CRISPR/Cas9-mediated mutation of OsSWEET14 in rice cv. Zhonghua11 confers resistance to Xanthomonas oryzae pv. oryzae without yield penalty.

BACKGROUND: Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating rice disease in Southeast Asia and West Africa. OsSWEET14, encoding a sugar transporter, is known to be a major susceptible gene of bacterial blight targeted by four different transcription activator-like (TAL) effectors from either Asian or African Xoo strains. However, the OsSWEET14 single knockout or promoter mutants in the Kitaake background are moderately resistant or even susceptible to African Xoo strains. Therefore, in this study, we knocked out OsSWEET14 in rice cv. Zhonghua 11 background for disease assessment. RESULTS: In this study, CRISPR/Cas9 was utilized to disrupt the function of OsSWEET14 by modifying its corresponding coding region in the genome of rice cv. Zhonghua 11 (CR-S14). In total, we obtained nine different OsSWEET14-mutant alleles. Besides conferring broad-spectrum resistance to Asian Xoo strains, tested mutant alleles also showed strong resistance to African Xoo strain AXO1947. Moreover, the expression of OsSWEET14 was detected in vascular tissues, including the stem, leaf sheath, leaf blade and root. The disruption of OsSWEET14 led to increased plant height without a reduction in yield. CONCLUSIONS: Disruption of OsSWEET14 in the Zhonghua 11 background is able to confer strong resistance to African Xoo strain AXO1947 and Asian Xoo strain PXO86. CR-S14 has normal reproductive growth and enhanced plant height under normal growth conditions. These results imply that CR-S14 may serve as a better tester line than sweet14 single-knockout mutant in the Kitaake background for the diagnostic kit for rice blight resistance. The genetic background and increased plant height need to be taken into consideration when utilizing OsSWEET14 for resistant rice breeding.

A conformational switch-based fluorescent biosensor for homogeneous detection of telomerase activity.

As a universal tumor biomarker, research on the activity and inhibition of telomerase is of great importance for cancer diagnosis and therapy. Herein, we demonstrate the conformational switch-based fluorescence detection of telomerase activity using a redesigned RNA aptamer Spinach. Briefly, the original Spinach aptamer was extended at its 5' end and folded into an inactive conformation, where association with the small molecule fluorophore, 5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) was prevented. Only in the presence of telomerase, (TTAGGG)n repeats were added to the 3' end of the telomerase substrate primer, and the elongation products hybridized with inactive Spinach molecules, triggering its conformational switch and refolding it into the active, DFHBI-binding conformation. Moreover, the fluorescence signal was further amplified through a target recycling circuit, where Ribonuclease H (RNase H) specifically hydrolyzed the phosphodiester bonds of RNA in the DNA-RNA hybrid. The released telomere products could then hybridize to new inactive Spinach molecules and initiate multiple amplification cycles. The proposed fluorescent biosensor presented great performance for telomerase activity detection from 100 to 5 × 104 Hela cells with a detection limit of 100 cells. Besides, this new assay offers a good biosensing platform for differentiation of cancer cell lines from normal cell line and evaluation the inhibition efficiency of telomere-binding ligand, which is of great importance for telomerase-related cancer diagnosis and therapy.

Determination of venlafaxine and its active metabolite O-desmethylvenlafaxine in human plasma by HPLC fluorescence.

BACKGROUND: Therapeutic drug monitoring guides clinical individualised medication by measuring plasma concentration, which could improve the curative effect, avoid drug overdose and reduce the incidence of adverse reactions. At present, there are few reports on the clinical detection of venlafaxine and its active metabolite O-desmethylvenlafaxine. In this paper, the detection method of venlafaxine and O-desmethylvenlafaxine in blood plasma was established, which provides an effective and convenient means for guiding clinical application of medication. AIM: To establish a method for determination of venlafaxine and its active metabolite O-desmethylvenlafaxine in human plasma by high-performance liquid chromatography with fluorescence detection. METHODS: Chromatographic separation was achieved on an Agilent Eclipse XDB-C18 Column (4.6 × 150 mm, 5 µm) with water containing sodium dihydrogen phosphate (0.05 mol/L) and acetonitrile (72:28) as the mobile phases. The following parameters were employed: flow rate 0.5 mL/min, column temperature 30°C, fluorescence excitation wavelength 276 nm and emission wavelength 598 nm. RESULTS: The method showed good linearity in the concentration range 10-1000 ng/mL. The regression equation for venlafaxine was R=0.0054C+0.0264, r2=0.99991. The regression equation for O-desmethylvenlafaxine was R=0.0034C+0.0272, r2=0.99969. The intraday and interday precisions (relative SD) were less than 10%, and the quantitative limit was 10 ng/mL. CONCLUSION: We established a sensitive, specific and simple method for the detection of venlafaxine and O-desmethylvenlafaxine. This method fully meets the needs of clinical trials of venlafaxine and the requirements of relevant guidelines. It provided a reference for the clinical detection of venlafaxine and O-desmethylvenlafaxine plasma concentrations and pharmacokinetic study.