Simultaneous quantitation of cytokinin bases and their glycoconjugates with stable isotope labelling ultrahigh performance liquid chromatography mass spectrometry.

Cytokinins (CKs) are one class of important phytohormones widely investigated in most aspects of plant life. Similar to other phytohormones, CKs and their glycoconjugates are hydrophilic. Their ionization efficiencies for mass spectrometry (MS) detection are rather poor, whereas their retention and separation on reverse phase liquid chromatography (RPLC) are often unsatisfying. Chemical isotope labelling LC-MS analysis methods have been developed for most other phytohormones, enhancing their LC separations and quantitative sensitivity. However, there are currently no reports for chemical-labelled CKs. Here, we report a new chemical isotope labelling LC-MS analytical method for one-pot derivatization of CK bases and their glycoconjugates, based on differential benzylation labelling of the adenine skeleton of CKs with benzyl bromide and its deuterium isotope-labelled reagent. Benzylation alters the hydrophilicity of CKs and their glycoconjugates, improving their retention and separation on RPLC. The developed method demonstrated enhanced sensitivity, as the CKs and their glycoconjugates could be analysed with LODs within the range of 0.62-25.9 pg/mL. The method also demonstrated good intra- and inter-day precisions with standard deviations in the range of 1.9%-13.0%, and acceptable accuracy with recoveries in the range of 84.0%-119.9%. The developed method was employed on the quantitation of CKs in the fresh roots of Astragalus membranaceus collected from both fertilized and unfertilized fields. The significant impact that fertilizers had on endogenous CKs metabolism was observed. As such, monitoring endogenous CKs and their metabolites might be promising to control fertilizer abuse.

The Application of Exosomes in the Development and Diagnosis and Treatment of Cervical Cancer / 中国生物化学与分子生物学报

Cervical cancer, as the second malignant female cancer, remains a major public health problem worldwide. Exosomes, nanometer-sized extracellular vesicles enveloped in a lipid bilayer membrane, are released by living cells and carry a variety of proteins, lipids, DNA, RNA(including mRNA, miRNA, lncRNA and circRNA) and other biologically active substances. As a novel intercellular communication molecule, exosomes are not only involved in biological processes such as signaling and substance exchange, but also play an important role in the process of the occurrence and development of cervical cancer such as regulating the tumor microenvironment to participate in HPV infection and the immune escape, promoting tumor cell proliferation and blood vessel formation, and regulating the invasion and metastasis of tumor. Exosomes with stability structure, are secreted by cervical cells of different lesion degrees, are ubiquitous in various biological fluids and can be enriched in cervicovaginal lavage sample fluid and plasma, thus it is expected to be a new type of liquid biopsy marker for the early diagnosis of cervical cancer. In addition, exosomes have the characteristics of low immunogenicity, good stability and strong penetration, which can overcome many shortcomings such as low bioavailability and enhance the targeting and drug effect of drugs while reducing non-targeted cytotoxicity and immunogenicity, thus it has the potential to be a new generation of drugs or drug carriers for tumor targeted therapy. This article reviews the latest research on the role of exosomes in the development of cervical cancer and the relevant clinical applications, aiming to provide the basis for novel biomarker for the clinical diagnosis and treatment of cervical cancer.

Inhibitory effect of Rnai on AML1 -ETO fusion gene expression in leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>By inhibiting AML1 -ETO fusion gene expression in Kasumi-1 cells with RNAi, to investigate the changes in cell proliferation and cell cycle.</p><p><b>METHODS</b>The small interference RNAs (siRNAs) specifically targeting the AML1 -ETO fusion gene were synthesized in vitro and transfected into Kasumi-1 cells by electroporation, the non-specific siRNAs transfected cells were taken as control. EGFP plasmid was transfected into Kasumi-1 cell and the transfection efficiency was detected by FCM. Inhibitory effect of siRNAs were detected by real-time RT-PCR and Western blots. Cell proliferation was measured by CCK-8 assay. DNA content was detected by PI assay.</p><p><b>RESULTS</b>The transfection efficiency was 44.5%. The AML1 -ETO specific siRNAs inhibited AML1 -ETO expression at both mRNA and protein levels. The cell proliferation rate in siRNAs treated group was lower than that in control group 72 h after transfection [(47.90 +/- 0.02)% vs (66.90 +/- 0.08)% , P < 0.05]. The cell cycle was blocked at G1 phase 72 h after siRNAs treatment, the cell proportion in G1 phase being 38.3% and 31.6% in control group, while in G2/M phase being 1.8% and 2.4% respectively.</p><p><b>CONCLUSIONS</b>The synthesized siRNAs can inhibit AML1 -ETO fusion gene expression. AML1 -ETO specific siRNA induced the decline of AML1 -ETO fusion protein in Kasumi-1 cell, and then caused the cell cycle blocked in G1 stage and eventually inhibited the cell proliferation.</p>

Inhibitory effect of RNA interference on chronic myeloid leukemia bcr/abl oncogene expression / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of RNA interference on chronic myeloid leukemia (CML) bcr/abl oncogene expression.</p><p><b>METHODS</b>The small interference RNAs (siRNAs) were synthesized in vitro. K562 cells stably expressing bcr/abl gene were transfected with the siRNA by electroporation, both the non-transfected cells and non-specific siRNAs transfected cells were taken as controls. The enhanced green fluorescent protein (EGFP) plasmid was used as positive control and the transfection efficiency was detected by flow cytometry. Inhibitory effect of siRNAs was demonstrated by real-time quantitative RT-PCR and Western blots. Cell proliferation was measured by MTT assay and apoptosis by Annexin V-FITC assay.</p><p><b>RESULTS</b>The transfection efficiency was about 70%. The synthesized siRNAs inhibited CML bcr/abl oncogene expression at both mRNA and protein levels. siRNAs could inhibit K562 cell proliferation to 47% and 56% at 24 h and 48 h after transfection, respectively, and induce cell apoptosis from 1.00% in control group to 15.05% and 19.4% at 24 h and 48 h respectively.</p><p><b>CONCLUSION</b>At the cell level, inhibition of CML bcr/abl oncogene expression by chemically synthesized siRNAs provides the new method for anti-leukemia study.</p>

Effects of acrylamide on DNA damage in human keratinocytes / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the toxic and DNA damaging effect of acrylamide (AA) on human keratinocytes and its mechanism.</p><p><b>METHODS</b>(1) After the keratinocyte cell line HaCaT cells were exposed to AA with different concentrations for 44 hours, cell survival rate was detected by MTT method. (2) The effects of DNA damage of exposed cells were detected by comet assay. (3) After treating the cells with 2.00 mmol/L of AA plus 0.50 mmol/L of 1-aminobenzotriazole (1-ABT), an inhibitor of cytochrome P-450 enzymes (CYP-450), for 4 hours, the relationship between DNA damage and CYP-450 was studied.</p><p><b>RESULTS</b>(1) Cytotoxicity measurement of AA showed that cell survival rate decreased significantly after 44-hour treatment. (2) Cytotoxicity was not detected after 4-hour AA treatment, but significant DNA damage was observed in all treatment groups, and the degree of damage increased with the concentration of AA. Moreover, the tail lengths of comet cells were in dose-effect relationship. As for cells treated by 1-ABT with 2 mmol/L AA, comet rate and tail length were 15.4% and (8.2 +/- 2.0) micro m respectively, which were decreased significantly (P < 0.01) when compared with 2 mmol/L AA treatment group [80.6% and (44.3 +/- 4.0) micro m].</p><p><b>CONCLUSIONS</b>Acrylamide has significant cytotoxicity and genotoxicity on HaCaT cells. AA-induced DNA damage may be related to the oxidative metabolite(s) of AA through CYP-450.</p>