Decontamination of DNA in Taq DNA polymerase reagents using nylon membranes for monitoring of GMOs

Zhao Shi Rong,Qu Bo,Kim Jae Kwang,Lee Bumkyu 한국식물생명공학회 2021 Plant biotechnology reports Vol.15 No.6

Polymerase chain reaction (PCR) is one of the most commonly used methods in diagnostic and molecular biology. However, DNA contamination can lead to incorrect results. This poses a major problem, especially in the monitoring of genetically modified organisms (GMOs). We investigated DNA contamination in 16 commercial Taq DNA polymerase reagents. Many of the Taq DNA polymerase reagents were contaminated with the 16S rRNA gene and an ampicillin resistance gene (bla). We attempted to decontaminate the Taq DNA polymerase reagents using UV, γ-ray, and electron-ray irradiation, as well as nylon membrane and FTA card treatment. UV irradiation reduced the activity of Taq DNA polymerase. Various wavelengths of γ-ray and electron-ray irradiation had no decontamination effect. However, 24-h treatment with three nylon membrane disks removed DNA contamination. FTA card treatment, which has membrane-like functions, also removed decontamina- tion but decreased enzyme activity. We further confirmed that the nylon membrane-treated Taq DNA polymerase reagent could be used in the detection of the bla and NPTII genes in genetically modified E. coli. These results are useful for future monitoring of GMOs.

Value of PAX1 Methylation Analysis by MS-HRM in the Triage of Atypical Squamous Cells of Undetermined Significance

Li, Shi-Rong,Wang, Zhen-Ming,Wang, Yu-Hui,Wang, Xi-Bo,Zhao, Jian-Qiang,Xue, Hai-Bin,Jiang, Fu-Guo Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.14

Background: Detection of cervical high grade lesions in patients with atypical squamous cells of undetermined significance (ASCUS) is still a challenge. Our study tested the efficacy of the paired boxed gene 1 (PAX1) methylation analysis by methylation-sensitive high-resolution melting (MS-HRM) in the detection of high grade lesions in ASCUS and compared performance with the hybrid capture 2 (HC2) human papillomavirus (HPV) test. Materials and Methods: A total of 463 consecutive ASCUS women from primary screening were selected. Their cervical scrapings were collected and assessed by PAX1 methylation analysis (MS-HRM) and high-risk HPV-DNA test (HC2). All patients with ASCUS were admitted to colposcopy and cervical biopsies. The Chisquare test was used to test the differences of PAX1 methylation or HPV infection between groups. Results: The specificity, sensitivity, and accuracy for detecting CIN2 + lesions were: 95.6%, 82.4%, and 94.6%, respectively, for the PAX1 MS-HRM test; and 59.7%, 64.7%, and 60.0% for the HC2 HPV test. Conclusions: The PAX1 methylation analysis by MS-HRM demonstrated a better performance than the high-risk HPV-DNA test for the detection of high grade lesions (CIN2 +) in ASCUS cases. This approach could screen out the majority of low grade cases of ASCUS, and thus reduce the referral rate to colposcopy.

MicroRNA-206 Reduces Osteosarcoma Cell Malignancy In Vitro by Targeting the PAX3-MET Axis

Qian-Rong Deng,Fang-Biao Zhan,Xian-Wei Zhang,Shi-Long Feng,Jun Cheng,You Zhang,Bo Li,Li-Zhong Xie 연세대학교의과대학 2019 Yonsei medical journal Vol.60 No.2

Purpose: This study was undertaken to explore how miR-206 represses osteosarcoma (OS) development. Materials and Methods: Expression levels of miR-206, PAX3, and MET mRNA were explored in paired OS and adjacent tissuespecimens. A patient-derived OS cell line was established. miR-206 overexpression and knockdown were achieved by lentiviraltransduction. PAX3 and MET overexpression were achieved by plasmid transfection. Treatment with hepatocyte growth factor(HGF) was utilized to activate c-Met receptor. Associations between miR-206 and PAX3 or MET mRNA in OS cells were verifiedby AGO2-RNA immunoprecipitation assay and miRNA pulldown assay. OS cell malignancy was evaluated in vitro by cell proliferation,metastasis, and apoptosis assays. PAX3 and MET gene expression in OS cells was assayed by RT-qPCR and Western blot. Activation of PI3K-AKT and MAPK-ERK in OS cells were assayed by evaluating Akt1 Ser473 phosphorylation and total threoninephosphorylation of Erk1/2, respectively. Results: Expression levels of miR-206 were significantly decreased in OS tissue specimens, compared to adjacent counterparts,and were inversely correlated with expression of PAX3 and MET mRNA. miR-206 directly interacted with PAX3 and MET mRNAin OS cells. miR-206 overexpression significantly reduced PAX3 and MET gene expression in OS cells in vitro, resulting in significantdecreases in Akt1 and Erk1/2 activation, cell proliferation, and metastasis, as well as increases in cell apoptosis, while miR-206 knockdown showed the opposite effects. The effects of miR-206 overexpression on OS cells were reversed by PAX3 or METoverexpression, but only partially attenuated by HGF treatment. Conclusion: miR-206 reduces OS cell malignancy in vitro by targeting PAX3 and MET gene expression.

Development of a Novel Spawn (Block Spawn) of an Edible Mushroom, Pleurotus ostreatus, in Liquid Culture and its Cultivation Evaluation

( Wei-rui Zhang ),( Sheng-rong Liu ),( Yun-bo Kuang ),( Shi-zhong Zheng ) 한국균학회 2019 Mycobiology Vol.47 No.1

Mushroom cultivation has gained increased attention in recent years. Currently, only four types of spawn, including sawdust spawn, grain spawn, liquid spawn, and stick spawn, are commonly available for mushroom cultivation. This limited spawn diversity has led to difficulty in selecting suitable inoculum materials in some cultivation. In this study, three small blocks of lignocellulosic agro-wastes and one block of a synthetic matrix were prepared as support for growing Pleurotus ostreatus in liquid medium. Mycelium-adsorbed blocks were then evaluated for their potential as block spawn for fructification. Our results indicated that the edible fungus was adsorbed and abundantly grew internally and externally on loofah sponge and synthetic polyurethane foam (PUF) supports and also has the ability to attach and grow on the surface of sugarcane bagasse and corncob supports. The mycelia of P. ostreatus adhered on corncob exhibited the highest metabolic activity, while those on the PUF showed the least activity. Mycelial extension rates of block spawns made of agro-waste materials were comparable to that of sawdust spawn, but the block spawn of PUF showed a significantly lower rate. No significant differences in cropping time and yield were observed among cultivations between experimental block spawns and sawdust spawns. Moreover, the corncob block spawn maintained its fruiting potential during an examined period of 6-month storage. The developed block spawn could be practically applied in mushroom cultivation.

<i>Premnagrandipaniculata</i> ( Lamiaceae , Premnoideae ), a remarkable new species from north Myanmar

Tan, Yun-Hong,Li, De-Rong,Zhou, Shi-Shun,Chen, Yong-Jun,Bramley, Gemma L.C.,Li, Bo Pensoft Publishers 2018 PhytoKeys Vol.94 No.-

<P>Abstract</P><P>A remarkable new <I>Premna</I> species from Myanmar, <I>P.grandipaniculata</I> Y.H.Tan & Bo Li (Lamiaceae), is here described and illustrated. It differs from all known congeneric taxa by having huge complicated panicles which have tertiary branches formed by spike-like thyrses. In <I>Premna</I>, such a spike-like thyrse is found in <I>P.bracteata</I> and <I>P.interrupta</I>, but those species can be easily distinguished from <I>P.grandipaniculata</I> by their habit, indumentum, leaf size and inflorescence structure.</P>

Different Catabolism Pathways Triggered by Various Methylxanthines in Caffeine-Tolerant Bacterium Pseudomonas putida CT25 Isolated from Tea Garden Soil

( Yi-xiao Ma ),( Xiao-han Wu ),( Hui-shi Wu ),( Zhan-bo Dong ),( Jian-hui Ye ),( Xin-qiang Zheng ),( Yue-rong Liang ),( Jian-liang Lu ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.7

The degradation efficiency and catabolism pathways of the different methylxanthines (MXs) in isolated caffeine-tolerant strain Pseudomonas putida CT25 were comprehensively studied. The results showed that the degradation efficiency of various MXs varied with the number and position of the methyl groups on the molecule (i.e., xanthine > 7-methylxanthine ≈ theobromine > caffeine > theophylline > 1-methylxanthine). Multiple MX catabolism pathways coexisted in strain CT25, and a different pathway would be triggered by various MXs. Demethylation dominated in the degradation of N-7-methylated MXs (such as 7- methylxanthine, theobromine, and caffeine), where C-8 oxidation was the major pathway in the catabolism of 1-methylxanthine, whereas demethylation and C-8 oxidation are likely both involved in the degradation of theophylline. Enzymes responsible for MX degradation were located inside the cell. Both cell culture and cell-free enzyme assays revealed that N-1 demethylation might be a rate-limiting step for the catabolism of the MXs. Surprisingly, accumulation of uric acid was observed in a cell-free reaction system, which might be attributed to the lack of activity of uricase, a cytochrome c-coupled membrane integral enzyme.

Characteristics of Refractive Index Profiles at Different Temperatures in LiNbO₃ and KTiOPO₄ Waveguide Formed by 350 keV Light Ions

Ke-Ming Wang,Feng Chen,Hui Hu,Hui-Hao Xia,Xue-Lin Wang,Bo-Rong Shi,Qing-Ming Lu 한국진공학회(ASCT) 2003 Applied Science and Convergence Technology Vol.12 No.S1

Both LiNbO₃ and KTiOPO₄ samples were implanted with 350 keV H^+ and He^+ ions at different doses ranging from 1×10^(16) to of 5×10^(16) ions/㎠. Single and multi-energy implantations were performed at room temperature. Mono-mode or a few modes in both LiNbO₃ and KTiOPO₄ waveguides were observed. The effect of temperature on the refractive index profiles of LiNbO₃ and KTiOPO₄ waveguids was studied. The temperature covered from room temperature, 200℃, 194.5 K (dry ice) and 77K (liquid nitrogen). Different mechanisms are needed to interpret the observed behavior. A n_e increased mono-mode LiNbO₃ waveguide was formed by multi-energy keV He^+ ions.