Biology subjects

Chan, C. K. F.

Publications and source records attributed to Chan, C. K. F..

4 recordsLinked to original sources

bioRxiv2023

Brain-Derived CCN3 Is An Osteoanabolic Hormone That Sustains Bone in Lactating Females

In lactating mothers, the high calcium (Ca2+) demand for milk production triggers significant bone resorption. While estrogen would normally counteract excessive bone loss and maintain sufficient bone formation during this postpartum period, this sex steroid drops precipitously after giving birth. Here, we report that brain-derived CCN3 (Cellular Communication Network factor 3) secreted from KISS1 neurons of the arcuate nucleus (ARCKISS1) fills this void and functions as a potent osteoanabolic factor to promote bone mass in lactating females. Using parabiosis and bone transplant methods, we first established that a humoral factor accounts for the female-specific, high bone mass previously observed by our group after deleting estrogen receptor alpha (ER) from ARCKISS1 neurons1. This exceptional bone phenotype in mutant females can be traced back to skeletal stem cells (SSCs), as reflected by their increased frequency and osteochondrogenic potential. Based on multiple assays, CCN3 emerged as the most promising secreted pro-osteogenic factor from ARCKISS1 neurons, acting on mouse and human SSCs at low subnanomolar concentrations independent of age or sex. That brain-derived CCN3 promotes bone formation was further confirmed by in vivo gain- and loss-of-function studies. Notably, a transient rise in CCN3 appears in ARCKISS1 neurons in estrogen-depleted lactating females coincident with increased bone remodeling and high calcium demand. Our findings establish CCN3 as a potentially new therapeutic osteoanabolic hormone that defines a novel female-specific brain-bone axis for ensuring mammalian species survival.

neuroscience↗

bioRxiv2023

Efficient and multiplexed tracking of single cells using whole-body PET/CT

In vivo molecular imaging tools are crucially important for elucidating how cells move through complex biological systems, however, achieving single-cell sensitivity over the entire body remains challenging. Here, we report a highly sensitive and multiplexed approach for tracking upwards of 20 single cells simultaneously in the same subject using positron emission tomography (PET). The method relies on a new tracking algorithm (PEPT-EM) to push the cellular detection threshold to below 4 Bq/cell, and a streamlined workflow to reliably label single cells with over 50 Bq/cell of 18F-fluorodeoxyglucose (FDG). To demonstrate the potential of method, we tracked the fate of over 70 melanoma cells after intracardiac injection and found they primarily arrested in the small capillaries of the pulmonary, musculoskeletal, and digestive organ systems. This study bolsters the evolving potential of PET in offering unmatched insights into the earliest phases of cell trafficking in physiological and pathological processes and in cell-based therapies. TEASERA novel PET imaging workflow reveals the fate of single cells across the body.

bioengineering↗

bioRxiv2022

Gla-domain mediated targeting of externalized phosphatidylserine for intracellular delivery

Phosphatidylserine (PS) is a negatively charged phospholipid normally localized to the inner leaflet of the plasma membrane of cells but is externalized onto the cell surface during apoptosis as well as in malignant and infected cells. Consequently, PS may comprise an important molecular target in diagnostics, imaging and targeted delivery of therapeutic agents. While an array of PS binding-molecules exist, their utility has been limited by their inability to recognize PS and internalize diagnostic or therapeutic payloads. We describe the generation, isolation, characterization, and utility of a PS binding motif comprised of a carboxylated glutamic acid (GLA) residue domain, that both recognizes cell surface-exposed PS and is internalized into these cells after binding. Internalization is independent of the traditional endosomal-lysosomal pathway, directly entering the cytosol of the target cell in a rapid and energy-independent fashion. We demonstrate that this PS recognition extends to extracellular vesicles and stem cells and that GLA-domain conjugated probes can be detected upon intravenous administration in animal models of infectious disease and cancer. GLA domain binding and internalization offers new opportunities for targeting specific cells for imaging and delivery of therapeutics.

cell biology↗

bioRxiv2017

Index Switching Causes "Spreading-Of-Signal" Among Multiplexed Samples In Illumina HiSeq 4000 DNA Sequencing

Illumina-based next generation sequencing (NGS) has accelerated biomedical discovery through its ability to generate thousands of gigabases of sequencing output per run at a fraction of the time and cost of conventional technologies. The process typically involves four basic steps: library preparation, cluster generation, sequencing, and data analysis. In 2015, a new chemistry of cluster generation was introduced in the newer Illumina machines (HiSeq 3000/4000/X Ten) called exclusion amplification (ExAmp), which was a fundamental shift from the earlier method of random cluster generation by bridge amplification on a non-patterned flow cell. The ExAmp chemistry, in conjunction with patterned flow cells containing nanowells at fixed locations, increases cluster density on the flow cell, thereby reducing the cost per run. It also increases sequence read quality, especially for longer read lengths (up to 150 base pairs). This advance has been widely adopted for genome sequencing because greater sequencing depth can be achieved for lower cost without compromising the quality of longer reads. We show that this promising chemistry is problematic, however, when multiplexing samples. We discovered that up to 5-10% of sequencing reads (or signals) are incorrectly assigned from a given sample to other samples in a multiplexed pool. We provide evidence that this \"spreading-of-signals\" arises from low levels of free index primers present in the pool. These index primers can prime pooled library fragments at random via complementary 3 ends, and get extended by DNA polymerase, creating a new library molecule with a new index before binding to the patterned flow cell to generate a cluster for sequencing. This causes the resulting read from that cluster to be assigned to a different sample, causing the spread of signals within multiplexed samples. We show that low levels of free index primers persist after the most common library purification procedure recommended by Illumina, and that the amount of signal spreading among samples is proportional to the level of free index primer present in the library pool. This artifact causes homogenization and misclassification of cells in single cell RNA-seq experiments. Therefore, all data generated in this way must now be carefully re-examined to ensure that \"spreading-of-signals\" has not compromised data analysis and conclusions. Re-sequencing samples using an older technology that uses conventional bridge amplification for cluster generation, or improved library cleanup strategies to remove free index primers, can minimize or eliminate this signal spreading artifact.

molecular biology↗