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1.
Nat Commun ; 11(1): 5078, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033266

RESUMO

Metabolic engineering facilitates chemical biosynthesis by rewiring cellular resources to produce target compounds. However, an imbalance between cell growth and bioproduction often reduces production efficiency. Genetic code expansion (GCE)-based orthogonal translation systems incorporating non-canonical amino acids (ncAAs) into proteins by reassigning non-canonical codons to ncAAs qualify for balancing cellular metabolism. Here, GCE-based cell growth and biosynthesis balance engineering (GCE-CGBBE) is developed, which is based on titrating expression of cell growth and metabolic flux determinant genes by constructing ncAA-dependent expression patterns. We demonstrate GCE-CGBBE in genome-recoded Escherichia coli Δ321AM by precisely balancing glycolysis and N-acetylglucosamine production, resulting in a 4.54-fold increase in titer. GCE-CGBBE is further expanded to non-genome-recoded Bacillus subtilis to balance growth and N-acetylneuraminic acid bioproduction by titrating essential gene expression, yielding a 2.34-fold increase in titer. Moreover, the development of ncAA-dependent essential gene expression regulation shows efficient biocontainment of engineered B. subtilis to avoid unintended proliferation in nature.


Assuntos
Acetilglucosamina/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Vias Biossintéticas , Escherichia coli/crescimento & desenvolvimento , Ácido N-Acetilneuramínico/metabolismo , Bacillus subtilis/metabolismo , Proliferação de Células , Escherichia coli/metabolismo , Código Genético , Proteínas de Fluorescência Verde/metabolismo , Engenharia Metabólica , Análise do Fluxo Metabólico , Regiões Promotoras Genéticas/genética , RNA de Transferência/genética , Tirosina/metabolismo
2.
Zhonghua Zhong Liu Za Zhi ; 42(8): 644-647, 2020 Aug 23.
Artigo em Chinês | MEDLINE | ID: mdl-32867455

RESUMO

Objective: To explore the application value of lung cancer-related gene methylation in lung cancer diagnosis. Methods: Sixty patients with lung cancer underwent surgery were selected as the case group, and 65 patients with benign lung lesions treated in the same period were recruited as the control group. The methylation levels of lung cancer-related genes including dying-associated protein kinase (DAPK), O-6-methylguanine-DNA methyltransferase (MGMT), APC gene promoter 1A (APC1A) and epithelial mucoprotein gene (ECAD) in the blood samples of two groups of patients were analyzed by methylated PCR-specific method. The relationship between methylation of lung cancer-related genes and lung cancer was analyzed and its diagnostic value in lung cancer was evaluated. Results: The methylation detection rates of DAPK, MGMT, APC1A and ECAD in the case group were 68.3%, 68.3%, 63.3% and 65.0%, respectively, all higher than those of the control group (all P<0.05). The multivariate Logistic regression analysis showed that DAPK (OR=0.709), MGMT (OR=0.793), APC1A (OR=0.163), and ECAD (OR=2.047) were all independent influencing factors for lung cancer (all P<0.05). Analysis of the receiver operating characteristic curve showed that, the area under the curve (AUC) of DAPK, MGMT, APC1A and ECAD methylation test for lung cancer predicting were 0.623, 0.680, 0.620 and 0.648, respectively, while the AUC of the combined four gene methylation for lung cancer predicting was 0.829, higher than the AUC of each gene (all P<0.05). Conclusion: The combined methylation detection of multiple lung cancer related genes can improve the diagnostic value of lung cancer, contribute to the early diagnosis of lung cancer, and have potentially clinical application value.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilação de DNA , Proteínas Quinases Associadas com Morte Celular/genética , Neoplasias Pulmonares/diagnóstico , O(6)-Metilguanina-DNA Metiltransferase/genética , Regiões Promotoras Genéticas/genética , Estudos de Casos e Controles , Detecção Precoce de Câncer , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Genética
3.
Anticancer Res ; 40(10): 5503-5508, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988873

RESUMO

BACKGROUND/AIM: Accumulating evidence shows that caspase-8 (Cas-8) rs3834129 genotypes determine susceptibility to various cancers, but their association with nasopharyngeal carcinoma (NPC) has not been examined. We aimed at investigating the association of Cas-8 rs3834129 with NPC risk. MATERIALS AND METHODS: Cas-8 rs3834129 genotypes and their associations with NPC risk were investigated among 176 NPC patients and 352 non-cancer subjects by the PCR-RFLP method. Additionally, the interaction of Cas-8 rs3834129 genotypes with smoking was examined. RESULTS: The II, ID and DD frequencies were 56.8, 36.9 and 6.3% among NPC patients and 54.8, 38.1 and 7.1% among control subjects (ptrend=0.8830). Allelic frequency distribution analysis also indicated that the D allele is not a risk factor for NPC (p=0.6183). There was no interaction between Cas-8 rs3834129 and smoking and NPC risk (p=0.8305). CONCLUSION: Cas-8 rs3834129 genotypes play a minor role in the risk for NPC.


Assuntos
Caspase 8/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Carcinoma Nasofaríngeo/genética , Alelos , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo/epidemiologia , Carcinoma Nasofaríngeo/patologia , Polimorfismo de Fragmento de Restrição/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Fatores de Risco , Fumar , Taiwan
4.
Anticancer Res ; 40(10): 5751-5756, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988902

RESUMO

BACKGROUND/AIM: A single study has shown positive association and genotype-phenotype correlation between metalloproteinase-9 (MMP-9) promoter genotypes and adult acute lymphocytic leukemia (ALL). However, there is no report about childhood ALL. Thus, this study aimed at examining the role of MMP-9 rs3918242 genotypes in childhood ALL risk. PATIENTS AND METHODS: A total of 266 childhood ALL cases and 266 healthy controls in Taiwan were examined for their MMP-9 rs3918242 genotypes via polymerase chain reaction-restriction fragment length polymorphism methodology. RESULTS: The MMP-9 rs3918242 CT or TT genotype carriers only had a slightly increased risk compared with CC carriers (p=0.6386 and 0.6005, respectively). The allelic frequency analysis also supported the idea that the variant T allele at MMP-9 rs3918242 is not differentially distributed between the case and control groups (p=0.4834). CONCLUSION: MMP-9 rs3918242 genotypes may indirectly influence the risk of childhood ALL. Further validations in other populations and analysis of the detail mechanisms are needed.


Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Metaloproteinase 9 da Matriz/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adulto , Alelos , Criança , Feminino , Genótipo , Heterozigoto , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Regiões Promotoras Genéticas/genética
5.
Nat Commun ; 11(1): 4544, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917861

RESUMO

Stratification of enhancers by signal strength in ChIP-seq assays has resulted in the establishment of super-enhancers as a widespread and useful tool for identifying cell type-specific, highly expressed genes and associated pathways. We examine a distinct method of stratification that focuses on peak breadth, termed hyperacetylated chromatin domains (HCDs), which classifies broad regions exhibiting histone modifications associated with gene activation. We find that this analysis serves to identify genes that are both more highly expressed and more closely aligned to cell identity than super-enhancer analysis does using multiple data sets. Moreover, genetic manipulations of selected gene loci suggest that some enhancers located within HCDs work at least in part via a distinct mechanism involving the modulation of histone modifications across domains and that this activity can be imported into a heterologous gene locus. In addition, such genetic dissection reveals that the super-enhancer concept can obscure important functions of constituent elements.


Assuntos
Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Loci Gênicos/genética , Ativação Transcricional , Acetilação , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Conjuntos de Dados como Assunto , Embrião de Mamíferos , Eritroblastos , Feminino , Feto , Código das Histonas/genética , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , RNA-Seq
6.
Nat Commun ; 11(1): 4634, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929078

RESUMO

The current opioid epidemic necessitates a better understanding of human addiction neurobiology to develop efficacious treatment approaches. Here, we perform genome-wide assessment of chromatin accessibility of the human striatum in heroin users and matched controls. Our study reveals distinct neuronal and non-neuronal epigenetic signatures, and identifies a locus in the proximity of the gene encoding tyrosine kinase FYN as the most affected region in neurons. FYN expression, kinase activity and the phosphorylation of its target Tau are increased by heroin use in the post-mortem human striatum, as well as in rats trained to self-administer heroin and primary striatal neurons treated with chronic morphine in vitro. Pharmacological or genetic manipulation of FYN activity significantly attenuates heroin self-administration and responding for drug-paired cues in rodents. Our findings suggest that striatal FYN is an important driver of heroin-related neurodegenerative-like pathology and drug-taking behavior, making FYN a promising therapeutic target for heroin use disorder.


Assuntos
Cromatina/metabolismo , Corpo Estriado/enzimologia , Dependência de Heroína/enzimologia , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Animais , Sequência de Bases , Comportamento Animal/efeitos dos fármacos , Sinais (Psicologia) , Genoma , Células HEK293 , Heroína/efeitos adversos , Humanos , Masculino , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Ratos Long-Evans , Autoadministração , Transcrição Genética/efeitos dos fármacos , Proteínas tau/metabolismo
7.
Medicine (Baltimore) ; 99(38): e22278, 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32957381

RESUMO

BACKGROUND: Diabetic nephropathy (DN) is a multifactorial disease with gene-environment interaction resulting in progressive renal function damage. Multiple studies have assessed the association between matrix metalloproteinase-9 (MMP-9) gene promoter polymorphism and DN susceptibility. However, the results are inconclusive. In the present study, we will conduct a meta-analysis to further examine this relationship more precisely. METHODS: Electronic databases (Pubmed, Web of Science, Embase, Google Scholar, Wanfang, China Biological Medicine and China National Knowledge Infrastructure) will be used to search clinical case-control studies about MMP-9 polymorphism and DN published until 18 August 2020. The language will be restricted to Chinese and English. Two reviewers will take charge of completing the selection of study, the extraction of data as well as the assessment of study quality independently. The Newcastle-Ottawa Scale will be used to evaluate the study quality. We will evaluate the association under 5 genetic models. Fixed-effects or random-effects models will be used to calculate the effect sizes of odds ratio and 95% confidence intervals. Afterwards, subgroup analysis will be conducted in terms of the ethnicity and genotyping method. Additionally, sensitivity analysis will be performed via sequentially omitting each of the included studies one at a time. The funnel plots, Egger regression test, and Begg rank correlation test will be used to test the potential publication bias. All the statistical analyses will be performed using Review Manager 5.3 and Stata 12.0. RESULTS: This protocol reported according to the Preferred Reporting ltems for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) statement. This study will provide a better understanding of the association between MMP-9 polymorphisms and DN risk. CONCLUSION: Publishing this protocol will minimize the potential bias related to data mining, thus contributing to generation of reliable evidence. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/H5FS4.


Assuntos
Nefropatias Diabéticas/genética , Metaloproteinase 9 da Matriz/genética , Metanálise como Assunto , Polimorfismo Genético , Revisões Sistemáticas como Assunto , Nefropatias Diabéticas/enzimologia , Predisposição Genética para Doença , Humanos , Regiões Promotoras Genéticas/genética , Fatores de Risco
8.
Nat Commun ; 11(1): 4126, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807804

RESUMO

Neisseria gonorrhoeae is an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association to identify loss-of-function (LOF) mutations in the efflux pump mtrCDE operon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical relative to urethral isolates. This enrichment holds true for LOF mutations in another efflux pump, farAB, and in urogenitally-adapted versus typical N. meningitidis, providing evidence for a model in which expression of these pumps in the female urogenital tract incurs a fitness cost for pathogenic Neisseria. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility.


Assuntos
Proteínas de Bactérias/metabolismo , Colo do Útero/microbiologia , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Animais , Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Testes de Sensibilidade Microbiana , Mutação/genética , Neisseria gonorrhoeae/metabolismo , Óperon/genética , Regiões Promotoras Genéticas/genética
9.
PLoS One ; 15(7): e0233582, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735620

RESUMO

The craniofacial developmental disorder Burn-McKeown Syndrome (BMKS) is caused by biallelic variants in the pre-messenger RNA splicing factor gene TXNL4A/DIB1. The majority of affected individuals with BMKS have a 34 base pair deletion in the promoter region of one allele of TXNL4A combined with a loss-of-function variant on the other allele, resulting in reduced TXNL4A expression. However, it is unclear how reduced expression of this ubiquitously expressed spliceosome protein results in craniofacial defects during development. Here we reprogrammed peripheral mononuclear blood cells from a BMKS patient and her unaffected mother into induced pluripotent stem cells (iPSCs) and differentiated the iPSCs into induced neural crest cells (iNCCs), the key cell type required for correct craniofacial development. BMKS patient-derived iPSCs proliferated more slowly than both mother- and unrelated control-derived iPSCs, and RNA-Seq analysis revealed significant differences in gene expression and alternative splicing. Patient iPSCs displayed defective differentiation into iNCCs compared to maternal and unrelated control iPSCs, in particular a delay in undergoing an epithelial-to-mesenchymal transition (EMT). RNA-Seq analysis of differentiated iNCCs revealed widespread gene expression changes and mis-splicing in genes relevant to craniofacial and embryonic development that highlight a dampened response to WNT signalling, the key pathway activated during iNCC differentiation. Furthermore, we identified the mis-splicing of TCF7L2 exon 4, a key gene in the WNT pathway, as a potential cause of the downregulated WNT response in patient cells. Additionally, mis-spliced genes shared common sequence properties such as length, branch point to 3' splice site (BPS-3'SS) distance and splice site strengths, suggesting that splicing of particular subsets of genes is particularly sensitive to changes in TXNL4A expression. Together, these data provide the first insight into how reduced TXNL4A expression in BMKS patients might compromise splicing and NCC function, resulting in defective craniofacial development in the embryo.


Assuntos
Processamento Alternativo , Atresia das Cóanas/patologia , Surdez/congênito , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Ribonucleoproteína Nuclear Pequena U5/deficiência , Spliceossomos/fisiologia , Apoptose , Diferenciação Celular , Técnicas de Reprogramação Celular , Atresia das Cóanas/genética , Células Clonais , Surdez/genética , Surdez/patologia , Transição Epitelial-Mesenquimal , Éxons/genética , Face/embriologia , Facies , Feminino , Cabeça/embriologia , Cardiopatias Congênitas/genética , Humanos , Crista Neural/citologia , Regiões Promotoras Genéticas/genética , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U5/genética , Deleção de Sequência , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Via de Sinalização Wnt
10.
Nat Commun ; 11(1): 3883, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753598

RESUMO

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Rearranjo Gênico , Glioma/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Idoso , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Adutos de DNA/efeitos dos fármacos , Adutos de DNA/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/prevenção & controle , Regiões Promotoras Genéticas/genética , RNA-Seq , Temozolomida/uso terapêutico , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Sequenciamento Completo do Genoma , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
11.
PLoS One ; 15(8): e0238032, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841304

RESUMO

Longan (Dimocarpus longan Lour.) is an important commercial fruit tree in southern China. The embryogenesis of longan affects the quality and yield of fruit. A large number of alternative splicing events occurs during somatic embryogenesis (SE), which is regulated by serine/arginine-rich (SR) proteins. However, the functions of SR proteins in longan are poorly understood. In this study, 21 Dlo-SR gene family members belonging to six subfamilies were identified, among which Dlo-RSZ20a, Dlo-SR30, Dlo-SR17, Dlo-SR53 and Dlo-SR32 were localized in the nucleus, Dlo-RSZ20b, Dlo-RSZ20c, Dlo-RSZ20d, Dlo-SC18, Dlo-RS2Z29, Dlo-SCL41, and Dlo-SR33 were localized in chloroplasts, and Dlo-RS43, Dlo-SC33, Dlo-SC37, Dlo-RS2Z33, Dlo-RS2Z16, Dlo-RS2Z24, Dlo-SCL43, Dlo-SR112, and Dlo-SR59 were localized in the nucleus and chloroplasts. The Dlo-SR genes exhibited differential expression patterns in different tissues of longan. The transcript levels of Dlo-RSZ20a, Dlo-SC18, Dlo-RS2Z29, DLo-SR59, Dlo-SR53, and Dlo-SR17 were low in all analyzed tissues, whereas Dlo-RS43, Dlo-RS2Z16, Dlo-RS2Z24, and Dlo-SR30 were highly expressed in all tissues. To clarify their function during SE, the transcript levels of Dlo-SR genes were analyzed at different four stages of SE, comprising non-embryonic callus (NEC), friable-embryogenic callus (EC), incomplete compact pro-embryogenic culture (ICpEC) and globular embryo (GE). Interestingly, the transcript levels of Dlo-RS2Z29 and Dlo-SR112 were increased in embryogenic cells compared with the NEC stage, whereas transcript levels of Dlo-RSZ20a, Dlo-RS43, Dlo-SC37, and Dlo-RS2Z16 were especially increased at the GE stage compared with the other stages. Alternative splicing events of Dlo-SR mRNA precursors (pre-mRNAs) was detected during SE, with totals of 41, 29, 35, and 44 events detected during NEC, EC, ICpEC, and GE respectively. Protein-protein interaction analysis showed that SR proteins were capable of interaction with each other. The results indicate that the alternative splicing of Dlo-SR pre-mRNAs occurs during SE and that Dlo-SR proteins may interact to regulate embryogenesis of longan.


Assuntos
Perfilação da Expressão Gênica , Genômica , Proteínas de Plantas/genética , Sapindaceae/genética , Motivos de Aminoácidos , Sequência Conservada , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Técnicas de Embriogênese Somática de Plantas , Regiões Promotoras Genéticas/genética , Mapeamento de Interação de Proteínas , Sapindaceae/metabolismo
12.
PLoS One ; 15(8): e0238021, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841306

RESUMO

Triple-negative breast cancer (TNBC) is typically treated with chemotherapeutic agents, including carboplatin (Cb), an DNA platinating agent. The O6-methylguanine-DNA-methyltransferase gene (MGMT) encodes for the protein O6-alkylguanine-DNA-alkyltransferase (MGMT protein). MGMT protein is involved in DNA repair mechanisms to remove mutagenic and cytotoxic adducts from O6-guanine in DNA. In glioblastoma multiforme, MGMT methylation status is a predictive biomarker for increased response to temozolomide therapy. It has been suggested, that MGMT protein may have relevance for cellular adaptation and could have an influence on resistance to carboplatin therapy. We investigated the influence of MGMT promoter methylation on pathologic complete response and survival of patients with TNBC treated in the neoadjuvant GeparSixto trial. In 174 of 210 available TNBC tumors a valid MGMT promoter methylation status was determined by pyrosequencing of 5 CpG islands. In 21.8%, we detected a mean MGMT promoter methylation >10%. Overall, MGMT promoter methylation was not significantly associated with pathological complete response (pCR) rate. After stratification for the two therapy arms with and without Cb no statistically significant differences in therapy response rates between the two MGMT promoter methylation groups could be observed. Our results show that different MGMT promoter methylation status is not related to different chemotherapy response rates in the TNBC setting in GeparSixto.


Assuntos
Ensaios Clínicos como Assunto , Ensaios Clínicos Fase II como Assunto , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Neoplasias de Mama Triplo Negativas/genética , Proteínas Supressoras de Tumor/genética , Biópsia , Estudos de Coortes , Ilhas de CpG/genética , Humanos , Estudos Retrospectivos , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/patologia
13.
Am J Hum Genet ; 107(3): 487-498, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32800095

RESUMO

The aggregation and joint analysis of large numbers of exome sequences has recently made it possible to derive estimates of intolerance to loss-of-function (LoF) variation for human genes. Here, we demonstrate strong and widespread coupling between genic LoF intolerance and promoter CpG density across the human genome. Genes downstream of the most CpG-rich promoters (top 10% CpG density) have a 67.2% probability of being highly LoF intolerant, using the LOEUF metric from gnomAD. This is in contrast to 7.4% of genes downstream of the most CpG-poor (bottom 10% CpG density) promoters. Combining promoter CpG density with exonic and promoter conservation explains 33.4% of the variation in LOEUF, and the contribution of CpG density exceeds the individual contributions of exonic and promoter conservation. We leverage this to train a simple and easily interpretable predictive model that outperforms other existing predictors and allows us to classify 1,760 genes-which are currently unascertained in gnomAD-as highly LoF intolerant or not. These predictions have the potential to aid in the interpretation of novel variants in the clinical setting. Moreover, our results reveal that high CpG density is not merely a generic feature of human promoters but is preferentially encountered at the promoters of the most selectively constrained genes, calling into question the prevailing view that CpG islands are not subject to selection.


Assuntos
Ilhas de CpG/genética , Genoma Humano/genética , Mutação com Perda de Função/genética , Regiões Promotoras Genéticas/genética , Metilação de DNA/genética , Éxons/genética , Humanos , RNA Polimerase II/genética , Sítio de Iniciação de Transcrição
14.
Brain Tumor Pathol ; 37(4): 154-158, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32749624

RESUMO

Telomerase reverse transcriptase promoter (TERTp) hotspot mutations are the most frequent mutations in primary glioblastomas (GBM). Previous studies have shown that the combination of TERTp and isocitrate dehydrogenase (IDH) status may serve as a useful diagnostic marker for oligodendroglioma and glioblastoma. In oligodendrogliomas, TERTp and IDH mutations, along with the 1p/19q codeletion, usually coexist and are likely to be founder mutations. However, in contrast to oligodendroglioma, the role of the TERTp status in GBM remains obscure. Here, we used Sanger sequencing, pyrosequencing, and digital PCR (dPCR) to examine the TERTp status in 15 pairs of frozen tissue samples from primary and recurrent IDH wild-type GBM, all of which were operated in a single institute. We showed that the TERTp status was stable between primary and recurrent GBM but this consistency was only detected by dPCR. The results suggest that dPCR is a powerful, highly sensitive tool to detect TERTp mutations, especially in a mixed cell population (e.g., a recurrent GBM tissue) where earlier treatment may have grossly altered the tumor microenvironment.


Assuntos
Neoplasias Encefálicas/genética , Análise Mutacional de DNA/métodos , Glioblastoma/genética , Mutação , Recidiva Local de Neoplasia/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas/genética , Telomerase/genética , Humanos , Isocitrato Desidrogenase/genética , Sensibilidade e Especificidade
15.
Life Sci ; 258: 118030, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32739470

RESUMO

The risk of atherosclerosis (AS) ascends among post-menopausal women, while current hormone replacement therapy exerts several adverse effects. Alisol B 23-acetate (AB23A), a tetracyclic triterpenoid isolated from the rhizome of Alisma orientale, was reported to show multiple physiological activities, including regulating lipid metabolism. According to molecular docking analysis, it was predicted to bind with estrogen receptor α (ERα). In this study, we aimed to observe the effect of AB23A on preventing post-menopausal AS and explore whether the mechanism was mediated by ERα. In vitro, free fatty acid (FFA) was applied to induce the abnormal lipid metabolism of L02 cells. In vivo, the ApoE-/- mice were ovariectomized to mimic the cessation of estrogen. The high-fat diet was also given to induce post-menopausal AS. We demonstrated AB23A attenuated the accumulation of total cholesterol and triglyceride induced by free fatty acids in hepatocytes. In high-fat diet-ovariectomy-treated ApoE-/- mice, AB23A eliminated lipids in blood and liver. AB23A not only reduced the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) through sterol-regulatory element binding proteins (SREBPs) but also suppressed the secretion of PCSK9 through silent information regulator 1 (SIRT1). Notably, AB23A promoted the expression of ERα in vivo and in vitro. The both ERα inhibitor and ERα siRNA were also applied in confirming whether the hepatic protective effect of AB23A was mediated by ERα. We found that AB23A significantly promoted the expression of ERα. AB23A could inhibit the synthesis and secretion of PCSK9 through ERα, lower the accumulation of triglyceride and cholesterol, and prevent post-menopausal AS.


Assuntos
Aterosclerose/patologia , Colestenonas/farmacologia , Receptor alfa de Estrogênio/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Pós-Menopausa/efeitos dos fármacos , Animais , Aterosclerose/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colestenonas/química , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Feminino , Lipoproteínas LDL/metabolismo , Camundongos , Ovariectomia , Regiões Promotoras Genéticas/genética , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Sirtuína 1/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Regulação para Cima/efeitos dos fármacos
16.
Gene ; 760: 145020, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32755656

RESUMO

Conserved sequences across species have always provided valuable insights to improve our understanding on the human genome's entity and the interplay among different loci. Lymphoma/leukemia related factor (LRF) is encoded by ZBTB7A gene and belongs to an evolutionarily conserved family of transcription factors, implicated in vital cellular functions. The present data, demonstrating the wide-spread and the high overlap of the LRF/ZBTB7A recognition sites with genomic segments identified as CpG islands in the human genome, suggest that its binding capacity strongly depends on a specific sequence-encoded feature within CpGs. We have previously shown that de-methylation of the CpG island 326 lying in the ZBTB7A gene promoter is associated with impaired pharmacological induction of fetal hemoglobin in ß-type hemoglobinopathies patients. Within this context we aimed to investigate the extent of the LRF/ZBTB7A conservation among primates and mouse genome, focusing our interest also on the CpG island flanking the gene's promoter region, in an effort to further establish its epigenetic regulatory role in human hematopoiesis and pharmacological involvement in hematopoietic disorders. Comparative analysis of the human ZBTB7A nucleotide and amino acid sequences and orthologous sequences among non-human primates and mouse, exhibited high conservation scores. Pathway analysis, clearly indicated that LRF/ZBTB7A influences conserved cellular processes. These data in conjunction with the high levels of expression foremost in hematopoietic tissues, highlighted LRF/ZBTB7A as an essential factor operating indisputably during hematopoiesis.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Doenças Hematológicas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Sequência Conservada/genética , Ilhas de CpG/genética , Bases de Dados Genéticas , Hemoglobina Fetal/genética , Hematopoese/genética , Humanos , Camundongos , Primatas/genética , Regiões Promotoras Genéticas/genética
17.
BMC Bioinformatics ; 21(1): 338, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32736515

RESUMO

BACKGROUND: Analysis of somatic mutations from tumor whole exomes has fueled discovery of novel cancer driver genes. However, ~ 98% of the genome is non-coding and includes regulatory elements whose normal cellular functions can be disrupted by mutation. Whole genome sequencing (WGS), on the other hand, allows for identification of non-coding somatic variation and expanded estimation of background mutation rates, yet fewer computational tools exist for specific interrogation of this space. RESULTS: We present MutEnricher, a flexible toolset for investigating somatic mutation enrichment in both coding and non-coding genomic regions from WGS data. MutEnricher contains two distinct modules for these purposes that provide customizable options for calculating sample- and feature-specific background mutation rates. Additionally, both MutEnricher modules calculate feature-level and local, or "hotspot," somatic mutation enrichment statistics. CONCLUSIONS: MutEnricher is a flexible software package for investigating somatic mutation enrichment that is implemented in Python, is freely available, can be efficiently parallelized, and is highly configurable to researcher's specific needs. MutEnricher is available online at https://github.com/asoltis/MutEnricher .


Assuntos
Genoma Humano , Mutação/genética , Neoplasias/genética , Software , Humanos , Regiões Promotoras Genéticas/genética , Sequenciamento Completo do Genoma
18.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1395-1404, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748597

RESUMO

By inserting microRNAs into the intron of EF1α promoter, we constructed a novel lentiviral vector knocking down PD-1 gene via microRNA and applied it to CAR-T cells. Lentiviral transduction efficiency and PD-1-silencing efficiency were detected by flow cytometry. PD-1 expression was detected by Western blotting. Relative expression of microRNA was measured by Q-PCR. Cytotoxicity of CAR-T cells based on this vector was tested by luciferase bioluminescence and flow cytometry. Compared with lentiviral vector with microRNA transcribed by U6 promotor, the transduction efficiency of lentiviral vector with microRNA which was inserted into the intron of EF1α promoter was more significant, and the knockdown rate of PD-1 was more than 90%, which was validated by flow cytometry and Western blotting. And the relative expression level of microRNA in Jurkat cells transduced with this novel lentiviral vector was shown by Q-PCR. Compared with normal CAR-T cells, CAR-T cells based on this vector showed stronger cytotoxicity against PD-L1 positive Raji cells. We successfully constructed a novel lentiviral vector that knocked down PD-1 via microRNA and verified the superiority of its transduction efficiency and knockdown efficiency of PD-1. CAR-T cells based on this vector can exert a more powerful cytotoxicity, thus providing theoretical support for the subsequent treatment of PD-L1 positive tumors.


Assuntos
Vetores Genéticos , Lentivirus , MicroRNAs , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Humanos , Lentivirus/genética , MicroRNAs/metabolismo , Receptor de Morte Celular Programada 1 , Regiões Promotoras Genéticas/genética
19.
BMC Evol Biol ; 20(1): 91, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727363

RESUMO

BACKGROUND: The SIAMESE (SIM) locus is a cell-cycle kinase inhibitor (CKI) gene that has to date been identified only in plants; it encodes a protein that promotes transformation from mitosis to endoreplication. Members of the SIAMESE-RELATED (SMR) family have similar functions, and some are related to cell-cycle responses and abiotic stresses. However, the functions of SMRs are poorly understood in maize (Zea mays L.). RESULTS: In the present study, 12 putative SMRs were identified throughout the entire genome of maize, and these were clustered into six groups together with the SMRs from seven other plant species. Members of the ZmSMR family were divided into four groups according to their protein sequences. Various cis-acting elements in the upstream sequences of ZmSMRs responded to abiotic stresses. Expression analyses revealed that all ZmSMRs were upregulated at 5, 20, 25, and 35 days after pollination. In addition, we found that ZmSMR9/11/12 may have regulated the initiation of endoreplication in endosperm central cells. Additionally, ZmSMR2/10 may have been primarily responsible for the endoreplication regulation of outer endosperm or aleurone cells. The relatively high expression levels of almost all ZmSMRs in the ears and tassels also implied that these genes may function in seed development. The effects of treatments with ABA, heat, cold, salt, and drought on maize seedlings and expression of ZmSMR genes suggested that ZmSMRs were strongly associated with response to abiotic stresses. CONCLUSION: The present study is the first to conduct a genome-wide analysis of members of the ZmSMR family by investigating their locations in chromosomes, identifying regulatory elements in their promoter regions, and examining motifs in their protein sequences. Expression analysis of different endosperm developmental periods, tissues, abiotic stresses, and hormonal treatments suggests that ZmSMR genes may function in endoreplication and regulate the development of reproductive organs. These results may provide valuable information for future studies of the functions of the SMR family in maize.


Assuntos
Evolução Molecular , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Família Multigênica , Zea mays/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos de Plantas/genética , Sequência Conservada/genética , Endosperma/genética , Duplicação Gênica , Genes de Plantas , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Análise de Regressão , Especificidade da Espécie , Estresse Fisiológico/efeitos dos fármacos , Sintenia/genética
20.
PLoS Genet ; 16(7): e1008917, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32628663

RESUMO

Mechanisms of transcriptional control in malaria parasites are still not fully understood. The positioning patterns of G-quadruplex (G4) DNA motifs in the parasite's AT-rich genome, especially within the var gene family which encodes virulence factors, and in the vicinity of recombination hotspots, points towards a possible regulatory role of G4 in gene expression and genome stability. Here, we carried out the most comprehensive genome-wide survey, to date, of G4s in the Plasmodium falciparum genome using G4Hunter, which identifies G4 forming sequences (G4FS) considering their G-richness and G-skewness. We show an enrichment of G4FS in nucleosome-depleted regions and in the first exon of var genes, a pattern that is conserved within the closely related Laverania Plasmodium parasites. Under G4-stabilizing conditions, i.e., following treatment with pyridostatin (a high affinity G4 ligand), we show that a bona fide G4 found in the non-coding strand of var promoters modulates reporter gene expression. Furthermore, transcriptional profiling of pyridostatin-treated parasites, shows large scale perturbations, with deregulation affecting for instance the ApiAP2 family of transcription factors and genes involved in ribosome biogenesis. Overall, our study highlights G4s as important DNA secondary structures with a role in Plasmodium gene expression regulation, sub-telomeric recombination and var gene biology.


Assuntos
Quadruplex G , Malária/genética , Motivos de Nucleotídeos/genética , Plasmodium falciparum/genética , Aminoquinolinas/farmacologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma/efeitos dos fármacos , Humanos , Malária/tratamento farmacológico , Malária/parasitologia , Ácidos Picolínicos/farmacologia , Plasmodium falciparum/patogenicidade , Regiões Promotoras Genéticas/genética , Ribossomos/efeitos dos fármacos , Ribossomos/genética
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