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1.
Insect Sci ; 27(1): 14-21, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31246335

RESUMO

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments. The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms. Driven by a given tissue-specific Gal4, expressing UAS-transgene or UAS-RNAi (RNA interference) could be used to up- or down-regulate target gene expression, respectively. However, the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock. Here, we describe a simple way to modulate optomotor blind (omb) expression levels in its endogenous expression region of the wing disc. We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch. The repression effect is more sensitive to temperature than that of overexpression. At low temperature, overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi. In contrast, at high temperature RNAi predominates in gene expression regulation. By this strategy, we could manipulate omb expression levels at a moderate level. It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Proteínas com Domínio T/genética , Asas de Animais/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Proteínas com Domínio T/metabolismo
2.
Medicine (Baltimore) ; 98(44): e17821, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31689868

RESUMO

Although many genetic variants related to anti-tuberculosis drug induced liver injury (ATDILI) have been identified, the prediction and personalized treatment of ATDILI have failed to achieve, indicating there remains an area for further exploration. This study aimed to explore the influence of single nucleotide polymorphisms (SNPs) in Bradykinin receptor B2 (BDKRB2), Teneurin transmembrane protein 2 (TENM2), transforming growth factor beta 2 (TGFB2), and solute carrier family 2 member 13 (SLC2A13) on the risk of ATDILI.The subjects comprised 746 Chinese tuberculosis (TB) patients. Custom-by-design 2x48-Plex SNPscanTM kit was employed to genotype 28 selected SNPs. The associations of SNPs with ATDILI risk and clinical phenotypes were analyzed according to the distributions of allelic and genotypic frequencies and different genetic models. The odds ratio (OR) with corresponding 95% confidence interval (CI) was calculated.Among subjects with successfully genotyped, 107 participants suffered from ATDILI during follow-up. In BDKRB2, patients with rs79280755 G allele or rs117806152 C allele were more vulnerable to ATDILI (PBonferronicorrection = .002 and .03, respectively). Rs79280755 increased the risk of ATDILI significantly whether in additive (OR = 3.218, 95% CI: 1.686-6.139, PBonferroni correction = .003) or dominant model (PBonferroni correction = .003), as well as rs117806152 (Additive model: PBonferroni correction = .05; dominant model: PBonferroni correction = .03). For TENM2, rs80003210 G allele contributed to the decreased risk of ATDILI (PBonferroni correction = .02), while rs2617972 A allele conferred susceptibility to ATDILI (PBonferroni correction = .01). Regarding rs2617972, significant findings were also observed in both additive (OR = 3.203, 95% CI: 1.487-6.896, PBonferroni correction = .02) and dominant model (PBonferroni correction = .02). Moreover, rs79280755 and rs117806152 in BDKRB2 significantly affected some laboratory indicators. However, no meaningful SNPs were observed in TGFB2 and SLC2A13.Our study revealed that both BDKRB2 and TENM2 genetic polymorphisms were interrogated in relation to ATDILI susceptibility and some laboratory indicators in the Western Chinese Han population, shedding a new light on exploring novel biomarkers and targets for ATDILI.


Assuntos
Antituberculosos/efeitos adversos , Sinalização do Cálcio/genética , Doença Hepática Induzida por Substâncias e Drogas/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Receptor B2 da Bradicinina/genética , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Feminino , Marcadores Genéticos , Predisposição Genética para Doença , Proteínas Facilitadoras de Transporte de Glucose/genética , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Estudos Prospectivos , Fator de Crescimento Transformador beta2/genética
3.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(11): 1111-1114, 2019 Nov 10.
Artigo em Chinês | MEDLINE | ID: mdl-31703138

RESUMO

OBJECTIVE: To explore the clinical features and molecular basis for a child featuring infantile epilepsy and developmental disorders. METHODS: Clinical data and peripheral blood samples of the child and his parents were collected. The coding regions of genes associated with nervous system development were subjected to target region capture sequencing. RESULTS: The child developed generalized spasm at 3 months and was diagnosed with epilepsy at 6 months of age. He was treated with Depakin but was diagnosed with mental retardation and developmental retardation at 3 years of age. A novel heterozygous c.3842T to G variant of the SYNE1 gene was detected. His father was found to carry the same variant and had a history of convulsions in infancy but with no mental or developmental anomalies. CONCLUSION: A novel variant of SYNE1 gene was identified in this child, and the prognosis may be poor.


Assuntos
Deficiências do Desenvolvimento/genética , Epilepsia/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Pré-Escolar , Proteínas do Citoesqueleto , Deficiências do Desenvolvimento/complicações , Epilepsia/complicações , Humanos , Lactente , Deficiência Intelectual/complicações , Deficiência Intelectual/genética , Masculino , Mutação , Convulsões
4.
Yi Chuan ; 41(10): 905-918, 2019 Oct 20.
Artigo em Chinês | MEDLINE | ID: mdl-31624053

RESUMO

Brain development diseases refer to a group of diseases that affect the development of the brain or the central nervous system. Autosomal recessive primary microcephaly (MCPH) is a typical neurodevelopmental disorder characterized by a decreased brain size, mental retardation and abnormal behaviors. To date, at least 25 genes have been discovered to cause MCPH when mutated. These genes were named MCPH1-25 according to the discovery order. MCPH proteins play important roles in regulating brain developmental signaling pathways. Here, we provide a timely review of the expression patterns, cellular localization, molecular functions, phenotypes, as well as animal models of these 25 MCPH proteins that will expedite our understanding of the pathogenesis of brain disorders at both molecular and cellular levels.


Assuntos
Proteínas de Ciclo Celular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microcefalia/genética , Proteínas do Tecido Nervoso/genética , Animais , Encéfalo , Humanos , Microcefalia/patologia , Mutação , Fenótipo
5.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(3): 298-304, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31631593

RESUMO

Objective: To determine the effect of Huntingtin-associated protein 1 ( Hap1) on fibroblast proliferation. Methods: Hap1 knockout ( Hap1 -/-) primary fibroblasts were isolated and cultured in vitro. The proliferation of Hap1 -/- fibroblasts was detected by EdU proliferation assay and cell flow assay. Transcriptome sequencing of the wild-type and Hap1 -/- fibroblasts was screened for proliferation-related genes. Real-time quantitative PCR (qPCR) was performed to verify changes in expressions of related genes. Skin repair was examined in Hap1 knockdown mice with skin wounds. The proliferation of fibroblasts during wound repair was detected by PCNA immunohistochemical staining. Results: Hap1 -/- fibroblasts were successfully cultured. Compared with WT, EdU-positive fibroblasts decreased in Hap1 -/-,with less cells entering the S phase. Transcriptome sequencing of primary fibroblasts identified genes of Cdc25C, E2f7, E2f8 and Ccl5. qPCR confirmed that Hap1 knockout increased E2f7 expression. Hap1 +/- mice had larger skin lesions, slower healing and lower positive density of fibroblast proliferation than those of wild type mice. Conclusion: Hap1 may positively regulate fibroblast proliferation by inhibiting the expression of cell cycle negative regulator E2f7.Its deletion inhibits fibroblasts entering the S phase, thereby reducing cell proliferation and affecting wound repair.


Assuntos
Proliferação de Células , Fibroblastos/citologia , Proteínas do Tecido Nervoso/genética , Cicatrização , Animais , Células Cultivadas , Técnicas de Inativação de Genes , Camundongos , Camundongos Knockout , Pele/patologia
6.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(9): 935-937, 2019 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-31515794

RESUMO

OBJECTIVE: To explore the genetic basis for a patient with autism. METHODS: High-throughput sequencing was carried out to detect copy number variations in the patient. RESULTS: DNA sequencing found that the patient has carried a 0.11 Mb deletion in distal 2p16.3 spanning from genomic position 50 820 001 to 50 922 000, which resulted removal of exon 6 and part of intron 7 of the NRXN1 gene. The same deletion was not found his parents and brother. CONCLUSION: Partial deletion of the NRXN1 gene may underlie the disease in this patient.


Assuntos
Transtorno Autístico/genética , Moléculas de Adesão Celular Neuronais/genética , Deleção de Genes , Proteínas do Tecido Nervoso/genética , Variações do Número de Cópias de DNA , Humanos , Masculino
7.
J Cancer Res Clin Oncol ; 145(11): 2713-2723, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31552488

RESUMO

BACKGROUND: During the development of tumors, tumors "educate" platelets causing changes in their mRNAs expression profiles and phenotypes, thereby, tumor-educated platelet (TEP) mRNA profile has the potential to diagnose lung cancer. The current study aimed to examine whether TEPs might be a potential biomarker for lung cancer diagnostics. METHODS: Platelet precipitation was obtained by low-speed centrifugation and subjected to Trizol for total RNA extraction. Platelet MAX, MTURN, and HLA-B mRNA were selected by microarray, validated by qPCR, and analyzed combined with related clinical factors. RESULTS: Our results showed that a three-platelet mRNA set: MAX, MTURN, and HLA-B was significantly up-regulated in lung cancer patients as well as in early-stage lung cancer patients compared with those from healthy donors, the area under the curve (AUC) was 0.734, 0.787, respectively, among which platelet MTURN mRNA processed a dramatically high diagnostic efficiency in female patients with lung cancer, its AUC for female was 0.825. More importantly, the three-platelet mRNA set: MAX, MTURN, and HLA-B was associated with chemotherapeutic effect, low mRNA expression of this three-platelet set was correlated with "favorable" first chemotherapy response. CONCLUSIONS: A three-platelet mRNA set: MAX, MTURN and HLA-B enables blood-based lung cancer diagnosis and chemotherapy response prediction.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Plaquetas/metabolismo , Antígenos HLA-B/genética , Neoplasias Pulmonares/diagnóstico , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Adenocarcinoma/sangue , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/sangue , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , Feminino , Perfilação da Expressão Gênica , Antígenos HLA-B/sangue , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/sangue , Prognóstico , RNA Mensageiro/sangue , Carcinoma de Pequenas Células do Pulmão/sangue , Carcinoma de Pequenas Células do Pulmão/genética
8.
Zh Nevrol Psikhiatr Im S S Korsakova ; 119(7. Vyp. 2): 74-82, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31532594

RESUMO

OBJECTIVE: To study clinical and neurophysiological data of early infantile epileptic encephalopathy type 14 caused by KCNT1 mutations. MATERIAL AND METHODS: For the period 2017 to 2019, 3 non-relative girls with clinical characteristics of epilepsy of infancy with migrating focal seizures (EIMFS) and mutations in the KCNT1 gene are identified and studied. DNA sequencing was performed using the Hereditary epilepsy panel (Next Generation Sequencing on platform IlluminaNextSeq 500, USA). Dynamical video-EEG monitoring was done with "Encephalan-Video" RM-19/26 ("Medicom MTD", Russia). RESULTS AND CONCLUSION: De novo KCNT1 mutations are identified and studied in three unrelated Russian girls: M.V., 3 years and 3 month old, T.V., 9 month old and M.A., 5 month old. M.V. has the previously unknown mutation in exon 12 (chr9:138656907C>T) with amino acid substitution Arg356Trp. T.V. has the previously described mutation in chromosome 9: 138651532G>A with amino acid substitution Gly288Ser (OMIM: 608167.0010). M.U. has the previously unknown mutation in exon 15 (chr9:138660712A>G) with amino acid substitution Asp480Gly. M.V. has seizure onset at the age of 4 month with behavioral arrest seizures and tonic versive seizures. T.V. developed seizures at 4,5 month in the manner of behavior arrest and ophthalmo-clonic seizures with hyperemia of face. M.U. has neonatal seizures with bilateral tonic-clonic seizures, cyanosis and further development of status epilepticus of alternating hemiconvulsive seizures. Further all the girls develop polymorphic seizures of multiregional genesis up to migrating status epilepticus with typical electro-clinical pattern of EIMFS. Therefore, KCNT1 is likely to be a major gene causing this rare and severe epileptic syndrome.


Assuntos
Epilepsia , Proteínas do Tecido Nervoso , Canais de Potássio , Convulsões , Espasmos Infantis , Eletroencefalografia , Feminino , Humanos , Lactente , Mutação , Proteínas do Tecido Nervoso/genética , Canais de Potássio/genética , Federação Russa , Convulsões/etiologia , Convulsões/genética , Espasmos Infantis/diagnóstico , Espasmos Infantis/genética
9.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(8): 817-820, 2019 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-31400136

RESUMO

OBJECTIVE: To explore clinical and genetic features of a pedigree affected with autosomal recessive neuromyotonia and axonal neuropathy (NMAN). METHODS: For the proband and her parents, clinical data was collected, genomic DNA was extracted from peripheral blood samples. Triplet primed-PCR was carried out to detect dynamic mutation of DMPK and ZNF9 genes, which are responsible for myotonic dystrophy, by capillary electrophoresis. High-throughput sequencing was used to screen variants of candidate genes for Mendelian disorders involving the nervous system. Candidate variants were confirmed by Sanger sequencing. The genotype of the variant was determined in the parents and 100 healthy controls. Pathogenicity of the variant was assessed by ACMG criterion. RESULTS: Mutation of DMPK and ZNF9 genes was excluded. DNA sequencing has identified a homozygous missense variant (c.335C>T, p.R119W) in the HINT1 gene. Both parents were found to carry the variant. The same variant was not found among the healthy controls. According to the ACMG criterion, the missense variant was classified as a pathogenic variant. CONCLUSION: The c.335C>T (p.R119W) of the HINT1 gene probably underlie the disease in this pedigree. Above finding provided further evidence for the connection between HINT1 and NMAN and enriched the mutation spectrum of HINT1 gene.


Assuntos
Síndrome de Isaacs/genética , Proteínas do Tecido Nervoso/genética , Feminino , Genótipo , Homozigoto , Humanos , Linhagem
10.
Nat Commun ; 10(1): 3407, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31431620

RESUMO

The worldwide incidence of pulmonary carcinoids is increasing, but little is known about their molecular characteristics. Through machine learning and multi-omics factor analysis, we compare and contrast the genomic profiles of 116 pulmonary carcinoids (including 35 atypical), 75 large-cell neuroendocrine carcinomas (LCNEC), and 66 small-cell lung cancers. Here we report that the integrative analyses on 257 lung neuroendocrine neoplasms stratify atypical carcinoids into two prognostic groups with a 10-year overall survival of 88% and 27%, respectively. We identify therapeutically relevant molecular groups of pulmonary carcinoids, suggesting DLL3 and the immune system as candidate therapeutic targets; we confirm the value of OTP expression levels for the prognosis and diagnosis of these diseases, and we unveil the group of supra-carcinoids. This group comprises samples with carcinoid-like morphology yet the molecular and clinical features of the deadly LCNEC, further supporting the previously proposed molecular link between the low- and high-grade lung neuroendocrine neoplasms.


Assuntos
Biomarcadores Tumorais/genética , Tumor Carcinoide/genética , Carcinoma de Células Grandes/genética , Neoplasias Pulmonares/genética , Carcinoma de Pequenas Células do Pulmão/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Tumor Carcinoide/mortalidade , Tumor Carcinoide/patologia , Carcinoma de Células Grandes/mortalidade , Carcinoma de Células Grandes/patologia , Hibridização Genômica Comparativa , Conjuntos de Dados como Assunto , Feminino , Genômica , Proteínas de Homeodomínio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Pulmão/patologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Aprendizado de Máquina , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Prognóstico , Carcinoma de Pequenas Células do Pulmão/mortalidade , Carcinoma de Pequenas Células do Pulmão/patologia , Taxa de Sobrevida , Adulto Jovem
11.
Photochem Photobiol Sci ; 18(10): 2509-2520, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31432859

RESUMO

We investigated changes in behavior, physiology and selected brain regions during the development of vernal migration and reproduction phenotypes in migratory redheaded buntings. We monitored 24 h activity-rest pattern and measured food intake, fat deposition, and body mass of buntings exposed for 12 weeks to short (SP, 8L : 16D) and long (LP, 13L : 11D) photoperiods at 22 ± 2 °C temperature. Under LP, not SP, buntings exhibited a photostimulated spring migration phenotype (hyperphagia, fat deposition and body mass gain). However, there were sex differences in the development of vernal migration, as shown by faster and earlier induction of Zugunruhe (nocturnal migratory restlessness) in males than in females. In the next experiment, increasing photoperiods over 12 weeks following the vernal equinox induced behavioural and physiological changes associated with vernal migration phenotypes in both male and female buntings, but in a sex-dependent manner. In a subsequent experiment over 8 weeks corresponding to the spring migration period we found an increased expression of CART, not NPY, in INc, and decreased expression of GnRH-I in POA in the brain by week 6 of the observation under increasing photoperiods. There was also an increased expression of doublecortin (a marker of neuronal incorporation) in the olfactory bulb and song control nuclei (Area X and HVC, higher vocal centre) in male birds. These results demonstrate changes in the brain peptides and neuronal recruitment along with changes in the behaviour and physiology, and give insights into the concurrent photoperiodic induction of the seasonal response at multiple levels in migratory songbirds.


Assuntos
Migração Animal/fisiologia , Neurônios/metabolismo , Passeriformes/fisiologia , Animais , Feminino , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fotoperíodo , Estações do Ano
12.
Nat Neurosci ; 22(8): 1223-1234, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31332372

RESUMO

Social deficit is a core clinical feature of autism spectrum disorder (ASD) but the underlying neural mechanisms remain largely unclear. We demonstrate that structural and functional impairments occur in glutamatergic synapses in the pyramidal neurons of the anterior cingulate cortex (ACC) in mice with a mutation in Shank3, a high-confidence candidate ASD gene. Conditional knockout of Shank3 in the ACC was sufficient to generate excitatory synaptic dysfunction and social interaction deficits, whereas selective enhancement of ACC activity, restoration of SHANK3 expression in the ACC, or systemic administration of an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-positive modulator improved social behavior in Shank3 mutant mice. Our findings provide direct evidence for the notion that the ACC has a role in the regulation of social behavior in mice and indicate that ACC dysfunction may be involved in social impairments in ASD.


Assuntos
Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/patologia , Giro do Cíngulo/patologia , Proteínas do Tecido Nervoso/genética , Comportamento Social , Animais , Dioxóis/farmacologia , Modelos Animais de Doenças , Ácido Glutâmico , Asseio Animal , Giro do Cíngulo/fisiopatologia , Relações Interpessoais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Optogenética , Piperidinas/farmacologia , Células Piramidais/patologia , Receptores de AMPA/agonistas , Sinapses/patologia
13.
Fish Shellfish Immunol ; 92: 680-689, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31271837

RESUMO

The Notch signaling pathway is known to regulate innate immunity by influencing macrophage function and interacting with the Toll-like receptor (TLR) signaling pathway. However, the comprehensive role of the Notch signaling pathway in the innate immune response remains unknown. To assess the function of Notch1a in immunity, we examined the innate immune responses to Vibrio parahaemolyticus strain Vp13 of wild-type (WT) and notch1a-/- zebrafish larvae generated using the clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system. The median lethal dose (LD50) of V. parahaemolyticus was significantly lower in notch1a-/- larvae than in WT larvae 3 days post fertilization (dpf). Transcriptome data analysis revealed 359 significantly differentially expressed genes (DEGs), including 246 significantly down-regulated genes and 113 significantly up-regulated genes, in WT infected groups compared with WT control groups. In contrast, 986 significantly DEGs were found in notch1a-/- infected groups compared with notch1a-/- control groups, of which 82 genes were significantly down-regulated and 904 genes were significantly up-regulated. These DEGs belonged to the tumor necrosis factor (TNF), complement, nuclear factor kappa B (NF-κB), cathepsin, interleukin (IL), chemokine, serpin peptidase inhibitor, matrix metallopeptidase, innate immune cells, pattern recognition receptor (PRR), and other cytokine families. Our results indicate that Notch1a plays roles in inhibiting many immunity-related genes and could comprehensively mediate the innate immune response by regulating TLRs, nucleotide-binding-oligomerization-domain-like receptors (NLRs), lectins, complement, ILs, chemokines, TNF, cathepsin, and serpin. Further studies are required to understand the specific mechanism of Notch1a in innate immunity.


Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Imunidade Inata/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Receptor Notch1/genética , Receptor Notch1/imunologia , Transdução de Sinais/imunologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Animais , Doenças dos Peixes/imunologia , Vibrioses/imunologia , Vibrioses/veterinária , Vibrio parahaemolyticus/fisiologia
14.
Neuron ; 103(4): 583-597.e8, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31272828

RESUMO

Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the "plug-and-play" nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.


Assuntos
Técnicas de Introdução de Genes/métodos , Genômica/métodos , Engenharia de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Dependovirus/genética , Edição de Genes/métodos , Vetores Genéticos/genética , Humanos , Imunoquímica/métodos , Inteínas , Camundongos , Mutagênese Insercional , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteômica , RNA Guia/genética , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência do Ácido Nucleico
15.
Toxicol Lett ; 313: 188-195, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31284022

RESUMO

Brucine is one of the main bioactive and toxic constituents of the herb drug Semen Strychni. Here we aimed to determine dosing time-dependent hepatotoxicity of brucine, and to investigate the role of metabolism in generation of brucine chronotoxicity. Brucine was administered to wild-type or Npas2-/- (a clock disrupted model) mice at different circadian time points for toxicity and pharmacokinetic characterization. The hepatotoxicity was evaluated by plasma alanine aminotransferase and aspartate aminotransferase measurements and histopathological analysis. The role of Cyp3a11 in brucine metabolism was determined by chemical inhibition assays and Cyp3a11-overexpressing HEK293 cells. Hepatic circadian Cyp3a11 mRNA and protein levels were determined by qPCR and Western blotting, respectively. The toxicity of brucine was more severe in the light phase [Zeitgeber time (ZT) 2 and ZT8] than in the dark phase (ZT14 and ZT20). Chemical inhibition and substrate metabolism assays suggested Cyp3a11 as a significant contributor to brucine metabolism. The Cyp3a11 mRNA, protein and activity in the livers of wild-type mice displayed significant circadian fluctuations. Npas2 ablation markedly down-regulated Cyp3a11 mRNA, protein and activity, and abrogated their circadian rhythms. The circadian time differences in brucine pharmacokinetics and liver distribution were lost in Npas2-/- mice, so were the time differences in brucine hepatotoxicity. In conclusion, chronotoxicity of brucine was determined by circadian variations in Cyp3a11 metabolism. The findings have implications in improving brucine (and possibly Semen Strychni) efficacy via dosing time optimization.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Ritmo Circadiano , Citocromo P-450 CYP3A/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Proteínas de Membrana/metabolismo , Fotoperíodo , Estricnina/análogos & derivados , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Ritmo Circadiano/genética , Cronoterapia Farmacológica , Células HEK293 , Humanos , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Estricnina/administração & dosagem , Estricnina/metabolismo , Estricnina/farmacocinética , Estricnina/toxicidade
16.
Nat Commun ; 10(1): 2983, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278301

RESUMO

Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug development.


Assuntos
Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Matriz Extracelular/metabolismo , Feminino , Adesões Focais/genética , Humanos , Microscopia Intravital , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Processamento de RNA/genética , RNA Interferente Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Transdução de Sinais/genética , Análise de Sobrevida , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/mortalidade
17.
Life Sci ; 232: 116630, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31279783

RESUMO

AIMS: Lung adenocarcinoma consists of multiple therapeutic targets, however, patients will inevitably progress to later stage diagnosis with Tyrosine Kinase Inhibitor treatment resistance. We aim to investigate the roles of non-coding TUSC7 in ordering the cell division tendency, helping to sensitize the resistance in a miRNA incorporating way. MATERIALS AND METHODS: Online study of bioinformatics analysis, molecular experiments of luciferase test, immunofluorescence staining and qRT-PCR were applied to dig out the mechanistic regulations. KEY FINDINGS: TUSC-7 inhibited the renewal ability of adenocarcinoma stem cells, yielding to asymmetric cell splitting. Informatics analysis and the luciferase testing confirmed the 3'UTR binding site, and revealed the post-transcriptional regulation of NUMB referring to miR-146. TUSC-7 sponged miR-146 and abolished its degradation toward to NUMB, and this integrated cascade made several genes become tangled to full functionality. SIGNIFICANCE: TUSC-7 was proved to be one strong suppressive lnc-RNA in lung adenocarcinoma stem cells, functioning through inactivating NOTCH signaling, and the turbulence on division modes precisely pointed to the key mechanisms of stem cells' renewal. The decreasing of tumor suppressive miR-146 was necessary in TUSC-7 conducted renewal repression, despite it alone could also reduce the renewal efficiency, indicating that more complicated non-coding genes may be involved in its regulation.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/metabolismo , RNA Longo não Codificante/fisiologia , Regiões 3' não Traduzidas , Adenocarcinoma de Pulmão/genética , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo
18.
Neoplasma ; 66(6): 908-917, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31307196

RESUMO

miRNA has shown its potential in the regulation of squamous cell carcinoma (SCC). However, the mechanism of such an effect was not quite clear. Therefore, we aimed to investigate whether miR-30a-5p participated in the regulation of cutaneous SCC (cSCC) and the possible mechanism involved. 5-Ethynyl-2'-deoxyuridine (EdU) and cell cycle were measured using flow cytometry. The formation of cell colony was tested by colony formation assay. The capacities of migration and invasion were tested by wound healing assay and Transwell invasion assay, respectively. The target of miR-30a-5p was predicted by bioinformatics and identified by luciferase assay. Western blot was used for the determination of proteins and qPCR was for mRNA levels. miR-30a-5p expression was lowered in SCL-1 and A431 cells, and its upregulation suppressed EdU positive cells, colony numbers, migration, invasion and Bcl-2 expression, and elevated Bcl-2-associated X protein (Bax) and cleaved Caspase-3 expressions, arresting cell cycle in G1 phase. Moreover, forkhead box protein G1 (FOXG1) was proved to be the target of miR-30a-5p, and FOXG1 overexpression partially offsets the decreased colony numbers, migration and invasion rates due to miR-30a-5p overexpression in SCL-1 and A431 cells. miR-30a-5p showed a regulatory role on the expression of FOXG1 and further modulated the progressing of cSCC cells, which could be a novel pathway intervening the development of cSCC.


Assuntos
Carcinoma de Células Escamosas/genética , Fatores de Transcrição Forkhead/genética , MicroRNAs/genética , Proteínas do Tecido Nervoso/genética , Neoplasias Cutâneas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Neoplasias Cutâneas/patologia
19.
Cancer Sci ; 110(9): 2973-2981, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31293054

RESUMO

Every year, approximately 1.2 million cases of colorectal carcinoma (CRC) are newly diagnosed worldwide. Although metastases to distant organs are often fatal complications of CRC, little information is known as to how such metastatic lesions are formed. To reveal the genetic profiles for CRC metastasis, we conducted whole-exome RNA sequencing on CRC tumors with liver metastasis (LM) (group A, n = 12) and clinical stage-matched larger tumors without LM (group B, n = 16). While the somatic mutation profiles were similar among the primary tumors and LM lesions in group A and the tumors in group B, the A-to-C nucleotide change in the context of "AAG" was only enriched in the LM regions in group A, suggesting the presence of a DNA damage process specific to metastasis. Genes already known to be associated with CRC were mutated in all groups at a similar frequency, but we detected somatic nonsynonymous mutations in a total of 707 genes in the LM regions, but not in the tumors without LM. Signaling pathways linked to such "LM-associated" genes were overrepresented for extracellular matrix-receptor interaction or focal adhesion. Further, fusions of the ADAP1 (ArfGAP with dual PH domain 1) were newly identified in our cohort (3 out of 28 patients), which activated ARF6, an ADAP1-substrate. Infrequently, mutated genes may play an important role in metastasis formation of CRC. Additionally, recurrent ADAP1 fusion genes were unexpectedly discovered. As these fusions activate small GTPase, further experiments are warranted to examine their contribution to CRC carcinogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais/genética , Fusão Gênica , Neoplasias Hepáticas/genética , Proteínas do Tecido Nervoso/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/genética , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Mutação Puntual , Sequenciamento Completo do Exoma
20.
Eur J Paediatr Neurol ; 23(4): 657-661, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31176596

RESUMO

GRIN1 encodes the obligate subunit (GluN1) of glutamate N-methyl-d-aspartate receptor (NMDAr). Pathogenic variants in GRIN1 are a well-known cause of infantile encephalopathy characterized by profound developmental delay (DD), variable epileptic phenotypes, and distinctive behavioral abnormalities. Recently, GRIN1 has also been implicated in the pathogenesis of polymicrogyria (PMG). We investigated two patients presenting with severe intellectual disability (ID), epilepsy, stereotyped movements, and abnormal ocular movements. They showed distinctive circadian rhythm alterations and sleep-wake patterns anomalies characterized by recurrent cyclic crying or laughing spells. Genetic analysis led to the identification of two distinct de novo variants in GRIN1 affecting the same amino acid residue of an important functional protein domain. Recent advances in circadian rhythm and sleep regulation suggest that abnormal GluN1 function might play a relevant pathogenetic role for the peculiar behavioral abnormalities observed in GRIN1 patients. Our cases highlight the relevance of circadian rhythm abnormalities in epileptic children as a clue toward GRIN1 encephalopathy and expand the complex phenotypic spectrum of this severe genetic disorder.


Assuntos
Encefalopatias/genética , Ritmo Circadiano/genética , Epilepsia/genética , Proteínas do Tecido Nervoso/genética , Receptores de N-Metil-D-Aspartato/genética , Transtornos do Sono-Vigília/genética , Adolescente , Encefalopatias/fisiopatologia , Feminino , Humanos , Lactente , Deficiência Intelectual/genética , Masculino
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