Biology subjects

Yao, X.-Q.

Publications and source records attributed to Yao, X.-Q..

3 recordsLinked to original sources

bioRxiv2025

CRISPR/Cas14a Combined with RPA for Visual Detection of Marek's Disease Virus

Mareks disease (MD), a highly contagious avian immunosuppressive disorder caused by the -herpesvirus MDV-1, poses a significant threat to poultry health. The development of rapid visual detection methods capable of distinguishing epidemic MDV-1 strains from vaccine strains is crucial for early disease warning, vaccine efficacy evaluation, and precise disease control. We developed a novel isothermal detection system that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas14a technology for the visual identification of epidemic MDV-1 strains. This method operates at a constant temperature of 37{degrees}C and allows for either real-time analysis or endpoint visual readout without the need for complex instrumentation. Our results showed no cross-reactivity with Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), MDV-1 vaccine strains, or herpesvirus of turkeys (HVT). Plasmid DNA standards were used to determine the sensitivity of the assay and the detection limit was 24.6 copies/L. Clinical evaluation using 24 field samples confirmed that the method successfully identified all MDV-positive cases, demonstrating its diagnostic reliability. In conclusion, we have developed a rapid, instrument-free, and highly specific nucleic acid detection platform for MDV-1 by combining the sensitivity of RPA with the specificity of CRISPR/Cas14a technology, offering promising potential for field-based diagnostics and disease surveillance. IMPORTANCEMareks disease virus (MDV-1) is a highly contagious and economically important avian pathogen. Existing diagnostic methods are unable to reliably distinguish between epidemic and vaccine strains in field settings, which hampers effective surveillance and evaluation of vaccination programs. To address this challenge, we developed a portable isothermal detection assay that combines recombinase polymerase amplification (RPA) with CRISPR/Cas14a technology. This approach enables highly sensitive (24.6 copies/L) and specific visual detection of epidemic MDV-1 strains without cross-reactivity with vaccine strains or related viruses. The assay demonstrated 100% agreement with reference methods when validated using clinical samples. As a cost-effective and instrument-free method, it offers a practical solution for rapid on-site diagnosis, facilitating enhanced outbreak control and improved poultry health management globally.

molecular biology↗

bioRxiv2023

ACKR3-arrestin2/3 complexes reveal molecular consequences of GRK-dependent barcoding

Atypical chemokine receptor 3 (ACKR3, also known as CXCR7) is a scavenger receptor that regulates extracellular levels of the chemokine CXCL12 to maintain responsiveness of its partner, the G protein-coupled receptor (GPCR), CXCR4. ACKR3 is notable because it does not couple to G proteins and instead is completely biased towards arrestins. Our previous studies revealed that GRK2 and GRK5 install distinct distributions of phosphates (or "barcodes") on the ACKR3 carboxy terminal tail, but how these unique barcodes drive different cellular outcomes is not understood. It is also not known if arrestin2 (Arr2) and 3 (Arr3) bind to these barcodes in distinct ways. Here we report cryo-electron microscopy structures of Arr2 and Arr3 in complex with ACKR3 phosphorylated by either GRK2 or GRK5. Unexpectedly, the finger loops of Arr2 and 3 directly insert into the detergent/membrane instead of the transmembrane core of ACKR3, in contrast to previously reported "core" GPCR-arrestin complexes. The distance between the phosphorylation barcode and the receptor transmembrane core regulates the interaction mode of arrestin, alternating between a tighter complex for GRK5 sites and heterogenous primarily "tail only" complexes for GRK2 sites. Arr2 and 3 bind at different angles relative to the core of ACKR3, likely due to differences in membrane/micelle anchoring at their C-edge loops. Our structural investigations were facilitated by Fab7, a novel Fab that binds both Arr2 and 3 in their activated states irrespective of receptor or phosphorylation status, rendering it a potentially useful tool to aid structure determination of any native GPCR-arrestin complex. The structures provide unprecedented insight into how different phosphorylation barcodes and arrestin isoforms can globally affect the configuration of receptor-arrestin complexes. These differences may promote unique downstream intracellular interactions and cellular responses. Our structures also suggest that the 100% bias of ACKR3 for arrestins is driven by the ability of arrestins, but not G proteins, to bind GRK-phosphorylated ACKR3 even when excluded from the receptor cytoplasmic binding pocket.

biochemistry↗

bioRxiv2018

Comparative structural dynamic analysis of GTPases

GTPases regulate a multitude of essential cellular processes ranging from movement and division to differentiation and neuronal activity. These ubiquitous enzymes operate by hydrolyzing GTP to GDP with associated conformational changes that modulate affinity for family-specific binding partners. There are three major GTPase superfamilies: Ras-like GTPases, heterotrimeric G proteins and protein-synthesizing GTPases. Although they contain similar nucleotide-binding sites, the detailed mechanisms by which these structurally and functionally diverse superfamilies operate remain unclear. Here we compare and contrast the structural dynamic mechanisms of each superfamily using extensive molecular dynamics (MD) simulations and subsequent network analysis approaches. In particular, dissection of the cross-correlations of atomic displacements in both the GTP and GDP-bound states of Ras, transducin and elongation factor EF-Tu reveals analogous dynamic features. This includes similar dynamic communities and subdomain structures (termed lobes). For all three proteins the GTP-bound state has stronger couplings between equivalent lobes. Network analysis further identifies common and family-specific residues mediating the state-specific coupling of distal functional sites. Mutational simulations demonstrate how disrupting these couplings leads to distal dynamic effects at the nucleotide-binding site of each family. Collectively our studies extend current understanding of GTPase allosteric mechanisms and highlight previously unappreciated similarities across functionally diverse families.\n\nAuthor SummaryGTPases are a large superfamily of essential enzymes that regulate a variety of cellular processes. They share a common core structure supporting nucleotide binding and hydrolysis, and are potentially descended from the same ancestor. Yet their biological functions diverge dramatically, ranging from cell division and movement to signal transduction and translation. It has been shown that conformational changes through binding to different substrates underlie the regulation of their activities. Here we investigate the conformational dynamics of three typical GTPases by in silico simulation. We find that these three GTPases possess overall similar substrate-associated dynamic features, beyond their distinct functions. Further identification of key common and family-specific elements in these three families helps us understand how enzymes are adapted to acquire distinct functions from a common core structure. Our results provide unprecedented insights into the functional mechanism of GTPases in general, which potentially facilitates novel protein design in the future.

bioinformatics↗