Biology subjects

Shen, M.-C.

Publications and source records attributed to Shen, M.-C..

6 recordsLinked to original sources

bioRxiv2025

Discovery and optimization of a guanylhydrazone-based small molecule to replace bFGF for cell culture applications

Replacing growth factors with a synthetic alternative molecule is an attractive opportunity to increase consistency, scalability and cost-effectiveness of cell-based products. Herein, we describe the discovery of a chemical class of FGFR1 agonists mimicking the action of basic fibroblast growth factor (bFGF), an essential component of cell-culture media. The guanylhydrazone-based molecule, TCB-32, was identified via structure-based virtual screening on the orthosteric binding site of FGFR1. It was shown to significantly increase cell proliferation by activating the FGFR1 signalling pathway like bFGF and exhibited enhanced thermostability over bFGF by containing activity over the course of several days. After extensive structure-activity relationship studies it was possible to increase potency and efficacy leading to three highly potent agonists. This finding has the potential to remove current bottlenecks in large-scale cell production as required for applications like cultivated meat or cell therapy. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/635029v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1d734aaorg.highwire.dtl.DTLVardef@1c28e0corg.highwire.dtl.DTLVardef@1fbdaa2org.highwire.dtl.DTLVardef@1cedd5e_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIDiscovery of a highly potent small molecule to replace bFGF in serum-free medium C_LIO_LIActivation of proliferation through the FGFR1-signalling pathway C_LIO_LIEnhanced thermal stability over bFGF C_LIO_LISAR led to three small molecules with decreased EC50, named TCB-494, TCB-541 and TCB-621 C_LI

biochemistry↗

bioRxiv2024

WheresWalker: a pipeline for rapid mutation mapping using whole genome sequencing

Forward genetic screening is a powerful approach to assign functions to genes and can be used to elucidate the many genes whose functions remain unknown. Chemical mutagenesis is an unbiased and efficient method for generating point mutations in the founding generation of animals in a forward genetic screening experiment. Missense and nonsense mutations induced by chemical mutagenesis can lead to the generation of partial function, gain-of-function, or null alleles that underlie compelling phenotypes, but positional cloning of the underlying causative single base pair changes can be laborious and time-consuming, especially in large polymorphic genomes. Current methods use a bioinformatic mapping-by-sequencing approach which often identifies large genomic regions which contain an intractable number of candidate genes for testing. Here, we describe WheresWalker, a modern mapping-by-sequencing algorithm that identifies a mutation-containing interval and then supports positional cloning to refine the interval which drastically reduces the number of potential candidates allowing for extremely rapid mutation identification. We validated this method using mutants from a forward genetic mutagenesis screen in zebrafish for modifiers of ApoB-lipoprotein metabolism. WheresWalker correctly maps and identifies novel zebrafish mutations in mttp, apobb.1, and mia2 genes, as well as a previously published mutation in maize. Further, we use WheresWalker to identify a previously unappreciated ApoB-lipoprotein metabolism-modifying locus, slc3a2a.

genetics↗

bioRxiv2023

A high-cholesterol zebrafish diet promotes hypercholesterolemia and fasting-associated liver triglycerides accumulation

Zebrafish are an ideal model organism to study lipid metabolism and to elucidate the molecular underpinnings of human lipid-associated disorders. In this study, we provide an improved protocol to assay the impact of a high-cholesterol diet (HCD) on zebrafish lipid deposition and lipoprotein regulation. Fish fed HCD developed hypercholesterolemia as indicated by significantly elevated ApoB-containing lipoproteins (ApoB-LP) and increased plasma levels of cholesterol and cholesterol esters. Feeding of the HCD to larvae (8 days followed by a 1 day fast) and adult female fish (2 weeks, followed by 3 days of fasting) was also associated with a fatty liver phenotype that presented as severe hepatic steatosis. The HCD feeding paradigm doubled the levels of liver triacylglycerol (TG), which was striking because our HCD was only supplemented with cholesterol. The accumulated liver TG was unlikely due to increased de novo lipogenesis or inhibited {beta}-oxidation since no differentially expressed genes in these pathways were found between the livers of fish fed the HCD versus control diets. However, fasted HCD fish had significantly increased lipogenesis gene fasn in adipose tissue and higher free fatty acids (FFA) in plasma. This suggested that elevated dietary cholesterol resulted in lipid accumulation in adipocytes, which supplied more FFA during fasting, promoting hepatic steatosis. In conclusion, our HCD zebrafish protocol represents an effective and reliable approach for studying the temporal characteristics of the physiological and biochemical responses to high levels of dietary cholesterol and provides insights into the mechanisms that may underlie fatty liver disease.

physiology↗

bioRxiv2022

Pla2g12b is Essential for Expansion of Nascent Lipoprotein Particles

SUMMARYTriglyceride-rich lipoproteins (TRLs) are micelle-like particles that enable efficient transport of lipids throughout the bloodstream, but also promote atherosclerotic cardiovascular disease. Despite this central relevance to cardiovascular disease, very little is known about how lipids are loaded onto nascent TRLs prior to secretion. Here we show that Pla2g12b, a gene with no previously described function, concentrates components of the TRL biogenesis machinery along the ER membrane to ensure efficient delivery of lipids to nascent TRLs. We find that the lipid-poor TRLs secreted in PLA2G12B-/- mice and zebrafish support surprisingly normal growth and physiology while conferring profound resistance to atherosclerosis, and demonstrate that these same processes are conserved in human cells. Together these findings shed new light on the poorly understood process of TRL expansion, ascribe function to the previously uncharacterized gene Pla2g12b, and reveal a promising new strategy to remodel serum lipoproteins to prevent cardiovascular disease.

cell biology↗

bioRxiv2019

Hedgehog signaling regulates neurogenesis in the larval and adult zebrafish hypothalamus

While neurogenesis in the adult hypothalamus is now known to be essential for proper function, the cell-cell signaling events that regulate neurogenesis in this evolutionarily conserved brain region remain poorly understood. Here we show that Hedgehog (Hh)/Gli signaling positively regulates hypothalamic neurogenesis in both larval and adult zebrafish and is necessary and sufficient for normal hypothalamic proliferation rates. Hedgehog-responsive cells are relatively rapidly proliferating pluripotent neural precursors that give rise to dopaminergic, serotonergic, and GABAergic neurons. in situ and transgenic reporter analyses revealed substantial heterogeneity in cell-cell signaling within the hypothalamic niche, with slow cycling Nestin-expressing cells residing among distinct and overlapping populations of Sonic Hh (Shh)-expressing, Hh-responsive, Notch-responsive, and Wnt-responsive radial glia. This work shows for the first time that Hh/Gli-signaling is a key component of the complex cell-cell signaling environment that regulates hypothalamic neurogenesis throughout life.

neuroscience↗

bioRxiv2017

mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces potentially functional transcripts

As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, allow frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of genomic compensation by means of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of genomic adaptations that can occur may inform the strategies of mutant generation.\n\nAuthor summaryThe recent rise of reverse genetic, gene targeting methods has allowed researchers to readily generate mutations in any gene of interest with relative ease. Should these mutations have the predicted effect on the mRNA and encoded protein, we would expect many more abnormal phenotypes than are typically being seen in reverse genetic screens. Here we set out to explore some of the reasons for this discrepancy by studying seven separate mutations in zebrafish. We present evidence that thorough cDNA sequence analysis is a key step in assessing the likelihood that a given mutation will produce hypomorphic or null alleles. This study reveals that alternative mRNA processing in the mutant background often produces transcripts that escape nonsense-mediated decay, thereby potentially preserving gene function. By understanding the ways that cells avoid the deleterious consequences of mutations, researchers can better design reverse genetic strategies to increase the likelihood of gene disruption.

molecular biology↗