RESUMO
Increasing epidemiological evidence has shown that PM2.5 exposure is significantly associated with the occurrence of osteoporosis. It has been well demonstrated that PM2.5 exposure enhanced the differentiation and function of osteoclasts by indirectly causing chronic inflammation, while the mechanism in osteoblasts remains unclear. In our study, toxic effects were evaluated by direct exposure of 20-80⯵g/ml PM2.5 to MC3T3-E1 cells and BMSCs. The results showed that PM2.5 exposure did not affect cell viability via proliferation and apoptosis, but significantly inhibited osteoblast differentiation in a dose-dependent manner. Osteogenic transcription factors Runx2 and Sp7 and other biomarkers Alp and Ocn decreased after PM2.5 exposure. RNA-seq revealed TGF-ß signaling was involved in PM2.5 exposure inhibited osteoblast differentiation, which led to P-Smad1/5 and P-Smad2 reduction in the nucleus by increasing the ubiquitination and degradation of Smad4. At last, the inflammation response increased in MC3T3-E1 cells with PM2.5 exposure. Moreover, the mRNA levels of Mmp9 increased in bone marrow-derived macrophage cells treated with the conditional medium collected from MC3T3-E1 cells exposed to PM2.5. Overall, these results indicated that PM2.5 exposure inhibits osteoblast differentiation and concurrently increases the maturation of osteoclasts. Our study provides in-depth mechanistic insights into the direct impact of PM2.5 exposure on osteoblast, which would indicate the unrecognized role of PM2.5 on osteoporosis.
Assuntos
Diferenciação Celular , Osteoblastos , Material Particulado , Proteína Smad4 , Ubiquitinação , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proteína Smad4/metabolismo , Proteína Smad4/genética , Camundongos , Material Particulado/toxicidade , Ubiquitinação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Poluentes Atmosféricos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Proteólise/efeitos dos fármacosRESUMO
In forensics, it is important to determine the time since deposition (TSD) of bloodstains, one of the most common types of biological evidence in criminal cases. However, no effective TSD inference methods have been established despite extensive attempts in forensic science. Our study investigated the changes in the blood transcriptome over time, and we found that degradation could be divided into four stages (days 0-2, 4-14, 21-56, and 84-168) at 4 °C. A random forest prediction model based on these transcriptional changes was trained on experimental samples and tested in separate test samples. This model was able to successfully predict TSD (area under the curve [AUC] = 0.995, precision = 1, and recall = 1). Thus, this proof-of-concept pilot study has practical significance for assessing physical evidence. Meanwhile, 11 upregulated and 13 downregulated transcripts were identified as potential time-marker transcripts, laying a foundation for further development of TSD analysis methods in forensic science and crime scene investigation.
Assuntos
Manchas de Sangue , Transcriptoma , Projetos Piloto , Medicina Legal/métodos , Perfilação da Expressão GênicaRESUMO
Cd/In-glycerate spheres are synthesized through a simple solvothermal method. After thermal treatment, these Cd/In-glycerates can be converted into CdIn2O4 spheres. Many characterization methods were performed to reveal the microstructure and morphology of the CdIn2O4. It was found that pure CdIn2O4 phase was obtained for the Cd/In starting materials at ratios of 1:1.6. The CdIn2O4 spheres are composed by a large number of nanoparticles subunits. The CdIn2O4 sphere-based sensor exhibited a low detection limit (1 ppm), high response (81.20 to 500 ppm n-butanol), fast response (4 s) and recovery (10 s) time, good selectivity, excellent repeatability, and stability at 280 °C. Our findings highlight the possibility to develop a novel gas sensor based on CdIn2O4 for application in n-butanol detection with high performance.
RESUMO
The human genome, like other mammalian genomes, encodes numerous natural antisense transcripts (NATs) that have been classified into head-to-head, tail-to-tail, or fully overlapped categories in reference to their sense transcripts. Evidence for NAT-mediated epigenetic silencing of sense transcription remains scanty. The DHRS4 gene encodes a metabolic enzyme and forms a gene cluster with its two immediately downstream homologous genes, DHRS4L2 and DHRS4L1, generated by gene duplication. We identified a head-to-head NAT of DHRS4, designated AS1DHRS4, which markedly regulates the expression of these three genes in the DHRS4 gene cluster. By pairing with ongoing sense transcripts, AS1DHRS4 not only mediates deacetylation of histone H3 and demethylation of H3K4 in cis for the DHRS4 gene, but also interacts physically in trans with the epigenetic modifiers H3K9- and H3K27-specific histone methyltransferases G9a and EZH2, targeting the promoters of the downstream DHRS4L2 and DHRS4L1 genes to induce local repressive H3K9me2 and H3K27me3 histone modifications. Furthermore, AS1DHRS4 induces DNA methylation in the promoter regions of DHRS4L2 by recruiting DNA methyltransferases. This study demonstrates that AS1DHRS4, as a long noncoding RNA, simultaneously controls the chromatin state of each gene within the DHRS4 gene cluster in a discriminative manner. This finding provides an example of transcriptional control over the multiple and highly homologous genes in a tight gene cluster, and may help explain the role of antisense RNAs in the regulation of duplicated genes as the result of genomic evolution.
Assuntos
Epigênese Genética/genética , Inativação Gênica , Oxirredutases/genética , RNA Antissenso/genética , Sequência de Bases , Carcinoma Hepatocelular , Cromatina/genética , Neoplasias Esofágicas , Duplicação Gênica/genética , Teste de Complementação Genética , Células HeLa , Hepatócitos/citologia , Humanos , Neoplasias Hepáticas , Dados de Sequência Molecular , Família Multigênica/genética , RNA não Traduzido/genéticaRESUMO
BACKGROUND: The human DHRS4 gene cluster consists of three genes, DHRS4, DHRS4L2 and DHRS4L1. Among them, DHRS4 encodes NADP(H)-dependent retinol dehydrogenase/reductase. In a previous study, we investigated the alternative splicing of DHRS4 and DHRS4L2. DHRS4L1 was added to the gene cluster recently, but little is known about its structure and expression. To reveal the regulatory mechanism of the DHRS4 gene cluster expression, we studied the structure and transcription of DHRS4L1 in the context of the transcriptional behaviors of the human DHRS4 gene cluster. Based on the results of bioinformatics analysis, we propose a possible mechanism for the transcriptional regulation of the human DHRS4 gene cluster. RESULTS: The homologous comparison analysis suggests that DHRS4, DHRS4L2 and DHRS4L1 are three homologous genes in human. DHRS4L1 and DHRS4L2 are paralogues of DHRS4, and DHRS4L2 is the most recent member of the DHRS4 gene cluster. In the minus strand of the human DHRS4 gene cluster, a gene transcribed in an antisense direction was found containing a 5' sequence overlapping the region of exon 1 and promoter of DHRS4. By cloning the full length of RNA variants through 5'RACE and 3'RACE, we identified two transcription start sites, within exon a2 and exon 1, of this newly named gene DHRS4L1 using neuroblastoma cell line BE(2)-M17. Analysis of exon composition in the transcripts of DHRS4 gene cluster revealed that exon 1 was absent in all the transcripts initiated from exon a1 of DHRS4L2 and exon a2 of DHRS4L1. CONCLUSIONS: Alternatively spliced RNA variants are prevalent in the human DHRS4 gene cluster. Based on the analysis of gene transcripts and bioinformatic prediction, we propose here that antisense transcription may be involved in the transcriptional initiation regulation of DHRS4 and in the posttranscriptional splicing of DHRS4L2 and DRHS4L1 for the homologous identity of DHRS4 gene cluster. Beside the alternative transcriptional start sites, the antisense RNA is novel possible factor serving to remove exon 1 from the transcripts initiated from exon a1 and exon a2.
Assuntos
Regulação da Expressão Gênica , Oxirredutases/genética , Transcrição Gênica , Processamento Alternativo , Linhagem Celular Tumoral , Biologia Computacional , Éxons , Humanos , Família Multigênica , Oxirredutases/metabolismo , Regiões Promotoras GenéticasRESUMO
In view of the weakness of antibiotics and the properties of antisense drugs, we applied DNAzymes to the field of drug resistance in bacteria. Two 10-23 mono-DNAzymes (Dz1, Dz2) and a di-DNAzyme (Dz1-2) targeted to beta-lactamase mRNA were designed to determine to what degree the growth of ampicillin-resistant bacteria (TEM-1, TEM-3) was inhibited. All three DNAzymes can play a role both in vitro and in vivo. In vitro, they exhibited high catalytic efficiency (kcat/KM) of 63.5, 91.1, and 30.8 pM(-1) min(-1), respectively, under multiple-turnover conditions. In vivo, after 9 hours' incubation, the degree of inhibition of Dz1, Dz2, and Dz1-2 for TEM-1 bacteria was 27.2%, 39.6%, and 57.7%, respectively, and that for TEM-3 bacteria was 39.1%, 44%, and 62.6%, respectively. Dz1-2 showed the greatest inhibiting effect, demonstrating in vivo activity may be increased by constructing multiple-target DNAzymes. The results indicated a potential possibility for DNAzymes to act as a new type of antibacterial or a tool of gene functional analysis for prokaryocytes.
Assuntos
Resistência a Ampicilina/genética , Bactérias/efeitos dos fármacos , Bactérias/genética , DNA Catalítico/farmacologia , Sistemas de Liberação de Medicamentos/métodos , RNA Mensageiro/metabolismo , beta-Lactamases/genética , Sequência de Bases , DNA Catalítico/biossíntese , Desenho de Fármacos , Eletroporação , Cinética , Dados de Sequência Molecular , RNA Mensageiro/genética , beta-Lactamases/biossínteseRESUMO
10-23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. However, the dependence on exogenous delivery limits its applications. The objective of this work is to establish a replicating DNAzyme in bacteria using a single-stranded DNA vector. By cloning the 10-23 DNAzyme into the M13mp18 vector, we constructed two circular DNAzymes, C-Dz7 and C-Dz482, targeting the beta-lactamase mRNA. These circular DNAzymes showed in vitro catalytic efficiencies (kcat/K(M)) of 7.82 x 10(6) and 1.36 x 10(7) M(-1) x min(-1), respectively. Their dependence on divalent metal ions is similar to that found with linear 10-23 DNAzyme. Importantly, the circular DNAzymes were not only capable of replicating in bacteria but also exhibited high activities in inhibiting beta-lactamase and bacterial growth. This study thus provides a novel strategy to produce replicating DNAzymes which may find widespread applications.