Phosphoantigens glue butyrophilin 3A1 and 2A1 to activate Vγ9Vδ2 T cells.

In both cancer and infections, diseased cells are presented to human Vγ9Vδ2 T cells through an 'inside out' signalling process whereby structurally diverse phosphoantigen (pAg) molecules are sensed by the intracellular domain of butyrophilin BTN3A11-4. Here we show how-in both humans and alpaca-multiple pAgs function as 'molecular glues' to promote heteromeric association between the intracellular domains of BTN3A1 and the structurally similar butyrophilin BTN2A1. X-ray crystallography studies visualized that engagement of BTN3A1 with pAgs forms a composite interface for direct binding to BTN2A1, with various pAg molecules each positioned at the centre of the interface and gluing the butyrophilins with distinct affinities. Our structural insights guided mutagenesis experiments that led to disruption of the intracellular BTN3A1-BTN2A1 association, abolishing pAg-mediated Vγ9Vδ2 T cell activation. Analyses using structure-based molecular-dynamics simulations, 19F-NMR investigations, chimeric receptor engineering and direct measurement of intercellular binding force revealed how pAg-mediated BTN2A1 association drives BTN3A1 intracellular fluctuations outwards in a thermodynamically favourable manner, thereby enabling BTN3A1 to push off from the BTN2A1 ectodomain to initiate T cell receptor-mediated γδ T cell activation. Practically, we harnessed the molecular-glue model for immunotherapeutics design, demonstrating chemical principles for developing both small-molecule activators and inhibitors of human γδ T cell function.

Phase separation at the nanoscale quantified by dcFCCS.

Liquid-liquid phase separation, driven by multivalent macromolecular interactions, causes formation of membraneless compartments, which are biomolecular condensates containing concentrated macromolecules. These condensates are essential in diverse cellular processes. Formation and dynamics of micrometer-scale phase-separated condensates are examined routinely. However, limited by commonly used methods which cannot capture small-sized free-diffusing condensates, the transition process from miscible individual molecules to micrometer-scale condensates is mostly unknown. Herein, with a dual-color fluorescence cross-correlation spectroscopy (dcFCCS) method, we captured formation of nanoscale condensates beyond the detection limit of conventional fluorescence microscopy. In addition, dcFCCS is able to quantify size and growth rate of condensates as well as molecular stoichiometry and binding affinity of client molecules within condensates. The critical concentration to form nanoscale condensates, identified by our experimental measurements and Monte Carlo simulations, is at least several fold lower than the detection limit of conventional fluorescence microscopy. Our results emphasize that, in addition to micrometer-scale condensates, nanoscale condensates are likely to play important roles in various cellular processes and dcFCCS is a simple and powerful quantitative tool to examine them in detail.

Not all edges are peers: Accurate structure-aware graph pooling networks.

Graph Neural Networks (GNNs) have achieved state-of-the-art performance in graph-related tasks. For graph classification task, an elaborated pooling operator is vital for learning graph-level representations. Most pooling operators derived from existing GNNs generate a coarsen graph through ordering the nodes and selecting some top-ranked ones. However, these methods fail to explore the fundamental elements other than nodes in graphs, which may not efficiently utilize the structure information. Besides, all edges attached to the low-ranked nodes are discarded, which destroys graphs' connectivity and loses information. Moreover, the selected nodes tend to concentrate on some substructures while overlooking information in others. To address these challenges, we propose a novel pooling operator called Accurate Structure-Aware Graph Pooling (ASPool), which can be integrated into various GNNs to learn graph-level representation. Specifically, ASPool adaptively retains a subset of edges to calibrate the graph structure and learns the abstracted representations, wherein all the edges are viewed as non-peers instead of simply connecting nodes. To preserve the graph's connectivity, we further introduce the selection strategy considering both top-ranked nodes and dropped edges. Additionally, ASPool performs a two-stage calculation process to promise that the sampled nodes are distributed throughout the graph. Experiment results on 9 widely used benchmarks show that ASPool achieves superior performance over the state-of-the-art graph representation learning methods.

Quantifying phase separation at the nanoscale by dual-color fluorescence cross-correlation spectroscopy (dcFCCS).

Liquid-liquid phase separation (LLPS) causes the formation of membraneless condensates, which play important roles in diverse cellular processes. Currently, optical microscopy is the most commonly used method to visualize micron-scale phase-separated condensates. Because the optical spatial resolution is restricted by the diffraction limit (~200 nm), dynamic formation processes from individual biomolecules to micron-scale condensates are still mostly unknown. Herein, we provide a detailed protocol applying dual-color fluorescence cross-correlation spectroscopy (dcFCCS) to detect and quantify condensates at the nanoscale, including their size, growth rate, molecular stoichiometry, and the binding affinity of client molecules within condensates. We expect that the quantitative dcFCCS method can be widely applied to investigate many other important phase separation systems.

Mechanisms of phase-separation-mediated cGAS activation revealed by dcFCCS.

Cyclic GMP-AMP synthase (cGAS), as a DNA sensor, plays an important role in cGAS-STING pathway, which further induces expression of type I interferon as the innate immune response. Previous studies reported that liquid-liquid phase separation (LLPS) driven by cGAS and long DNA is essential to promote catalytic activity of cGAS to produce a second messenger, cyclic GMP-AMP (cGAMP). However, the molecular mechanism of LLPS promoting cGAS activity is still unclear. Here, we applied dual-color fluorescence cross-correlation spectroscopy (dcFCCS), a highly sensitive and quantitative method, to characterize phase separation driven by cGAS and DNA from miscible individual molecule to micronscale. Thus, we captured nanoscale condensates formed by cGAS at close-to-physiological concentration and quantified their sizes, molecular compositions and binding affinities within condensates. Our results pinpointed that interactions between DNA and cGAS at DNA binding sites A, B, and C and the dimerization of cGAS are the fundamental molecular basis to fully activate cGAS in vitro. Due to weak binding constants of these sites, endogenous cGAS cannot form stable interactions at these sites, leading to no activity in the absence of LLPS. Phase separation of cGAS and DNA enriches cGAS and DNA by 2 to 3 orders of magnitude to facilitate these interactions among cGAS and DNA and to promote cGAS activity as an on/off switch. Our discoveries not only shed lights on the molecular mechanisms of phase-separation-mediated cGAS activation, but also guided us to engineer a cGAS fusion, which can be activated by 15 bp short DNA without LLPS.

Pseudoknot length modulates the folding, conformational dynamics, and robustness of Xrn1 resistance of flaviviral xrRNAs.

To understand how RNA dynamics is regulated and connected to its function, we investigate the folding, conformational dynamics and robustness of Xrn1 resistance of a set of flaviviral xrRNAs using SAXS, smFRET and in vitro enzymatic assays. Flaviviral xrRNAs form discrete ring-like 3D structures, in which the length of a conserved long-range pseudoknot (PK2) ranges from 2 bp to 7 bp. We find that xrRNAs' folding, conformational dynamics and Xrn1 resistance are strongly correlated and highly Mg2+-dependent, furthermore, the Mg2+-dependence is modulated by PK2 length variations. xrRNAs with long PK2 require less Mg2+ to stabilize their folding, exhibit reduced conformational dynamics and strong Xrn1 resistance even at low Mg2+, and tolerate mutations at key tertiary motifs at high Mg2+, which generally are destructive to xrRNAs with short PK2. These results demonstrate an unusual regulatory mechanism of RNA dynamics providing insights into the functions and future biomedical applications of xrRNAs.

Controlled Synthesis and Structure Engineering of Transition Metal-based Nanomaterials for Oxygen and Hydrogen Electrocatalysis in Zinc-Air Battery and Water-Splitting Devices.

Electrocatalytic energy conversion plays a crucial role in realizing energy storage and utilization. Clean energy technologies such as water electrolysis, fuel cells, and metal-air batteries heavily depend on a series of electrochemical redox reactions occurring on the catalysts surface. Therefore, developing efficient electrocatalysts is conducive to remarkably improved performance of these devices. Among numerous studies, transition metal-based nanomaterials (TMNs) have been considered as promising catalysts by virtue of their abundant reserves, low cost, and well-designed active sites. This Minireview is focused on the typical clean electrochemical reactions: hydrogen evolution reaction, oxygen evolution reaction, and oxygen reduction reaction. Recent efforts to optimize the external morphology and the internal electronic structure of TMNs are described, and beginning with single-component TMNs, the active sites are clarified, and strategies for exposing more active sites are discussed. The summary about multi-component TMNs demonstrates the complementary advantages of integrating functional compositions. A general introduction of single-atom TMNs is provided to deepen the understanding of the catalytic process at an atomic scale. Finally, current challenges and development trends of TMNs in clean energy devices are summarized.

Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for investigating the dynamic properties of biomacromolecules. However, the success of protein smFRET relies on the precise and efficient labeling of two or more fluorophores on the protein of interest (POI), which has remained highly challenging, particularly for large membrane protein complexes. Here, we demonstrate the site-selective incorporation of a novel unnatural amino acid (2-amino-3-(4-hydroselenophenyl) propanoic acid, SeF) through genetic expansion followed by a Se-click reaction to conjugate the Bodipy593 fluorophore on calmodulin (CaM) and ß-arrestin-1 (ßarr1). Using this strategy, we monitored the subtle but functionally important conformational change of ßarr1 upon activation by the G-protein coupled receptor (GPCR) through smFRET for the first time. Our new method has broad applications for the site-specific labeling and smFRET measurement of membrane protein complexes, and the elucidation of their dynamic properties such as transducer protein selection.

Correction: Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction.

[This corrects the article DOI: 10.1039/D1SC02653D.].

[Study of hoses esophagoscopy].

OBJECTIVE: To explore a method of extracting the esophageal foreign body more effectively and safely, so as to reduce its complication and mortality. METHOD: The hoses, the endoscope (gastrofibroscope or electron-icgastroscope) and the tongs were used to make up the hoses esophagoscopy, using the technique of hoses esophagoscopy to extract the esophageal foreign body. RESULT: Hoses esophagoscopy was successful in removing the impaction in 30 of 33 adults. The other three patients with longer and sharp foreign body were treated by hoses and rigid esophagoscopy. No perforation or complication occurred. CONCLUSION: The technique of hoses esophagoscopy have the advantage of flexible endoscope and rigid esophagoscopy. It is a more effective and safe method for removing esophageal impactions.