Biology subjects

Chong, Y.

Publications and source records attributed to Chong, Y..

5 recordsLinked to original sources

bioRxiv2021

Whole-ExM: Expansion microscopy imaging of all anatomical structures of whole larval zebrafish

Nanoscale resolution imaging of whole vertebrates is required for a systematic understanding of human diseases, but this has yet to be realized. Expansion microscopy (ExM) is an attractive option for achieving this goal, but the expansion of whole vertebrates has not been demonstrated due to the difficulty of expanding hard body components. Here, we demonstrate whole-body ExM, which enables nanoscale resolution imaging of anatomical structures, proteins, and endogenous fluorescent proteins (FPs) of whole zebrafish larvae and mouse embryos by expanding them fourfold. We first show that post-digestion decalcification and digestion kinetics matching are critical steps in the expansion of whole vertebrates. Then, whole-body ExM is combined with the improved pan-protein labeling approach to demonstrate the three-dimensional super-resolution imaging of antibody- or FP-labeled structures and all major anatomical structures surrounding them. We also show that whole-body ExM enables visualization of the nanoscale details of neuronal structures across the entire body.

developmental biology↗

bioRxiv2020

Converging preganglionic axons refine without synaptic transmission when targets downstream of sympathetic nerves are active

Synaptic activity plays several roles as developing neurons make connections with their targets. It acts locally at synapses to influence the expression of genes needed to establish and maintain synaptic contacts. And, downstream it provides the necessary activity to strengthen and refine connections. Many studies have demonstrated how synaptic activity alters synaptic strength and increases synapse numbers. Much less is known, however, about the long-term consequences when a circuit develops without synaptic activity. To address this, we developed a mosaic model of sympathetic ganglia where synaptically-active and synaptically-inactive sympathetic neurons develop side-by-side in vivo. This model allowed us to address two issues. One is the relationship between activity and the refinement of converging inputs; the second is how synaptic activity contributes to a neurons gene expression profile. Our results indicate that converging presynaptic inputs to synaptically-silent neurons do not require postsynaptic activity to refine, provided these neurons share targets with synaptically-active neurons. Second, we demonstrate with single-cell RNA sequencing experiments that the expression of many genes by sympathetic neurons is independent of endogenous activity or local signals immediately downstream of excitatory postsynaptic potentials. An exception are genes required for neurotransmitter metabolism: We found that for a large sub-population of sympathetic neurons, synaptic activity increases the expression of adrenergic genes and supresses the expression of cholinergic genes. We conclude that signals generated locally at synapses do not initiate refinement of converging inputs, and that synaptic activitys influence on a neurons gene expression profiles is complex and depends on context.

neuroscience↗

bioRxiv2020

DGAT-onco: A powerful method to detect oncogenes by integrating differential mutational analysis and functional impacts of somatic mutations

MotivationOncogenes are genes whose malfunctions play critical roles in cancer development, and their discovery is a major aim of cancer mechanisms study. By counting the mutation frequency, oncogenes have been identified with frequent mutations, while it is believed that many more oncogenes could be discovered by differential mutational profile analysis. However, it is common that current methods only utilize mutations in the cancer population, which have an obvious bias in background mutation modelling. MethodsTo predict oncogenes efficiently, we developed a method, DGAT-onco that analyzed the frequency distribution and functional impacts of mutations in both cancer and natural population. Our method can capture the mutational difference of two population, and provide a comprehensive view of genomics basis underlying cancer development. DGAT-onco was constructed by germline mutations from the 1000 Genomes project and somatic mutations of 33 cancer types from the Cancer Genome Atlas (TCGA) dataset. Its reliability was verified on an independent test set including 19 cancers from other sources. ResultsWe demonstrated that our method is more effective than alternative methods in oncogenes discovering. Using this approach achieves higher classification performance in oncogene discovery than 6 alternative methods, and 22.8% significant genes identified by our method were verified as oncogenes by the Cancer Gene Census (CGC). AvailabilityDGAT-onco is available at https://github.com/zhanghaoyang0/DGAT-onco. Contactyangyd25@mail.sysu.edu.cn or zhaohy8@mail.sysu.edu.cn

bioinformatics↗

bioRxiv2019

The stepwise endonuclease activity of a thermophilic Argonaute protein

Thermophilic Argonaute proteins (Agos) can function as endonucleases via specific guide-target base-pairing cleavage for host defense. The ability to cleave target DNA sequences at any arbitrary sites endows them with reprogramed DNA capacity. Here, we identify that an Ago from the hyperthermophilic archaeon Pyrococcus furiosus (PfAgo) shows a stepwise endonuclease activity, which is demonstrated by the double strand DNA cleavage directed by a single guide DNA rather than canonical one pair of guide DNAs. We reveal that the cleavage products with 5-phosphorylated ends can used as the renewed guide which is capable to induce next round of cleavage to complementary sequences of target DNA. By combining the PfAgo stepwise endonuclease activity followed by target DNA amplification, we establish a rapid and specific platform for the unambiguously multiplex gene detection, termed RADAR (Renewed-gDNA Assisted DNA-cleavage by Argonaute). In the end, RADAR was applied to distinguish of human papillomavirus of serotypes in patient samples in a single reaction, suggesting that our technique would be adopted for diagnosing application.

bioengineering↗

bioRxiv2019

A-Star, an Argonaute-directed System for Rare SNV Enrichment and Detection

The ability to detect rare mutations has revolutionized diagnosis and monitoring of tumors, but is limited by the shortage of sensitive, cost-effective and high coverage methods for identification of extremely low abundant mutations. Here, we establish a single-tube multiplex PCR system by employing thermophilic Argonaute-derived DNA-guided nuclease for highly efficient rare mutation detection, referred to as A-Star (Argonaute-directed specific target enrichment and detection), that combines the selective cleavage of the wild type DNA in the DNA denaturation step and the followed amplification of mutant DNA during PCR. A-Star enables easy detection and quantitation of rare mutations originally as low as 0.01% in allele frequency with a [>=] 5500-fold efficiency. We also demonstrate the feasibility of A-Star for detecting oncogenic mutations in complex biological systems such as solid tumors tissues and blood samples. Remarkably, A-Star could achieve the detection of multiple oncogenic genes simultaneously through a simple single-tube reaction. Taken together, our work illustrates a supersensitive and rapid nucleic acid detection system, thereby extending the utility for both research and therapeutic applications.

bioengineering↗