Jove-Journal of Visualized Experiments
·
2019
·
IF 1.108
Yafeng Zhang,Xun Liao,Peihua Luo,Kui Peng,Wenyi Ye,Tengxiang Lian,Qibin Ma,Hai Nian
In plant mitochondria, some steady-state transcripts have 5' triphosphate derived from transcription initiation (primary transcripts), while the others contain 5' monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5' triphosphate. However, the triphosphate at primary 5' termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5' polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5' triphosphate.In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5' polyphosphatase, which converts 5' triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5' polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5' end and is insensitive to 5' polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot.By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.
研究领域:
植物学
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分子生物学
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DOI
PubMed
高影响因子
TALANTA
·
2023
·
IF 6.1
Haodong Cui,Wenxing Song,Xiaoling Ru,Wen Fu,Ling Ji,Wenhua Zhou,Zhen Zhao,Guangbo Qu,Xue-Feng Yu,Guibin Jiang
The ongoing outbreak of the novel coronavirus disease 2019 (COVID-19) draws worldwide concerns due to its long incubation period and strong infectivity. Although RT-PCR-based methods are being widely applied for clinical diagnosis, timely and accurate diagnosis towards COVID-19 causing virus, the SARS-CoV-2, is still limited due to labor-intensive and time-consuming operations. Herein, we report a new viral RNA extraction method based on poly-(amino ester) with carboxyl group (PC)-coated magnetic nanoparticles (pcMNPs) for the sensitive detection of SARS-CoV-2. This method combines the lysis and binding steps into one step, and refines multiple washing steps into one step, giving a turnaround time of less than 9 min. Furthermore, the extracted pcMNP-RNA complexes can be directly introduced into subsequent RT-PCR reactions without elution. This simplified viral RNA method could be well adapted in fast manual and automated high-throughput nucleic acids extraction protocols suitable for different scenarios. A high sensitivity down to 100 copies/mL and a linear correlation between 100 and 10 6 copies/mL of SARS-CoV-2 pseudovirus particles are achieved in both protocols. Benefitting from the simplicity and excellent performances, this new method can dramatically improve the efficiency and reduce operational requirements for the early clinical diagnosis and large-scale SARS-CoV-2 nucleic acid screening.
研究领域:
分子生物学
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医学诊断
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纳米技术
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传染病研究
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2012
·Vol. 13
·期 12
·17006-17018
·
IF 2.598
Beihua Bao,Ting Geng,Yudan Cao,Weifeng Yao,Li Zhang,Anwei Ding
The aim of this study was to find out whether Schizonepetin influences the pharmacokinetics of the main substrates drugs of CYP1A2, CYP3A1/2, CYP2E1, CYP2C19 and CYP2D6 in rats; the influence on the levels of CYP mRNA was also studied. Phenacetin, dapsone, chlorzoxazone, omeprazole and metoprolol were selected as probe substrates for CYP1A2, CYP3A1/2, CYP2E1, CYP2C19 and CYP2D6 respectively. HPLC methods were employed for the determination of these substrates in plasma and the pharmacokinetic parameters were calculated. Real-time RT-PCR was used to determine the effects of Schizonepetin on the mRNA expression of CYP3A1, CYP1A2 and CYP2E1 in the rat liver. After the rats were orally administrated with Schizonepetin once a day for seven consecutive days, there were significant differences in plasma concentration of phenacetin, dapsone, chlorzoxazone and metoprolol, but not omeprazole, as compared with pre-administration. In addition, Schizonepetin induced the expression of CYP3A1, CYP1A and CYP2E1 at dosages of 24 and 48 mg/kg. Our results indicated that Schizonepetin had significant induction effects on CYP3A1/2 and inhibition effects on CYP1A2, CYP2E1 or CYP2D6 as oriented from the pharmacokinetic profiles of the substrates. Moreover, in the mRNA expression levels, Schizonepetin could induce the mRNA expression of CYP3A1, CYP1A and CYP2E1. In conclusion, co-administration of some CYP substrates with Schizonepetin may lead to an undesirable herb-drug interaction.Keywords:Schizonepetin;Cytochrome P450;substrates drugs;pharmacokinetic parameters;mRNA;real-time RT-PCR
研究领域:
药理学
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分子生物学
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DOI
PubMed
高影响因子
Cancer Reports
·
2022
·Vol. 6
·期 2
·e1688
·
IF 0
Ying Liang,Yan Liu,Pin Zhang,Mengxin Zhang,Bang Du,Weyland Cheng,Zhidan Yu,Lifeng Li,Huanmin Wang,Guangjun Hou,Xianwei Zhang,Wancun Zhang
The postoperative recurrence of neuroblastoma (NB) patients is an essential reason for the high mortality of NB due to the lack of early, non-invasive, and dynamic strategies for monitoring NB recurrence. Therefore, whether the plasma circulating cell-free MYCN gene as an indicator for monitoring of NB recurrence was systematically evaluated. The MYCN copy number and NAGK (reference gene) copy number (M/N) ratio in plasma and corresponding tumor tissues of NB patients was detected using an economical, sensitive, and specific single-tube dual RT-PCR approach developed in this study. The plasma M/N ratio of the MYCN gene amplification (MNA) group ( N = 25, median M/N ratio = 4.90) was significantly higher than that of the non-MNA group ( N = 71, median M/N ratio = 1.22), p < .001. The M/N ratio in NB plasma ( N = 60) was positively correlated with the M/N ratio in NB tumor tissue ( N = 60), with a correlation coefficient of 0.9496. In particular, the results of dynamic monitoring of postoperative plasma M/N ratio of NB patients showed that an abnormal increase in M/N ratio could be detected 1–2 months before recurrence in NB patients. In summary, the single-tube double RT-PCR approach can be used to quantitatively detect MYCN copy number. The copy number of MYCN in the tissue and plasma of NB patients is consistent with each other. More importantly, the circulating cell-free MYCN gene of NB patients can be used as a monitoring indicator for early, non-invasive, and dynamic monitoring of NB recurrence.
研究领域:
肿瘤学
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分子生物学
·
遗传学
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DOI
PubMed
Evidence-based Complementary and Alternative Medicine
·
2019
·
IF 1.984
Liwen Han,Haotian Kong,Fasheng Liu,Xiaobin Li,Shanshan Zhang,Xuanming Zhang,Yong Wu,Hua Yang,Aiping Zhang,Kechun Liu
The aim of the study is to explore the protective effect of new gelatin (NG, Xin’ejiao in China) on hematopoietic injury caused by chemotherapy. Zebrafish, at 48 hours post fertilization (hpf), was treated with different chemotherapeutic drugs to establish the zebrafish hematopoietic damage model with reduced thrombocytes and erythrocytes. The protecting effects of NG on the thrombocytes and erythrocytes were observed, respectively, on zebrafish models. Then, the RT-PCR method was used to detect the change of mRNA level of the hematopoiesis-related cytokines scl1, c-myb, pu.1, GATA1, and runx1 genes. The results showed that 50 μg·mL−1 and 100 μg·mL−1 NG rescued and increased the thrombocytes numbers induced by vinorelbine (NVB) and chloramphenicol (CHL) and the erythrocytes numbers induced by methotrexate (MTX), doxorubicin (ADM), and mechlorethamine hydrochloride (MH) in zebrafish models. Meanwhile, the mRNA expression of scl1, c-myb, and GATA1 genes in the NG treatment group was raised compared with the MTX treatment group. Also, the mRNA expression of pu.1 and Runx1 in the NG treatment group was reduced compared with the MTX treatment group. In consequence, traditional Chinese medicine NG showed a certain degree protective effect on hematopoiesis injury induced by chemotherapy in this study, which may depend on the promotion of erythrocytes proliferation and the regulation of the hematopoietic genes level.
研究领域:
药理学
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血液学
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分子生物学
·
斑马鱼研究
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DOI
PubMed
高影响因子
Frontiers in Cell and Developmental Biology
·
2020
·
IF 5.186
Jiang Ying,Song Hanbing,Jiang Ling,Qiao Yu,Yang Dan,Wang Donghua,Li Ji
Background: Silybin was known to exert inhibition in prostate cancer, but the underlying mechanism remained largely unknown. This study was designed to find out the potential target of Silybin on prostate cancer and explore the relative mechanisms.Methods: Firstly, we screened the possible targets of Silybin through the PubChem database and Subpathway – GM. Then DU145 cells were transferred to investigate the correction about related targets, magnetic bead sorting and flow cytometry were used to sort and identify the cells. Proliferation, migration and invasion ability of DU145 cells were detected by MTT assay, Transwell assay, plate clonality and sphere formation assay. BALB/c nude mice were constructed models with implanted sarcoma and measured the tumor volume every 5 days as wells tumor weight. The levels of proteins were detected by Western blot and immunocytochemistry. RT-PCR was selected to test the expression of protein’s mRNA.Results: It was screened out the ALDH1A1 was highly correlated with subpathways of the Silybin risk metabolic pathway. And ALDH1A1 expression was positively correlated RARα with Ets1 by interfering with the ALDH1A1 gene. Importantly, ALDH1A1(+) cells showed proliferation, migration and invasion ability. In addition, it showed that Silybin exerted the inhibition on prostate cells by suppressed the proliferation, migration and invasion ability of cells in vitro experiment. Silybin also reduced the tumor volume and weight. And Silybin displayed obviously reduced the proteins and mRNA of ALDH1A1, RARα, Ets1 and MMP9 expressions.Conclusion: Our results indicated that Silybin showed inhibition of prostate cancer and the mechanism was involving with downregulating ALDH1A1 expression, thereby inhibiting the activation of RARα and preventing the activation of Ets1 to inhibit the growth and invasion of prostate cancer.
研究领域:
肿瘤学
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药理学
·
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
BIOMACROMOLECULES
·
2021
·
IF 6.988
Lei Xu,Fenbo Ma,Jun Huang,Ka Li Frankie Leung,Chenghe Qin,William Weijia Lu,X. Edward Guo,Bin Tang
Cartilage lesion is a common tissue defect and is challenging in clinical practice. Trauma-induced cellular senescence could decrease the chondrocyte capability of maintaining cartilage tissue regeneration. A previous investigation showed that, by controlling the cellular senescence, the cartilage regeneration can be significantly accelerated. Based on this finding, we design a novel hydrogel, Alg/MH-Sr, that combines metformin, an established drug for inhibiting senescence, and strontium, an effective anti-inflammatory material for cartilage tissue engineering. A RT-PCR test suggests the significant inhibitory effect of the hydrogel on senescent, apoptotic, oxidative, and inflammatory genes’ expression. Histological examinations demonstrate that the Alg/MH-Sr hydrogel accelerated cartilage repairment, and chondrocyte senescence was significantly inhibited. Our study demonstrates that the Alg/MH-Sr hydrogel is effective for cartilage defect treatment and provides a new clue in accelerating tissue repairment by inhibiting the senescence of cells and tissues.
研究领域:
生物医学工程
·
药剂学
·
组织工程
·
再生医学
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DOI
PubMed
高影响因子
JOURNAL OF THE INDIAN CHEMICAL SOCIETY
·
2022
·
IF 0.243
Xiao-Na Liang,Li-Ying Xu,Ming-Li Zhai,Jie Zhang
In the current research, through employing the dual ligand method, a novel coordination polymer has been produced in success via the reaction between H 2 glu and In(NO 3 ) 3 ·6H 2 O in the existence of the nitrogen-donor chelating 2,2′-bpy ligand in CH 3 CN In(III) and DMF mixed solvent, and its chemical formula is In(glu)(Hglu)(2,2′-bpy) ( 1 , 2,2′-bpy is 2,2′-bipyridine and H 2 glu is glutaric acid). Its application values on the acute stroke were assessed and the corresponding mechanism was investigated simultaneously. Firstly, the levels of inflammatory response in the astrocytes were evaluated by ELISA by measuring the content of inflammatory cytokines released into cerebrospinal fluid. Additionally, the miRNA199a relative expression levels in astrocytes were measured via exploiting real time RT-PCR. Molecular docking simulation demonstrated that synthesized In ion complex exhibited excellent biological activities, multiple binding interactions were formed by the carboxyl groups on the In ion complex.
研究领域:
药理学
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神经科学
·
分子生物学
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DOI
高影响因子
Nanoscale
·
2023
·
IF 6.7
Haipeng Cui,Wenwei Pan,Tiechuan Li,Xiaotian Shen,Ye Chang,Wei Pang,Xuexin Duan
Virus infections remain one of the principal causes of morbidity and mortality worldwide. The current gold standard approach for diagnosing pathogens requires access to reverse transcription–polymerase chain reaction (RT‒PCR) technology. However, separation and enrichment of the targets from complex and diluted samples remains as a major challenge. In this work, we proposed a micromotor-based sample preparation concept for the efficient separation and concentration of target viral particles before PCR. The micromotors are functionalized with antibodies with a 3D polymer linker and are capable of self-propulsion by the catalytic generation of oxygen bubbles for selective and positive virus enrichment. This strategy significantly improves the enrichment efficiency and recovery rate of virus (up to 80% at 104 tu mL-1 in a 1 mL volume within just 6 min) without external mixing equipment. The method allows the Ct value in regular PCR tests appearing 6-7 cycles earlier and a detection limit of 1 tu mL-1 for target virus from swap samples. A point-of-need test kit is designed based on the micromotors which can be readily applied to pretreat a large volume of samples.
研究领域:
生物技术
·
病毒学
·
分析化学
·
医学诊断
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DOI
PubMed
高影响因子
AMERICAN JOURNAL OF POTATO RESEARCH
·
2020
·Vol. 97
·期 4
·367-375
·
IF 0.858
Daurov Dias,Daurova Ainash,Karimov Aibek,Tolegenova Dana,Volkov Dmitriy,Raimbek Daniyar,Zhambakin Kabyl,Shamekova Malika
Potato ( Solanum tuberosum L.) is the fourth most common food crop worldwide and one of the main food products in Kazakhstan. One of the factors leading to crop loss in potato is viral disease. There are more than 30 viruses known to cause diseases in potato. Therefore, using virus-free planting material is an important aspect of growing potato. It is relatively difficult to eliminate viruses from plants, despite the application of isolated apical meristems. In this study, we used the most effective methods to eliminate viral infections, such as electrotherapy, chemotherapy (40 and 50 mg/l), cryotherapy, and thermotherapy. Complete elimination of certain viral infections, such as PVM, PVS, PVX, and PVY, was achieved after chemotherapy with 40 mg/l of ribavirin. A multiplex RT-PCR method was used for efficient monitoring of viral infections, which is more precise than the enzyme-linked immunosorbent assay (DAS-ELISA). The obtained virus-free planting material will subsequently be used for industrial virus-free potato seed production in Kazakhstan.
研究领域:
农业
·
植物病理学
·
病毒学
·
生物技术
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DOI
高影响因子
LAB ON A CHIP
·
2021
·Vol. 21
·期 9
·1759-1770
·
IF 6.799
Wenwen Chen,Rongkai Cao,Wentao Su,Xu Zhang,Yuhai Xu,Peng Wang,Zhongqiao Gan,Yingying Xie,Hongjing Li,Jianhua Qin
Tumor-derived exosomes have been recognized as promising biomarkers for early-stage cancer diagnosis, tumor prognosis monitoring and individual medical treatment. However, it is a huge challenge to separate exosomes from trace biological samples in clinics for disease diagnosis. Herein, we propose a simple, quick, and label-free method for isolating circulating exosomes from serum of patients. The strategy synergistically integrates chitosan electrostatic-adsorption, micro-patterned substrates, and microfluidic shuttle flow control to enable the capture/release of circulating exosomes in a simple manner. Using this microchip, we can isolate exosomes from trace samples (10 μl) with relative purity over 90% and high RNA recovery ratio over 84% within 15 minutes, which is impossible for traditional ultracentrifugation methods. We then validate the application of the microchip using 24 serum samples from clinical breast cancer and breast fibroma patients. The isolated exosomes are subjected to miRNA sequencing and RT-PCR, followed by pathway prediction analysis. The results showed that exosomes were relevant to the invasion and metastasis of breast cancer cells and hsa-miR-18a-3p might have the potential to become a new biomarker for distinguishing breast cancer from breast fibroma (AUC = 0.83, P value = 0.019). This established method is simple, quick and easy to operate with integration. And it may pave a new way for clinical research on exosomes and tumor relevant diagnosis.
研究领域:
生物医学工程
·
癌症生物学
·
分子生物学
·
临床诊断
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产品关联
DOI
PubMed
高影响因子
Aquaculture Reports
·
2021
·
IF 3.216
Dongyong Fu,Yadong Hu,Peng Chu,Tao Wang,Mingxu Chu,Yonghai Shi,Shaowu Yin,Yongxiang Zhu,Yaohui Wang,Zhenglong Guo
Copper nanoparticles (Cu-NPs) are water pollutants, which has significant toxic environmental effects. However, the effects of Cu-NPs on Takifugu fasciatus are still relatively unknown. Compared with the control (no add CuSO 4 and Cu-NPs). This study aims to describe the pathological changes in the gill and liver of pufferfish exposed to CuSO 4 (Cu 2+ for short) and Cu-NPs dissolved at a concentration of either 20 or 100 μg/L, in a semi-static waterborne exposure regime for 28 days. All Cu treatments resulted in organ injuries, and the pathologies observed following exposure to Cu-NPs were broadly of the same type as those observed following exposure to Cu 2+ . In the gill, these included lamellar fusion, clubbed tips, and mucocytes swollen, and in the liver included oedema, aggregation of blood cells, and necrosis. Furthermore, enzyme activity assays revealed that Ca 2+ -ATP activity significantly increased under the influence of Cu, whilst Na + -K + -ATPase activity was significantly inhibited. In this study, the CRT in T. fasciatus (referred to as Tf CRT) was identified and full-length cDNA sequences were characterized. Subsequently, RT-PCR and western blot analysis were carried out to assess the expressions of Tf CRT in the gill and liver at both the mRNA and protein levels. Under both the Cu-NPs and Cu 2+ treatments, both the mRNA and protein expressions of Tf CRT were considerably higher in the gill and liver compared to that of the control treatment. Furthermore, there were no significant differences between Cu-NPs and Cu 2+ treatment (at the same treatment concentration) in the expressions of Tf CRT at the mRNA or protein levels. Overall, both Cu 2+ and Cu-NPs had negative effects on T. fasciatus , and Cu-NPs had the toxic effects similar to dissolved Cu. Our results provide the ecological safety data service of Cu-NPs in water, and provide a reference for the health T. fasciatus aquaculture.
研究领域:
环境毒理学
·
水生毒理学
·
分子生物学
·
鱼类生理学
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DOI
高影响因子
危险品化学品经营许可证（带存储）