Structural mechanism of cooperative activation of the human calcium-sensing receptor by Ca2+ ions and L-tryptophan

The human calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) responsible for maintaining Ca²⁺ homeostasis in the blood. The general consensus is that extracellular Ca²⁺ is the principal agonist of CaSR. Aliphatic and aromatic L-amino acids, such as L-Phe and L-Trp, increase the sensitivity of CaSR towards Ca²⁺ and are considered allosteric activators. Crystal structures of the extracellular domain (ECD) of CaSR dimer have demonstrated Ca²⁺ and L-Trp binding sites and conformational changes of the ECD upon Ca²⁺/L-Trp binding. However, it remains to be understood at the structural level how Ca²⁺/L-Trp binding to the ECD leads to conformational changes in transmembrane domains (TMDs) and consequent CaSR activation. Here, we determined the structures of full-length human CaSR in the inactive state, Ca²⁺- or L-Trp-bound states, and Ca²⁺/L-Trp-bound active state using single-particle cryo-electron microscopy. Structural studies demonstrate that L-Trp binding induces the closure of the Venus flytrap (VFT) domain of CaSR, bringing the receptor into an intermediate active state. Ca²⁺ binding relays the conformational changes from the VFT domains to the TMDs, consequently inducing close contact between the two TMDs of dimeric CaSR, activating the receptor. Importantly, our structural and functional studies reveal that Ca²⁺ ions and L-Trp activate CaSR cooperatively. Amino acids are not able to activate CaSR alone, but can promote the receptor activation in the presence of Ca²⁺. Our data provide complementary insights into the activation of class C GPCRs and may aid in the development of novel drugs targeting CaSR.Subject terms: Cryoelectron microscopy, Calcium signalling     

Interaction between Staphylococcus Agr virulence and neutrophils regulates pathogen expansion in the skin

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MiR-200c-3p increased HDMEC proliferation through the notch signaling pathway

Excessive proliferation of vascular endothelial cells can cause hemangioma. Although typically benign, hemangiomas can become life-threatening. The microRNA miR-200c-3p is abnormally expressed in some types of tumors, but its expression, biological role, and mechanism of action in infantile hemangioma remain to be fully elucidated. The expression levels of miR-200c-3p in hemangioma tissue were compared with those in adjacent healthy tissue by using bioinformatics analyses and TargetScan. Western blot, enzyme-linked immunosorbent assay, and Cell Counting Kit 8 analyses were used to determine the biological function and site of action of miR-200c-3p in human dermal microvascular endothelial cells (HDMECs). MiR-200c-3p was one of the top 10 differentially expressed genes between healthy tissue, and hemangiomas tissues, having markedly decreased expression in hemangioma tissue. Reduction of miR-200c-3p expression in HDMECs through the transfection of a miR-200c-3p inhibitor significantly increased HDMEC proliferation. The addition of the Notch signaling pathway inhibitor DAPT to HDMECs transfected with the miR-200c-3p inhibitor eliminated the inhibitor-induced enhancement of proliferation in HDMECs. These findings indicate that miR-200c-3p targets the Notch signaling pathway to promote the proliferation of vascular endothelial cells, suggesting that miR-200c-3p plays an important role in the pathogenesis of hemangioma.     

Role of tumor-derived exosomes in cancer metastasis

The highlights of cancer research include the discovery of exosomes, which are small (30-100?nm) sized vesicular nanoparticles released virtually by all cells. Tumor-derived exosomes (TDEs) are notoriously known for orchestrating the invasion-metastasis cascade via systemic pathways that we have previously proposed (1), resulting in a paradigm shift of our understanding about the pathobiology of metastases. In principle, exosomes serve as transport medium for proteins, mRNAs and miRNAs to transmit targeted cues from the primary cell to distant sites via horizontal transfer or cell-receptor interaction. In this chapter, we seek to explore in-depth the mechanisms engendering TDE in the metastatic cascade, along with experimental models to augment our understanding. The aforementioned has also paved way for parallel advancements in the therapeutic armamentarium, as evident from pronounced efforts to exploit the metastatic process for therapeutic targeting. In this light, we aim to examine potential anti-metastatic therapeutic opportunities derived from exosomal research. Lastly, exosomes may play a crucial role in the contemporary era of “liquid biopsies”, given the array of molecular information with diagnostic and predictive indications. We thus intend to end this chapter off by exploring future applications of exosomes that could illuminate shortcomings and propel advancements in biomarker research.     

Asd-based balanced-lethal system in attenuated Edwardsiella tarda to express a heterologous antigen for a multivalent bacterial vaccine

Edwardsiella tarda is an enteric Gram-negative invasive intracellular pathogen, which causes enteric septicemia in fish. It could be potentially used to develop a recombinant attenuated E. tarda vaccine for the aquaculture industry. Because live vaccine strains can potentially be released into the environment upon vaccination, medical and environmental safety issues must be considered. Deletion of the asdB gene in E. tarda resulted in a diaminopimelic acid (DAP)-dependent mutant. The wild type asdB gene was inserted in place of the antibiotic-resistance gene in the plasmid, and the resultant non-antibiotic resistant vector was transformed into the attenuated and DAP-dependent E. tarda vaccine strain (WEDΔasdB) to obtain a balanced-lethal system for heterologous antigen expression. The balanced-lethal expression system was further optimized by comparing plasmid replicons with different Shine–Dalgarno sequences and start codons for the asdB gene. Utilizing the optimized balanced-lethal expression system, the protective antigen gene gapA34 from the fish pathogen Aeromonas hydrophila LSA34 was expressed in the attenuated E. tarda to generate the multivalent vaccine candidate WEDΔasdB/pUTta4DGap. This vaccine was shown to evoke an effective immune response against both E. tarda and A. hydrophila LSA34 by vaccinating turbot via a simple immersion route. This multivalent E. tarda vector vaccine has great potential for broad applications in aquaculture.     

A novel mutant PIK3R1EY451delinsD breast cancer patient resistant to HER2-targeted therapy treated with everolimus: a case report

Molecular Biology Reports - Resistance to HER2-targeted therapy is a critical issue in breast cancer that must be addressed immediately. PIK3R1 mutations are more common in Chinese breast cancer...