Transcriptomic and Metabolomic Analysis Reveal Possible Molecular Mechanisms Regulating Tea Plant Growth Elicited by Chitosan Oligosaccharide.

Chitosan oligosaccharide (COS) plays an important role in the growth and development of tea plants. However, responses in tea plants trigged by COS have not been thoroughly investigated. In this study, we integrated transcriptomics and metabolomics analysis to understand the mechanisms of chitosan-induced tea quality improvement and growth promotion. The combined analysis revealed an obvious link between the flourishing development of the tea plant and the presence of COS. It obviously regulated the growth and development of the tea and the metabolomic process. The chlorophyll, soluble sugar, and amino acid content in the tea leaves was increased. The phytohormones, carbohydrates, and amino acid levels were zoomed-in in both transcript and metabolomics analyses compared to the control. The expression of the genes related to phytohormones transduction, carbon fixation, and amino acid metabolism during the growth and development of tea plants were significantly upregulated. Our findings indicated that alerted transcriptomic and metabolic responses occurring with the application of COS could cause efficiency in substrates in pivotal pathways and hence, elicited plant growth.

Design, synthesis and biological evaluation of anthranilamide derivatives as potent SMO inhibitors.

A series of anthranilamide derivatives were designed and synthesized as novel smoothened (SMO) inhibitors based on the SMO inhibitor taladegib (LY2940680), which can also inhibit the SMO-D473H mutant, via a ring-opening strategy. The phthalazine core in LY2940680 was replaced with anthranilamide, which retained the inhibitory activity towards the hedgehog (Hh) signaling pathway as evidenced by a dual luciferase reporter gene assay. Compound 12a displayed the best inhibitory activity against the Hh signaling pathway with IC50 value of 34.09 nM, and exhibited better proliferation inhibitory activity towards the Daoy cell line (IC50 = 0.48 µM) than LY2940680 (IC50 = 0.79 µM).

Design, synthesis and biological evaluation of novel 5-fluoro-1H-benzimidazole-4-carboxamide derivatives as potent PARP-1 inhibitors.

A series of novel 5-fluorine-benzimidazole-4-carboxamide analogs were designed and synthesized. All target compounds were evaluated for their PARP-1 inhibitory activity. Compounds possessed high intrinsic PARP-1 inhibitory potency have been evaluated in vitro cellular assays to measure the potentiation effect of cytotoxic agents against cancer cell line. These efforts led to the identification of compound 10f, which displayed strong inhibition against the PARP-1 enzyme with an IC50 of 43.7nM, excellent cell inhibitory activity in HCT116 cells (IC50=7.4µM) and potentiation of temozolomide cytotoxicity in cancer cell line A549 (PF50=1.6).

An engineered influenza virus to deliver antigens for lung cancer vaccination.

The development of cancer neoantigen vaccines that prime the anti-tumor immune responses has been hindered in part by challenges in delivery of neoantigens to the tumor. Here, using the model antigen ovalbumin (OVA) in a melanoma model, we demonstrate a chimeric antigenic peptide influenza virus (CAP-Flu) system for delivery of antigenic peptides bound to influenza A virus (IAV) to the lung. We conjugated attenuated IAVs with the innate immunostimulatory agent CpG and, after intranasal administration to the mouse lung, observed increased immune cell infiltration to the tumor. OVA was then covalently displayed on IAV-CPG using click chemistry. Vaccination with this construct yielded robust antigen uptake by dendritic cells, a specific immune cell response and a significant increase in tumor-infiltrating lymphocytes compared to peptides alone. Lastly, we engineered the IAV to express anti-PD1-L1 nanobodies that further enhanced regression of lung metastases and prolonged mouse survival after rechallenge. Engineered IAVs can be equipped with any tumor neoantigen of interest to generate lung cancer vaccines.

Site-specific tethering nanobodies on recombinant adeno-associated virus vectors for retargeted gene therapy.

Recombinant adeno-associated viruses (rAAVs) have been extensively studied for decades as carriers for delivering therapeutic genes. However, designing rAAV vectors with selective tropism for specific cell types and tissues has remained challenging. Here, we introduce a strategy for redirecting rAAV by attaching nanobodies with desired tropism at specific sites, effectively replacing the original tropism. To demonstrate this concept, we initially modified the genetic code of rAAV2 to introduce an azido-containing unnatural amino acid at a precise site within the capsid protein. Following a screening process, we identified a critical site (N587+1) where the introduction of unnatural amino acid eliminated the natural tropism of rAAV2. Subsequently, we successfully redirected rAAV2 by conjugating various nanobodies at the N587+1 site, using click and SpyTag-Spycatcher chemistries to form nanobody-AAV conjugates (NACs). By investigating the relationship between NACs quantity and effect and optimizing the linker between rAAV2 and the nanobody using a cathepsin B-susceptible valine-citrulline (VC) dipeptide, we significantly improved gene delivery efficiency both in vitro and in vivo. This enhancement can be attributed to the facilitated endosomal escape of rAAV2. Our method offers an exciting avenue for the rational modification of rAAV2 as a retargeting vehicle, providing a convenient platform for precisely engineering various rAAV2 vectors for both basic research and therapeutic applications. STATEMENT OF SIGNIFICANCE: AAVs hold great promise in the treatment of genetic diseases, but their clinical use has been limited by off-target transduction and efficiency. Here, we report a strategy to construct NACs by conjugating a nanobody or scFv to an rAAV capsid site, specifically via biorthogonal click chemistry and a spy-spycatcher reaction. We explored the structure-effect and quantity-effect relationships of NACs and then optimized the transduction efficiency by introducing a valine-citrulline peptide linker. This approach provides a biocompatible method for rational modification of rAAV as a retargeting platform without structural disruption of the virus or alteration of the binding capacity of the nanobody, with potential utility across a broad spectrum of applications in targeted imaging and gene delivery.

Proximity-enabled covalent binding of IL-2 to IL-2Rα selectively activates regulatory T cells and suppresses autoimmunity.

Interleukin-2 (IL-2) is a pleiotropic cytokine that orchestrates bidirectional immune responses via regulatory T cells (Tregs) and effector cells, leading to paradoxical consequences. Here, we report a strategy that exploited genetic code expansion-guided incorporation of the latent bioreactive artificial amino acid fluorosulfate-L-tyrosine (FSY) into IL-2 for proximity-enabled covalent binding to IL-2Rα to selectively promote Treg activation. We found that FSY-bearing IL-2 variants, such as L72-FSY, covalently bound to IL-2Rα via sulfur-fluoride exchange when in proximity, resulting in persistent recycling of IL-2 and selectively promoting the expansion of Tregs but not effector cells. Further assessment of L72-FSY-expanded Tregs demonstrated that L72-FSY maintained Tregs in a central memory phenotype without driving terminal differentiation, as demonstrated by simultaneously attenuated expression of lymphocyte activation gene-3 (LAG-3) and enhanced expression of programmed cell death protein-1 (PD-1). Subcutaneous administration of L72-FSY in murine models of pristane-induced lupus and graft-versus-host disease (GvHD) resulted in enhanced and sustained therapeutic efficacy compared with wild-type IL-2 treatment. The efficacy of L72-FSY was further improved by N-terminal PEGylation, which increased its circulatory retention for preferential and sustained effects. This proximity-enabled covalent binding strategy may accelerate the development of pleiotropic cytokines as a new class of immunomodulatory therapies.

Evaluation of Eleven Plant Species as Potential Banker Plants to Support Predatory Orius sauteri in Tea Plant Systems.

Tea green leafhoppers and thrips are key pests in tea plantations and have widely invaded those of Asian origin. Pesticides are currently a favorable control method but not desirable for frequent use on tea plants. To meet Integrated Pest Management (IPM) demand, biological control with a natural enemy is viewed as the most promising way. Orius sauteri are slated to be a natural enemy to tea pests. However, more knowledge of rearing O. sauteri and selecting banker plant systems is strongly needed. The reproductive biology evaluation of the egg oviposition and population life parameters of O. sauteri under laboratory conditions were examined, and the supporting ability of 11 plant species-motherwort, white clover, red bean, mung bean, peanut, soybean, kidney bean, herba violae, bush vetch, smooth vetch, and common vetch-in a greenhouse was assessed. Most of the selected plants, except for herba violae, performed relatively well with high oviposition quantity and survival. The mean fecundity per female on red bean and motherwort was 148.75 eggs and 148.25 eggs, respectively, and 90.20 eggs for tea plants (the smallest); there also were significant differences. In an experiment to determine the life parameters of O. sauteri, all the tested plants, except herba violae, were found to be able to complete the growth and development of the life cycle; there also were significant differences. The intrinsic rate of increase of motherwort and red bean was 1.18 and 1.17, respectively, and higher compared to that of the other plants, including tea plants (1.13). This result of the O. sauteri population development index was also confirmed in a greenhouse with the number of motherwort and red beans being as high as 113.33 and 112.67. Since motherwort was found to be susceptible to aphids and powdery mildew in each trial, it cannot be used for intercropping in tea gardens. Among the 11 plants, red bean was found to be the most suitable to support O. sauteri in tea plantations.

Functional Response and Control Potential of Orius sauteri (Hemiptera: Anthocoridae) on Tea Thrips (Dendrothrips minowai Priesner).

This study aimed to clarify the functional response and control potential of O. sauteri in relation to tea thrips. The functional response, interference response, and control potential of O. sauteri on adult tea thrips, in different insect stages and environment temperatures, were studied. The results showed that the predation of O. sauteri against tea thrips was positively correlated with prey density, while the effects of searching for O. sauteri on the adult tea thrips were negatively correlated with prey density. The predation effects of O. sauteri on tea thrips were also influenced by prey density, which indicated that there was an intra-specific interference response from predators to tea thrips. The population density of tea thrips was significantly decreased, and O. sauteri showed a remarkable ability to control them when the benefit-to-harm ratio was 3:100.

Site-specific PEGylation of interleukin-2 enhances immunosuppression via the sustained activation of regulatory T cells.

The preferential activation of regulatory T (Treg) cells by interleukin-2 (IL-2), which selectively binds to the trimeric IL-2 receptor (IL-2R) on Treg cells, makes this cytokine a promising therapeutic for the treatment of autoimmune diseases. However, IL-2 has a narrow therapeutic window and a short half-life. Here, we show that the pharmacokinetics and half-life of IL-2 can be substantially improved by orthogonally conjugating the cytokine to poly(ethylene glycol) (PEG) moieties via a copper-free click reaction through the incorporation of azide-bearing amino acids at defined sites. Subcutaneous injection of a PEGylated IL-2 that optimally induced sustained Treg-cell activation and expansion over a wide range of doses through highly selective binding to trimeric IL-2R led to enhanced therapeutic efficacy in mouse models of lupus, collagen-induced arthritis and graft-versus-host disease without compromising the immune defences of the host against viral infection. Site-specific PEGylation could be used more generally to engineer cytokines with improved therapeutic performance for the treatment of autoimmune diseases.

Discovery of potent, orally bioavailable ERK1/2 inhibitors with isoindolin-1-one structure by structure-based drug design.

Constitutive activation of MAPK (RAS/RAF/MEK/ERK) pathway is frequently observed in many tumors and thus has become an interesting therapeutic target for cancer therapy. Despite the successful development of BRAF and MEK inhibitors in clinic treatment, resistance often appears to re-enhance ERK1/2 signaling. Inspired by the central role of the ERK1/2 signaling cascade in cancer, we describe the scaffold-hopping generation of a series of isoindolin-1-one ERK1/2 inhibitors. Our new compounds could inhibit proliferation of KRAS and BRAF mutant cells lines at low nanomolar concentrations. Compound 22a possesses acceptable pharmacokinetic profiles and showed considerable in vivo antitumor efficacy in a HCT-116 xenograft model, providing a promising basis for further optimization towards clinical ERK1/2 inhibitors.

Metal-Free Remote C-H Bond Amidation of 8-Amidoquinolines on the C5 Position under Mild Conditions.

An efficient and facile process was developed for the remote C-H bond amidation of 8-aminoquinoline scaffolds on the C5 position which is geometric. The method only made use of PhI(OAc)2 as a mediator and showed good tolerance toward numerous dibenzenesulfonimides and amides, giving the corresponding products in moderate to excellent yield.

Efficient Palladium-Catalyzed C-H Fluorination of C(sp3)-H Bonds: Synthesis of ß-Fluorinated Carboxylic Acids.

A novel and facile process for direct fluorination of unactivated C(sp3)-H bonds at the ß position of carboxylic acids was accomplished by a palladium(II)-catalyzed C-H activation. The addition of Ag2O and pivalic acid was found to be crucial for the success of this transformation. This reaction provides a versatile strategy for the synthesis of ß-fluorinated carboxylic acids.