The Streptococcus pyogenes CRISPR-Cas9 system effectively mediates RNA-guided DNA double-strand breaks and is used for genome editing in many different organisms, including plants （Puchta, 2016）. ...CRISPR-Cas9 is a two-component system in which the Cas9 protein is expressed from a Pol II promoter （Lowder et al., 2015）. In contrast, the sgRNAs are typically expressed from Pol III promoters, such as U6 and U3.
MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in plant development and stress responses. Loss-of-function analysis of miRNA genes has been traditionally challenging due to ...lack of appropriate knockout tools. In this study, single miRNA genes (OsMIR408 and OsMIR528) and miRNA gene families (miR815a/b/c and miR820a/b/c) in rice were targeted by CRISPR-Cas9. We showed single strand conformation polymorphism (SSCP) is a more reliable method than restriction fragment length polymorphism (RFLP) for identifying CRISPR-Cas9 generated mutants. Frequencies of targeted mutagenesis among regenerated T0 lines ranged from 48 to 89% at all tested miRNA target sites. In the case of miRNA528, three independent guide RNAs (gRNAs) all generated biallelic mutations among confirmed mutant lines. When targeted by two gRNAs, miRNA genes were readily to be deleted at a frequency up to 60% in T0 rice lines. Thus, we demonstrate CRISPR-Cas9 is an effective tool for knocking out plant miRNAs. Single-base pair (bp) insertion/deletion mutations (indels) in mature miRNA regions can lead to the generation of functionally redundant miRNAs. Large deletions at either the mature miRNA or the complementary miRNA
were found to readily abolish miRNA function. Utilizing mutants of
and
, we find that knocking out a single miRNA can result in expression profile changes of many other seemingly unrelated miRNAs. In a case study on
, we reveal it is a positive regulator in salt stress. Our work not only provides empirical guidelines on targeting miRNAs with CRISPR-Cas9, but also brings new insights into miRNA function and complex cross-regulation in rice.
MiRNA-induced gene silencing (MIGS) technology is a special kind of RNA interference technology that uses miR173 to mediate the production of
-acting siRNA (ta-siRNA) to achieve target gene ...silencing. This technique has successfully mediated the silencing of interested genes in plants such as Arabidopsis, tobacco, petunia, etc. In order to establish the MIGS technology system in monocots such as rice, we constructed the MIGS backbone vectors pZHY930, pZHY931, pZHY932, and pZHY933 with different with promoters to regulate the expression of miR173 and miR173_ts. The rice OsPDS reporter gene was selected to compare the efficiency of four MIGS backbone vectors by the ratio of albino plants. The results showed that all the four backbone vectors could effectively mediate the target gene silencing, and pZHY932 showed highest efficiency up to 90%. Through MIGS silencing of endogenous OsROC5 and OsLZAY1 in rice, we successfully obtained rice mutant plants with rice leaf roll and tillering angles increasing, and further confirmed that MIGS backbone vector can efficiently mediate target gene silencing in rice. On the other hand, in order to verify the efficiency of MIGS-mediated multi-gene silencing in rice, we constructed two double-gene silencing vectors OsPDS and OsROC5, OsPDS and OsLZAY1, based on pZHY932 backbone vector. Double mutant rice plants with increased leaf and albino tiller angles. And we successfully obtained bladed leaf albino seedling and increased tillering angle albino seedling double-silencing mutations. We further constructed a MIGS-OsGBSS gene silencing vector and obtained rice materials with significantly reduced amylose content. This result indicated that MIGS could be an efficient method in monocots gene silencing and gene function analysis.
The aqueous zinc-ion batteries (ZIBs) have been considered as a promising energy storage device. However, the ion transfer at the Zn metal anode–electrolyte interface is limited by the sluggish ...kinetics resulting in high interface resistance. Herein, mesoporous TiO2 (m-TiO2) is coated on the Zn foil (Zn-TiO2) driving the ion’s faster transfer to reduce interface resistance (70.1 Ω vs 799.3 Ω of bare Zn). The lower interface resistance is ascribed to shortening the ion transfer path provided by the mesoporous structure and the smaller Zn2+ absorption energy barrier of the surface of the Zn-TiO2 anode as well as the unobstructed ion transfer path at the crystal planes (100) of TiO2, which have been supported by the density functional theory (DFT) calculation and experiments. Therefore, the Zn-TiO2 anodes in the symmetrical cells display a low voltage hysteresis (36.5 mV) and long-term cycling stability (500 h at 4.4 mA cm–2). Especially, the Zn-TiO2/MnO2 full cells show superior cycling performance with a high capacity of 269.8 mAh g–1 after 50 cycles at 0.2 A g–1 and 210.9 mAh g–1 after 1000 cycles at 0.5 A g–1. The analysis of ion-transfer kinetics at the interface provides deep enlightenment and reference for the study of the metal anodes.
Acyclovir (Acyclovir, ACV) is an antiviral drug which, despite its favorable safety profile, can have systemic adverse effects, following improper use. This study prepared a clay-like two-dimensional ...transition metal carbide Ti3C2 by etching Ti3AlC2 with LiF and HCl. Carboxylated multi walled carbon nanotubes (Carboxylated multi-walled carbon nanotubes, MWCNTs-COOH) were combined with Ti3C2 using chitosan (Chitosan, CS) as solvent and ultrasonic treatment. The nanocomposite was then modified onto the surface of a glassy carbon electrode (Glassy carbon electrode, GCE) to develop an electrochemical sensor for the detection of ACV. The Ti3C2-CS-MWCNTs-COOH/GCE nanocomposite was characterized by X-ray diffractometry (X-ray diffractometry, XRD), scanning electron microscope (Scanning electron microscope，SEM), and cyclic voltammetry (Cyclic voltammetry，CV), as well as the electrochemical impedance method (Electrochemical impedance，EIS). The successful preparation of Ti3C2-CS-MWCNTs-COOH, and the good electrochemical performance of the electrode were verified. Linear scanning voltammetry (Linear scan voltammetry，LSV) was used to quantify the ACV. The linear range of the method is wider than other works (0.05 ∼ 20.0 and 20.0 ∼ 500.0 μM), with a low detection limit (0.088 μM). The sensor achieves sensitive detection of ACV and can be used for the analysis of ACV in pharmaceutical and urine dosage forms, with recoveries of 95.5∼97.6% and 95.6∼97.5%, respectively. For the first time, this sensor utilizes clay-like Ti3C2 with a larger specific surface area in combination with MWCNTs-COOH for ACV detection, which exhibits excellent immunity to interference, repeatability, reproducibility, and stability. This method provides a rapid and sensitive method for the determination of ACV, and lays foundations for developing miniaturized field rapid test detectors.
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•Ti3C2-CS-MWCNTs-COOH/GCE sensor is for electrochemical determination of acyclovir.•Clay-like Ti3C2 has higher conductivity, larger specific surface area and higher security than accordion-like Ti3C2.•Wide linear range (0.05–20.0 µM and 20.0–500.0 µM), low detection limit (0.088 µM) are obtained.•The developed sensor could be used for the detection of acyclovir in urine and pharmaceuticals.
Bacillus tequilensis DZY 6715 was isolated from healthy leaves in Camellia oleifera, and the strain DZY 6715 significantly inhibited anthracnose disease resulting from Colletotrichum fructicola in C. ...oleifera, besides, its associated mechanism of disease resistance was explored. B. tequilensis DZY 6715 treatment controlled mycelial growth of C. fructicola in C. oleifera, and significantly decreased C. oleifera anthracnose incidence and disease index compared with the control group. B. tequilensis DZY 6715 has strong biofilm forming ability, and also secretes extracellular β-1, 3-glucanase and chitinase, which could cause cell membranes damage and increased cellular compound leakage. C.oleifera treated with DZY 6715 also effectively enhanced enzyme activities and stimulated the synthesis the substances related to phenylpropane metabolism and reactive oxygen metabolism. Moreover, transcript profiling analysis revealed more differentially expressed genes related to phenylpropanoid pathway metabolism and antioxidant system inducing by DZY 6715 compared with the control in C. oleifera. Thus, it can be concluded that B. tequilensis DZY 6715 is a suitable bio-control agent to control anthracnose disease in C. oleifera.
•Bacillus tequilensis DZY 6715 inhibits anthracnose disease in Camellia oleifera.•B. tequilensis DZY 6715 damage pathogenic cell membrane.•B. tequilensis DZY 6715 enhances the defense-related enzyme activity in C. oleifera.•B. tequilensis DZY 6715 induces defense-related expression in C. oleifera.
This review comprehensively discusses the performance of various electrochemical sensors using different detection recognition methods, based on nanomaterials, MIPs, aptamer, etc. The portable ...electrochemical sensors using smartphone, USB and disk are summarized in detail. The future research direction and challenges of using portable devices for the detection of mycotoxins are highlighted.
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•Recent progress in electrochemical sensors is summarized for various mycotoxins detection in food.•The main electrochemical recognition methods for mycotoxins detection are discussed.•On-site portable electrochemical devices are reviewed.•Future research trends and perspectives on portable electrochemical sensors are presented.
The mycotoxins commonly found in food may pose a significant threat to human health. Therefore, the development of portable electrochemical sensing chip methods is very important. In addition, due to the need to study various pollutants when conducting food analysis, which involves multiple and complex fields, the use of disposable portable sensing chips can improve detection efficiency. In this review, we have summarized the significant work on electrochemical sensors for mycotoxins detection in food samples. Although earlier reviews have covered this topic, the mechanisms and portable use of toxin detection have not yet been addressed. This paper’s novelty is that it has reviewed recent advances in various electrochemical sensors and portable applications, for mycotoxins detection. This provides a new perspective on future trends in the development of portable electrochemical sensors.
e23515 Background: Imatinib (IM) has significantly enhanced the prognosis of patients (pts) with advanced gastrointestinal stromal tumors (GISTs). The clinical outcomes may correlate with IM ...exposure. However, the efficacy threshold, particularly based on different primary KIT mutant, remains undefined. This study aimed to establish the efficacy threshold of IM plasma trough concentration (C min ) at steady-state in Chinese pts with advanced GIST, additionally to define subgroup thresholds based on various primary KIT mutations. Methods: This was a single-center, prospective, observational cohort study. Pts with histologically confirmed recurrent/metastatic GIST who received regular IM treatment and had undergone at least two measurements of steady-state IM C min were enrolled from Jul 2017 to Jun 2022. Clinical outcomes including objective response rate (ORR), survival, and IM C min were assessed. Results: A total of 168 pts with a median age of 52y were enrolled. The primary lesion locations comprised stomach, small intestine, rectum and others. Metastatic lesions were found in the liver, peritoneum, both liver and peritoneum and others. Median sum of longest diameter of target lesions was 10.7cm. Of 168 pts, 117 (69.6%) harbored KIT 11, 27 (16.1%) harbored KIT 9, 5 (3.0%) harbored PDGFRA, and 9 (5.4%) were KIT/PDGFRA wild type. During IM response period, 104 pts combined with surgical treatment (surgery or interventional therapy). After a median follow-up duration was 50.6m (range, 16.3–189.8), 72 pts had disease progression and 20 died. Estimated median progression-free survival (mPFS) for all patients was 79.4m (95%CI 55.7–103.1). The median overall survival was not reached. A C min threshold of ≥738ng/ml defined by log-rank test was associated with longer PFS for the whole population (P = 0.002) irrespective of other clinical characteristics. Pts with an IM C min below 738ng/ml exhibited a shorter mPFS (25.3m, 95%CI 41.9–97.1) compared to those surpassing this threshold (96.7m, 95%CI 65.8–127.6). The ORR for the whole population was 73.0% (CR 2.2%, PR 70.8%), with no significant difference observed between pts with IM C min below and above 738ng/ml. For pts with KIT 11 and KIT 9 mutations, the efficacy thresholds for IM C min were ≥680ng/ml and ≥1877ng/ml, respectively. Multivariate Cox regression analysis revealed that IM Cmin ≥738ng/ml, primary gastric origin, liver-only metastasis, sum of target lesions diameter ≤5cm, KIT 11 mutation, and combined with surgical treatment were favorable prognostic factors. Conclusions: IM exposure significantly correlates with clinical benefits in advanced GISTs, with an IM C min above 738ng/ml associated with a longer PFS in Chinese population. Considering different primary KIT mutant, a threshold of 680 ng/ml could be considered for efficacy in KIT 11 mutations, while a higher C min of 1877 ng/ml was necessary for KIT 9 mutation.
Preparation of MnS/Co3S4 ErGO/GCE and schematic diagram of its electrochemical response to DA. This protocol used a two-step ion exchange method to prepare MnS/Co3S4 heterostructures, with self-made ...cobalt chloride bicarbonate (CO (CO3) 0.35C0.20 (OH) 1.10) as the precursor, sodium disulfide monohydrate as the sulfur source, and manganese chloride as the manganese source. The obtained MnS/Co3S4-ErGO was modified on a glassy carbon electrode, exhibiting electrochemically ultrasensitive ability for the redox reaction of DA determination at nanomolar concentration.
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•An ultrasensitive DA determination sensor at nanomolar level is presented.•High selectivity, selective and good reproducibility of the sensor is obtained.•Good linear concentration ranges of DA with low LOD (2.0 nM) is observed.•The sensor is successfully applied to the detection of DA in real samples.
An ultrasensitive and selective electrochemical sensor has been constructed for the quantitative assay of dopamine (DA) based on hollow tubular manganese-cobalt sulfide (MnS/Co3S4) hybrids embedded on electrochemically reduced graphene oxide (ErGO). The MnS/Co3S4 heterostructures were created by two-step ion exchange method, of which Co9S8 with hollow tubular structures was fabricated by solvothermal method using the self-made cobalt chloride carbonate hydroxide (CO(CO3)0.35Cl0.20(OH)1.10) as precursor and sodium disulfide monohydrate as sulfur source and manganese chloride as manganese source. The MnS/Co3S4-ErGO/GCE could exhibit strong catalytic ability for the redox reaction of DA. Under the optimal conditions, the developed sensor exhibits a wide linear response for DA ranging from 6.0 nM to 20.0 μM by second-order derivative linear sweep voltammetry (SDLSV), with a detection limit of 2.0 nM. In addition, the developed method exhibited high sensitivity and selectivity, good reproducibility and low-cost. It is a very promising analytical method for the determination of DA in pharmaceutical and biological samples.
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