Biology subjects

Hui, K.

Publications and source records attributed to Hui, K..

7 recordsLinked to original sources

bioRxiv2026

Brainwide dopamine dynamics across sleep-wake transitions

Dopaminergic signaling plays a critical role in regulating arousal and transitions between sleep states, yet brainwide dynamics underlying these transitions remain incompletely characterized. Here, we employed multi-site fiber photometry with the dopamine sensor GRAB-DA2m, combined with electroencephalogram (EEG)/electromyogram (EMG) recordings, to systematically assess regional dopamine (DA) dynamics across sleep-wake states in the medial prefrontal cortex (mPFC), striatal subregions, central amygdala (CeA), and midbrain nuclei in mice. We found that DA levels prominently increased in the mPFC, dorsolateral striatum (DLS), ventral tegmental area (VTA), substantia nigra pars compacta (SNc), and dorsal raphe nucleus (DRN) during transitions from non-rapid eye movement (NREM) sleep to wakefulness (WAKE), supporting their role in arousal. Conversely, DA decreased in the CeA and nucleus accumbens lateral parts (NAc-L) during these transitions. During transitions from NREM to rapid eye movement sleep (REM), DA elevations were observed in the CeA and middle accumbens subregion (NAc-M), rather than NAc-L, while other regions exhibited decreases. Cross-regional DA correlations revealed synchronized network activity across sleep-wake transitions. Optogenetic activation of VTA and DRN dopamine neurons induced robust DA release in cortical and subcortical regions, and chemogenetic activation promoted wakefulness selectively via VTA and DRN, but not SNc DA neurons. These results elucidate distinct brainwide DA dynamics across state transitions and highlight differential roles for DA signaling in modulating sleep-wake states.

neuroscience↗

bioRxiv2025

Evaluating Microglial Contributions to the Neurovascular Unit in Health and Neurodegeneration Using Human In Vitro Models

BackgroundMicroglia are emerging as critical regulators of neurovascular function in health and Alzheimers disease (AD), yet their interactions with the human neurovascular unit (NVU), particularly brain endothelial cells, remain incompletely understood. Current in vitro NVU platforms typically exclude microglia and lack perfusable vascular networks with physiologically relevant architecture. Here, we established complementary two-dimensional (2D) and three-dimensional (3D) NVU models to investigate microglia-endothelial and microglia-neurovascular interactions. MethodsHuman induced pluripotent stem cell derived-neurons (iNs), astrocytes (iAs), and microglia-like cells (iMGLs) were incorporated into a soft-lithography based engineered microvessel system to establish a multicellular neuroimmune-vascular model. To specifically evaluate iMGL-endothelial cell (EC) interactions, iMGL were co-cultured with primary human brain microvascular endothelial cells (HBMECs) and junctional protein localization was evaluated using immunofluorescence. The barrier integrity of engineered microvessels containing iMGL was evaluated using dextran permeability. Our 2D and 3D systems were stimulated with tumor necrosis factor- (TNF) to evaluate whether iMGL would promote or attenuate EC inflammation and barrier breakdown. ResultsIncorporation of iNs, iAs, and iMGLs into a perfusable vascular model enabled a more complete representation of NVU cellular diversity and promoted neuronal health. In monolayer co-culture with iMGL, HBMECs enhanced the junctional localization of tight and adherens junction proteins through both contact-dependent and paracrine mechanisms. Following an inflammatory challenge, iMGLs reduced endothelial inflammatory activation, suggesting a protective role in response to AD-relevant inflammatory conditions. Finally, when embedded in 3D collagen matrices surrounding perfusable endothelialized lumen networks, iMGLs reduced dextran permeability and preserved endothelial barrier integrity following TNF challenge. ConclusionsTogether, these findings establish a 3D perfusable neuroimmune-vascular model that enables the dissection of microglial contributions to neurovascular function in health and disease.

cell biology↗

bioRxiv2025

Population structure and antimicrobial resistance of Corynebacterium diphtheriae in Victoria, Australia

Corynebacterium diphtheriae, the main aetiological agent of diphtheria, is a re-emerging bacterial pathogen of public health concern, yet remains largely understudied globally. In this study, we analysed the population structure and antimicrobial resistance (AMR) of 210 C. diphtheriae isolates from Victoria, Australia including 103 historical (1950-1970) and 107 contemporary clinical isolates (2004-2023), using whole-genome sequencing and phenotypic susceptibility testing. The diphtheria toxin gene (tox) was detected in 89 isolates, the majority of which (n=83; 93.3%) were historical. Population structure comprised two primary phylogenetic lineages, Mitis and Gravis, each containing multiple sublineages. Multi-locus sequence type (MLST) analysis revealed a highly diverse population structure with multiple novel MLST profiles and alleles. When placed within a global phylogenetic framework, Australian isolates were broadly distributed, reflecting substantial genetic diversity. Phenotypic susceptibility testing against eleven antimicrobials revealed that several contemporary isolates were resistant to multiple agents, including penicillin and erythromycin, first-line treatments of Corynebacterium infections. Eight contemporary isolates were multidrug-resistant (resistant to [≥]3 antimicrobial classes), including five with resistance to both penicillin and erythromycin. Genomic analysis identified multiple genes and mutations conferring resistance among contemporary isolates. In contrast, no antimicrobial resistance phenotypes or genotypes were observed in historical isolates. Analysis of historical genomes provides valuable insights into a period of heightened diphtheria activity in Victoria prior to widespread immunisation. Overall, these findings establish a baseline for ongoing genomic surveillance in the face of increasing global outbreaks, support informed empiric treatment strategies, and contribute to the knowledge of global population structure of C. diphtheriae. Impact statementCorynebacterium diphtheriae is a re-emerging pathogen of public health concern with limited genomic data available to support surveillance and public health interventions. This study provides a contemporary understanding of C. diphtheriae from Australia in the era of genomic surveillance and helps us better understand baseline genomic diversity and antimicrobial resistance. Additionally, these findings enhance preparedness for potential future incursions or outbreaks, including those involving drug-resistant strains, as recently observed in parts of Africa and Europe. Data summaryGenome sequences are deposited in GenBank under BioProject PRJNA870170. Sample data and accession numbers are included in the Supplementary Table S1. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

genomics↗

bioRxiv2024

Histologic variants in bladder cancer harbor aggressive molecular features including TM4SF1 expression and a CA125+ cell state

Histologic variant (HV) subtypes of bladder cancer are clinically aggressive tumors that are more resistant to standard therapy compared to conventional urothelial carcinoma (UC). Little is known about the transcriptional programs that account for the morphological and biological differences in HV tumors. To investigate the tumor biology of HV bladder cancers, we generated a single cell RNA sequencing (scRNA- seq) atlas of nine HV tumors and three UC tumors. Our analyses revealed a tumor cell state specific to HVs that is characterized by expression of MUC16 (CA125), KRT24, and WISP2. This CA125+ cell state bears transcriptional hallmarks of epithelial-mesenchymal transition, is enriched in metastases, is predicted to be highly chemotherapy resistant, and is linked with poor survival, suggesting that this cell state plays an important role in the aggressive biology of HV tumors. Our analyses also provide novel evidence of transcriptional "mimicry" between HVs and histologically similar non-urothelial cell types. Lastly, we identified higher expression of TM4SF1, a cell surface protein associated with cancer metastasis, in HV tumor cells compared to UC tumor cells. Finally, CAR T cells engineered against TM4SF1 protein demonstrated in vitro and in vivo activity against bladder cancer cell lines in a TM4SF1 expression- dependent manner, highlighting its potential as a therapeutic target in bladder cancer. One sentence summarySingle cell RNA sequencing of primary bladder cancers identified a CA125+ cell state specific to histologic variants that is associated with aggressive biological features and TM4SF1 as a novel therapeutic target for histologic variant subtypes of bladder cancer which can be targeted by anti- TM4SF1 CAR T cells.

cancer biology↗

bioRxiv2023

Single cell transcriptional profiling of benign prostatic hyperplasia reveals a progenitor-like luminal epithelial cell state within an inflammatory microenvironment

Benign prostatic hyperplasia (BPH) is characterized by excessive cell proliferation and inflammation and affects most aging men. The development of new therapies for BPH requires a deeper understanding of the underlying pathophysiology and cellular components of BPH. Here, we characterize at single cell resolution the cellular states of BPH and identify cell populations enriched in BPH that contribute to cell proliferation and inflammation. Single-cell RNA-sequencing was performed on prostate tissue from 15 patients undergoing holmium laser enucleation of the prostate for treatment of BPH. Clustering and differential expression analysis on aligned single cell RNA-seq data was performed to annotate all cell types. Pseudotime, gene set enrichment, gene ontology, and ligand-receptor analyses were performed. 16,234 cells were analyzed and specific stromal, epithelial, and immune subgroups were found to be strongly associated with inflammation. A rare luminal subgroup was identified and pseudotime analysis indicated this luminal subgroup was more closely related to club and basal cells. Using a gene set derived from epithelial stem cells, we found that this luminal subgroup had a significantly higher stem cell signature score than all other epithelial subgroups, suggesting this subgroup is a luminal precursor state. Ligand-receptor interactions between stromal, epithelial, and immune cells were explored with CellPhoneDB. Unique interactions highlighting MIF, a pro-inflammatory cytokine that promotes epithelial cell growth and inflammation in the prostate, were found between fibroblasts and the progenitor luminal subgroup. This luminal subgroup also interacted with neutrophils and macrophages through MIF. Our single-cell profiling of BPH provides a roadmap for inflammation-linked cell subgroups and highlights a novel luminal progenitor subgroup interacting with other cell groups via MIF that may contribute to the inflammation and cell proliferation phenotype associated with BPH.

genomics↗

bioRxiv2022

CDKL5 regulates p62-mediated selective autophagy and host antiviral defense

Virophagy, the selective autophagosomal engulfment and degradation of viral components, is crucial for antiviral immunity. However, the mechanisms leading to viral antigen recognition and autophagy induction remain poorly understood. Here, we identify a novel kinase, Cyclin-dependent kinase-like 5 (CDKL5), as an essential regulator of virophagy. Deletion of CDKL5 or abrogation of its kinase activity reduced virophagy of Sindbis virus (SINV), a neurotropic RNA virus, and increased intracellular accumulation of SINV capsid proteins and cellular cytotoxicity. Mechanistically, through direct phosphorylation of the selective autophagy receptor p62, CDKL5 promoted formation of p62 inclusion bodies that bound capsid. Loss of CDKL5 disrupted the capsid-p62 interaction, and a p62 phosphomimetic mutant rescued the interaction. CDKL5 knockout mice demonstrated increased neuronal cell death after SINV infection and enhanced lethality after infection with several human viruses. Overall, these findings identify a cell-autonomous innate immune mechanism for autophagy activation to clear toxic viral capsid aggregates during infection.

microbiology↗

bioRxiv2018

Highly Multiplexed Genome Engineering Using CRISPR/Cas9 gRNA Arrays

The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limiting due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.

bioengineering↗