathogenic or likely pathogenic variants. SPTB variants were detected in six patients, five of which were classified as pathogenic or likely pathogenic variants. We detected g6pd pathogenic and spta1 likely pathogenic variants in two patients and one patient, respectively. Whole-gene deletions in both HBA1 and HBA2 were detected in two patients, while only HBA2 deletion was detected in one patient. One likely pathogenic variant in PLKR was detected in one patient, and one likely pathogenic variant in ALAS2 was detected in another. CONCLUSION: Here, NGS played a critical role in definitive diagnosis in 18 out of 29 patients (62.07%) with suspected HHA. Thus, its incorporation into the diagnostic workflow is crucial.
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Anemia Hemolítica Congênita , Humanos , Criança , Estudos Retrospectivos , Anemia Hemolítica Congênita/diagnóstico , Anemia Hemolítica Congênita/genética , Eritrócitos , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas do Citoesqueleto , 5-Aminolevulinato SintetaseRESUMO
Circulating tumor DNA (ctDNA) is a promising biomarker for clear cell renal cell carcinoma (ccRCC); however, its characteristics in small renal masses of ccRCC remain unclear. In this pilot study, we explored the characteristics of ctDNA in pT1a ccRCC. Plasma samples were collected preoperatively from 53 patients with pT1a ccRCC. The ctDNA of pT1a ccRCC was profiled using next-generation sequencing and compared with that of higher-stage ccRCC. The association of ctDNA in pT1a ccRCC with clinicopathological features was investigated. The positive relationship of mutations between ctDNA and matched tissues was evaluated. In pT1a ccRCC, the ctDNA detection rate, cell-free DNA concentration, and median variant allele frequency were 20.8%, 5.8 ng/mL, and 0.38%, respectively, which were significantly lower than those in metastatic ccRCC. The ctDNA gene proportions in pT1a samples differed from those in metastatic ccRCC samples. The relationships between ctDNA and tumor size, tumor grade, and patient age were not elucidated. The positive concordance between ctDNA and matched tissues was poor for pT1a ccRCC. Strategies are needed to increase sensitivity while eliminating noise caused by clonal hematopoiesis to increase the clinical utility of ctDNA analysis in small renal masses of ccRCC.
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Next-generation sequencing (NGS) facilitates comprehensive molecular analyses that help with diagnosing unsolved disorders. In addition to detecting single-nucleotide variations and small insertions/deletions, bioinformatics tools can identify copy number variations (CNVs) in NGS data, which improves the diagnostic yield. However, due to the possibility of false positives, subsequent confirmation tests are generally performed. Here, we introduce Copy-number Analysis by BAse-level NormAlization (CABANA), a visualization tool that allows users to intuitively identify candidate CNVs using the normalized single-base-level read depth calculated from NGS data. To demonstrate how CABANA works, NGS data were obtained from 474 patients with neuromuscular disorders. CNVs were screened using a conventional bioinformatics tool, ExomeDepth, and then we normalized and visualized those data at the single-base level using CABANA, followed by manual inspection by geneticists to filter out false positives and determine candidate CNVs. In doing so, we identified 31 candidate CNVs (7%) in 474 patients and subsequently confirmed all of them to be true using multiplex ligation-dependent probe amplification. The performance of CABANA was deemed acceptable by comparing its diagnostic yield with previous data about neuromuscular disorders. Despite some limitations, we expect CABANA to help researchers accurately identify CNVs and reduce the need for subsequent confirmation testing.
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Variações do Número de Cópias de DNA , Humanos , Variações do Número de Cópias de DNA/genéticaAssuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Células Clonais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
Non-alcoholic fatty liver disease (NAFLD) is the major cause of chronic liver disease, yet cost-effective and non-invasive diagnostic tools to monitor the severity of the disease are lacking. We aimed to investigate the metabolomic changes in NAFLD associated with therapeutic responses. It was conducted in 63 patients with NAFLD who received either ezetimibe plus rosuvastatin or rosuvastatin monotherapy. The treatment response was determined by MRI performed at baseline and week 24. The metabolites were measured at baseline and week 12. In the combination group, a relative decrease in xanthine was associated with a good response to liver fat decrease, while a relative increase in choline was associated with a good response to liver stiffness. In the monotherapy group, the relative decreases in triglyceride (TG) 20:5_36:2, TG 18:1_38:6, acetylcarnitine (C2), fatty acid (FA) 18:2, FA 18:1, and docosahexaenoic acid were associated with a decrease in liver fat, while hexosylceramide (d18:2/16:0) and hippuric acid were associated with a decrease in liver stiffness. Models using the metabolite changes showed an AUC of >0.75 in receiver operating curve analysis for predicting an improvement in liver fat and stiffness. This approach revealed the physiological impact of drugs, suggesting the mechanism underlying the development of this disease.
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Defective primary cilia cause a range of diseases known as ciliopathies, including hearing loss. The etiology of hearing loss in ciliopathies, however, remains unclear. We analyzed cochleae from three ciliopathy mouse models exhibiting different ciliogenesis defects: Intraflagellar transport 88 (Ift88), Tbc1d32 (a.k.a. bromi), and Cilk1 (a.k.a. Ick) mutants. These mutants showed multiple developmental defects including shortened cochlear duct and abnormal apical patterning of the organ of Corti. Although ciliogenic defects in cochlear hair cells such as misalignment of the kinocilium are often associated with the planar cell polarity pathway, our results showed that inner ear defects in these mutants are primarily due to loss of sonic hedgehog signaling. Furthermore, an inner ear-specific deletion of Cilk1 elicits low-frequency hearing loss attributable to cellular changes in apical cochlear identity that is dedicated to low-frequency sound detection. This type of hearing loss may account for hearing deficits in some patients with ciliopathies.
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Polaridade Celular/fisiologia , Ciliopatias/fisiopatologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Perda Auditiva/fisiopatologia , Proteínas Hedgehog/metabolismo , Animais , Cílios/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos/metabolismo , Células Ciliadas Auditivas/metabolismo , Camundongos , Via de Sinalização Wnt/fisiologiaRESUMO
BACKGROUND: Electroencephalography (EEG)-based brain-computer interface (BCI) systems are mainly divided into three major paradigms: motor imagery (MI), event-related potential (ERP), and steady-state visually evoked potential (SSVEP). Here, we present a BCI dataset that includes the three major BCI paradigms with a large number of subjects over multiple sessions. In addition, information about the psychological and physiological conditions of BCI users was obtained using a questionnaire, and task-unrelated parameters such as resting state, artifacts, and electromyography of both arms were also recorded. We evaluated the decoding accuracies for the individual paradigms and determined performance variations across both subjects and sessions. Furthermore, we looked for more general, severe cases of BCI illiteracy than have been previously reported in the literature. RESULTS: Average decoding accuracies across all subjects and sessions were 71.1% (± 0.15), 96.7% (± 0.05), and 95.1% (± 0.09), and rates of BCI illiteracy were 53.7%, 11.1%, and 10.2% for MI, ERP, and SSVEP, respectively. Compared to the ERP and SSVEP paradigms, the MI paradigm exhibited large performance variations between both subjects and sessions. Furthermore, we found that 27.8% (15 out of 54) of users were universally BCI literate, i.e., they were able to proficiently perform all three paradigms. Interestingly, we found no universally illiterate BCI user, i.e., all participants were able to control at least one type of BCI system. CONCLUSIONS: Our EEG dataset can be utilized for a wide range of BCI-related research questions. All methods for the data analysis in this study are supported with fully open-source scripts that can aid in every step of BCI technology. Furthermore, our results support previous but disjointed findings on the phenomenon of BCI illiteracy.
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Interfaces Cérebro-Computador , Eletroencefalografia , Potenciais Evocados Visuais/fisiologia , Potenciais Evocados/fisiologia , Adulto , Algoritmos , Feminino , Humanos , Masculino , Movimento/fisiologiaRESUMO
OBJECTIVE: The mouse is the most popular animal model in olfactory research. Behavior tests with odorants are essential for determining olfactory phenotype. To the best of our knowledge, the mouse olfactory behavior test has not been standardized, making the results vulnerable to inter-observer variation. We sought to develop a new mouse olfactory behavior test assessed by an automatic video tracking system with minimal inter-observer variation. METHODS: A video-tracking system was used to automatically track mouse behavior in standard breeding cages with C57BL/6N mice. We tested two odorants (peanut butter for the preference test, 2MB acid for the avoidance test) and distilled water (for a control). Mouse behavior was recorded for 3min and analyzed. For the preference test, investigation time was measured. For the avoidance test, time spent in sectors away from the odorant zone was measured. To confirm our experimental settings, we also evaluated an anosmia mouse model prepared with intranasal administration of ZnSO4. RESULTS: All strains of mice showed reproducible behavior patterns of preference or avoidance for the odorants. The anosmia mouse model, as expected, failed to show an olfactory ability for preference or avoidance, and this was well-matched by histologic changes caused by the ZnSO4 treatment. The automatic video tracking system successfully tracked and automatically calculated mouse behavior with good reproducibility. CONCLUSION: Our olfactory behavior test offers a simple and accurate method to evaluate olfactory function in mice. This test can be utilized as a possible standard method to search for features of olfactory phenotypes in mice.
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Comportamento Apetitivo , Comportamento Animal , Camundongos , Modelos Animais , Olfato/fisiologia , Animais , Escala de Avaliação Comportamental , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Odorantes , Transtornos do Olfato , Mucosa Olfatória/anatomia & histologia , Gravação em VídeoRESUMO
During the differentiation of the amoeba Naegleria pringsheimi into a flagellate, a transient complex containing γ-tubulin, pericentrin-like protein, and myosin II (GPM complex) is formed, and subsequently a pair of basal bodies is assembled from the complex. It is not understood, however, how a single GPM is formed nor how the capability to form this complex is acquired by individual cells. We hypothesized that the GPM is formed from a precursor complex and developed an antibody that recognizes Naegleria (Ng)-transacylase, a component of the precursor complex. Immunostaining of differentiating cells showed that Ng-transacylase is concentrated at a site in the amoeba and that γ-tubulin is transiently co-concentrated at the site, suggesting that the GPM is formed from a precursor, GPMp, which contains Ng-transacylase and is already present in the amoeba. Immunostaining of growing N. pringsheimi with Ng-transacylase antibody revealed the presence of one GPMp in interphase cells, but two GPMps in mitotic cells, suggesting that N. pringsheimi maintains one GPMp per cell by duplicating and segregating the complex according to its cell cycle. Our results demonstrate the existence of a cell cycle-dependent duplicating complex that provides a site for the de novo assembly of the next generation of basal bodies.
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Corpos Basais/metabolismo , Naegleria/citologia , Naegleria/fisiologia , Antígenos/metabolismo , Ciclo Celular , Diferenciação Celular , Miosina Tipo II/metabolismo , Multimerização Proteica , Tubulina (Proteína)/metabolismoRESUMO
Endocrine-cerebro-osteodysplasia (ECO) syndrome is a recessive genetic disorder associated with multiple congenital defects in endocrine, cerebral, and skeletal systems that is caused by a missense mutation in the mitogen-activated protein kinase-like intestinal cell kinase (ICK) gene. In algae and invertebrates, ICK homologs are involved in flagellar formation and ciliogenesis, respectively. However, it is not clear whether this role of ICK is conserved in mammals and how a lack of functional ICK results in the characteristic phenotypes of human ECO syndrome. Here, we generated Ick knockout mice to elucidate the precise role of ICK in mammalian development and to examine the pathological mechanisms of ECO syndrome. Ick null mouse embryos displayed cleft palate, hydrocephalus, polydactyly, and delayed skeletal development, closely resembling ECO syndrome phenotypes. In cultured cells, down-regulation of Ick or overexpression of kinase-dead or ECO syndrome mutant ICK resulted in an elongation of primary cilia and abnormal Sonic hedgehog (Shh) signaling. Wild-type ICK proteins were generally localized in the proximal region of cilia near the basal bodies, whereas kinase-dead ICK mutant proteins accumulated in the distal part of bulged ciliary tips. Consistent with these observations in cultured cells, Ick knockout mouse embryos displayed elongated cilia and reduced Shh signaling during limb digit patterning. Taken together, these results indicate that ICK plays a crucial role in controlling ciliary length and that ciliary defects caused by a lack of functional ICK leads to abnormal Shh signaling, resulting in congenital disorders such as ECO syndrome.
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Anormalidades Múltiplas/patologia , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Anormalidades Múltiplas/genética , Animais , Western Blotting , Padronização Corporal/genética , Padronização Corporal/fisiologia , Córtex Cerebral/embriologia , Córtex Cerebral/patologia , Cílios/genética , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Sistema Endócrino/embriologia , Sistema Endócrino/patologia , Proteínas Hedgehog/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica , Sistema Musculoesquelético/embriologia , Sistema Musculoesquelético/patologia , Células NIH 3T3 , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , SíndromeRESUMO
Pax3 mutations result in malformed inner ears in Splotch mutant mice and hearing loss in humans with Waardenburg's syndrome type I. In the inner ear, Pax3 is thought to be involved mainly in the development of neural crest. However, recent studies have shown that Pax3-expressing cells contribute extensively to multiple inner ear structures, some of which were considered to be derived from the otic epithelium. To examine the specific functions of Pax3 during inner ear development, fate mapping of Pax3 lineage was performed in the presence or absence of functional Pax3 proteins using Pax3(Cre) knock-in mice bred to Rosa26 reporter (R26R) line. ß-gal-positive cells were widely distributed in Pax3(Cre/+); R26R inner ears at embryonic day (E) 15.5, including the endolymphatic duct, common crus, cristae, maculae, cochleovestibular ganglion, and stria vascularis. In the absence of Pax3 in Pax3(Cre/Cre); R26R inner ears, ß-gal-positive cells disappeared from regions with melanocytes such as the stria vascularis of the cochlea and dark cells in the vestibule. Consistently, the expression of Dct, a melanoblast marker, was also absent in the mutant inner ears. However, when examined at E11.5, ß-gal positive cells were present in Pax3(Cre/Cre) mutant otocysts, whereas Dct expression was absent, suggesting that Pax3 lineage with a melanogenic fate migrated to the inner ear, yet failed to differentiate and survive without Pax3 function. Gross inner ear morphology was generally normal in Pax3(Cre/Cre) mutants, unless neural tube defects extended to the cranial region. Taken together, these results suggest that despite the extensive contribution of Pax3-expressing cells to multiple inner ear tissues, Pax3 function is required specifically for inner ear components with melanogenic fates.
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Orelha Interna/anormalidades , Melanócitos/citologia , Fatores de Transcrição Box Pareados/metabolismo , Animais , Diferenciação Celular , Movimento Celular , Orelha Interna/embriologia , Orelha Interna/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Perda Auditiva/genética , Humanos , Melanócitos/metabolismo , Camundongos , Mutação , Crista Neural/anormalidades , Crista Neural/embriologia , Crista Neural/metabolismo , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genéticaRESUMO
The de novo formation of basal bodies in Naegleria gruberi was preceded by the transient formation of a microtubule (MT)-nucleating complex containing gamma-tubulin, pericentrin, and myosin II complex (GPM complex). The MT-nucleating activity of GPM complexes was maximal just before the formation of visible basal bodies and then rapidly decreased. The regulation of MT-nucleating activity of GPM complexes was accomplished by a transient phosphorylation of the complex. Inhibition of dephosphorylation after the formation of basal bodies resulted in the formation of multiple flagella. 2D-gel electrophoresis and Western blotting showed a parallel relationship between the MT-nucleating activity of GPM complexes and the presence of hyperphosphorylated gamma-tubulin in the complexes. These data suggest that the nucleation of MTs by GPM complexes precedes the de novo formation of basal bodies and that the regulation of MT-nucleating activity of GPM complexes is essential to the regulation of basal body number.