Biology subjects

Wu, H.-J.

Publications and source records attributed to Wu, H.-J..

21 records · Page 2Linked to original sources

bioRxiv2021

Higher-order interaction of brain microstructural and functional connectome

Despite a relatively fixed anatomical structure, the human brain can support rich cognitive functions, triggering particular interest in investigating structure-function relationships. Myelin is a vital brain microstructure marker, yet the individual microstructure-function relationship is poorly understood. Here, we explore the brain microstructure-function relationships using a higher-order framework. Global (network-level) higher-order microstructure-function relationships negatively correlate with male participants personality scores and decline with aging. Nodal (node-level) higher-order microstructure-function relationships are not aligned uniformly throughout the brain, being stronger in association cortices and lower in sensory cortices, showing gender differences. Notably, higher-order microstructure-function relationships are maintained from the whole-brain to local circuits, which uncovers a compelling and straightforward principle of brain structure-function interactions. Additionally, targeted artificial attacks can disrupt these higher-order relationships, and the main results are robust against several factors. Together, our results increase the collective knowledge of higher-order structure-function interactions that may underlie cognition, individual differences, and aging.

neuroscience↗

bioRxiv2020

A simple and highly efficient method for multi-allelic CRISPR-Cas9 editing in primary cell cultures

ABSTRACTBackground CRISPR-Cas9-based technologies have revolutionized experimental manipulation of mammalian genomes. None-the-less, limitations of the delivery and efficacy of these technologies restrict their application in primary cells.Aims To create an optimized protocol for penetrant, reproducible, and fast targeted insertiondeletion mutation (indel) formation in cell cultures derived from primary cells, using patient-derived glioblastoma (GBM) stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs) for proof-of-concept experiments.Methods We employed transient nucleofection of Cas9:sgRNA ribonucleoprotein complexes using chemically synthesized 2’-O-methyl 3’phosphorothioate-modified sgRNAs and purified Cas9 protein. Indel frequency and size distribution were measured via computational deconvolution of Sanger sequencing trace data. Western blotting was used to evaluate protein loss. RNA-seq in edited NSCs was used to assess gene expression changes resulting from knockout of tumor suppressors commonly altered in GBM.Results We found that with this optimized technique, we can routinely achieve >90% indel formation in only 3 days, without the need to create clonal lines for simple loss-of-function experiments. We observed near-total protein loss of target genes in cell pools. Additionally, we found that this approach allows for the creation of targeted genomic deletions. We also demonstrated the utility of this method for quickly creating a series of gene knockouts that allow for the study of oncogenic activities.Conclusion Our data suggest that this relatively simple method can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells (such as GSCs). This represents an extremely useful tool for the cancer research community when wishing to inactivate not only coding genes, but also non-coding RNAs, UTRs, enhancers, and promoters. This method can be readily applied to diverse cell types by varying the nucleofection conditions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2018

Alternative splicing and translation play important roles in parallel with transcriptional regulation during rice hypoxic germination

Post-transcriptional mechanisms, including alternative splicing (AS) and alternative translation initiation (ATI), have been used to explain the protein diversity involved in plant developmental processes and stress responses. Rice germination under hypoxia conditions is a classical model system for the study of low oxygen stress. It is known that there is transcriptional regulation during rice hypoxic germination, but the potential roles of AS and ATI in this process are not well understood. In this study, a proteogenomic approach was used to integrate the data from RNA sequencing, qualitative and quantitative proteomics to discover new players or pathways in the response to hypoxia stress. The improved analytical pipeline of proteogenomics led to the identification of 10,253 intron-containing genes, 1,729 of which were not present in the current annotation. Approximately 1,741 differentially expressed AS (DAS) events from 811 genes were identified in hypoxia-treated seeds in comparison to controls. Over 95% of these were not present in the list of differentially expressed genes (DEG). In particular, regulatory pathways such as spliceosome, ribosome, ER protein processing and export, proteasome, phagosome, oxidative phosphorylation and mRNA surveillance showed substantial AS changes under hypoxia, suggesting that AS responses are largely independent of traditional transcriptional regulation. Massive AS changes were identified, including the preference usage of certain non-conventional splice sites and enrichment of splicing factors in the DAS datasets. In addition, using self-constructed protein libraries by 6-frame translation, thousands of novel proteins/peptides contributed by ATI were identified. In summary, these results provide deeper insights towards understanding the underlying mechanisms of AS and ATI during rice hypoxic germination.

plant biology↗