Biology subjects

Leong, K. W.

Publications and source records attributed to Leong, K. W..

22 records · Page 2Linked to original sources

bioRxiv2021

Mapping morphological malformation to genetic dysfunction in blood vessel organoids with 22q11.2 Deletion Syndrome

DiGeorge Syndrome, or 22q11.2 deletion syndrome (22q11.2 DS), is a genetic disorder caused by microdeletions in chromosome 22, impairing the function of endothelial cells (EC) and/or mural cells and leading to deficits in blood vessel development such as abnormal aortic arch morphology, tortuous retinal vessels, and tetralogy of Fallot. The mechanism by which dysfunctional endothelial cells and pericytes contribute to the vasculopathy, however, remains unknown. In this study, we used human blood vessel organoids (VOs) generated from iPSC of 22q11.2 DS patients to model the vascular malformations and genetic dysfunctions. We combined high-resolution lightsheet imaging and single-cell transcriptome analysis to link the genetic profile and vascular phenotype at the single-cell level. We developed a comprehensive analytical methodology by integrating deep learning-mediated blood vessel segmentation, network graph construction, and tessellation analysis for automated morphology characterization. We report that 22q11.2DS VOs demonstrate a smaller size with increased angiogenesis/sprouting, suggesting a less stable vascular network. Overall, clinical presentations of smaller vascular diameter, less connected vasculature, and increased branch points were recapitulated in 22q11.2DS VOs. Single-cell transcriptome profiling showed heterogeneity in both 22q11.2DS and control VOs, but the former demonstrated alterations in endothelial characteristics that are organ-specific and suggest a perturbation in the vascular developmental process. Intercellular communication analysis indicated that the vascular dysfunctions in 22q11.2 deletion were due to a lower cell-cell contact and upregulated extracellular matrix organization involving collagen and fibronectin. Voronoi diagram-based tessellation analysis also indicated that the colocalization of endothelial tubes and mural cells was different between control and 22q11.2 VOs, indicating that alterations in EC and mural interactions might contribute to the deficits in vascular network formation. This study illustrates the utility of VO in revealing the pathogenesis of 22q11.2DS vasculopathy.

bioengineering↗

bioRxiv2021

A programmable probiotic encapsulation system enhances therapeutic delivery in vivo

Recent advances in therapeutic modulation of human microbiota have driven new efforts to engineer living microbial medicines using synthetic biology. However, a long-standing challenge for live bacterial therapies is balancing the high dose required to achieve robust efficacy with the potential for sepsis. Here, we developed a genetically encoded microbial encapsulation system with tunable and dynamic expression of surface capsular polysaccharides to enhance therapeutic delivery. Following a synthetic small RNA knockdown screen of the capsular biosynthesis pathway, we constructed synthetic gene circuits that regulate bacterial encapsulation based on sensing the levels of environmental inducer, bacterial density, and blood pH. The induced encapsulation system enabled tunable immunogenicity and survivability of the probiotic Escherichia coli, resulting in increased maximum tolerated dose and enhanced efficacy in murine cancer models. Furthermore, triggering in situ encapsulation was found to increase microbial translocation between mouse tumors, leading to efficacy in distal tumors. The programmable encapsulation system demonstrates a new approach to control microbial therapeutic profiles in vivo using synthetic biology.

synthetic biology↗

bioRxiv2021

Chronic opioid treatment arrests neurodevelopment and alters synaptic activity in human midbrain organoids

The impact of long-term opioid exposure on the embryonic brain is crucial to healthcare due to the surging number of pregnant mothers with an opioid dependency. Current studies on the neuronal effects are limited due to human brain inaccessibility and cross-species differences among animal models. Here, we report a model to assess cell-type specific responses to acute and chronic fentanyl treatment, as well as fentanyl withdrawal, using human induced pluripotent stem cell (hiPSC)-derived midbrain organoids. Single cell mRNA sequencing (25,510 single cells in total) results suggest that chronic fentanyl treatment arrests neuronal subtype specification during early midbrain development and alters the pathways associated with synaptic activities and neuron projection. Acute fentanyl treatment, however, increases dopamine release but does not induce significant changes in gene expressions of cell lineage development. To date, our study is the first unbiased examination of midbrain transcriptomics with synthetic opioid treatment at the single cell level.

neuroscience↗

bioRxiv2021

MAESTRO affords 'breadth and depth' for mutation testing

The ability to assay large numbers of low-abundance mutations is crucial in biomedicine. Yet, the technical hurdles of sequencing multiple mutations at extremely high depth and accuracy remain daunting. For sequencing low-level mutations, its either depth or breadth but not both. Here, we report a simple and powerful approach to accurately track thousands of distinct mutations with minimal reads. Our technique called MAESTRO (minor allele enriched sequencing through recognition oligonucleotides) employs massively-parallel mutation enrichment to empower duplex sequencing--one of the most accurate methods--to track up to 10,000 low-frequency mutations with up to 100-fold less sequencing. In example use cases, we show that MAESTRO could enable mutation validation from cancer genome sequencing studies. We also show that it could track thousands of mutations from a patients tumor in cell-free DNA, which may improve detection of minimal residual disease from liquid biopsies. In all, MAESTRO improves the breadth, depth, accuracy, and efficiency of mutation testing.

molecular biology↗