A plastic microfluidic device that integrates a filter disc as a DNA capture phase was successfully developed for low-cost, rapid and automated DNA extraction and PCR amplification from various raw samples. The microdevice was constructed by sandwiching a piece of Fusion 5 filter, as well as a PDMS (polydimethylsiloxane) membrane, between two PMMA (poly(methyl methacrylate)) layers. An automated DNA extraction from 1 μL of human whole blood can be finished on the chip in 7 minutes by sequentially aspirating NaOH, HCl, and water through the filter. The filter disc containing extracted DNA was then taken out directly for PCR. On-chip DNA purification from 0.25-1 μL of human whole blood yielded 8.1-21.8 ng of DNA, higher than those obtained using QIAamp® DNA Micro kits. To realize DNA extraction from raw samples, an additional sample loading chamber containing a filter net with an 80 μm mesh size was designed in front of the extraction chamber to accommodate sample materials. Real-world samples, including whole blood, dried blood stains on Whatman® 903 paper, dried blood stains on FTA™ cards, buccal swabs, saliva, and cigarette butts, can all be processed in the system in 8 minutes. In addition, multiplex amplification of 15 STR (short tandem repeat) loci and Sanger-based DNA sequencing of the 520 bp GJB2 gene were accomplished from the filters that contained extracted DNA from blood. To further prove the feasibility of integrating this extraction method with downstream analyses, "in situ" PCR amplifications were successfully performed in the DNA extraction chamber following DNA purification from blood and blood stains without DNA elution. Using a modified protocol to bond the PDMS and PMMA, our plastic PDMS devices withstood the PCR process without any leakage. This study represents a significant step towards the practical application of on-chip DNA extraction methods, as well as the development of fully integrated genetic analytical systems.

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, has become one of the most serious disease problems in oilseed rape-growing areas in Australia. Sources of resistance to this disease have been sought worldwide. In this study, germplasm comprising 42 Brassica napus and 12 Brassica juncea accessions from China and Australia, was screened for resistance to Sclerotinia stem rot under Western Australian field conditions. Resistance was confirmed in some germplasm from China and new sources of resistance were identified in germplasm from Australia. Furthermore, our study found that the severity of stem lesions was related to stem diameter and percentage of the host plants that were dead. It was evident that both stem lesion length and percentage of plant death were at the lowest level when the stem diameter was approximately 10 mm. Smaller or greater stem diameter resulted both in increased stem lesion length and plant death. Stem diameter may be a useful parameter in breeding cultivars of oilseed Brassicas with Sclerotinia resistance.

In this study, to systematically identify the most stably expressed genes for internal reference in zebrafish Danio rerio investigations, 37 D. rerio transcriptomic datasets (both RNA sequencing and microarray data) were collected from gene expression omnibus (GEO) database and unpublished data, and gene expression variations were analysed under three experimental conditions: tissue types, developmental stages and chemical treatments. Forty-four putative candidate genes were identified with the c.v. <0·2 from all datasets. Following clustering into different functional groups, 21 genes, in addition to four conventional housekeeping genes (eef1a1l1, b2m, hrpt1l and actb1), were selected from different functional groups for further quantitative real-time (qrt-)PCR validation using 25 RNA samples from different adult tissues, developmental stages and chemical treatments. The qrt-PCR data were then analysed using the statistical algorithm refFinder for gene expression stability. Several new candidate genes showed better expression stability than the conventional housekeeping genes in all three categories. It was found that sep15 and metap1 were the top two stable genes for tissue types, ube2a and tmem50a the top two for different developmental stages, and rpl13a and rp1p0 the top two for chemical treatments. Thus, based on the extensive transcriptomic analyses and qrt-PCR validation, these new reference genes are recommended for normalization of D. rerio qrt-PCR data respectively for the three different experimental conditions.

In the last few decades use of metal-based nanoparticles (MNPs) has been increased significantly that eventually contaminating agricultural land and limiting crop production worldwide. Moreover, contamination of food chain with MNPs has appeared as a matter of public concern due to risk of potential health hazard. Brassinosteroid has been shown to play a critical role in alleviating heavy metal stress; however, its function in relieving zinc oxide nanoparticles (ZnO NPs)-induced phytotoxicity remains unknown. In this study, we investigated the potential role of 24-epibrassinolide (BR) in mitigating ZnO NPs-induced toxicity in tomato seedlings. Seedling growth, biomass production, and root activity gradually decreased, but Zn accumulation increased with increasing ZnO NPs concentration (10–100 mg/L) in growth media (½ MS). The augmentation of BR (5 nM) in media significantly ameliorated 50 mg/L ZnO NPs-induced growth inhibition. Visualization of hydrogen peroxide (H2O2), and quantification of H2O2 and malondialdehyde (MDA) in tomato roots confirmed that ZnO NPs induced an oxidative stress. However, combined treatment with BR and ZnO NPs remarkably reduced concentration of H2O2 and MDA as compared with ZnO NPs only treatment, indicating that BR supplementation substantially reduced oxidative stress. Furthermore, the activities of key antioxidant enzymes such as superoxide dismutase (SOD), catalase, ascorbate peroxidase and glutathione reductase were increased by combined treatment of BR and ZnO NPs compared with ZnO NPs only treatment. BR also increased reduced glutathione (GSH), but decreased oxidized glutathione (GSSG)] and thus improved cellular redox homeostasis by increasing GSH:GSSG ratio. The changes in relative transcript abundance of corresponding antioxidant genes such as Cu/Zn SOD, CAT1, GSH1, and GR1 were in accordance with the changes in those antioxidants under different treatments. More importantly, combined application of BR and ZnO NPs significantly decreased Zn content in both shoot and root of tomato seedlings as compared with ZnO NPs alone. Taken together, this study, for the first time, showed that BR could not only improve plant tolerance to ZnO NPs but also reduce the excess zinc content in tomato seedlings. Such a finding may have potential implication in safe vegetable production in the MNPs-polluted areas.

Previously we have developed a transgenic zebrafish line (LiPan) with liver-specific red fluorescent protein (DsRed) expression under the fabp10a promoter. Since red fluorescence in the liver greatly facilitates the observation of liver in live LiPan fry, we envision that the LiPan zebrafish may provide a useful tool in analyses of hepatotoxicity based on changes of liver red fluorescence intensity and size. In this study, we first tested four well-established hepatotoxins (acetaminophen, aspirin, isoniazid and phenylbutazone) in LiPan fry and demonstrated that these hepatotoxins could significantly reduce both liver red fluorescence and liver size in a dosage-dependent manner, thus the two measurable parameters could be used as indicators of hepatotoxicity. We then tested the LiPan fry with nine other chemicals including environmental toxicants and human drugs. Three (mefenamic acid, lindane, and arsenate) behave like hepatotoxins in reduction of liver red fluorescence, while three others (17β-estradiol, TCDD [2,3,7,8-tetrachlorodibenzo-p-dioxin] and NDMA [N-nitrosodimethylamine]) caused increase of liver red fluorescence and the liver size. Ethanol and two other chemicals, amoxicillin (antibiotics) and chlorphenamine (pain killer) did not resulted in significant changes of liver red fluorescence and liver size. By quantitative RT-PCR analysis, we found that the changes of red fluorescence intensity caused by different chemicals correlated to the changes of endogenous fabp10a RNA expression, indicating that the measured hepatotoxicity was related to fatty acid transportation and metabolism. Finally we tested a mixture of four hepatotoxins and observed a significant reduction of red fluorescence in the liver at concentrations below the lowest effective concentrations of individual hepatotoxins, suggesting that the transgenic zebrafish assay is capable of reporting compound hepatotoxicity effect from chemical mixtures. Thus, the LiPan transgenic fry provide a rapid and convenient in vivo hepatotoxicity assay that should be applicable to high-throughput hepatotoxicity test in drug screening as well as in biomonitoring environmental toxicants.

No abstract

Despite great progress in aluminum ion batteries (AIBs), the commercialization and performance improvement of AIBs‐based carbon cathodes is greatly impeded by sluggish intercalation/extraction and redox kinetics due to large‐sized AlCl4− anions. Phosphates with tunnel channels and much larger d‐spacing than the radius of Al3+ could be an alternative candidate as a cathode for potential high‐performance AIBs. Herein, elaborately designed porous tunnel structured Co3(PO4)2@C composites derived from ZIF‐67 as AIBs cathodes are demonstrated, showing increased active sites, high ionic mobility, and high Al3+ ion diffusion coefficient, leading to remarkably enhanced discharge–charge redox reaction kinetics. Furthermore, the carbon shell and porous structure performs as armor to alleviate volume change and maintain the structure integrity of the cathodes. As expected, the rationally constructed Co3(PO4)2@C composite exhibits a superior capacity of 111 mA h g−1 at a high current density of 6 A g−1 and 151 mA h g−1 at 2 A g−1 after 500 cycles with capacity decay of 0.02% per cycle. This innovative strategy could be a big step forward for long‐term cycle stable AIBs and reveals significant insights into the redox reaction mechanism for high‐performance AIBs based on Al3+ rather than large‐sized AlCl4−.

No abstract