bZIP transcription factor responds to changes in light quality and affects saponins synthesis in Eleutherococcus senticosus.

Light quality considerably influences plant secondary metabolism, yet the precise mechanism underlying its impact on Eleutherococcus senticosus remains elusive. Comprehensive metabolomic and transcriptomic analyses revealed that varying light quality alters the biosynthesis of triterpene saponins by modulating the expression of genes involved in the process in E. senticosus. Through correlation analysis of gene expression and saponin biosynthesis, we identified four light-responsive transcription factors, namely EsbZIP1, EsbZIP2, EsbZIP4, and EsbZIP5. EsbZIP transcription factors function in the nucleus, with light quality-dependent promoter activity. Except for EsbZIP2, the other EsbZIP transcription factors exhibit transcriptional self-activation. Furthermore, EsbZIP can bind to the promoter areas of genes that encode important enzymes (EsFPS, EsSS, and EsSE) involved in triterpene saponin biosynthesis, thereby regulating their expression. Overexpression of EsbZIP resultes in significant down-regulation of most downstream target genes，which leads to a decrease in saponin content. Overall, varying light quality enhances the content of triterpene saponins by suppressing the expression of EsbZIP. This study thus elucidates the molecular mechanism by which E. senticosus adjusts triterpene saponin levels in response to changes in light quality.

Identification of the AP2/ERF transcription factor family of Eleutherococcus senticosus and its expression correlation with drought stress.

In this study, through analysis of the genome of Eleutherococcus senticosus (ES). 228 AP2/ERF genes were identified and classified into 5 groups AP2 (47 genes), ERF (108 genes), RAV (6 genes), DREB (64 genes), and soloist (3 genes). According to the AP2/ERF classification of Arabidopsis thaliana, the ES AP2/ERF proteins were subdivided into 15 groups. The gene structure and motifs of each group of AP2/ERF in ES were highly similar, which confirmed the conservation of AP2/ERF genes. The ES AP2/ERF genes were unevenly distributed on chromosomes, and a total of four pairs of tandem repeats, and 84 co-linear gene pairs were found, so the AP2/ERF genes expanded in a fragment replication manner, and dominated by pure selection during evolution. By analyzing the transcriptome data of ES under different drought stress conditions, 87 AP2/ERF genes with differential expression were obtained, of which 10 genes with highly significant differences were further analyzed and screened for qRT-PCR validation. To the best of our knowledge, this is the first report on the AP2/ERF gene of Eleutherococcus senticosus, and the bioinformatics analysis and experimental validation provided valuable information about them, which is of great significance for further research on the molecular mechanisms of ES in response to drought stress.

Reprogrammed siTNFα/neutrophil cytopharmaceuticals targeting inflamed joints for rheumatoid arthritis therapy.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by severe synovial inflammation and cartilage damage. Despite great progress in RA therapy, there still lacks the drugs to completely cure RA patients. Herein, we propose a reprogrammed neutrophil cytopharmaceuticals loading with TNFα-targeting-siRNA (siTNFα) as an alternative anti-inflammatory approach for RA treatment. The loaded siTNFα act as not only the gene therapeutics to inhibit TNFα production by macrophages in inflamed synovium, but also the editors to reprogram neutrophils to anti-inflammatory phenotypes. Leveraging the active tendency of neutrophils to inflammation, the reprogrammed siTNFα/neutrophil cytopharmaceuticals (siTNFα/TP/NEs) can rapidly migrate to the inflamed synovium, transfer the loaded siTNFα to macrophages followed by the significant reduction of TNFα expression, and circumvent the pro-inflammatory activity of neutrophils, thus leading to the alleviated synovial inflammation and improved cartilage protection. Our work provides a promising cytopharmaceutical for RA treatment, and puts forward a living neutrophil-based gene delivery platform.

Transcriptome and Metabolome Analysis of the Synthesis Pathways of Allelochemicals in Eupatorium adenophorum.

Eupatorium adenophorum (Crofton weed) is an invasive weed in more than 30 countries. It inhibits the growth of surrounding plants by releasing allelochemicals during its invasion. However, the synthetic pathways and molecular mechanisms of its allelochemicals have been rarely reported. In this study, the related genes and pathways of allelochemicals in E. adenophorum were analyzed. Transcriptome analysis showed that differentially expressed genes (DEGs) were mainly enriched in the phenylpropanoid biosynthetic pathway and flavonoid biosynthetic pathway. Thirty-three DEGs involved in the synthesis of allelochemicals were identified, and 30 DEGs showed significant differences in blades and stems. Six allelochemicals were identified from blades and stems by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Correlation analysis of genes and metabolites showed a strong correlation between the five genes and allelochemicals. In addition, this study supplemented the biosynthetic pathway of Eupatorium adenophorum B (HHO). It was found that acyclic sesquiterpene synthase (NES), δ-cadinene synthase (TPS), and cytochrome P450 (P450) were involved in the synthesis of HHO. These findings provide a dynamic spectrum consisting of allelochemical metabolism and a coexpression network of allelochemical synthesis genes in E. adenophorum.

Anisamide-functionalized pH-responsive amphiphilic chitosan-based paclitaxel micelles for sigma-1 receptor targeted prostate cancer treatment.

Controlled release and tumor-selective distribution are highly desirable for anticancer nanomedicines. Here, we design and synthesize an anisamide-conjugated N-octyl-N,O-maleoyl-O-phosphoryl chitosan (a-OMPC) which can form amphiphilic micelles featuring pH-responsive release and high affinity to sigma-1 receptor-overexpressed tumors for paclitaxel (PTX) delivery. Thereinto, maleoyl and phosphoryl groups cooperatively contribute to pH-responsive drug release due to a conversion from hydrophile to hydrophobe in the acidic microenvironment of endo/lysosomes. We demonstrated that PTX-loaded a-OMPC micelles (PTX-aM) enhanced the cellular internalization via the affinity between anisamide and sigma-1 receptor, rapidly released drug in endo/lysosomes and elevated the cytotoxicity against PC-3 cells. The in vivo studies further verified that PTX-aM could largely accumulate at the tumor site even after 24 h of administration, resulting in obvious inhibition effect and prolonged survival period in PC-3 tumor xenograft-bearing mice. Moreover, OMPC showed no obvious hemolytic and acute toxicity. Collectively, this chitosan derivate holds a promising potential in application of prostate cancer-targeted drug delivery system.

Co-delivery of TRAIL and siHSP70 using hierarchically modular assembly formulations achieves enhanced TRAIL-resistant cancer therapy.

The combined therapy of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and heat shock protein 70-targeting siRNA (siHSP70) has shown an improved anti-tumor effect on TRAIL-resistant tumor. However, vehicles to co-deliver these two biopharmaceuticals are challenging because of the distinct location of their targets on the cell surface and in the cytosol. Here we developed a hierarchically modular assembly formulation (TH-s-RSC) via the copper-free click reaction to co-encapsulate the positively-charged TRAIL and negatively-charged siHSP70 and release them in the extracellular space and cytoplasm. We demonstrate that TH-s-RSC can protect the packaged biopharmaceuticals through its hyaluronic acid shell in vivo, and sequentially release TRAIL in response to extracellular molecular including hyaluronidase (HAase) and matrix metalloproteinase 2 (MMP2), followed by the release of siHSP70 triggered by the reductive conditions in the cytoplasm. We showed that the complementary activity of TRAIL and siHSP70 exhibited superior synergistic anticancer efficacy in both A549 lung cancer xenograft models and 4T1 lung metastatic breast cancer models, compared to either treatment alone. Our strategy provides a promising platform for safe and effective co-delivery and dual-site targeting of biopharmaceuticals in cancer treatment that may be applicable in the future.