RESUMO
RNF111/Arkadia is an E3 ubiquitin ligase that activates the transforming growth factor-ß (TGF-ß) pathway by degrading transcriptional repressors SKIL/SnoN and SKI. Truncations of the RING C-terminal domain of RNF111 that abolish its E3 function and subsequently activate TGF-ß signaling are observed in some cancers. In the present study, we sought to perform a comprehensive analysis of RNF111 endogenous substrates upon TGF-ß signaling activation using an integrative proteomic approach. In that aim, we carried out label-free quantitative proteomics after the enrichment of ubiquitylated proteins (ubiquitylome) in parental U2OS cell line compared with U2OS CRISPR engineered clones expressing a truncated form of RNF111 devoid of its C-terminal RING domain. We compared two methods of enrichment for ubiquitylated proteins before proteomics analysis by mass spectrometry, the diGlycine (diGly) remnant peptide immunoprecipitation with a K-ε-GG antibody, and a novel approach using protein immunoprecipitation with a ubiquitin pan nanobody that recognizes all ubiquitin chains and monoubiquitylation. Although we detected SKIL ubiquitylation among 108 potential RNF111 substrates with the diGly method, we found that the ubiquitin pan nanobody method also constitutes a powerful approach because it enabled the detection of 52 potential RNF111 substrates including SKI, SKIL, and RNF111. Integrative comparison of the RNF111-dependent proteome and ubiquitylomes enabled the identification of SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 E3 ligase function in the presence of TGF-ß. Our results indicate that lysine 343 localized in the SAND domain of SKIL constitutes a target for RNF111 ubiquitylation and demonstrate that RNF111 E3 ubiquitin ligase function specifically targets SKI and SKIL ubiquitylation and degradation upon TGF-ß pathway activation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Proteoma/metabolismo , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Mutations in transcription factor p63 are associated with developmental disorders that manifest defects in stratified epithelia including the epidermis. The underlying cellular and molecular mechanism is however not yet understood. We established an epidermal commitment model using human induced pluripotent stem cells (iPSCs) and characterized differentiation defects of iPSCs derived from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients carrying p63 mutations. Transcriptome analyses revealed stepwise cell fate transitions during epidermal commitment: Specification from multipotent simple epithelium to basal stratified epithelia and ultimately to the mature epidermal fate. Differentiation defects of EEC iPSCs caused by p63 mutations occurred during the specification switch from the simple epithelium to the basal-stratified epithelial fate. Single-cell transcriptome and pseudotime analyses of cell states identified mesodermal activation that was associated with the deviated commitment route of EEC iPSCs. Integrated analyses of differentially regulated genes and p63-dependent dynamic genomic enhancers during epidermal commitment suggest that p63 directly controls epidermal gene activation at the specification switch and has an indirect effect on mesodermal gene repression. Importantly, inhibitors of mesodermal induction enhanced epidermal commitment of EEC iPSCs. Our findings demonstrate that p63 is required for specification of stratified epithelia, and that epidermal commitment defects caused by p63 mutations can be reversed by repressing mesodermal induction. This study provides insights into disease mechanisms underlying stratified epithelial defects caused by p63 mutations and suggests potential therapeutic strategies for the disease.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Displasia Ectodérmica/genética , Epitélio/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Epiderme/embriologia , Epiderme/metabolismo , Epitélio/embriologia , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Queratinócitos/metabolismo , Mutação , Análise de Sequência de RNA , Análise de Célula Única , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Background There are conflicting data regarding the diagnostic performance of chest CT for COVID-19 pneumonia. Disease extent at CT has been reported to influence prognosis. Purpose To create a large publicly available data set and assess the diagnostic and prognostic value of CT in COVID-19 pneumonia. Materials and Methods This multicenter, observational, retrospective cohort study involved 20 French university hospitals. Eligible patients presented at the emergency departments of the hospitals involved between March 1 and April 30th, 2020, and underwent both thoracic CT and reverse transcription-polymerase chain reaction (RT-PCR) testing for suspected COVID-19 pneumonia. CT images were read blinded to initial reports, RT-PCR, demographic characteristics, clinical symptoms, and outcome. Readers classified CT scans as either positive or negative for COVID-19 based on criteria published by the French Society of Radiology. Multivariable logistic regression was used to develop a model predicting severe outcome (intubation or death) at 1-month follow-up in patients positive for both RT-PCR and CT, using clinical and radiologic features. Results Among 10 930 patients screened for eligibility, 10 735 (median age, 65 years; interquartile range, 51-77 years; 6147 men) were included and 6448 (60%) had a positive RT-PCR result. With RT-PCR as reference, the sensitivity and specificity of CT were 80.2% (95% CI: 79.3, 81.2) and 79.7% (95% CI: 78.5, 80.9), respectively, with strong agreement between junior and senior radiologists (Gwet AC1 coefficient, 0.79). Of all the variables analyzed, the extent of pneumonia at CT (odds ratio, 3.25; 95% CI: 2.71, 3.89) was the best predictor of severe outcome at 1 month. A score based solely on clinical variables predicted a severe outcome with an area under the curve of 0.64 (95% CI: 0.62, 0.66), improving to 0.69 (95% CI: 0.6, 0.71) when it also included the extent of pneumonia and coronary calcium score at CT. Conclusion Using predefined criteria, CT reading is not influenced by reader's experience and helps predict the outcome at 1 month. ClinicalTrials.gov identifier: NCT04355507 Published under a CC BY 4.0 license. Online supplemental material is available for this article. See also the editorial by Rubin in this issue.
Assuntos
COVID-19/diagnóstico por imagem , Tomografia Computadorizada por Raios X/métodos , Idoso , Estudos de Coortes , Feminino , Humanos , Pulmão/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Estudos Retrospectivos , SARS-CoV-2 , Sensibilidade e EspecificidadeRESUMO
Background The role and performance of chest CT in the diagnosis of the coronavirus disease 2019 (COVID-19) pandemic remains under active investigation. Purpose To evaluate the French national experience using chest CT for COVID-19, results of chest CT and reverse transcription polymerase chain reaction (RT-PCR) assays were compared together and with the final discharge diagnosis used as the reference standard. Materials and Methods A structured CT scan survey (NCT04339686) was sent to 26 hospital radiology departments in France between March 2, 2020, and April 24, 2020. These dates correspond to the peak of the national COVID-19 epidemic. Radiology departments were selected to reflect the estimated geographic prevalence heterogeneities of the epidemic. All symptomatic patients suspected of having COVID-19 pneumonia who underwent both initial chest CT and at least one RT-PCR test within 48 hours were included. The final discharge diagnosis, based on multiparametric items, was recorded. Data for each center were prospectively collected and gathered each week. Test efficacy was determined by using the Mann-Whitney test, Student t test, χ2 test, and Pearson correlation coefficient. P < .05 indicated a significant difference. Results Twenty-six of 26 hospital radiology departments responded to the survey, with 7500 patients entered; 2652 did not have RT-PCR test results or had unknown or excess delay between the RT-PCR test and CT. After exclusions, 4824 patients (mean age, 64 years ± 19 [standard deviation], 2669 male) were included. With final diagnosis as the reference, 2564 of the 4824 patients had COVID-19 (53%). Sensitivity, specificity, negative predictive value, and positive predictive value of chest CT in the diagnosis of COVID-19 were 2319 of 2564 (90%; 95% CI: 89, 91), 2056 of 2260 (91%; 95% CI: 91, 92), 2056 of 2300 (89%; 95% CI: 87, 90), and 2319 of 2524 (92%; 95% CI: 91, 93), respectively. There was no significant difference for chest CT efficacy among the 26 geographically separate sites, each with varying amounts of disease prevalence. Conclusion Use of chest CT for the initial diagnosis and triage of patients suspected of having coronavirus disease 2019 was successful. © RSNA, 2021 Online supplemental material is available for this article.
Assuntos
COVID-19/diagnóstico por imagem , COVID-19/epidemiologia , Radiografia Torácica/métodos , Tomografia Computadorizada por Raios X/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , França/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , SARS-CoV-2 , Sensibilidade e Especificidade , Adulto JovemRESUMO
Dermal papilla cells (DPCs) play a pivotal role in the regulation of hair follicle (HF) growth, formation, and cycling, mainly through paracrine mechanisms. In the last decade, extracellular vesicles (EVs) have been recognized as a new paracrine mechanism that can modify the physiological state of recipient cells by transferring biological material. Herein, we investigated the effect of EVs isolated from stimulated human dermal fibroblasts (DFs) on DPC activation and HF growth. We found that these EVs (st-EVs) enhanced HF growth ex vivo. Comparative transcriptomic analysis on DPCs identified specific activation of the NDP gene, encoding the non-Wnt ligand Norrin. We found that Norrin was secreted by st-EVs-stimulated DPCs activating in a noncell autonomous manner ß-catenin pathway in follicular keratinocytes (human HF keratinocyte [HHFK]) and hair growth ex vivo. Although Norrin-specific receptor Frizzled4 was barely detected in HHFK, we found its presence in DF-EVs. Accordingly, DF-EVs provided Frizzled4 to potentiate Norrin effects ex vivo. Our study identifies DF-EVs as efficient activators of DPCs and Norrin as a novel modulatory player in HF physiopathology. Stem Cells 2019;37:1166-1175.
Assuntos
Proliferação de Células/genética , Derme/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas do Olho/genética , Fibroblastos/metabolismo , Folículo Piloso/metabolismo , Proteínas do Tecido Nervoso/genética , Linhagem Celular , Células Cultivadas , Derme/citologia , Proteínas do Olho/metabolismo , Fibroblastos/citologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Folículo Piloso/citologia , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Proteínas do Tecido Nervoso/metabolismoRESUMO
PURPOSE: To determine the impact of the COVID-19 on the CT activities in French radiological centers during the epidemic peak. MATERIALS AND METHODS: A cross-sectional prospective CT scan survey was conducted between March 16 and April 12, 2020, in accordance with the local IRB. Seven hundred nine radiology centers were invited to participate in a weekly online survey. Numbers of CT examinations related to COVID-19 including at least chest (CTcovid) and whole chest CT scan activities (CTchest) were recorded each week. A sub-analysis on French departments was performed during the 4 weeks of the study. The impact of the number of RT-PCRs (reverse transcriptase polymerase chain reactions) on the CT workflow was tested using two-sample t test and Pearson's test. RESULTS: Five hundred seventy-seven structures finally registered (78%) with mean response numbers of 336 ± 18.9 (323; 351). Mean CTchest activity per radiologic structure ranged from 75.8 ± 133 (0-1444) on week 12 to 99.3 ± 138.6 (0-1147) on week 13. Mean ratio of CTcovid on CTchest varied from 0.36 to 0.59 on week 12 and week 14 respectively. There was a significant relationship between the number of RT-PCR performed and the number of CTcovid (r = 0.73, p = 3.10-16) but no link with the number of positive RT-PCR results. CONCLUSION: In case of local high density COVID-19, CT workflow is strongly modified and redirected to the management of these specific patients. KEY POINTS: ⢠Over the 4-week survey period, 117,686 chest CT (CTtotal) were performed among the responding centers, including 61,784 (52%) CT performed for COVID-19 (CTcovid). ⢠Across the country, the ratio CTcovid/CTtotal varied from 0.36 to 0.59 and depended significantly on the local epidemic density (p = 0.003). ⢠In clinical practice, in a context of growing epidemic, in France, chest CT was used as a surrogate to RT-PCR for patient triage.
Assuntos
Betacoronavirus , Infecções por Coronavirus/diagnóstico , Pandemias , Pneumonia Viral/diagnóstico , Tomografia Computadorizada por Raios X/métodos , Triagem/métodos , Adulto , COVID-19 , Infecções por Coronavirus/epidemiologia , Estudos Transversais , Feminino , França/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Pneumonia Viral/epidemiologia , Estudos Prospectivos , SARS-CoV-2 , Inquéritos e QuestionáriosRESUMO
BACKGROUND: DICER1 syndrome is an autosomal dominant disorder that predisposes individuals to develop benign or malignant tumours from infancy to adulthood. There is low-to-moderate penetrance of tumour development, which is sex- and age-dependent. Multinodular goitre (MNG) is among the most highly penetrant phenotype of the disorder, especially in females. PATIENTS AND METHODS: We report a series of eight families referred for childhood-onset of MNG or DICER1-related tumours with familial history of MNG in relatives. No additional families with these criteria stated were identified during the same date. We screened DNA samples from the probands and members of their family (40) for constitutional DICER1 variants using Next Generation Sequencing tools. RESULTS: Germline pathogenic DICER1 gene variants were identified in all probands and several of their relatives: 64% presented with MNG/thyroidectomy as the phenotypic expression of the syndrome. DICER1 gene variants were identified in the RNAseIII and the PAZ domains. All tumour tissues studied presented clonal pathogenic variants in hotspot regions. Early identification of DICER1 variant carriers has permitted diagnosis and therapeutic scheme correction for two patients and cascade testing in relatives. CONCLUSIONS: Multinodular goitre is uncommon in children. Childhood-onset MNG, multiple occurrences of the disease within the same family, or its association with rare benign or malignant tumours should raise suspicions of anomalies in the DICER1 gene, as proposed by recent international recommendations. Early detection of DICER1 pathogenic variants has important consequences in terms of therapeutic strategy, early tumour screening, and genetic counselling.
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RNA Helicases DEAD-box/genética , Bócio Nodular/metabolismo , Bócio Nodular/patologia , Ribonuclease III/genética , Adolescente , Criança , Feminino , Predisposição Genética para Doença , Bócio Nodular/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação/genética , Síndromes Neoplásicas Hereditárias/genética , Síndromes Neoplásicas Hereditárias/metabolismo , Síndromes Neoplásicas Hereditárias/patologia , LinhagemRESUMO
Heterozygous PAX6 gene mutations leading to haploinsufficiency are the main cause of congenital aniridia, a rare and progressive panocular disease characterized by reduced visual acuity. Up to 90% of patients suffer from aniridia-related keratopathy (ARK), caused by a combination of factors including limbal epithelial stem cell (LSC) deficiency, impaired healing response and abnormal differentiation of the corneal epithelium. It usually begins in the first decade of life, resulting in recurrent corneal erosions, sub-epithelial fibrosis, and corneal opacification. Unfortunately, there are currently no efficient treatments available for these patients and no in vitro model for this pathology. We used CRISPR/Cas9 technology to introduce into the PAX6 gene of LSCs a heterozygous nonsense mutation found in ARK patients. Nine clones carrying a p.E109X mutation on one allele were obtained with no off-target mutations. Compared with the parental LSCs, heterozygous mutant LSCs displayed reduced expression of PAX6 and marked slow-down of cell proliferation, migration and detachment. Moreover, addition to the culture medium of recombinant PAX6 protein fused to a cell penetrating peptide was able to activate the endogenous PAX6 gene and to rescue phenotypic defects of mutant LSCs, suggesting that administration of such recombinant PAX6 protein could be a promising therapeutic approach for aniridia-related keratopathy. More generally, our results demonstrate that introduction of disease mutations into LSCs by CRISPR/Cas9 genome editing allows the creation of relevant cellular models of ocular disease that should greatly facilitate screening of novel therapeutic approaches. Stem Cells 2018;36:1421-1429.
Assuntos
Aniridia/genética , Sistemas CRISPR-Cas/fisiologia , Epitélio Corneano/metabolismo , Edição de Genes/métodos , Fator de Transcrição PAX6/genética , Aniridia/patologia , HumanosRESUMO
To assess risk factors of recurrent bronchial obstruction and allergic sensitization 3 years after an episode of acute bronchiolitis, whether after ambulatory care treatment or hospitalization. A monocentric prospective longitudinal study including infants aged under 1 year with acute bronchiolitis was performed, with clinical (severity score), biological (serum Krebs von den Lungen 6 antigen), and viral (14 virus by naso-pharyngeal suction detection) assessments. Follow-up included a quaterly telephone interview, and a final clinical examination at 3 years. Biological markers of atopy were also measured in peripheral blood, including specific IgEs towards aero- and food allergens. Complete data were available for 154 children. 46.8% of them had recurrent wheezing (RW). No difference was found according to initial severity, care at home or in the hospital, respiratory virus involved, or existence of co-infection. A familial history of atopy was identified as a risk factor for recurrent bronchial obstruction (60% for RW infants versus 39%, P = 0.02), as living in an apartment (35% versus 15%, P = 0.002). 18.6% of the infants were sensitized, with 48.1% of them sensitized to aeroallergens and 81.5% to food allergens. Multivariate analysis confirmed that a familial history of atopy (P = 0.02) and initial co-infection RSV-hRV (P = 0.02) were correlated with the risk of sensitization to aeroallergens at 3 years. Familial history of atopy and RSV-hRV co-infection are risk factors for recurrent bronchial obstruction and sensitization.
Assuntos
Obstrução das Vias Respiratórias/epidemiologia , Bronquiolite/complicações , Coinfecção/complicações , Infecções por Picornaviridae/complicações , Infecções por Vírus Respiratório Sincicial/complicações , Obstrução das Vias Respiratórias/patologia , Animais , Asma/epidemiologia , Asma/patologia , Bronquiolite/patologia , Bronquiolite/virologia , Pré-Escolar , Coinfecção/virologia , Feminino , Seguimentos , Hospitalização , Humanos , Lactente , Recém-Nascido , Estudos Longitudinais , Masculino , Faringe/virologia , Estudos Prospectivos , Recidiva , Vírus Sincicial Respiratório Humano/isolamento & purificação , Rhinovirus/isolamento & purificação , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Protein S100ß (PS100 ß) and neuron specific enolase (NSE) have been described as biological markers of neuronal damage. The purpose of our study was to assess the prognosis thresholds of these biomarkers in subarachnoid aneurysmal hemorrhage (SAH). METHODS: Forty eight patients admitted following SAH were treated by endovascular coiling. Initial neurologic severity was assessed using the World Federation Neurologic Surgeons (WFNS), Fisher grades, initial Glasgow coma scale (GCS) and SAPS II. PS100ß and NSE plasma concentration were measured daily within the first week. The primary endpoint of the study was the 6-month Glasgow Outcome Score (GOS) dichotomized as poor (GOS 1-3) or good (GOS 4-5). RESULTS: A poor outcome at 6-months was associated with significant higher levels of S100ß value from day 1 to day 7, whereas NSE values were significantly higher from day 5 to day 7. Best threshold value, for prognosis, was obtained at day 5 for PS100ß >0.13 µg/L (specificity 0.95 95% confidence interval (CI) 0.74-1; sensitivity 0.83 95% CI 0.65-0.93) and day 7 for NSE >14.5 µg/L (specificity 0.90 95% CI 0.67-0.98); sensitivity (0.69 95% CI 0.51-0.83)). After multivariate logistic analysis, only PS100ß at day 5 and SAPS II enabled to predict neurological outcome at 6 months (p<0.05). CONCLUSION: PS100ß >0.13 µg/L at day 5 is an independent predicting factor of poor neurological outcome at 6 months following SAH. This result could support the use of this biomarker at the acute phase of SAH to help physician determine the prognosis.
Assuntos
Avaliação de Resultados da Assistência ao Paciente , Subunidade beta da Proteína Ligante de Cálcio S100/sangue , Hemorragia Subaracnóidea/mortalidade , Adulto , Idoso , Biomarcadores , Feminino , Escala de Coma de Glasgow , Escala de Resultado de Glasgow , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Crescimento Neural/sangue , Prognóstico , Estudos Prospectivos , Índice de Gravidade de DoençaRESUMO
Ectodermal dysplasia is a group of congenital syndromes affecting a variety of ectodermal derivatives. Among them, ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome is caused by single point mutations in the p63 gene, which controls epidermal development and homeostasis. Phenotypic defects of the EEC syndrome include skin defects and limbal stem-cell deficiency. In this study, we designed a unique cellular model that recapitulated major embryonic defects related to EEC. Fibroblasts from healthy donors and EEC patients carrying two different point mutations in the DNA binding domain of p63 were reprogrammed into induced pluripotent stem cell (iPSC) lines. EEC-iPSC from both patients showed early ectodermal commitment into K18(+) cells but failed to further differentiate into K14(+) cells (epidermis/limbus) or K3/K12(+) cells (corneal epithelium). APR-246 (PRIMA-1(MET)), a small compound that restores functionality of mutant p53 in human tumor cells, could revert corneal epithelial lineage commitment and reinstate a normal p63-related signaling pathway. This study illustrates the relevance of iPSC for p63 related disorders and paves the way for future therapy of EEC.
Assuntos
Fenda Labial/tratamento farmacológico , Fenda Labial/patologia , Fissura Palatina/tratamento farmacológico , Fissura Palatina/patologia , Displasia Ectodérmica/tratamento farmacológico , Displasia Ectodérmica/patologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/patologia , Quinuclidinas/farmacologia , Sítios de Ligação/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Fenda Labial/genética , Fenda Labial/metabolismo , Fissura Palatina/genética , Fissura Palatina/metabolismo , Displasia Ectodérmica/genética , Displasia Ectodérmica/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio Corneano/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação Puntual , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Alopecia-neurological defects-endocrinopathy (ANE) syndrome is a rare inherited hair disorder, which was shown to result from decreased expression of the RNA-binding motif protein 28 (RBM28). In this study, we attempted to delineate the role of RBM28 in hair biology. First, we sought to obtain evidence for the direct involvement of RBM28 in hair growth. When RBM28 was downregulated in human hair follicle (HF) organ cultures, we observed catagen induction and HF growth arrest, indicating that RBM28 is necessary for normal hair growth. We also aimed at identifying molecular targets of RBM28. Given that an RBM28 homologue was recently found to regulate miRNA biogenesis in C. elegans and given the known pivotal importance of miRNAs for proper hair follicle development, we studied global miRNA expression profile in cells knocked down for RBM28. This analysis revealed that RBM28 controls the expression of miR-203. miR-203 was found to regulate in turn TP63, encoding the transcription factor p63, which is critical for hair morphogenesis. In conclusion, RBM28 contributes to HF growth regulation through modulation of miR-203 and p63 activity.
Assuntos
Alopecia/metabolismo , Doenças do Sistema Endócrino/metabolismo , Regulação da Expressão Gênica , Folículo Piloso/metabolismo , Deficiência Intelectual/metabolismo , MicroRNAs/fisiologia , Proteínas de Ligação a RNA/fisiologia , Fatores de Transcrição/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Alopecia/fisiopatologia , Células Cultivadas , Doenças do Sistema Endócrino/fisiopatologia , Genes Reporter , Cabelo/crescimento & desenvolvimento , Folículo Piloso/crescimento & desenvolvimento , Humanos , Deficiência Intelectual/fisiopatologia , Queratinócitos/metabolismo , Morfogênese , Técnicas de Cultura de Órgãos , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Transfecção , Regulação para CimaRESUMO
The influence of the generalization of cystic fibrosis newborn screening (CFNBS) in France on sweat test (ST) prescription is unknown. In this French retrospective, descriptive, and multicenter study, we studied the indications, number, methods, and results of STs prescribed by a pediatric pulmonologist in children who had a negative CFNBS and an ST for respiratory symptoms in 2012. We included 502 children with 523 STs, performed with four different methods. The main indication was asthma (71.3 %), then chronic cough (52.4 %), atypical lower airway infections (42.2 %), and bronchiectasis (7 %). Four children had a diagnosis of CF (0.8 %), all presenting with chronic productive cough and recurrent respiratory infections. CONCLUSION: Asthma is the most frequent indication of ST in our cohort. Because of the systematic CFNBS in France, some prescriptions should be avoided, particularly in case of severe or moderate asthma with no other associated symptom. Moreover, methods of STs often do not follow the guidelines and need standardization. WHAT IS KNOWN: ⢠Newborn screening (NBS) has become the most frequent circumstance of the diagnosis of cystic fibrosis (CF) in France after its generalization. ⢠The prescription of sweat test (ST) in children with respiratory symptoms who already had a negative NBS has not been studied. WHAT IS NEW: ⢠In children with a negative CF NBS referred to a university hospital for respiratory diseases, despite important variations of ST methods, only 4 children among 502 have been diagnosed as CF. ⢠Despite recommendations, ST prescription should be avoided in children with moderate to severe asthma and no other associated symptom.
Assuntos
Fibrose Cística/diagnóstico , Triagem Neonatal/métodos , Suor/química , Criança , Pré-Escolar , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , França , Humanos , Lactente , Recém-Nascido , Masculino , Pneumologia , Estudos RetrospectivosAssuntos
Mutação , Hipertensão Arterial Pulmonar/genética , Proteínas com Domínio T/genética , Adulto , Biópsia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Ecocardiografia , Feminino , Fibrose/diagnóstico por imagem , Fibrose/genética , Humanos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Hipertensão Arterial Pulmonar/diagnóstico por imagem , Enfisema Pulmonar/diagnóstico por imagem , Enfisema Pulmonar/genética , Tomografia Computadorizada por Raios XRESUMO
Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1(+)/c-Kit(+)/Lin(-) cells and interfered with their enhanced chemotactic migration to stromal cell-derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met-mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.
Assuntos
Movimento Celular/fisiologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Quimiocina CXCL12/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Fator de Crescimento de Hepatócito/metabolismo , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Approximately 6 million people worldwide are suffering from severe visual impairments or blindness due to corneal diseases. Corneal allogeneic transplantation is often required to restore vision; however, shortage in corneal grafts and immunorejections remain major challenges. The molecular basis of corneal diseases is poorly understood largely due to lack of appropriate cellular models. Here, we described a robust differentiation of human-induced pluripotent stem cells (hiPSCs) derived from hair follicles or skin fibroblasts into corneal epithelial-like cells. We found that BMP4, coupled with corneal fibroblast-derived conditioned medium and collagen IV allowed efficient corneal epithelial commitment of hiPSCs in a manner that recapitulated corneal epithelial lineage development with high purity. Organotypic reconstitution assays suggested the ability of these cells to stratify into a corneal-like epithelium. This model allowed us identifying miR-450b-5p as a molecular switch of Pax6, a major regulator of eye development. miR-450b-5p and Pax6 were reciprocally distributed at the presumptive epidermis and ocular surface, respectively. miR-450b-5p inhibited Pax6 expression and corneal epithelial fate in vitro, altogether, suggesting that by repressing Pax6, miR-450b-5p triggers epidermal specification of the ectoderm, while its absence allows ocular epithelial development. Additionally, miR-184 was detectable in early eye development and corneal epithelial differentiation of hiPSCs. The knockdown of miR-184 resulted in a decrease in Pax6 and K3, in line with recent findings showing that a point mutation in miR-184 leads to corneal dystrophy. Altogether, these data indicate that hiPSCs are valuable for modeling corneal development and may pave the way for future cell-based therapy.
Assuntos
Linhagem da Célula/fisiologia , Córnea/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , MicroRNAs/biossíntese , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular/fisiologia , Córnea/citologia , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Camundongos , MicroRNAs/genética , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/biossíntese , Fatores de Transcrição Box Pareados/genética , Células-Tronco Pluripotentes/citologia , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genéticaRESUMO
The mechanisms through which hematopoietic cytokines accelerate revascularization are unknown. Here, we show that the magnitude of cytokine-mediated release of SDF-1 from platelets and the recruitment of nonendothelial CXCR4+ VEGFR1+ hematopoietic progenitors, 'hemangiocytes,' constitute the major determinant of revascularization. Soluble Kit-ligand (sKitL), thrombopoietin (TPO, encoded by Thpo) and, to a lesser extent, erythropoietin (EPO) and granulocyte-macrophage colony-stimulating factor (GM-CSF) induced the release of SDF-1 from platelets, enhancing neovascularization through mobilization of CXCR4+ VEGFR1+ hemangiocytes. Although revascularization of ischemic hindlimbs was partially diminished in mice deficient in both GM-CSF and G-CSF (Csf2-/- Csf3-/-), profound impairment in neovascularization was detected in sKitL-deficient Mmp9-/- as well as thrombocytopenic Thpo-/- and TPO receptor-deficient (Mpl-/-) mice. SDF-1-mediated mobilization and incorporation of hemangiocytes into ischemic limbs were impaired in Thpo-/-, Mpl-/- and Mmp9-/- mice. Transplantation of CXCR4+ VEGFR1+ hemangiocytes into Mmp9-/- mice restored revascularization, whereas inhibition of CXCR4 abrogated cytokine- and VEGF-A-mediated mobilization of CXCR4+ VEGFR1+ cells and suppressed angiogenesis. In conclusion, hematopoietic cytokines, through graded deployment of SDF-1 from platelets, support mobilization and recruitment of CXCR4+ VEGFR1+ hemangiocytes, whereas VEGFR1 is essential for their angiogenic competency for augmenting revascularization. Delivery of SDF-1 may be effective in restoring angiogenesis in individuals with vasculopathies.
Assuntos
Quimiocinas CXC/metabolismo , Citocinas/sangue , Neovascularização Fisiológica , Receptores CXCR4/metabolismo , Regeneração , Células-Tronco/fisiologia , Animais , Plaquetas/metabolismo , Quimiocina CXCL12 , Quimiocinas CXC/genética , Humanos , Isquemia/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CXCR4/genética , Fator de Células-Tronco/metabolismo , Trombocitopenia/metabolismo , Trombopoetina/sangue , Trombopoetina/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Background: Embryonal rhabdomyosarcomas (ERMS) of the uterine cervix and corpus are rare pediatric tumors usually associated with a late age of onset and frequent somatic DICER1 mutation. It may also develop in the context of a familial predisposition such as DICER1 syndrome requiring specific medical care for children and young adults at risk for a broad range of tumors. Case presentation: This is a case of a prepubescent 9-year-old girl who was presented to our department for metrorrhagias due to a vaginal cervical mass, initially classified as a müllerian endocervical polyp on negative myogenin immunostaining. The patient subsequently manifested growth retardation (-2DS) and learning disabilities leading to genetic explorations and the identification of a germline pathogenic DICER1 variant. The family history revealed thyroid diseases in the father, aunt and paternal grandmother before the age of 20. Conclusion: Rare tumors such as cervical ERMS associated with a family history of thyroid disease during infancy could be related to DICER1 syndrome. Identifying at-risk relatives is challenging but necessary to detect early DICER1 spectrum tumors in young patients.
RESUMO
CONTEXT: Pituitary stalk interruption syndrome (PSIS) is rare in the pediatric population. It combines ectopic posterior pituitary stalk interruption and anterior pituitary hypoplasia with hormonal deficiencies. The phenotype is highly heterogeneous and obesity/overweight seems to be underreported in the literature. OBJECTIVE: To identify patients with PSIS and obesity or overweight, describe their phenotype, and compare them with patients with PSIS without overweight/obesity. METHODS: Sixty-nine children and young adults with PSIS in a Toulouse cohort from 1984 to 2019 were studied. We identified 25 obese or overweight patients (OB-OW group), and 44 were nonobese/overweight (NO group). Then the groups were compared. RESULTS: All cases were sporadic. The sex ratio was 1.6. The main reason for consultation in both groups was growth retardation (61% in OB-OW group, 77% in NO group). History of neonatal hypoglycemia was more common in the OB-OW than in the NO group (57% vs 14%, P = .0008), along with extrapituitary malformations (64% vs 20%, P < 0001). The incidence of caesarean section was higher in the OB-OW group (52%) than in the NO group (23%), although not significant (P = .07). CONCLUSION: Patients with PSIS who are obese/overweight display interesting phenotypic differences that suggest hypothalamic defects. Studies are needed that include additional information on hormonal levels, particularly regarding oxytocin and ghrelin.