ANALYTICAL CHEMISTRY
·
2020
·
IF 6.785
Moxuan Ji,Xinan Wang,Haifeng Zheng,Wenjie Mao,Xiaorui Shi,Si Chen,Chu Tang,Fu Wang
Pyroptotic cell death is a phenomenon that runs through all life activities and plays an important role in physiological and pathological processes of the body’s metabolism. It is of big biological significance to understand the phenomenon and nature of cell pyroptosis. In the process of cell pyroptosis, the pore-forming effector gasdermin D (GSDMD) is cleaved to form oligomers, which are inserted into the cell membrane, causing rapid cell death. However, the effective cell death induced by GSDMD complicates our ability to understand the behavior of pyroptosis. In this work, we performed molecular mutagenesis to develop a genetically encoded pyroptotic reporter, where a secreted Gaussia luciferase (Gluc) was strategically placed in the p30-p20 tolerated junction of GSDMD to support natural pyrophosphorylation and promote live imaging of cell pyroptosis. In addition, we demonstrated that this fused Gluc-GSDMD reporter executed inflammatory body-dependent pyroptosis in response to extracellular stimuli, and that the lysed p30-GSDMD can be secreted out of the cell and can be detected in the culture medium and animal blood. Therefore, our study provides a valuable tool that not only noninvasive and real-time monitoring of cell pyroptosis, but also affords a high-throughput functional screening of pyroptosis-targeted compounds in cultured cells and animal models.
研究领域:
免疫学
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分子生物学
·
细胞生物学
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DOI
PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2020
·Vol. 412
·期 29
·8127-8134
·
IF 3.637
Su Yuchen,Liu Chunlan,Jiang Xuemei,Wei Weili
Although luminescent bacteria-based bioluminescence inhibition assay has been widely used in the toxicity assessment of environmental pollutants, the response of a luminescent bacterium usually lacks specificity to a target analyte. Recently, some specific analyte inductive promoters were fused to the lux genes for the purpose of selective bioluminescent sensing, and suits of specific promoters were fused to lux genes to compose a bioluminescent array sensor for simultaneous identification of multiple analytes. However, specific promoter-based methods still suffer from drawbacks including limited selectivity, slow responding time, expensive to construct different promoters involved plasmids, and laborious to find new promoters. Herein, we proposed a novel strategy to construct a lux reporter array sensor by directly transforming the natural lux genes in different bacterial hosts without the involvement of any specific promoters. Due to the distinct pathways of signal production, the responding time of the current different bacterial host (DBH)-based lux reporter array has nearly an order of magnitude faster than with specific promoter-based methods. The DBH-based lux reporter array was successfully used for simultaneous identification, quantification, and toxicity/bioactivity assessment of multiple metal ions. Obviously, all the chemical synthetic material-based metal ion sensing methods cannot simultaneously achieve analysis and toxicity evaluation. This approach possessed additional advantages of facile construction, easy operation, high selectivity, fast response, and strong adaptability to other analytes. Graphical abstract
A different bacterial host-based lux reporter array was established for simultaneous analysis and toxicity assessment of multiple metal ions.
研究领域:
环境科学
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微生物学
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生物技术
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2017
·
IF 6.32
Huateng Zhang,Jing Su,Yuxin Lin,Haixiu Bai,Jiaxiang Liu,Hui Chen,Lupei Du,Lichuan Gu,Minyong Li
Chalcone refers to an aromatic ketone and an enone that constitutes the central core for various important biological compounds in drug discovery. Moreover, the firefly luciferase (Fluc) as the bioluminescent reporter has been widely used in life science research and high-throughput screening (HTS). However, Fluc might suffer from direct inhibition by HTS compounds resulting in the occurrence of “false positives.” In the current research, we discovered a series of chalcone compounds as Fluc inhibitors with favorable potency both in vitro and in vivo. Moreover, our compound 3i showed remarkable systemic inhibition in transgenic mice. Both enzymatic kinetics study and cocrystal structure demonstrated that compound 3i is competitive for substrate aminoluciferin, while noncompetitive for ATP. Besides, compound 3i exhibited excellent selectivity as a promising quenching agent in a simulated dual-luciferase reporter assay. We believed that our research would contribute to improving scientists’ awareness of the Fluc inhibitors, pay attention to the bias results, and even expand the utilization of bioluminescence in life science research.
研究领域:
药理学
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分子生物学
·
生命科学
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DOI
PubMed
高影响因子
PLANT MOLECULAR BIOLOGY
·
2025
·Vol. 115
·期 4
·1-21
·
IF 3.8
Xu Yao,Li En,Cao Weiping,Zhang Yingchao,Zhang Xinfang,Liu Zhaoyu,Cai Shanshan,Wang Jiayu,Cheng Fansheng,Chen Ruibing,Gao Ting
Scutellaria baicalensis , a traditional medicinal plant originating in China, is widely cultivated for its therapeutic properties. The main bioactive substances in S. baicalensis are flavonoids, which exhibit extensive antibacterial and antiviral activities. However, the contents of these valuable natural product ingredients are relatively low in the plant. MYB transcription factors play crucial roles in regulating plant secondary metabolism, including flavonoid biosynthesis. While the regulation of MYB transcription factors has been extensively studied in various species, research on their role in S. baicalensis remains relatively scarce. In this study, we identified SbMYB111, belonging to the S7 subgroup of R2R3-MYB transcription factors, which functions as a transcriptional activator and is localized in the nucleus. Through heterologous overexpression of SbMYB111 in Arabidopsis thaliana and suppression expression in S. baicalensis , we demonstrated that SbMYB111 acts as a positive regulator in the biosynthesis of flavonoids. Furthermore, the yeast one-hybrid and dual-luciferase reporter gene assays validated that SbMYB111 activates the expression of SbC4H2 , a key enzyme gene in the flavonoid biosynthesis pathway. This study provides a theoretical basis for understanding the transcriptional regulation mechanism of flavonoid synthesis and further developing medicinal resources of S. baicalensis .
研究领域:
植物学
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生物化学与分子生物学
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遗传学
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产品关联
DOI
PubMed
高影响因子
BIOTECHNOLOGY LETTERS
·
2023
·Vol. 45
·期 3
·401-410
·
IF 2.7
Tian Li,Yan Bingyu,Huo Dandan,Sun Wenhui,Cui Sufang,Li Xiaojing,Zhang Xiangmei,Dong Huijun
Objectives To develop a modified CRISPR/Cas9 system with the β-glucuronidase (GusA) reporter and a dual sgRNA cassette for Nonomuraea gerenzanensis ( N. gerenzanensis ). Results With the aid of a visual GusA reporter, the complicated and tedious process of cloning and gene identification could be abandoned entirely in the genetic editing of N. gerenzanensis . Moreover, introducing a dual sgRNA cassette into the CRISPR/Cas9 system significantly improved gene deletion efficiency compared to the single sgRNA element. Furthermore, the length of the homologous flanking sequences set to the lowest value of 500 bp in this system could still reach the relatively higher conjugation transfer frequency. Conclusions The enhanced CRISPR/Cas9 system could efficiently perform genetic manipulation on the rare actinomycete N. gerenzanensis .
研究领域:
微生物学
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分子生物学
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遗传工程
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产品关联
DOI
PubMed
高影响因子
Clinical & Translational Oncology
·
2021
·Vol. 23
·期 9
·1866-1873
·
IF 3.405
Bai Z. M.,Li X. F.,Yang Y.,Yang Y. F.,Lv D. R.,Tang L. L.
Purpose The aim of the present study was to elucidate the functional role of hsa-miR-328-3p/STAT3 pathway in the effects of propofol on gastric cancer proliferation. Methods Bioinformatics was used to analyze the molecular expression differences of hsa-miR-328-3p/STAT3 axis in stomach adenocarcinoma ( n = 435) and normal samples ( n = 41) from TCGA database. The expression of the above molecules in gastric cancer cells SGC-7901 and normal gastric mucosal cells GES-1 was verified via qPCR. The dual-luciferase assay was carried out to confirm the interaction between hsa-miR-328-3p and STAT3. Subsequently, the cell proliferation and the expression of the above molecules in SGC-7901 and GES-1 cells were evaluated after 10 μM propofol treatment. Finally, we analyzed whether propofol still inhibited the proliferation of gastric cancer by suppressing STAT3 pathway after hsa-miR-328-3p down-regulation. Results Compared with normal samples, the expression of hsa-miR-328-3p was significantly down-regulated in stomach adenocarcinoma samples, while the expression of STAT3 and downstream target genes (MMP2, CCND1 and COX2) was up-regulated. The results were consistent with those in GES-1 and SGC-7901 cell lines. Meanwhile, we found that hsa-miR-328-3p can bind to the 3′-UTR of the potential target gene STAT3. Furthermore, propofol significantly inhibited the proliferation of gastric cancer cell line SGC-7901, where hsa-miR-328-3p was up-regulated and the expression of STAT3 and downstream proliferation-related target genes were down-regulated. However, the growth inhibition of propofol on SGC-7901 cell was significantly reversed after the inhibition of hsa-miR-328-3p. Conclusions To sum up, propofol suppressed the STAT3 pathway via up-regulating hsa-miR-328-3p to inhibit gastric cancer proliferation.
研究领域:
肿瘤学
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分子生物学
·
药理学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2023
·
IF 6.1
Yun Huang,Junyue Zheng,Peizhuo Wu,Yu Zhang,Lihong Qiu
Cytochrome P450 CYP6B7 has previously been proved to be associated with fenvalerate-resistance in Helicoverpa armigera. Here, how CYP6B7 is regulated and involved in the resistance of H. armigera is studied. Seven base differences (M1–M7) were found in CYP6B7 promoter between a fenvalerate-resistant (HDTJFR) and a susceptible (HDTJ) strain of H. armigera. M1–M7 sites in HDTJFR were mutated into the corresponding base in HDTJ, and pGL3-CYP6B7 reporter genes with different mutation sites were constructed. Fenvalerate-induced activities of reporter genes mutated at M3, M4, and M7 sites were significantly reduced. Transcription factors Ubx and Br, whose binding sites contain M3 and M7, respectively, were overexpressed in HDTJFR. Knockdown of Ubx and Br results in significant expression inhibition of CYP6B7 and other resistance-related P450 genes, and increase of sensitivity of H. armigera to fenvalerate. These results indicate that Ubx and Br regulate the expression of CYP6B7 to mediate the fenvalerate-resistance in H. armigera.
研究领域:
昆虫学
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分子生物学
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遗传学
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产品关联
DOI
PubMed
高影响因子
Bioengineered
·
2021
·
IF 3.269
Yi Sun,Yulong Shen,Xing Li
Long non-coding RNA (LncRNA) AGAP2-AS1 has been demonstrated to involve in various malignancies. However, the expression and biological effect of AGAP2-AS1 on glioma remain enigmatic. We aimed to explore the effects of AGAP2-AS1 on glioma. Expressions and relationship of AGAP2-AS1 and microRNA-497-5p (miR-497-5p) in different grades of glioma tissues and cell lines as well as normal ones were assessed by quantitative real-time polymerase chain reaction (qRT-PCR), starBase, and dual-luciferase reporter assay. In addition, the effect of AGAP2-AS1 on the cell biological behaviors, epithelial-mesenchymal transition (EMT)-related markers, and miR-497-5p level was detected by cell functional experiments, western blot, and qRT-PCR. After transfection with miR-497-5p mimic (M), inhibitor (I), and AGAP2-AS1 knockdown, miR-497-5p level, cell biological behaviors, and EMT-related markers were detected again. AGAP2-AS1 expression was increased while miR-497-5p expression was decreased in glioma tissues and cell lines, and increase of AGAP2-AS1 expression or reduction of miR-497-5p expression was also correlated with clinicopathological grades of glioma. Furthermore, AGAP2-AS1 knockdown repressed cell biological behaviors and EMT-related markers expressions. Mechanically, AGAP2-AS1 targeted miR-497-5p and AGAP2-AS1 knockdown led to elevation of miR-497-5p expression. In addition, rescue experiments were conducted to validate the vital influence of miR-497-5p on the AGAP2-AS1-regulated proliferation and metastasis of glioma. AGAP2-AS1 may serve as an oncogene in the tumorigenesis of glioma by inhibiting miR-497-5p expression, showing its potential as a prognostic biomarker and a therapeutic target for glioma.
研究领域:
肿瘤学
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分子生物学
·
癌症研究
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产品关联
DOI
PubMed
高影响因子
Tomography
·
2017
·Vol. 3
·期 1
·1-8
·
IF 0
E. Mark Haacke
A unique hybrid protein ferritin–enhanced green fluorescent protein (EGFP) was built to serve as an endogenous dual reporter for both fluorescence and magnetic resonance imaging (MRI). It consists of a human ferritin heavy chain (an iron-storage protein) at the N terminus, a flexible polypeptide in the middle as a linker, and an EGFP at the C terminus. Through antibiotic screening, we established stable human glioma U251 cell strains that expressed ferritin–EGFP under the control of tetracycline. These cells emitted bright green fluorescence and were easily detected by a fluorescent microscope. Ferritin–EGFP overexpression proved effective in triggering obvious intracellular iron accumulation as shown by Prussian blue staining and by MRI. Further, we found that ferritin–EGFP overexpression did not cause proliferation differences between experimental and control group cells when ferritin–EGFP was expressed for <96 hours. Application of this novel ferritin–EGFP chimera has a promising future for combined optical and MRI approaches to study in vivo imaging at a cellular level.
研究领域:
医学影像学
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分子生物学
·
神经科学
·
癌症研究
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DOI
PubMed
Biology Direct
·
2024
·Vol. 19
·期 1
·1-14
·
IF 5.7
Sun Tong,Yang Zhiguang
Six-transmembrane epithelial antigen of prostate 3 (STEAP3) is associated with the progression of several human malignancies. However, its role in lung squamous cell carcinoma (LUSC) remains unclear. We measured STEAP3 expression in LUSC cell lines and tissues. LUSC cells with stable STEAP3 overexpression and knockdown were obtained through G418 selection. Multiple assays were used to evaluate the malignant phenotypes of LUSC cells and the activation of the β-catenin signaling. The potential transcriptional regulatory factors of STEAP3 were predicted using the JASPAR database, and the correlation between transcription factor AP-2 gamma (TFAP2C) and STEAP3 was analyzed through the GEPIA database. The study evaluated the regulatory relationship between a potential transcription factor and STEAP3 through ChIP and luciferase reporter assays. Additionally, rescue assays were utilized to ascertain whether TFAP2C serves as the upstream regulatory factor of STEAP3, contributing to LUSC progression. Finally, tumor growth and metastasis were evaluated in vivo. STEAP3 expression was notably higher in LUSC, and its overexpression was linked to a poor prognosis. Moreover, STEAP3 overexpression activated the β-catenin pathway, thereby accelerating cell proliferation and metastasis. Conversely, STEAP3 knockdown had an anti-tumor effect in LUSC. Additionally, TFAP2C bound directly to the STEAP3 promoter and positively regulate its expression in LUSC. The anti-tumor effects of TFAP2C knockdown were partially reversed by STEAP3 overexpression. The study indicates that the TFAP2C/STEAP3 axis may be a therapeutic target for LUSC treatment. This enhances our understanding of lung carcinogenesis.
研究领域:
肿瘤学
·
分子生物学
·
细胞生物学
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产品关联
DOI
PubMed
高影响因子
PHYTOMEDICINE
·
2021
·
IF 5.34
Ying-yuan Zhao,Xin-peng Yao,Ting-ying Jiao,Jia-ning Tian,Yue Gao,Shi-cheng Fan,Pan-pan Chen,Yi-ming Jiang,Yan-ying Zhou,Yi-xin Chen,Xiao Yang,Min Huang,Hui-chang Bi
Background Schisandrol B (SolB) is one of the bioactive components from a traditional Chinese medicine Schisandra chinensis or Schisandra sphenanthera . It has been demonstrated that SolB exerts hepatoprotective effects against drug-induced liver injury and promotes liver regeneration . It was found that SolB can induce hepatomegaly but the involved mechanisms remain unknown. Purpose This study aimed to explore the mechanisms involved in SolB-induced hepatomegaly. Methods Male C57BL/6 mice were injected intraperitoneally with SolB (100 mg/kg) for 5 days. Serum and liver samples were collected for biochemical and histological analyses. The mechanisms of SolB were investigated by qRT-PCR and western blot analyses, luciferase reporter gene assays and immunofluorescence. Results SolB significantly increased hepatocyte size and proliferation, and then promoted liver enlargement without liver injury and inflammation. SolB transactivated human PXR , activated PXR in mice and upregulated hepatic expression of its downstream proteins, such as CYP3A11 , CYP2B10 and UGT1A1. SolB also significantly enhanced nuclear translocation of PXR and YAP in human cell lines. YAP signal pathway was activated by SolB in mice. Conclusion These findings demonstrated that SolB can significantly induce liver enlargement, which is associated with the activation of PXR and YAP pathways.
研究领域:
药理学
·
分子生物学
·
肝病学
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产品关联
DOI
PubMed
高影响因子
ACTA BIOCHIMICA ET BIOPHYSICA SINICA
·
2020
·Vol. 52
·期 8
·842-852
·
IF 2.836
Zhang Liang,Wang Xin,Liu Xin,Lv Mingyue,Shen Erdong,Zhu Guolian,Sun Zhe
Esophageal cancer (EC) is one of the most common aggressive malignant diseases worldwide. miR-28-5p plays important regulatory roles in many cancers including human EC. However, the molecular mechanism and potential role of miR-28-5p in EC remain uncertain. In this study, qRT-PCR and western blot analysis revealed that miR-28-5p expression was up-regulated and metastasis suppressor-1 (MTSS1) was down-regulated in EC tissues relative to matched para-cancer tissues. Cell counting kit-8 (CCK-8) assay demonstrated that miR-28-5p mimics increased cell viability, and miR-28-5p inhibitor decreased it. Flow cytometry (FCM) assay indicated that miR-28-5p mimics promoted cell cycle entry, while miR-28-5p inhibitor reduced it and induced cell apoptosis. Moreover, miR-28-5p mimics up-regulated the expressions of cyclin A, cyclin dependent kinase 2 (CDK2), cyclin D1, and cyclin E but down-regulated the expressions of cleaved caspase-3 and cleaved caspase-9, which was abolished by miR-28-5p inhibitor. Furthermore, luciferase reporter assay verified that miR-28-5p directly targeted MTSS1 3′UTR and down-regulated its expression. MTSS1 overexpression in TE-1 cells inhibited cell proliferation and promoted apoptosis induced by miR-28-5p mimics, whereas silencing of MTSS1 reversed cell progression induced by miR-28-5p inhibitor. We also demonstrated that miR-28-5p could promote esophageal tumor formation in vivo. Hematoxylin–eosin staining, immunohistochemistry, and TUNEL assays confirmed that miR-28-5p antagomir inhibited cell growth and accelerated apoptosis. Our results suggest that miR-28-5p may induce cell proliferation and suppress apoptosis to promote EC tumor formation via decreasing MTSS1 expression. Thus, miR-28-5p may be a potential target for human EC therapy.
研究领域:
肿瘤学
·
分子生物学
·
细胞生物学
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）