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1.
Nat Commun ; 11(1): 4903, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994412

RESUMO

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.


Assuntos
Sistemas CRISPR-Cas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Descoberta de Drogas/métodos , Edição de Genes/métodos , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína 9 Associada à CRISPR/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Transgênicos , RNA Guia/genética , Recombinação Genética/efeitos dos fármacos , Reprodutibilidade dos Testes , Ativação Transcricional/efeitos dos fármacos , Transfecção/métodos , Transgenes/genética
2.
J Toxicol Sci ; 45(9): 549-558, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32879254

RESUMO

Trimethyltin chloride (TMT) is a stabilizer by-product in the process of manufacturing plastic, which is a kind of very strong toxic substance, and has acute, cumulative and chronic toxicity. TMT may cause bradycardia in patients with occupational poisoning, the mechanism of which has not been reported. This study explored the mechanism of TMT resulting in bradycardia of C57BL/6 mice. TMT was administered to mice to measure heart rate, serum succinate dehydrogenase (SDH) level, and myocardial Na+/K+-ATPase activity and expression. The effects of TMT on myocardial apoptosis were observed by changing the expressions of caspase-3, Bax and Bcl-2 in myocardium. It was found that the heart rate and SDH activity in serum of mice gradually decreased with the increase of TMT dose compared with the control group. The activity and the expression of Na+/K+-ATPase in the heart tissue of mice exposed to TMT was measured and gradually decreased with the increase of dose and time. We measured the expression of Bcl-2, Bax, caspase-3 and cleaved caspase-3 in the heart tissues of TMT exposed mice and found that the expressions of Bax, caspase-3 and cleaved caspase-3 increased and the expressions of Bcl-2 decreased in the heart tissues of the TMT-exposed mice at different doses. With the extension of TMT exposure time, the expression of Bax and caspase-3 increased and the expression of Bcl-2 decreased in the heart tissues of TMT exposed mice. Our findings suggest the mechanisms of TMT resulting in bradycardia may be associated with the inhibited activity and decreased content of Na+/K+-ATPase, thus further leading to the changes of Bcl-2, Bax, caspase-3 and cleaved caspase-3 in the mice's ventricular tissues.


Assuntos
Apoptose/efeitos dos fármacos , Bradicardia/etiologia , Miocárdio/metabolismo , Miocárdio/patologia , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Compostos de Trimetilestanho/toxicidade , Animais , Apoptose/genética , Bradicardia/genética , Caspase 3/genética , Caspase 3/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
3.
Gene ; 760: 145025, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32758582

RESUMO

Numerous cell lines for human alveolar rhabdomyosarcoma (ARMS) have been developed and are widely used to study biological processes of this myogenic cancer. The present study investigated the resemblance of commonly used ARMS cell lines to primary tumors in regards to gene expression. RNA-sequencing data was retrieved from published datasets for 4 commonly used ARMS cell lines and 35 ARMS primary tumors. The genes with most variable expression across primary tumors were used to calculate rank-based Spearman's correlation. The observed median correlations ranged from 0.36 to 0.61. RH-41 showed the highest median correlation while KYM-1 was the least correlated cell line. A significant number of genes dysregulated between tumors and non-tumors also exhibited similar expression patterns between tumors and cell lines, including The findings suggest that ARMS cell lines exhibit changes in gene expression compared to primary tumors and may not be completely representative of the disease process.


Assuntos
RNA Mensageiro/genética , Rabdomiossarcoma Alveolar/genética , Transcriptoma/genética , Linhagem Celular Tumoral , Bases de Dados Genéticas , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Modelos Biológicos , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologia , Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/metabolismo , Rabdomiossarcoma Embrionário/patologia , Análise de Sequência de RNA/métodos , Estatísticas não Paramétricas
4.
Nat Commun ; 11(1): 3871, 2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32747712

RESUMO

Relapses in multiple sclerosis can result in irreversible nervous system tissue injury. If these events could be detected early, targeted immunotherapy could potentially slow disease progression. We describe the use of engineered biomaterial-based immunological niches amenable to biopsy to provide insights into the phenotype of innate immune cells that control disease activity in a mouse model of multiple sclerosis. Differential gene expression in cells from these niches allow monitoring of disease dynamics and gauging the effectiveness of treatment. A proactive treatment regimen, given in response to signal within the niche but before symptoms appeared, substantially reduced disease. This technology offers a new approach to monitor organ-specific autoimmunity, and represents a platform to analyze immune dysfunction within otherwise inaccessible target tissues.


Assuntos
Encefalomielite Autoimune Experimental/terapia , Imunoterapia/métodos , Monitorização Fisiológica/métodos , Esclerose Múltipla/terapia , Animais , Modelos Animais de Doenças , Progressão da Doença , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Feminino , Expressão Gênica/genética , Expressão Gênica/imunologia , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos Endogâmicos , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Recidiva , Resultado do Tratamento
5.
Medicine (Baltimore) ; 99(32): e21702, 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769939

RESUMO

Hepatocellular carcinoma (HCC) is a malignant tumor with unsatisfactory prognosis. The abnormal genes expression is significantly associated with initiation and poor prognosis of HCC. The aim of the present study was to identify molecular biomarkers related to the initiation and development of HCC via bioinformatics analysis, so as to provide a certain molecular mechanism for individualized treatment of hepatocellular carcinoma.Three datasets (GSE101685, GSE112790, and GSE121248) from the GEO database were used for the bioinformatics analysis. Differentially expressed genes (DEGs) of HCC and normal liver samples were obtained using GEO2R online tools. Gene ontology term and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis were conducted via the Database for Annotation, Visualization, and Integrated Discovery online bioinformatics tool. The protein-protein interaction (PPI) network was constructed by the Search Tool for the Retrieval of Interacting Genes database and hub genes were visualized by Cytoscape. Survival analysis and RNA sequencing expression were conducted by UALCAN and Gene Expression Profiling Interactive Analysis.A total of 115 shared DEGs were identified, including 30 upregulated genes and 85 downregulated genes in HCC samples. P53 signaling pathway and cell cycle were the major enriched pathways for the upregulated DEGs whereas metabolism-related pathways were the major enriched pathways for the downregulated DEGs. The PPI network was established with 105 nodes and 249 edges and 3 significant modules were identified via molecular complex detection. Additionally, 17 candidate genes from these 3 modules were significantly correlated with HCC patient survival and 15 of 17 genes exhibited high expression level in HCC samples. Moreover, 4 hub genes (CCNB1, CDK1, RRM2, BUB1B) were identified for further reanalysis of KEGG pathway, and enriched in 2 pathways, the P53 signaling pathway and cell cycle pathway.Overexpression of CCNB1, CDK1, RRM2, and BUB1B in HCC samples was correlated with poor survival in HCC patients, which could be potential therapeutic targets for HCC.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma Hepatocelular/diagnóstico , Biologia Computacional/estatística & dados numéricos , Programas de Rastreamento/normas , Prognóstico , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/fisiopatologia , China/epidemiologia , Análise por Conglomerados , Expressão Gênica/genética , Humanos , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Programas de Rastreamento/métodos , Mapas de Interação de Proteínas/genética , Análise de Sobrevida
6.
PLoS One ; 15(8): e0226235, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32797046

RESUMO

Plant-derived fuels and chemicals from renewable biomass have significant potential to replace reliance on petroleum and improve global carbon balance. However, plant biomass contains significant fractions of oligosaccharides that are not usable natively by many industrial microorganisms, including Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis. Even after chemical or enzymatic hydrolysis, some carbohydrate remains as non-metabolizable oligosaccharides (e.g., cellobiose or longer cellulose-derived oligomers), thus reducing the efficiency of conversion to useful products. To begin to address this problem for Z. mobilis, we engineered a strain (Z. mobilis GH3) that expresses a glycosyl hydrolase (GH) with ß-glucosidase activity from a related α-proteobacterial species, Caulobacter crescentus, and subjected it to an adaptation in cellobiose medium. Growth on cellobiose was achieved after a prolonged lag phase in cellobiose medium that induced changes in gene expression and cell composition, including increased expression and extracellular release of GH. These changes were reversible upon growth in glucose-containing medium, meaning they did not result from genetic mutation but could be retained upon transfer of cells to fresh cellobiose medium. After adaptation to cellobiose, our GH-expressing strain was able to convert about 50% of cellobiose to glucose within 24 h and use it for growth and ethanol production. Alternatively, pre-growth of Z. mobilis GH3 in sucrose medium enabled immediate growth on cellobiose. Proteomic analysis of cellobiose- and sucrose-adapted strains revealed upregulation of secretion-, transport-, and outer membrane-related proteins, which may aid release or surface display of GHs, entry of cellobiose into the periplasm, or both. Our two key findings are that Z. mobilis can be reprogrammed to grow on cellobiose as a sole carbon source and that this reprogramming is related to a natural response of Z. mobilis to sucrose that promotes sucrase production.


Assuntos
Celobiose/metabolismo , Zymomonas/crescimento & desenvolvimento , Zymomonas/metabolismo , Adaptação Fisiológica/fisiologia , Biomassa , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Celulose/metabolismo , Expressão Gênica/genética , Glucose/metabolismo , Hidrolases/metabolismo , Proteômica , Sacarase/metabolismo , Sacarose/metabolismo , Zymomonas/genética , beta-Glucosidase/metabolismo
7.
PLoS One ; 15(8): e0234892, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817668

RESUMO

The mosquito Aedes aegypti vectors the arboviral diseases yellow fever, dengue, Zika and chikungunya. Larvae are usually found developing in freshwater; however, more recently they have been increasingly found in brackish water, potential habitats which are traditionally ignored by mosquito control programs. Aedes aegypti larvae are osmo-regulators maintaining their hemolymph osmolarity in a range of ~ 250 to 300 mOsmol l-1. In freshwater, the larvae must excrete excess water while conserving ions while in brackish water, they must alleviate an accumulation of salts. The compensatory physiological mechanisms must involve the transport of ions and water but little is known about the water transport mechanisms in the osmoregulatory organs of these larvae. Water traverses cellular membranes predominantly through transmembrane proteins named aquaporins (AQPs) and Aedes aegypti possesses 6 AQP homologues (AaAQP1 to 6). The objective of this study was to determine if larvae that develop in freshwater or brackish water have differential aquaporin expression in osmoregulatory organs, which could inform us about the relative importance and function of aquaporins to mosquito survival under these different osmotic conditions. We found that AaAQP transcript abundance was similar in organs of freshwater and brackish water mosquito larvae. Furthermore, in the Malpighian tubules and hindgut AaAQP protein abundance was unaffected by the rearing conditions, but in the gastric caeca the protein level of one aquaporin, AaAQP1 was elevated in brackish water. We found that AaAQP1 was expressed apically while AaAQP4 and AaAQP5 were found to be apical and/or basal in the epithelia of osmoregulatory organs. Overall, the results suggest that aquaporin expression in the osmoregulatory organs is mostly consistent between larvae that are developing in freshwater and brackish water. This suggests that aquaporins may not have major roles in adapting to longterm survival in brackish water or that aquaporin function may be regulated by other mechanisms like post-translational modifications.


Assuntos
Aedes/genética , Aquaporinas/genética , Osmorregulação/genética , Aedes/fisiologia , Animais , Aquaporinas/metabolismo , Infecções por Arbovirus , Transporte Biológico , Ecossistema , Água Doce , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Hemolinfa/metabolismo , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Osmorregulação/fisiologia , Osmose , Águas Salinas , Salinidade , Água/metabolismo
8.
BMC Bioinformatics ; 21(1): 288, 2020 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-32631229

RESUMO

BACKGROUND: Cancer is a highly heterogeneous disease with varying responses to anti-cancer drugs. Although several attempts have been made to predict the anti-cancer therapeutic responses, there remains a great need to develop highly accurate prediction models of response to the anti-cancer drugs for clinical applications toward a personalized medicine. Patient derived xenografts (PDXs) are preclinical cancer models in which the tissue or cells from a patient's tumor are implanted into an immunodeficient or humanized mouse. In the present study, we develop a bioinformatics analysis pipeline to build a predictive gene expression model (GEM) for cancer patients' drug responses based on gene expression and drug activity data from PDX models. RESULTS: Drug sensitivity biomarkers were identified by performing an association analysis between gene expression levels and post-treatment tumor volume changes in PDX models. We built a drug response prediction model (called PDXGEM) in a random-forest algorithm by using a subset of the drug sensitvity biomarkers with concordant co-expression patterns between the PDXs and pretreatment cancer patient tumors. We applied the PDXGEM to several cytotoxic chemotherapies as well as targeted therapy agents that are used to treat breast cancer, pancreatic cancer, colorectal cancer, or non-small cell lung cancer. Significantly accurate predictions of PDXGEM for pathological response or survival outcomes were observed in extensive independent validations on multiple cancer patient datasets obtained from retrospective observational studies and prospective clinical trials. CONCLUSION: Our results demonstrated the strong potential of using molecular profiles and drug activity data of PDX tumors in developing a clinically translatable predictive cancer biomarkers for cancer patients. The PDXGEM web application is publicly available at http://pdxgem.moffitt.org .


Assuntos
Biomarcadores Tumorais/metabolismo , Expressão Gênica/genética , Neoplasias/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Feminino , Humanos , Masculino , Estudos Prospectivos , Estudos Retrospectivos
9.
J Pharmacol Sci ; 144(2): 69-75, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32713799

RESUMO

The mechanism of the papaverine (PV) for the treatment of cerebral ischemia remains unclear. A total of 42 mice induced with focal cerebral ischemia were randomly divided into three groups: PV,baicalin (BA)and vehicle group. Both PV and BA could significantly reduce the ischemic infarct volume (P < 0.05). Pathway enrichment analysis was performed on MetaCore™ to search the molecular pathways associated with the gene expression profile of PV, compared with vehicle and BA. Compared with vehicle, we found that 60% of the top 10 pathways in PV group were related to immune response. The top 10 biological processes of the targeted pathways were mainly related to the multiple immunomodulatory process of neuro-vascular inflammation, including immune_Th17-deried cytokins, regulation of angiogenesis, cell adhesion_Leucocyte chemotaxis, antigen presentation, cell adhesion_synaptic contact, and inflammation related to Amphoterin signaling. Moreover, compared with BA, 17 pathways of PV were identified, and 58.82% (10/17) were also related to immune response, especially, half of the top 10 pathways with the lower p-value. In these top 10 pathways, 4 were the cytokine-mediated signaling pathways, which play key role in inflammation, like IL-17 signaling pathways with the activation of G-CSF,IL-23,RANKL, p38MAPK and NF-κB.These findings indicate that PV may be an efficacious pluripotent anti-inflammatory agent against cerebral ischemic-reperfusion injury by targeting on multiple immunomodulatory process of neuro-vascular inflammation.


Assuntos
Anti-Inflamatórios , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/genética , Fatores Imunológicos , Papaverina/farmacologia , Papaverina/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/genética , Animais , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Inflamação , Interleucina-17/genética , Interleucina-17/metabolismo , Masculino , Camundongos Endogâmicos , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
10.
RNA ; 26(10): 1320-1333, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32554554

RESUMO

Human CD4+ T cells are often subdivided into distinct subtypes, including Th1, Th2, Th17, and Treg cells, that are thought to carry out distinct functions in the body. Typically, these T-cell subpopulations are defined by the expression of distinct gene repertoires; however, there is variability between studies regarding the methods used for isolation and the markers used to define each T-cell subtype. Therefore, how reliably studies can be compared to one another remains an open question. Moreover, previous analysis of gene expression in CD4+ T-cell subsets has largely focused on gene expression rather than alternative splicing. Here we take a meta-analysis approach, comparing eleven independent RNA-seq studies of human Th1, Th2, Th17, and/or Treg cells to determine the consistency in gene expression and splicing within each subtype across studies. We find that known master-regulators are consistently enriched in the appropriate subtype; however, cytokines and other genes often used as markers are more variable. Importantly, we also identify previously unknown transcriptomic markers that appear to consistently differentiate between subsets, including a few Treg-specific splicing patterns. Together this work highlights the heterogeneity in gene expression between samples designated as the same subtype, but also suggests additional markers that can be used to define functional groupings.


Assuntos
Linfócitos T CD4-Positivos/fisiologia , Expressão Gênica/genética , Processamento de RNA/genética , Subpopulações de Linfócitos T/fisiologia , Transcriptoma/genética , Adulto , Células Cultivadas , Citocinas/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
11.
Nucleic Acids Res ; 48(13): e76, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32479612

RESUMO

The control of gene expression noise is important for improving drug treatment and the performance of synthetic biological systems. Previous work has tuned gene expression noise by changing the rate of transcription initiation, mRNA degradation, and mRNA translation. However, these methods are invasive: they require changes to the target genetic components. Here, we create an orthogonal system based on CRISPR-dCas9 to tune gene expression noise. Specifically, we modulate the gene expression noise of a reporter gene in Escherichia coli by incorporating CRISPR activation and repression (CRISPRar) simultaneously in a single cell. The CRISPRar uses a single dCas9 that recognizes two different single guide RNAs (sgRNA). We build a library of sgRNA variants with different expression activation and repression strengths. We find that expression noise and mean of a reporter gene can be tuned independently by CRISPRar. Our results suggest that the expression noise is tuned by the competition between two sgRNAs that modulate the binding of RNA polymerase to promoters. The CRISPRar may change how we tune expression noise at the genomic level. Our work has broad impacts on the study of gene functions, phenotypical heterogeneity, and genetic circuit control.


Assuntos
Sistemas CRISPR-Cas/genética , Clonagem Molecular/métodos , Escherichia coli/genética , Expressão Gênica/genética , RNA Guia/genética , Regulação Bacteriana da Expressão Gênica , Genes Reporter/genética
12.
Gene ; 755: 144884, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32562739

RESUMO

The aim of this work was to study changes in gene expression levels of 7 ER-resident selenoproteins under ER-stress caused by the action of a selenium-containing compound of organic nature, methylselenic acid using three human cancer cell lines DU 145 (prostate carcinoma), MCF 7 (breast adenocarcinoma)and HT-1080 (fibrosarcoma). According to the obtained results, we can speak of a synchronous changes in the expression of SELT and SEP15 mRNA depending on the concentration of MSA for 24 h, while the pattern of SELM expression was completely opposite and was radically different from other selenoproteins. It should be noted that in HT-1080 cells, the expression pattern of SELM differed from the expression pattern in two other cancer cells, while the expression patterns of other ER-resident selenoproteins (SELT, SEP15, SELK, SELS, SELN and DIO2) differed slightly depending on the cell line. Also we investigated the molecular mechanisms of UPR caused by MSA-induced ER stress in three cancer cell lines. According to the obtained results, it can be assumed that in DU 145 cells, MSA promotes activation of the PERK signaling pathway of UPR. In fibrosarcoma cells MSA was promoted the activation of ATF-6 UPR signaling pathway. In MCF 7 cells, MSA promoted the activation of two pro-apoptotic UPR signaling pathways at once: IRE1 and ATF-6.The results of this work once again demonstrate that the mechanisms of ER-stress regulation caused by the same agent, in this case, MSA, lead to the activation of different UPR signaling pathways in different cancer cells, and about their relationship.


Assuntos
Estresse do Retículo Endoplasmático/genética , Compostos Organosselênicos/metabolismo , Selenoproteínas/genética , Apoptose/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Humanos , RNA Mensageiro/genética , Selenoproteínas/metabolismo , Transdução de Sinais/genética , Resposta a Proteínas não Dobradas/genética
13.
Gene ; 755: 144908, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32565322

RESUMO

Large-scale -omics data now allows for investigating genome-wide functional elements. Using RNA-Seq data, we tested the expression of 134 pseudogenes which do not show duplicated protein coding genes in the M. smegmatis genome. We observe significant expression and translation of 28 pseudogenes. Further examination using RNA-Seq reads suggested the sequencing errors in many pseudogenes. These include some of the functionally relevant genes such as recN and manB. We propose that the analysis of transcriptional and translational landscape using multi-dimensional -omics data could shed light on the current annotations of the bacterial pseudogenes.


Assuntos
Mycobacterium smegmatis/genética , Pseudogenes/genética , Análise de Sequência de RNA/métodos , Cromatografia Líquida/métodos , Biologia Computacional/métodos , Expressão Gênica/genética , Mycobacteriaceae/genética , RNA/genética , Espectrometria de Massas em Tandem/métodos , Transcrição Genética/genética , Transcriptoma/genética
14.
PLoS One ; 15(6): e0234306, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555615

RESUMO

Moraxella catarrhalis is a human-adapted, opportunistic bacterial pathogen of the respiratory mucosa. Although asymptomatic colonization of the nasopharynx is common, M. catarrhalis can ascend into the middle ear, where it is a prevalent causative agent of otitis media in children, or enter the lower respiratory tract, where it is associated with acute exacerbations of chronic obstructive pulmonary disease in adults. Phase variation is the high frequency, random, reversible switching of gene expression that allows bacteria to adapt to different host microenvironments and evade host defences, and is most commonly mediated by simple DNA sequence repeats. Bioinformatic analysis of five closed M. catarrhalis genomes identified 17 unique simple DNA sequence repeat tracts that were variable between strains, indicating the potential to mediate phase variable expression of the associated genes. Assays designed to assess simple sequence repeat variation under conditions mimicking host infection demonstrated that phase variation of uspA1 (ubiquitous surface protein A1) from high to low expression occurs over 72 hours of biofilm passage, while phase variation of uspA2 (ubiquitous surface protein A2) to high expression variants occurs during repeated exposure to human serum, as measured by mRNA levels. We also identify and confirm the variable expression of two novel phase variable genes encoding a Type III DNA methyltransferase (modO), and a conserved hypothetical permease (MC25239_RS00020). These data reveal the repertoire of phase variable genes mediated by simple sequence repeats in M. catarrhalis and demonstrate that modulation of expression under conditions mimicking human infection is attributed to changes in simple sequence repeat length.


Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Moraxella catarrhalis/genética , Aderência Bacteriana/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Expressão Gênica/genética , Humanos , Repetições de Microssatélites/genética , Moraxella catarrhalis/patogenicidade , Infecções por Moraxellaceae , Otite Média/microbiologia , Sequências Repetitivas de Ácido Nucleico/genética
15.
PLoS One ; 15(6): e0234328, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579617

RESUMO

FABP4 is a candidate gene for carcass and meat quality traits in livestock and poultry. However, the effects of FABP4 have not been examined in the Yanbian yellow cattle, an economically important local cattle breed in China. In this study, we characterized single nucleotide polymorphisms (SNPs) in FABP4 in this cattle breed and their associations with meat quality traits. Six SNPs (referred to as SNP1-6) were identified in FABP4 by direct sequencing and polymerase chain reaction-restriction fragment length polymorphism. The six SNPs were significantly correlated with meat quality traits. In particular, the GG and GA genotypes of SNP1 were significantly associated with water and fat contents and GG and AA genotypes of SNP1 were significantly associated with protein contents (P < 0.05). The fat content and marbling in heterozygous individuals at SNP2-6 were significantly higher than those in wild-type or mutant individuals (P < 0.05), while protein content was significantly higher in wild-type and mutant individuals than in heterozygous individuals (P < 0.05). A gene expression analysis indicated that the lipid metabolism-related genes FABP4, PPARγ, ANGPTL4, and LPL show similar expression patterns with respect to FABP4 genotypes, with the highest levels in wild-type individuals and the lowest levels in mutants. In conclusion, FABP4 SNPs can be used for marker-assisted selection in Yanbian yellow cattle breeding.


Assuntos
Bovinos/genética , Proteínas de Ligação a Ácido Graxo/genética , Metabolismo dos Lipídeos/genética , Animais , China , Feminino , Qualidade dos Alimentos , Expressão Gênica/genética , Frequência do Gene/genética , Estudos de Associação Genética/métodos , Genótipo , Masculino , Mutação/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Carne Vermelha/análise , Análise de Sequência de DNA/métodos
16.
Leg Med (Tokyo) ; 46: 101736, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32563979

RESUMO

Individuals harbouring specific genetic variations might trend towards suffering sudden cardiac death. Cystathionine-γ-lyase is one of the key enzymes of endogenous hydrogen sulfide production, and a key factor on the expression regulation of hydrogen sulfide in human heart. Compelling studies have suggested the cardioprotective effects of hydrogen sulfide, while it remains controversial whether cystathionine-γ-lyase and hydrogen sulfide are beneficial to cardiovascular diseases. In this study, we performed a candidate-gene-based study to evaluate the association of the Indel polymorphism rs113044851 within the 3' untranslated region of Cystathionine-γ-lyase gene and risk of sudden cardiac death in a Chinese Han population. Logistic regression analysis showed that the insertion allele of rs113044851 significantly decreased the risk of sudden cardiac death [odds ratio = 0.58; 95% confidence interval:0.38-0.88; P = 0.0076]. Further genotype-phenotype association analysis indicated that the insertion allele was significantly associated with lower expression of cystathionine-γ-lyase in myocardium tissues. The subsequently in-silico predication revealed that compared with the deletion allele, the binding of the insertion allele with miR-1324 matched better. Finally, dual-luciferase activity assay validated the prediction that the gene transcriptional activity indicated by firefly luciferase activity with ins/ins genotype was lower than that with del/del genotype. In summary, our data suggested that rs113044851 might contribute to susceptibility of sudden cardiac death via regulating gene expression at post-transcriptional level. This indel has the potential to become a molecular diagnosis marker and genetic counseling of sudden cardiac death.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Cistationina gama-Liase/genética , Morte Súbita Cardíaca/etiologia , Estudos de Associação Genética , Predisposição Genética para Doença/genética , Polimorfismo Genético , Expressão Gênica/genética , Humanos , Risco
17.
Arch Virol ; 165(8): 1827-1835, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32507978

RESUMO

Human cytomegalovirus (HCMV) infection causes high morbidity and mortality among immunocompromised patients and can remain in a latent state in host cells. Expression of the immediate-early (IE) genes sustains HCMV replication and reactivation. As a novel genome-editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been extensively utilized to modify and edit genomic DNA. In the present study, the CRISPR/Cas9 system was used to target the IE region of the HCMV genome via specific single-guide RNAs (sgRNAs). Infection with CRISPR/Cas9/sgRNA lentiviral constructs significantly reduced viral gene expression and virion production in HFF primary fibroblasts and inhibited viral DNA production and reactivation in the THP-1 monocytic cell line. Thus, the CRISPR/Cas9/sgRNA system can accurately and efficiently target HCMV replication and reactivation and represents a novel therapeutic strategy against latent HCMV infection.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citomegalovirus/genética , Endonucleases/genética , Genes Virais/genética , Replicação Viral/genética , Linhagem Celular , Infecções por Citomegalovirus/virologia , DNA Viral/genética , Fibroblastos/virologia , Edição de Genes/métodos , Expressão Gênica/genética , Células HEK293 , Humanos , RNA Guia/genética , Células THP-1
18.
Nat Commun ; 11(1): 2953, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528005

RESUMO

The West Africa Ebola outbreak was the largest outbreak ever recorded, with over 28,000 reported infections; this devastating epidemic emphasized the need to understand the mechanisms to counteract virus infection. Here, we screen a library of nearly 400 interferon-stimulated genes (ISGs) against a biologically contained Ebola virus and identify several ISGs not previously known to affect Ebola virus infection. Overexpression of the top ten ISGs attenuates virus titers by up to 1000-fold. Mechanistic studies demonstrate that three ISGs interfere with virus entry, six affect viral transcription/replication, and two inhibit virion formation and budding. A comprehensive study of one ISG (CCDC92) that shows anti-Ebola activity in our screen reveals that CCDC92 can inhibit viral transcription and the formation of complete virions via an interaction with the viral protein NP. Our findings provide insights into Ebola virus infection that could be exploited for the development of therapeutics against this virus.


Assuntos
Proteínas de Transporte/metabolismo , Ebolavirus/patogenicidade , Interferons/farmacologia , Animais , Western Blotting , Proteínas de Transporte/genética , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Chlorocebus aethiops , Ebolavirus/metabolismo , Imunofluorescência , Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Vero , Proteínas Virais/metabolismo , Internalização do Vírus , Replicação Viral/genética , Replicação Viral/fisiologia
19.
Am J Hum Genet ; 107(1): 164-172, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32553196

RESUMO

CNOT1 is a member of the CCR4-NOT complex, which is a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. We report on 39 individuals with heterozygous de novo CNOT1 variants, including missense, splice site, and nonsense variants, who present with a clinical spectrum of intellectual disability, motor delay, speech delay, seizures, hypotonia, and behavioral problems. To link CNOT1 dysfunction to the neurodevelopmental phenotype observed, we generated variant-specific Drosophila models, which showed learning and memory defects upon CNOT1 knockdown. Introduction of human wild-type CNOT1 was able to rescue this phenotype, whereas mutants could not or only partially, supporting our hypothesis that CNOT1 impairment results in neurodevelopmental delay. Furthermore, the genetic interaction with autism-spectrum genes, such as ASH1L, DYRK1A, MED13, and SHANK3, was impaired in our Drosophila models. Molecular characterization of CNOT1 variants revealed normal CNOT1 expression levels, with both mutant and wild-type alleles expressed at similar levels. Analysis of protein-protein interactions with other members indicated that the CCR4-NOT complex remained intact. An integrated omics approach of patient-derived genomics and transcriptomics data suggested only minimal effects on endonucleolytic nonsense-mediated mRNA decay components, suggesting that de novo CNOT1 variants are likely haploinsufficient hypomorph or neomorph, rather than dominant negative. In summary, we provide strong evidence that de novo CNOT1 variants cause neurodevelopmental delay with a wide range of additional co-morbidities. Whereas the underlying pathophysiological mechanism warrants further analysis, our data demonstrate an essential and central role of the CCR4-NOT complex in human brain development.


Assuntos
Deficiências do Desenvolvimento/genética , Expressão Gênica/genética , Transtornos do Neurodesenvolvimento/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , RNA/genética , Receptores CCR4/genética , Fatores de Transcrição/genética , Alelos , Feminino , Variação Genética/genética , Haploinsuficiência/genética , Heterozigoto , Humanos , Masculino , Malformações do Sistema Nervoso/genética , Fenótipo , Estabilidade Proteica
20.
Immunol Med ; 43(3): 121-129, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32546118

RESUMO

The roles of interleukin-22 (IL-22) in carcinogenesis have been proposed in various neoplasms. Increased expression of IL-22 has been observed in oral squamous cell carcinoma (OSCC) lesions as well as in other cancers. OSCC is still associated with poor prognosis and a high mortality rate because of its invasiveness and frequent lymph node metastasis. In the present study, we investigated the effects of IL-22 on OSCC cells. The human OSCC cell lines Ca9-22 and SAS were stimulated with IL-22 (1-10 ng/mL), and their migration abilities were examined using a cell scratch assay. A Matrigel invasion assay was performed to evaluate the invasion abilities of OSCC cells. Signal transducer and activator of transcription 3 (STAT3) phosphorylation, matrix metalloproteinase (MMP) and epithelial-mesenchymal transition (EMT)-related genes and proteins were also examined. IL-22 treatment promoted the migration and invasion abilities of OSCC cells without increasing their viability. IL-22 stimulation also induced STAT3 phosphorylation, MMP-9 activity and EMT-related genes and proteins. Our findings suggest that IL-22 has possible roles in the development of OSCC.


Assuntos
Carcinoma de Células Escamosas/patologia , Movimento Celular/efeitos dos fármacos , Interleucinas/efeitos adversos , Interleucinas/fisiologia , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Humanos , Metástase Linfática/genética , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Invasividade Neoplásica/genética , Fosforilação/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo
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