Bitter taste TAS2R14 activation by intracellular tastants and cholesterol.

Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents1,2. TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications3. Here we present cryogenic electron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and Gi1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery.

Study on the Design, Synthesis, Bioactivity and Translocation of the Conjugates of Phenazine-1-carboxylic Acid and N-Phenyl Alanine Ester.

The natural pesticide phenazine-1-carboxylic acid (PCA) is known to lack phloem mobility, whereas Metalaxyl is a representative phloem systemic fungicide. In order to endow PCA with phloem mobility and also enhance its antifungal activity, thirty-two phenazine-1-carboxylic acid-N-phenylalanine esters conjugates were designed and synthesized by conjugating PCA with the active structure N-acylalanine methyl ester of Metalaxyl. All target compounds were characterized by 1H NMR, 13C NMR and HRMS. The antifungal evaluation results revealed that several target compounds exhibited moderate to potent antifungal activities against Sclerotinia sclerotiorum, Bipolaris sorokiniana, Phytophthora parasitica, Phytophthora citrophthora. In particular, compound F7 displayed excellent antifungal activity against S. sclerotiorum with an EC50 value of 6.57 µg/mL, which was superior to that of Metalaxyl. Phloem mobility study in castor bean system indicated good phloem mobility for the target compounds F1-F16. Particularly, compound F2 exhibited excellent phloem mobility; the content of compound F2 in the phloem sap of castor bean was 19.12 µmol/L, which was six times higher than Metalaxyl (3.56 µmol/L). The phloem mobility tests under different pH culture solutions verified the phloem translocation of compounds related to the "ion trap" effect. The distribution of the compound F2 in tobacco plants further suggested its ambimobility in the phloem, exhibiting directional accumulation towards the apical growth point and the root. These results provide valuable insights for developing phloem mobility fungicides mediated by exogenous compounds.

Design, Synthesis, Antibacterial, and Antifungal Evaluation of Phenylthiazole Derivatives Containing a 1,3,4-Thiadiazole Thione Moiety.

To effectively control the infection of plant pathogens, we designed and synthesized a series of phenylthiazole derivatives containing a 1,3,4-thiadiazole thione moiety and screened for their antibacterial potencies against Ralstonia solanacearum, Xanthomonas oryzae pv. oryzae, as well as their antifungal potencies against Sclerotinia sclerotiorum, Rhizoctonia solani, Magnaporthe oryzae and Colletotrichum gloeosporioides. The chemical structures of the target compounds were characterized by 1H NMR, 13C NMR and HRMS. The bioassay results revealed that all the tested compounds exhibited moderate-to-excellent antibacterial and antifungal activities against six plant pathogens. Especially, compound 5k possessed the most remarkable antibacterial activity against R. solanacearum (EC50 = 2.23 µg/mL), which was significantly superior to that of compound E1 (EC50 = 69.87 µg/mL) and the commercial agent Thiodiazole copper (EC50 = 52.01 µg/mL). Meanwhile, compound 5b displayed the most excellent antifungal activity against S. sclerotiorum (EC50 = 0.51 µg/mL), which was equivalent to that of the commercial fungicide Carbendazim (EC50 = 0.57 µg/mL). The preliminary structure-activity relationship (SAR) results suggested that introducing an electron-withdrawing group at the meta-position and ortho-position of the benzene ring could endow the final structure with remarkable antibacterial and antifungal activity, respectively. The current results indicated that these compounds were capable of serving as promising lead compounds.

Recent research and applications in lipid-based food and lipid-incorporated bioink for 3D printing.

Three-dimensional (3D) printing, as an emerging digital production technology, has recently been receiving increasing attention in food processing. It is important to understand the effect of key ingredients of food materials on the printing, which makes it possible to achieve a wider range of structures using few nozzles and to provide tailored nutrition and personalization. This comprehensive review delves into the latest research on 3D-printed lipid-based foods, encompassing a variety of products such as chocolate, processed cheese, as well as meat. It also explores the development and application of food bioinks that incorporate lipids as a pivotal component, including those based on starch, protein, oleogels, bigels, and emulsions, as well as emulsion gels. Moreover, this review identifies the current challenges and presents an outlook on future research directions in the field of 3D food printing, especially the research and application of lipids in food 3D printing.

Protective effect of rubber seed oil on human endothelial cells.

OBJECTIVE: This study was conducted to characterize the antioxidant and anti-inflammatory properties of Rubber Seed Oil (RSO) against atherosclerosis (AS) through the study of the protective effects and mechanisms on human umbilical vein endothelial cells (HUVECs) injury induced by oxidized low-density lipoprotein (ox-LDL). METHODS: HUVECs were treated with RSO, ox-LDL, RSO + ox-LDL, respectively, followed by cell activity testing, levels of IL-1ß, IL-6, IL-10, TNF-α, ROS, NO, the mRNA expression of eNOS and protein expression of MCP-1, VCAM-1, eNOS, TLR4, NF-κB p65、p-NF-κB p65. RESULTS: Compared with the ox-LDL group, cell viability, NO level and the expression of eNOS mRNA significantly increased. and the levels of pro-inflammatory factors such as IL-1ß, IL-6, TNF-α, IL-10, ROS were significantly decreased, which was accompanied by decreases in TLR4 mRNA, TLR4, MCP-1, VCAM-1 protein expression, as well as the ratio of NF-κB p-p65/p65 in the group treated with 250 µg/ml ox-LDL + 50 µg/ml RSO, 250 µg/ml ox-LDL + 100 µg/ml RSO, 250 µg/ml ox-LDL + 150 µg/ml RSO. CONCLUSIONS: RSO can reduce the expression of pro-inflammatory mediators, oxidative factors involved in injured vascular endothelial cells, exhibiting anti-inflammatory and antioxidant properties HUVECs exposed to ox-LDL. In addition, it may alleviate endothelial cell damage by inhibiting the TLR4/NF-κB signaling pathway.

Unveiling the structural mechanisms of nonpeptide ligand recognition and activation in human chemokine receptor CCR8.

The human CC chemokine receptor 8 (CCR8) is an emerging therapeutic target for cancer immunotherapy and autoimmune diseases. Understanding the molecular recognition of CCR8, particularly with nonpeptide ligands, is valuable for drug development. Here, we report three cryo-electron microscopy structures of human CCR8 complexed with Gi trimers in the ligand-free state or activated by nonpeptide agonists LMD-009 and ZK 756326. A conserved Y1.39Y3.32E7.39 motif in the orthosteric binding pocket is shown to play a crucial role in the chemokine and nonpeptide ligand recognition. Structural and functional analyses indicate that the lack of conservation in Y1143.33 and Y1724.64 among the CC chemokine receptors could potentially contribute to the selectivity of the nonpeptide ligand binding to CCR8. These findings present the characterization of the molecular interaction between a nonpeptide agonist and a chemokine receptor, aiding the development of therapeutics targeting related diseases through a structure-based approach.

Characterization and application of active human α2,6-sialyltransferases ST6GalNAc V and ST6GalNAc VI recombined in Escherichia coli.

Eukaryotic sialyltransferases play key roles in many physiological and pathological events. The expression of active human recombinant sialyltransferases in bacteria is still challenging. In the current study, the genes encoding human N-acetylgalactosaminide α2,6-sialyltransferase V (hST6GalNAc V) and N-acetylgalactosaminide α2,6-sialyltransferase VI (hST6GalNAc VI) lacking the N-terminal transmembrane domains were cloned into the expression vectors, pET-32a and pET-22b, respectively. Soluble and active forms of recombinant hST6GalNAc V and hST6GalNAc VI when coexpressed with the chaperone plasmid pGro7 were successfully achieved in Escherichia coli. Further, lactose (Lac), Lacto-N-triose II (LNT II), lacto-N-tetraose (LNT), and sialyllacto-N-tetraose a (LSTa) were used as acceptor substrates to investigate their activities and substrate specificities. Unexpectedly, both can transfer sialic acid onto all those substrates. Compared with hST6GalNAc V expressed in the mammalian cells, the recombinant two α2,6-sialyltransferases in bacteria displayed flexible substrate specificities and lower enzymatic efficiency. In addition, an important human milk oligosaccharide disialyllacto-N-tetraose (DSLNT) can be synthesized by both human α2,6-sialyltransferases expressed in E. coli using LSTa as an acceptor substrate. To the best of our knowledge, these two active human α2,6-sialyltransferases enzymes were expressed in bacteria for the first time. They showed a high potential to be applied in biotechnology and investigating the molecular mechanisms of biological and pathological interactions related to sialylated glycoconjugates.

A framework for Frizzled-G protein coupling and implications to the PCP signaling pathways.

The ten Frizzled receptors (FZDs) are essential in Wnt signaling and play important roles in embryonic development and tumorigenesis. Among these, FZD6 is closely associated with lens development. Understanding FZD activation mechanism is key to unlock these emerging targets. Here we present the cryo-EM structures of FZD6 and FZD3 which are known to relay non-canonical planar cell polarity (PCP) signaling pathways as well as FZD1 in their G protein-coupled states and in the apo inactive states, respectively. Comparison of the three inactive/active pairs unveiled a shared activation framework among all ten FZDs. Mutagenesis along with imaging and functional analysis on the human lens epithelial tissues suggested potential crosstalk between the G-protein coupling of FZD6 and the PCP signaling pathways. Together, this study provides an integrated understanding of FZD structure and function, and lays the foundation for developing therapeutic modulators to activate or inhibit FZD signaling for a range of disorders including cancers and cataracts.

Automated chemoenzymatic modular synthesis of human milk oligosaccharides on a digital microfluidic platform.

Glycans, along with proteins, nucleic acids, and lipids, constitute the four fundamental classes of biomacromolecules found in living organisms. Generally, glycans are attached to proteins or lipids to form glycoconjugates that perform critical roles in various biological processes. Automatic synthesis of glycans is essential for investigation into structure-function relationships of glycans. In this study, we presented a method that integrated magnetic bead-based manipulation and modular chemoenzymatic synthesis of human milk oligosaccharides (HMOs), on a DMF (Digital Microfluidics) platform. On the DMF platform, enzymatic modular reactions were conducted in solution, and purification of products or intermediates was achieved by using DEAE magnetic beads, circumventing the intricate steps required for traditional solid-phase synthesis. With this approach, we have successfully synthesized eleven HMOs with highest yields of up to >90% on the DMF platform. This study would not only lay the foundation for OPME synthesis of glycans on the DMF platform, but also set the stage for developing automated enzymatic glycan synthesizers based on the DMF platform.

Cadmium exposure induced neuronal ferroptosis and cognitive deficits via the mtROS-ferritinophagy pathway.

Exposure to environmental cadmium (Cd) is known to cause neuronal death and cognitive decline in humans. Ferroptosis, a novel iron-dependent type of regulated cell death, is involved in various neurological disorders. In the present study, Cd exposure triggered ferroptosis in the mouse hippocampus and in the HT22 murine hippocampal neuronal cell line, as indicated by significant increases in ferroptotic marker expression, intracellular iron levels, and lipid peroxidation. Interestingly, ferroptosis of hippocampal neurons in response to Cd exposure relied on the induction of autophagy since the suppression of autophagy by 3-methyladenine (3-MA) and chloroquine (CQ) substantially ameliorated Cd-induced ferroptosis. Furthermore, nuclear receptor coactivator 4 (NCOA4)-mediated degradation of ferritin was required for the Cd-induced ferroptosis of hippocampal neurons, demonstrating that NCOA4 knockdown decreased intracellular iron levels and lipid peroxidation and increased cell survival, following Cd exposure. Moreover, Cd-induced mitochondrial reactive oxygen species (mtROS) generation was essential for the ferritinophagy-mediated ferroptosis of hippocampal neurons. Importantly, pretreatment with the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively attenuated Cd-induced hippocampal neuronal death and cognitive impairment in mice. Taken together, these findings indicate that ferroptosis is a novel mechanism underlying Cd-induced neurotoxicity and cognitive impairment and that the mtROS-ferritinophagy axis modulates Cd-induced neuronal ferroptosis.

Novel Cocrystal Structures of Peptide Antagonists Bound to the Human Melanocortin Receptor 4 Unveil Unexplored Grounds for Structure-Based Drug Design.

Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934 and SBL-MC-31. These peptides differ from SHU9119 by substituting His6 with Pro6 and inserting Gly10 or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119 potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+ channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders.