scholarly journals Transcriptomics Reveals the ERF2-bHLH2-CML5 Module Responses to H2S and ROS in Postharvest Calcium Deficiency Apples

2021 ◽  
Vol 22 (23) ◽  
pp. 13013
Author(s):  
Hong-Ye Sun ◽  
Wei-Wei Zhang ◽  
Hai-Yong Qu ◽  
Sha-Sha Gou ◽  
Li-Xia Li ◽  
...  
Keyword(s):  
Principal Component ◽  
Calcium Content ◽  
Calcium Deficiency ◽  
Potential Interaction ◽  
Postharvest Quality ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Physiological Analysis ◽  
Apple Peels ◽  
H2s Production

Calcium deficiency usually causes accelerated quality deterioration in postharvest fruit, whereas the underlining mechanism is still unclear. Here, we report that calcium deficiency induced the development of bitter pit on the surface of apple peels compared with the healthy appearance in control apples during postharvest storage. Physiological analysis indicates that calcium-deficient peels contained higher levels of superoxide anion (O2•−), malondialdehyde (MDA), total phenol, flavonoid contents and polyphenol oxidase (PPO) activity, and reduced calcium, H2S production, anthocyanin, soluble protein content, and peroxidase (POD) activity compared with those in calcium-sufficient peels. The principal component analysis (PCA) results show that calcium content, ROS, and H2S production were the main factors between calcium-deficient and calcium-sufficient apple peels. Transcriptome data indicated that four calmodulin-like proteins (CMLs), seven AP2/ERFs, and three bHLHs transcripts were significantly differentially expressed in calcium-deficient apple peels. RT-qPCR and correlation analyses further revealed that CML5 expression was significantly positively correlated with the expression of ERF2/17, bHLH2, and H2S production related genes. In addition, transcriptional co-activation of CML5 by ERF2 and bHLH2 was demonstrated by apple transient expression assays and dual-luciferase reporter system experiments. Therefore, these findings provide a basis for studying the molecular mechanism of postharvest quality decline in calcium-deficient apples and the potential interaction between Ca2+ and endogenous H2S.

scholarly journals Overexpression of microRNA gga-miR-1650 decreases the replication of avian leukosis virus subgroup J in infected cells

2013 ◽  
Vol 94 (10) ◽  
pp. 2287-2296 ◽  
Author(s):  
Qi Wang ◽  
Xiaolin Ji ◽  
Yulong Gao ◽  
Xiaole Qi ◽  
Xiaojun Wang ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Avian Leukosis Virus ◽  
Potential Interaction ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Reporter System ◽  
Cellular Mirnas ◽  
Gag Protein ◽  
Subgroup J ◽  
Avian Leukosis Virus Subgroup

MicroRNAs (miRNAs) are a class of small regulatory non-coding RNAs that modulate gene expression at the post-transcriptional level, playing a crucial role in cell differentiation and development. Recently, some reports have demonstrated that a number of cellular miRNAs play a role during viral infection. In this study, a luciferase-reporter system carrying the 5′ untranslated region (5′ UTR) and 3′ UTR of avian leukosis virus subgroup J (ALV-J) was used to determine whether cellular miRNAs are involved in ALV-J infection. The miRNA gga-miR-1650 was screened for its potential interaction with the 5′ UTR of ALV-J and the ability to suppress luciferase-reporter activity. A mutational analysis of predicted gga-miR-1650-binding sites showed that the 5′ and 3′ ends of gga-miR-1650 contributed to the interaction between gga-miR-1650 and its target located at the 5′ UTR. Overexpression of miRNA gga-miR-1650 was shown to downregulate the expression of the Gag protein and influence the replication of ALV-J through binding to the 5′ UTR. Overall, this report provides the basis for the development of new strategies for anti-ALV-J intervention.

scholarly journals Structure-guided engineering of adenine base editor with minimized RNA off-targeting activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jianan Li ◽  
Wenxia Yu ◽  
Shisheng Huang ◽  
Susu Wu ◽  
Liping Li ◽  
...  
Keyword(s):  
Rna Editing ◽  
Potential Interaction ◽  
Reporter System ◽  
Independent Manner ◽  
Robust Detection ◽  
Guide Rna ◽  
Highly Efficient ◽  
Adenine Base

AbstractBoth adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.

Detection and Functional Evaluation of −262A/T and −188A/G Polymorphisms of SLAM Gene in Patients with Systemic Lupus Erythematosus

2010 ◽  
Vol 37 (11) ◽  
pp. 2268-2272 ◽  
Author(s):  
YI YOU ◽  
ZHE WANG ◽  
GUO-HONG DENG ◽  
YI LIU ◽  
FEI HAO
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Lupus Erythematosus ◽  
Mononuclear Cells ◽  
Gene Promoter ◽  
Chinese Patients ◽  
Luciferase Reporter ◽  
Functional Evaluation ◽  
Reporter System ◽  
Systemic Lupus ◽  
Peripheral Blood Mononuclear

Objective.Signaling lymphocytic activation molecule (SLAM) has been related to the pathology of systemic lupus erythematosus (SLE) through regulation of T cell-dependent humoral immune responses. We investigated the functional associations of the −262A/T and −188A/G polymorphisms of SLAM in Chinese patients with SLE.Methods.Genotyping of −262A/T (rs2295614) and −188A/G (rs2295613) in SLAM was carried out in 248 cases and 278 controls. Promoter activities of haplotypes on the SLAM gene were evaluated with the dual-luciferase reporter system. The mRNA expressions of SLAM on peripheral blood mononuclear cells (PBMC) of SLE patients with different genotypes were determined by real-time polymerase chain reaction.Results.Frequencies of −262A allele and −188G allele were significantly higher in SLE patients than in controls. Haplotype analysis and multifactorial logistic regression analysis showed that individuals with the AG/AG haplotype had increased susceptibility to SLE (p = 0.002, OR 1.478, 95% CI 1.152–1.897). In response to PHA stimulation, the SLAM mRNA expression on PBMC of SLE patients was significantly higher in −262A-188G haplotype homozygotes compared with −262A-188G heterozygotes and individuals with other genotypes.Conclusion.Our findings suggest that −262A-188G haplotype in the SLAM gene promoter contributes to the risk of SLE by increasing the expression of SLAM.

scholarly journals The nuclear factor κB inhibitor (E)-2-fluoro-4′-methoxystilbene inhibits firefly luciferase

2012 ◽  
Vol 32 (6) ◽  
pp. 531-537 ◽  
Author(s):  
Albert Braeuning ◽  
Silvia Vetter
Keyword(s):  
Photon Emission ◽  
Gene Promoter ◽  
Firefly Luciferase ◽  
Nuclear Factor Κb ◽  
Signalling Pathway ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Reporter System ◽  
Reporter Assays

Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

scholarly journals miR-299-3p suppresses cell progression and induces apoptosis by downregulating PAX3 in gastric cancer

2021 ◽  
Vol 16 (1) ◽  
pp. 266-276
Author(s):  
Zhenfen Wang ◽  
Qing Liu ◽  
Ping Huang ◽  
Guohao Cai
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Western Blot Assay ◽  
Normal Tissues ◽  
Cell Progression ◽  
Luciferase Reporter System ◽  
Proliferation And Invasion

Abstract Gastric cancer (GC) is ranked the fourth leading cause of cancer-related death, with an over 75% mortality rate worldwide. In recent years, miR-299-3p has been identified as a biomarker in multiple cancers, such as acute promyelocytic leukemia, thyroid cancer, and lung cancer. However, the regulatory mechanism of miR-299-3p in GC cell progression is still largely unclear. Cell viability and apoptosis tests were performed by CCK8 and flow cytometry assay, respectively. Transwell assay was recruited to examine cell invasion ability. The interaction between miR-299-3p and PAX3 was determined by the luciferase reporter system. PAX3 protein level was evaluated by western blot assay. The expression of miR-299-3p was downregulated in GC tissues and cell lines (MKN-45, AGS, and MGC-803) compared with the normal tissues and cells. Besides, overexpression of miR-299-3p significantly suppressed proliferation and invasion and promoted apoptosis in GC. Next, we clarified that PAX3 expression was regulated by miR-299-3p using a luciferase reporter system, qRT-PCR, and western blot assay. Additionally, downregulation of PAX3 repressed GC cell progression. The rescue experiments indicated that restoration of PAX3 inversed miR-299-3p-mediated inhibition on cell proliferation and invasion. miR-299-3p suppresses cell proliferation and invasion as well as induces apoptosis by regulating PAX3 expression in GC, representing desirable biomarkers for GC diagnosis and therapy.

without the necessity for cultivation of the cells (Rattray et al.1990). The light output is indicative of a metabolically active population of cells, since luciferase enzymes are dependent on cellular activity reserves, or reducing equivalents for bioluminescence. If the cells are growing, the light output is proportional to the number of cells in the sample. However, after long-term incubation in harsh environments, microbial cells often become starved or stressed and the light production from luciferase enzymes declines as a response to the change in cellular energy status (Duncan et al.1994). Therefore, in situbioluminescence is not a reliable indicator of microbial biomass under starvation conditions. The substrate and energy limitations for the luciferase reporter system may be overcome by adding nutrients to the sample in order to activate the microbial population (Meikle et al.1992, Duncan et al.1994). Alternatively, total protein from the environmental sample may be extracted. Then, the energy source can be added directly to the protein extract in vitroand the luciferase enzyme activity can be correlated to the specific luciferase-tagged microbial biomass in the sample (Moller et al.1995).

2003 ◽  
pp. 236-236
Keyword(s):  
Microbial Biomass ◽  
Light Output ◽  
Luciferase Reporter ◽  
Energy Status ◽  
Reporter System ◽  
Active Population ◽  
Light Production ◽  
Luciferase Enzyme ◽  
Starvation Conditions

scholarly journals miRNA-24 Down-Regulates Endothelial Nitric Oxide Synthase Expression and Inhibits Endothelial Cell Tube Formation

2020 ◽  
Author(s):  
Xuelan Luo ◽  
Wei Chen ◽  
Yuwang Qin ◽  
Na Gan ◽  
Dongning Lvy ◽  
...  
Keyword(s):  
Umbilical Vein ◽  
Cell Number ◽  
Tube Formation ◽  
High Expression ◽  
Luciferase Reporter ◽  
Tube Length ◽  
Control Group ◽  
Reporter System ◽  
Enos Expression ◽  
High Expression Group

Abstract Background: This study is to investigate the effects of miR-24 on expression of eNOS and Sp1, the proliferation, migration, and tube formation abilities of human umbilical vein endothelial cells (HUVECs). Results: After transfection with miR-24 overexpression plasmid or anti-miR-24, miR-24 was successfully over-expressed or inhibited. Compared with the control group, the HUVEC proliferation and cell number in the miR-24 high expression group was significantly decreased. Moreover, for the miR-24 high expression group, the cell motility was slower, and the migrating cells were significantly decreased by 61.20%, with very few capillaries in the Matrigel assay. Furthermore, the mRNA and protein expression levels of eNOS were decreased by 44.44% and 47.00 %, respectively. Meanwhile, the mRNA and protein levels of Sp1 were significantly decreased by 34.88% and 68.00%, respectively. In the miR-24 interference group, the above indexes were decreased compared with control group, while significantly increased compared with the miR-24 high expression group, especially concerning the number of branches and the tube length. Moreover, the Sp-1 and eNOS mRNAs were found to be the direct targets of miR-24 by a luciferase reporter system.Conclusion: Over-expression of miR-24 significantly suppresses cell proliferation, migration, and tube formation ability of HUVECs, via regulating eNOS expression. The transcription factor Sp1, a target of miR-24, might contribute to the eNOS expression regulation and the inhibiting effects on HUVECs.

scholarly journals Effect of UV-C radiation and hot water on the calcium content and postharvest quality of apples

2007 ◽  
Vol 5 (4) ◽  
pp. 559 ◽  
Author(s):  
S. Hemmaty ◽  
S. Hemmaty ◽  
N. Moallemi ◽  
L. Naseri
Keyword(s):  
Calcium Content ◽  
Hot Water ◽  
Postharvest Quality ◽  
Uv C

scholarly journals A Cell-Based High-Throughput Assay for the Screening of Small-Molecule Inhibitors of p53–MDM2 Interaction

2011 ◽  
Vol 16 (4) ◽  
pp. 450-456 ◽  
Author(s):  
Jing Li ◽  
Shuyong Zhang ◽  
Linghuan Gao ◽  
Ying Chen ◽  
Xin Xie
Keyword(s):  
High Throughput ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Direct Inhibition ◽  
A Cell ◽  
Luciferase Reporter System ◽  
Novel Strategy ◽  
High Throughput Assay ◽  
Two Hybrid ◽  
Rule Out

The p53 tumor suppressor is a potent transcription factor that regulates cell growth inhibition and apoptosis. The oncoprotein MDM2 suppresses p53 activity by direct inhibition of its transcriptional activity and enhances the degradation of p53 via the ubiquitin–proteosome pathway. Overexpression of MDM2, found in many human tumors, impairs p53-mediated cell death effectively. Inhibition of the p53–MDM2 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. To search for new inhibitors of the p53–MDM2 interaction, the authors developed a cell-based high-throughput assay system based on mammalian two-hybrid technology. They also used a dual-luciferase reporter system to rule out false- positive hits due to the cytotoxic effect of compounds. Using this assay, they screened a library consisting of 3840 compounds and identified one compound that activates p53 pathway and induces growth arrest in tumor cells.

Abstract 1384: Osteogenic Transcription Factor Runx2 Regulates Expression of RANKL during VSMC Calcification

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Chang Hyun Byon ◽  
Jay McDonald ◽  
Yabing Chen
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Molecular Mechanism ◽  
Luciferase Reporter ◽  
Rankl Expression ◽  
Reporter System ◽  
Mobility Shift ◽  
Atherosclerotic Lesions ◽  
Flanking Region

The expression of receptor activator of nuclear factor κ B (RANKL) is up-regulated in calcified atherosclerotic lesions, whereas it is frequently undetectable in normal vessels. The underlying molecular mechanism of increased expression of RANKL in calcified vessels is not known. We have previously demonstrated that oxidative stress induces calcification of vascular smooth muscle cells (VSMC) in vitro . Therefore, we determined whether oxidative stress regulates RANKL expression in VSMC and the underlying molecular mechanism. Consistent with previous observations in vivo , we found that the expression of RANKL in VSMC isolated from mouse. However, hydrogen peroxide (H 2 O 2 ), which induces VSMC calcification, induced a 33-fold increase in the transcripts of RANKL as determined by real-time PCR. Increased expression of RANKL protein was further confirmed by ELISA. Using flow cytometry, we demonstrated that membrane-bound RANKL was increased by oxidative stress. To characterize the molecular mechanism underlying H 2 O 2 -induced RANKL expression, we employed the luciferase reporter system with a series of deletion mutants of the RANKL 5′-flanking region. The H 2 O 2 responsive region is located between −200 to −400 in the 5′-flanking region of RANKL gene. Analyses of the sequence of this region identified multiple binding sites for the key osteogenic transcription factor, Runx2, which we previously reported to be an essential regulator of VSMC calcification. Electrophoretic mobility shift analyses demonstrated increased binding of Runx2 on the RANKL promoter sequence in nuclear extracts from VSMC exposed to H 2 O 2 . To further determine the role of Runx2 in regulating RANKL expression, we generated stable Runx2 knockdown VSMC with the use of lentivirus-carrying shRNA for Runx2 gene. H 2 O 2 -induced RANKL expression was abrogated in VSMC with Runx2 knockdown. In addition, adenovirus-mediated overexpression of Runx2 in VSMC induced the expression of RANKL. In summary, we have demonstrated that H 2 O 2 induces the expression of RANKL in VSMC, which is regulated by the osteogenic transcription factor Runx2. These observations provide novel molecular insights into the regulation of RANKL and its role on the pathogenesis of calcified atherosclerotic lesions.

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