Development of Novel Formulation for Sustained Release of Drug to Prevent Swainsonine-Containing Plants Poisoning in Livestock.

Swainsonine-containing plants contain swainsonine which has been shown to cause neurological signs and pathological changes in farm animals. It causes a large number of livestock poisonings every year resulting in economic losses to the livestock industry. At present, "Jifang E" is used in the prevention of swainsonine-containing plants poisoning livestock, and the preventive effects have been well-documented. However, "Jifang E" is typically administered in drinking water, making it difficult to control the administered dosage, because of feeding difficulties and it may cause certain side effects that are unique to the water-dissolved powder. To overcome these difficulties, we developed a temperature-sensitive gel for injection and the optimal ratio of each formula of sustained-release injection is P407 (24%), P188 (6%), Vitamin C (1%), PEG4000 (0.5%), and "Jifang E" (10%). Our results suggest that novel formulation makes the micellar system more stable and the particles are uniformly dispersed. Colloidal morphological studies showed that each group formed a homogeneous pore structure after gelling, and the structure became more dense with the addition of "Jifang E". The rheological study shows that "Jifang E" is a pseudoplastic fluid, and the addition of "Jifang E" reduces the viscosity of the formula, which is beneficial to the injection. In vitro and in vivo release rate studies have shown that the effective concentration of "Jifang E" can be maintained for 3 to 5 days. The acute toxicity test in SPF Kunming mice showed that its LD50 was 828.323 mg/kg, with confidence limits of 676.706-1013.911 mg/kg, which is a safe dosage (LD50 > 200 mg/kg). There were no observed reactions of muscle irritation or subcutaneous tissue irritation with the dosage used for New Zealand rabbits. In summary, we successfully developed the sustained-release injection formulation of "Jifang E" for the prevention of swainsonine-containing plants poisoning livestock, which provides the basis for subsequent field extension trials and the further study of its detoxification mechanism.

Model-Based Chance-Constrained Reinforcement Learning via Separated Proportional-Integral Lagrangian.

Safety is essential for reinforcement learning (RL) applied in the real world. Adding chance constraints (or probabilistic constraints) is a suitable way to enhance RL safety under uncertainty. Existing chance-constrained RL methods, such as the penalty methods and the Lagrangian methods, either exhibit periodic oscillations or learn an overconservative or unsafe policy. In this article, we address these shortcomings by proposing a separated proportional-integral Lagrangian (SPIL) algorithm. We first review the constrained policy optimization process from a feedback control perspective, which regards the penalty weight as the control input and the safe probability as the control output. Based on this, the penalty method is formulated as a proportional controller, and the Lagrangian method is formulated as an integral controller. We then unify them and present a proportional-integral Lagrangian method to get both their merits with an integral separation technique to limit the integral value to a reasonable range. To accelerate training, the gradient of safe probability is computed in a model-based manner. The convergence of the overall algorithm is analyzed. We demonstrate that our method can reduce the oscillations and conservatism of RL policy in a car-following simulation. To prove its practicality, we also apply our method to a real-world mobile robot navigation task, where our robot successfully avoids a moving obstacle with highly uncertain or even aggressive behaviors.

Genome Editing Enables Next-Generation Hybrid Seed Production Technology.

The next-generation hybrid seed technology enables the successful production of sortable hybrid seeds from genic male sterile (GMS) lines and maintainers; however, it requires multiple laborious and complicated steps. Here, we designed a simple next-generation hybrid seed production strategy that takes advantage of the CRISPR/Cas9 technology to create a Manipulated GMS Maintainer (MGM) system via a single transformation. Under this schema, the maize male fertility gene ZmMS26 was nullified by removal of its fifth exon using the CRISPR/Cas9 system on a vector, and a second vector carrying a functional ZmMS26 cDNA was co-transformed to restore fertility. The second vector also contains a male gametophyte inactivation gene (ZmAA1) encoding maize α-amylase driven by the pollen-specific promoter PG47 and an endosperm fluorescent marker (DsRED) driven by the barley endosperm aleurone-specific promoter Ltp2. The derived single-copy hemizygous MGM lines bore a mutated MS26 gene, leading to complete male sterility but normal vegetative growth and grain yield. The MGM system could prevent genetic transmission of the MGM elements via male gametophytes, providing an efficient method for sorting maintainer seeds labeled by DsRED. This strategy can be extended to any GMS gene and to hybrid crops other than maize.

[Advances in targeted replacement genome editing in plants].

Targeted replacement genome editing refers to DNA modification and engineering technology that could induce and achieve mutations of targeted gene replacement or knockin at a target gene or DNA region. In this review, the principles, implementation methods, factors that influence efficiency and accuracy, and applications of gene replacement editing were summarized and discussed. It provides the reference for gene functional characterization and genetic improvements through gene replacement strategies in higher plant especially crops.

The maize WRKY transcription factor ZmWRKY17 negatively regulates salt stress tolerance in transgenic Arabidopsis plants.

MAIN CONCLUSION: We cloned and characterized the ZmWRKY17 gene from maize. Overexpression of ZmWRKY17 in Arabidopsis led to increased sensitivity to salt stress and decreased ABA sensitivity through regulating the expression of some ABA- and stress-responsive genes. The WRKY transcription factors have been reported to function as positive or negative regulators in many different biological processes including plant development, defense regulation and stress response. This study isolated a maize WRKY gene, ZmWRKY17, and characterized its role in tolerance to salt stress by generating transgenic Arabidopsis plants. Expression of the ZmWRKY17 was up-regulated by drought, salt and abscisic acid (ABA) treatments. ZmWRKY17 was localized in the nucleus with no transcriptional activation in yeast. Yeast one-hybrid assay showed that ZmWRKY17 can specifically bind to W-box, and it can activate W-box-dependent transcription in planta. Heterologous overexpression of ZmWRKY17 in Arabidopsis remarkably reduced plant tolerance to salt stress, as determined through physiological analyses of the cotyledons greening rate, root growth, relative electrical leakage and malondialdehyde content. Additionally, ZmWRKY17 transgenic plants showed decreased sensitivity to ABA during seed germination and early seedling growth. Transgenic plants accumulated higher content of ABA than wild-type (WT) plants under NaCl condition. Transcriptome and quantitative real-time PCR analyses revealed that some stress-related genes in transgenic seedlings showed lower expression level than that in the WT when treated with NaCl. Taken together, these results suggest that ZmWRKY17 may act as a negative regulator involved in the salt stress responses through ABA signalling.

An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design.

Precision DNA/gene replacement is a promising genome-editing tool that is highly desirable for molecular engineering and breeding by design. Although the CRISPR/Cas9 system works well as a tool for gene knockout in plants, gene replacement has rarely been reported. Towards this end, we first designed a combinatory dual-sgRNA/Cas9 vector (construct #1) that successfully deleted miRNA gene regions (MIR169a and MIR827a). The deletions were confirmed by PCR and subsequent sequencing, yielding deletion efficiencies of 20% and 24% on MIR169a and MIR827a loci, respectively. We designed a second structure (construct #2) that contains sites homologous to Arabidopsis TERMINAL FLOWER 1 (TFL1) for homology-directed repair (HDR) with regions corresponding to the two sgRNAs on the modified construct #1. The two constructs were co-transformed into Arabidopsis plants to provide both targeted deletion and donor repair for targeted gene replacement by HDR. Four of 500 stably transformed T0 transgenic plants (0.8%) contained replaced fragments. The presence of the expected recombination sites was further confirmed by sequencing. Therefore, we successfully established a gene deletion/replacement system in stably transformed plants that can potentially be utilized to introduce genes of interest for targeted crop improvement.

Genome-wide analysis of the IQD gene family in maize.

IQD gene family plays important roles in plant developmental processes and stress responses. To date, no systematic characterization of this gene family has been carried out in maize. In this study, 26 IQD genes, from ZmIQD1 to ZmIQD26, were identified using Blast search tools. The phylogenetic analysis showed these genes were divided into four subfamilies (IQD I-IV) and members within the same subfamily shared conserved exon/intron distribution and motif composition. The 26 ZmIQD genes are distributed unevenly on 8 of the 10 chromosomes, with 9 segmental duplication events, suggesting that the expansion of IQDs in maize was due to the segmental duplication. The analysis of Ka/Ks ratios showed that the duplicated ZmIQDs had primarily undergone strong purifying selection. In addition, the 26 ZmIQDs displayed different expression patterns at different developmental stages of maize based on transcriptome analysis. Further, quantitative real-time PCR analysis showed that all 26 ZmIQD genes were responsive to drought treatment, suggesting their crucial roles in drought stress response. Yeast two-hybrid assay proved that ZmIQD2 and ZmIQD15 can interact with ZmCaM2 and IQ or I in IQ motif is required for ZmIQD15 to combine with CaM2. Our results present a comprehensive overview of the maize IQD gene family and lay an important foundation for further analysis aimed at uncovering the biological functions of ZmIQDs in growth and development.