Optimized prime editing efficiently generates heritable mutations in maize.

Low efficiency is the main obstacle to using prime editing in maize (Zea mays). Recently, prime-editing efficiency was greatly improved in mammalian cells and rice (Oryza sativa) plants by engineering prime-editing guide RNAs (pegRNAs), optimizing the prime editor (PE) protein, and manipulating cellular determinants of prime editing. In this study, we tested PEs optimized via these three strategies in maize. We demonstrated that the ePE5max system, composed of PEmax, epegRNAs (pegRNA-evopreQ. 1), nicking single guide RNAs (sgRNAs), and MLH1dn, efficiently generated heritable mutations that conferred resistance to herbicides that inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), acetolactate synthase (ALS), or acetyl CoA carboxylase (ACCase) activity. Collectively, we demonstrate that the ePE5max system has sufficient efficiency to generate heritable (homozygous or heterozygous) mutations in maize target genes and that the main obstacle to using PEs in maize has thus been removed.

miR-34 modulates wing polyphenism in planthopper.

Polyphenism is a successful strategy adopted by organisms to adapt to environmental changes. Brown planthoppers (BPH, Nilaparvata lugens) develop two wing phenotypes, including long-winged (LW) and short-winged (SW) morphs. Though insulin receptor (InR) and juvenile hormone (JH) have been known to regulate wing polyphenism in BPH, the interaction between these regulators remains largely elusive. Here, we discovered that a conserved microRNA, miR-34, modulates a positive autoregulatory feedback loop of JH and insulin/IGF signaling (IIS) pathway to control wing polyphenism in BPH. Nlu-miR-34 is abundant in SW BPHs and suppresses NlInR1 by targeting at two binding sites in the 3'UTR of NlInR1. Overexpressing miR-34 in LW BPHs by injecting agomir-34 induces the development towards SW BPHs, whereas knocking down miR-34 in SW BPHs by injecting antagomir-34 induces more LW BPHs when another NlInR1 suppressor, NlInR2, is also suppressed simultaneously. A cis-response element of Broad Complex (Br-C) is found in the promoter region of Nlu-miR-34, suggesting that 20-hydroxyecdysone (20E) might be involved in wing polyphenism regulation. Topic application of 20E downregulates miR-34 expression but does not change wing morphs. On the other hand, JH application upregulates miR-34 expression and induces more SW BPHs. Moreover, knocking down genes in IIS pathway changes JH titers and miR-34 abundance. In all, we showed that miRNA mediates the cross talk between JH, 20E and IIS pathway by forming a positive feedback loop, uncovering a comprehensive regulation mechanism which integrates almost all known regulators controlling wing polyphenism in insects.

Identification and Analysis of MicroRNAs Associated with Wing Polyphenism in the Brown Planthopper, Nilaparvata lugens.

Many insects are capable of developing two types of wings (i.e., wing polyphenism) to adapt to various environments. Though the roles of microRNAs (miRNAs) in regulating animal growth and development have been well studied, their potential roles in modulating wing polyphenism remain largely elusive. To identify wing polyphenism-related miRNAs, we isolated small RNAs from 1st to 5th instar nymphs of long-wing (LW) and short-wing (SW) strains of the brown planthopper (BPH), Nilaparvata lugens. Small RNA libraries were then constructed and sequenced, yielding 158 conserved and 96 novel miRNAs. Among these, 122 miRNAs were differentially expressed between the two BPH strains. Specifically, 47, 2, 27 and 41 miRNAs were more highly expressed in the 1st, 3rd, 4th and 5th instars, respectively, of the LW strain compared with the SW strain. In contrast, 47, 3, 29 and 25 miRNAs were more highly expressed in the 1st, 3rd, 4th and 5th instars, respectively, of the SW strain compared with the LW strain. Next, we predicted the targets of these miRNAs and carried out Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. We found that a number of pathways might be involved in wing form determination, such as the insulin, MAPK, mTOR, FoxO and thyroid hormone signaling pathways and the thyroid hormone synthesis pathway. Thirty and 45 differentially expressed miRNAs targeted genes in the insulin signaling and insect hormone biosynthesis pathways, respectively, which are related to wing dimorphism. Among these miRNAs, Nlu-miR-14-3p, Nlu-miR-9a-5p and Nlu-miR-315-5p, were confirmed to interact with insulin receptors (NlInRs) in dual luciferase reporter assays. These discoveries are helpful for understanding the miRNA-mediated regulatory mechanism of wing polyphenism in BPHs and shed new light on how insects respond to environmental cues through developmental plasticity.

Formation of cyclic structure at amino-terminus of glucagon-like peptide-1 exhibited a prolonged half-life in vivo.

The multiple physiological characterizations of glucagon-like peptide-1 (GLP-1) make it a promising drug candidate for the therapy of type 2 diabetes. However, the biological half-life of GLP-1 is short in vivo due to degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. The stabilization of GLP-1 is critical for its utility in drug development. In this study, several GLP-1 mutants containing an N-terminal cyclic conformation were prepared in that the existence of cyclic conformation is predicted to increase the stabilization of GLP-1 in vivo. In this study, the binding capacities of the mutants were determined, the stabilities of the mutants were investigated and the physiological functions of the mutants were compared with those of wild-type GLP-1 in animals. The results indicated that the mutant (GLP1N8) remarkably raised the half-life in vivo; it also showed better glucose tolerance and higher HbA(1c) reduction than GLP-1 and exendin-4 in rodents. These results suggest that the GLP-1 analog (GLP1N8) which contains an N-terminal cyclic structure might be utilized as possible potent anti-diabetic drugs in the treatment of type 2 diabetes mellitus.

Self-assembled peptide (CADY-1) improved the clinical application of doxorubicin.

CADY-1 is an amphipathic peptide that possesses cell-penetrating activity. As an amphipathic peptide, CADY-1 is capable of forming complexes by self-assembly, and they are these complexes that possess cell-penetrating activity. This distinct characteristic of CADY-1 makes it a potent cell-penetrating drug delivery system. Doxorubicin is a widely used cytotoxic anti-cancer drug but is limited by its toxicity. Although the liposomal formulation of doxorubicin ameliorates its toxicity, its complicated synthesis remains an obstacle to its wide clinical use. In this study, our findings revealed that CADY-1 and doxorubicin form a stable complex at optimised molar ratios in a self-assembling manner. Formation of the complex extended the blood residence time of doxorubicin in a similar fashion to that of liposomal doxorubicin. In addition, the complex was capable of carrying doxorubicin across the cell membrane, which increased the therapeutic index of doxorubicin. Experimental animals treated with a CADY-1/doxorubicin complex exhibited better tolerance and anti-tumour activity than animals treated with either liposomal doxorubicin or the free form of doxorubicin. Collectively, the findings in this study support the advantages of using complexes formed by the self-assembled peptide CADY-1 and suggest that CADY-1 is a potent drug delivery system.