TALANTA
·
2019
·
IF 4.916
Xuan Wang,Caicheng Long,Xiaoyan Xu,Taiping Qing,Peng Zhang,Bo Feng
Micrococcal nuclease (MNase) is a nonspecific endo-exonuclease that digests single-stranded/double-stranded DNA and RNA. The existence of MNase can serves as an important diagnostic biomarker of Staphylococcus aureus ( S. aureus ) infection. However, most of the substrates in MNase-based sensors are single-stranded DNA, which could also be digested by exonuclease I or S1 nuclease and interfere the MNase detection. In this work, we developed a highly selective fluorescent method for MNase detection using a specific dsDNA and nucleic acid dye SYBR Green I (SGI) as the indicator. After rational design, an AT-rich dsDNA with 3′ protruding termini was screened as the high-specific substrate of MNase assay and efficient enhancer of SGI. The AT-rich dsDNA substrate can resist the digestion of other exonuclease and greatly enhance the fluorescence of SGI. This high-specific substrate-based probe can detect MNase in buffer as well as biological sample with highly selectivity . Moreover, this method was also applied to monitor the MNase secreted by S. aureus . Thus, the proposed MNase-based assay has a strong potential to identify S. aureus in food safety and microbial infection due to its excellent analytical sensitivity and high selectivity.
研究领域:
微生物学
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分子生物学
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医学诊断
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DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2017
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IF 5.401
Dongxia Li,Jin Qin,Guiqin Yan
To solve interference from background fluorescence and scattering light of real biological samples, we developed a room-temperature phosphorescence (RTP) sensor to detect Micrococcal nuclease (MNase). The sensor was based on the phosphorescent resonance energy transfer (PRET) between 3-mercaptopropionic acid (MPA)-capped Mn-doped ZnS quantum dots (QDs) and single-marked oligonucleotide (DNA-ROX). Specifically, the poly-(diallyldimethylammonium chloride) (PDAD)-modified QDs (PDAD-QDs) were prepared as the energy donor, and the DNA-ROX was chosen as the energy receptor. The DNA-ROX could be adsorbed to the surface of the PDAD-QDs through electrostatic interaction, which induced PRET and quenched the RTP of PDAD-QDs. The MNase could efficiently degrade the DNA-ROX into small DNA segments, which were less prone to electrostatic interaction with PDAD-QDs, and thus the PRET efficiency decreased. The RTP intensity of PDAD-QDs was gradually enhanced with the increment of MNase concentration. Under the optimal conditions, the change of RTP intensity was proportional to the logarithm of MNase concentration in the range from 2 × 10 −3 to 8.0 × 10 −2 U mL −1 , with a high correlation coefficient of 0.993 and a detection limit of 6 × 10 −4 U mL −1 . This proposed RTP sensor can avoid interferences from the background fluorescence or scattering light of the matrix that are encountered in spectrofluorometry. Thus, this biosensor can be applied to detect MNase in culture media.
研究领域:
生物化学
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分析化学
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产品关联
DOI
高影响因子
Current Protocols
·
2025
·Vol. 5
·期 7
·e70179
·
IF 2.2
Bingyan Yuan,Jingjun Hong
In eukaryotic cells, nucleosomes are octameric structures composed of DNA and histones, and their tandem arrangement ultimately forms chromosomes. Nucleosome assembly is governed in vivo by a series of molecular mechanisms, particularly histone chaperones such as Swr1, which is indispensable for H2A.Z-nucleosome formation. The assessment of nucleosome assembly using micrococcal nuclease (MNase) digestion has been widely accepted. We describe a simple and reproducible protocol for analyzing the chaperone activity of SRCAP (Snf2-related CREBBP activator protein), the histone H2A.Z chaperone. This approach involves purifying a complex of H2A.Z, H2B, and SRCAP via fast protein liquid chromatography (FPLC) only, followed by its assembly with DNA-(H3.1-H4 A15C ) 2 . The formation of nucleosomes is then determined in vitro using electrophoretic mobility shift assay (EMSA) and MNase digestion. This approach can also be adapted to evaluate whether other histone chaperones or regulatory factors promote nucleosome assembly activity. © 2025 Wiley Periodicals LLC. Basic Protocol : In vitro nucleosome assembly facilitated by the histone chaperone SRCAP
研究领域:
分子生物学
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生物化学
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细胞生物学
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表观遗传学
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DOI
PubMed
高影响因子
BIODEGRADATION
·
2023
·Vol. 34
·期 4
·371-381
·
IF 3.6
Yue Caixu,Jia Nan,Lv Xueru,Wang Shenghui
3, 5, 6-Trichloro-2-pyridinol (TCP) is a metabolite of the insecticide chlorpyrifos and the herbicide triclopyr, and it is higher toxic than the parent compounds. Microbially-mediated mineralization appears to be the primary degradative pathway and the important biological process of detoxification. However, little information is available on TCP complete metabolic pathways and mechanisms. In this study, the degradation of TCP was studied with a novel strain Micrococcus luteus ML isolated from a stable TCP degrading microbiota. Strain ML was capable of degrading 61.6% of TCP (50 mg/L) and 35.4% of chlorpyrifos (50 mg/L) at 24 h and 48 h under the optimal conditions (temperature: 35 °C; pH: 7.0), respectively. It could also degrade 3, 5-dichloro-2-pyridone, 6-chloropyridin-2-ol, 2-hydroxypyridine and phoxim when provided as sole carbon and energy sources. Seven TCP intermediate metabolites were detected in strain ML and two possible degradation pathways of TCP were proposed on the basis of LC–MS analysis. Both the hydrolytic-oxidative dechlorination pathway and the denitrification pathway might be involved in TCP biodegradation by strain ML. To the best of our knowledge, this is the first report on two different pathways responsible for TCP degradation in one strain, and this finding also provides novel information for studying the metabolic mechanism of TCP in pure culture.
研究领域:
环境微生物学
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生物修复
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代谢组学
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产品关联
DOI
PubMed
高影响因子
ECOTOXICOLOGY
·
2024
·
IF 2.4
Wang Jie,Huang Hexi,Yang Hailin,Wang Su,Li Mengnan,Zhu Zhengyang,Trumble John T.,Di Ning,Zang Liansheng
Cadmium (Cd), a widely distributed environmental pollutant in agroecosystems, causes negative effects on crops and herbivores through bottom-up processes. The gut microbial community of an insect can play a critical role in response to metal stress. To understand how microbiota affect the stress responses of organisms to heavy metals in agroecosystems, we initially used 16S rRNA sequencing to characterize the larval gut microbiota of Chilo suppressalis , an important agricultural pest, exposed to a diet containing Cd. The species richness, diversity, and composition of the gut microbial community was then analyzed. Results revealed that while the richness (Chao1 and ACE) of gut microbiota in larvae exposed to Cd was not significantly affected, diversity (Shannon and Simpson) was reduced due to changes in species distribution and relative abundance. Overall, the most abundant genus was Enterococcus , while the abundance of the genera Micrococcaceae and Faecalibaculum in the control significantly superior to that in Cd-exposed pests. Phylogenetic investigation of microbial communities by the reconstruction of unobserved states (PICRUSt) showed that the intestinal microorganisms appear to participate in 34 pathways, especially those used in environmental information processing and the metabolism of the organism. This study suggests that the gut microbiota of C. suppressalis are significantly impacted by Cd exposure and highlights the importance of the gut microbiome in host stress responses and negative effects of Cd pollution in agroecosystems.
研究领域:
环境科学
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昆虫学
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微生物学
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生态毒理学
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产品关联
DOI
PubMed
高影响因子
Jove-Journal of Visualized Experiments
·
2024
·
IF 1.2
Yushu Tang,Ning Hao
Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and sideline products. Therefore, the selection of B. licheniformis with high production of bacitracin is of great importance. In this experimental protocol, Bacillus with a high yield of bacitracin was isolated, purified, and identified from the fresh feces of healthy pigs. The inhibitory effect of secondary metabolite bacitracin on Micrococcus luteus was also tested. Thin-layer chromatography and high-performance liquid chromatography were used for the qualitative and quantitative detection of bacitracin. The physiological and biochemical characteristics of B. licheniformis were determined by relevant kits. The phylogenetic relationships of B. licheniformis were determined and constructed using gene sequence detection. This protocol describes and introduces the standard isolation, purification, and identification process of B. licheniformis from animal fresh feces from multiple perspectives, providing a method for the large-scale utilization of B. licheniformis and bacitracin in factories.
研究领域:
微生物学
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药理学
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农业科学
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水产养殖
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生物技术
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产品关联
DOI
PubMed
高影响因子
ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH
·
2021
·Vol. 29
·期 2
·1824-1835
·
IF 4.223
Chen Yuan,Chen Yuancai
At present, there are many studies on microbial reduction of Pd (II), although few of these studies assess the bacterial toxicity of Pd (II) before and after reduction by microorganisms. In this study, the toxicity of Pd (II) to Bacillus wiedmannii MSM ( B. wiedmannii MSM) was assessed before and after reduction by live B. wiedmannii MSM cells (referred to as “Pd (0)-loaded cells”) and after biosorption by dead B. wiedmannii MSM cells (referred to as “Pd (II)-loaded cells”). B. wiedmannii MSM is a widely occurring, nonpathogenic aerobic bacterium. Compared with Pd (II), the EC 20 , EC 50 , and EC 80 of Pd (0)-loaded cells increased by 77.73-, 112.75-, and 163.91-fold, respectively, while the EC 20 , EC 50 , and EC 80 of Pd (II)-loaded cells increased by only 11.55-, 9.77-, and 8.29-fold, respectively. The sterilization contribution rates of the mechanisms of action of Pd (0)-loaded cells were ranked in the order of: remaining Pd (II) > oxidative stress > physical puncture. Pd (II) was found to increase cell membrane permeability, glutathione S-transferase (GST) enzyme activity, and reactive oxygen species levels in cells and decrease the cell membrane potential. XPS results indicated that Pd (II) increased the content of polysaccharides and peptides and decreased the content of hydrocarbons in cells. These findings reveal the bactericidal mechanism of toxicity of Pd (II) and Pd (0)-loaded cells on Bacillus wiedmannii MSM and provide an environmentally friendly and inexpensive method for Pd (II) detoxification.
研究领域:
环境科学
·
微生物学
·
毒理学
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产品关联
DOI
PubMed
高影响因子
TALANTA
·
2020
·
IF 5.339
Yue Wu,Shan Jiang,Zhifeng Fu
Based on the specific affinity behavior of teicoplanin towards Gram-positive (G + ) bacteria, teicoplanin-coated magnetic particles were employed to separate G + bacteria. Moreover, intracellular catalase released by catalase-positive bacteria led to catalyzed hydrolysis of H 2 O 2 and inhibition of chemiluminescent signal of luminol-H 2 O 2 system. By combining the separation capability of teicoplanin-coated magnetic particles and the inhibition of luminol chemiluminescence by catalase, a facile method was proposed for quantifying catalase-positive G + bacteria. In this proof-of-principle work, Staphylococcus aureus , Bacillus subtilis and Micrococcus luteus were adopted as model bacteria to evaluate its quantification performance. The three bacteria were quantified within the dynamic ranges of 5.0 × 10 2 –5.0 × 10 7 CFU mL −1 , 4.0 × 10 2 –4.0 × 10 7 CFU mL −1 and 2.0 × 10 2 –2.0 × 10 7 CFU mL −1 , with detection limits of 156 CFU mL −1 , 86 CFU mL −1 and 44 CFU mL −1 , respectively. Gram-negative bacteria and catalase-negative G + bacteria both displayed minor interference. The results for quantifying bacteria in milk, human urine and physiological saline samples demonstrated its reliability for real application. It provided a promising technique tool for food safety, medical diagnosis as well as drug quality control.
研究领域:
微生物学
·
分析化学
·
生物技术
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产品关联
DOI
PubMed
高影响因子
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY
·
2021
·
IF 3.636
Yijing Han,Qianqian Zhang,Lizhu Chen,Dinglong Yang,Jianmin Zhao
The formation of extracellular traps (ETs) is an important innate immune mechanism that serves to combat different invading pathogens. In this study, zymosan significantly induced the formation of ETs in the hemocytes of Ruditapes philippinarum , and this effect was accompanied by translocation of the mitochondria to the cell surface. Zymosan stimulation clearly induced an increase in intracellular ROS and MPO production and an overexpression of ROS-related genes ( PI3K , AKT and HIF ). In response to the ROS burst, the mitochondrial membrane potential decreased, and the mitochondrial permeability transition pore opened. Conversely, mitochondrial superoxide inhibitor (Mito-TEMPO) significantly inhibited the formation of ETs, suggesting that mitochondrial ROS were necessary for the formation of ETs. In addition, we found that zymosan-induced ETs showed antibacterial activities against gram-negative and gram-positive bacteria, such as Vibrio anguillarum , Vibrio harveyi , Escherichia coli and Micrococcus luteus . Taken together, these findings elucidated a new antibacterial approach for R. philippinarum and highlighted the role of mitochondria in the formation of zymosan-induced ETs.
研究领域:
免疫学
·
海洋生物学
·
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）