Biology subjects

Gong, S.

Publications and source records attributed to Gong, S..

62 records · Page 4Linked to original sources

bioRxiv2019

Mapping Human Pluripotent Stem Cell Derived Erythroid Differentiation by Single-Cell Transcriptome Analysis

There is an imbalance between the supply and demand of functional red blood cells (RBCs) in clinical applications. This imbalance can be addressed by regenerating RBCs using several in vitro methods. Induced pluripotent stem cells (iPSCs) can handle the low supply of cord blood and the ethical issues in embryonic stem cell research and provide a promising strategy to eliminate immune rejection. However, no complete single-cell level differentiation pathway exists for the iPSC-derived RBC differentiation system. In this study, we used iPSC line BC1 to establish a RBCs regeneration system. The 10x genomics single-cell transcriptome platform was used to map the cell lineage and differentiation trajectories on day 14 of the regeneration system. We observed that iPSCs differentiation was not synchronized during embryoid body (EB) culture. The cells (day 14) mainly consisted of mesodermal and various blood cells, similar to the yolk sac hematopoiesis. We identified six cell classifications and characterized the regulatory transcription factors (TFs) networks and cell-cell contacts underlying the system. iPSCs undergo two transformations during the differentiation trajectory, accompanied by the dynamic expression of cell adhesion molecules and estrogen-responsive genes. We identified different stages of erythroid cells such as burst-forming unit erythroid (BFU-E) and orthochromatic erythroblasts (ortho-E) and found that the regulation of TFs (e.g., TFDP1 and FOXO3) is erythroid-stage specific. Immune erythroid cells were identified in our system. This study provides systematic theoretical guidance for optimizing the iPSCs-derived RBCs differentiation system, and this system is a useful model for simulating in vivo hematopoietic development and differentiation.

developmental biology↗

bioRxiv2019

Basis for discrimination by engineered CRISPR/Cas9 enzymes

CRISPR/Cas9 is a programmable genome editing tool that has been widely used for biological applications. While engineered Cas9s have been reported to increase discrimination against off-target cleavage compared with wild type Streptococcus pyogenes (SpCas9) in vivo, the mechanism for enhanced specificity has not been extensively characterized. To understand the basis for improved discrimination against off-target DNA containing important mismatches at the distal end of the guide RNA, we performed kinetic analyses on the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants. While DNA unwinding is the rate-limiting step for on-target cleavage by SpCas9, we show that chemistry is seriously impaired by more than 100-fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the rate of chemistry without increasing the rate of DNA rewinding--the kinetic partitioning favors release rather than cleavage of a bound off-target substrate because chemistry is slow. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.

biochemistry↗

bioRxiv2019

Cloning, Biochemical Characterization and inhibition of Alanine racemase from Streptococcus iniae

Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species, as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5'-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from the pathogenic strain of S. iniae, determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The optimal enzyme activity occurred at 35{degrees}C and a pH of 9.5. The enzyme belongs to the PLP dependent enzymes family and is highly specific to L-alanine. S.iniae Alr can be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters Km and Vmax of the enzyme were 33.11 mM, 2426 units/mg for L-alanine and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors, were determined and found to be effective against both gram positive and gram negative bacteria employed in this study. The important role of alanine racemase as a target of developing new antibiotics against S. iniae highlighted the usefulness of the enzyme for new antibiotics discovery.

biochemistry↗

bioRxiv2019

PERK inhibition mitigates restenosis and thrombosis - a potential low-thrombogenic anti-restenotic paradigm

BackgroundDrug-eluting stents (DES) represent the main-stream management of restenosis following treatments of occlusive cardiovascular diseases. However, DES cannot eliminate instent restenosis yet exacerbate thrombogenic risks. To achieve dual inhibition of restenotic smooth muscle cell (SMC) de-differentiation/proliferation and thrombogenic endothelial cell (EC) dysfunction, a common target in both cell types, has been long-sought after. We evaluated the potential of protein kinase RNA-like endoplasmic reticulum kinase (PERK) as such a target for low-thrombogenic anti-restenotic intervention.\n\nMethods and ResultsWe used a rat angioplasty model of restenosis and a FeCl3-induced mouse model of thrombosis. Loss-or gain-of-function was achieved by PERK inhibition (GSK2606414, siRNA) or overexpression (adenovirus). Restenosis was robustly mitigated by GSK2606414 administered either via injected (i.v.) lesion-homing platelet membrane-coated nanoclusters or a perivascular hydrogel; it was enhanced by PERK transgene. Whereas PERK inhibition blocked, its overexpression exacerbated PDGF-induced human aortic SMC de-differentiation (reduced smooth muscle -actin or SMA) and proliferation (BrdU incorporation). Further, PERK activity promoted STAT3 activation but inhibited SRF transcriptional (luciferase) activity; its protein co-immunoprecipitated with STAT3 and also MRTF-A, the SRF activator for SMA transcription. Importantly, PERK inhibition also prevented TNF-induced impairment of human EC growth and upregulation of thrombogenic tissue factor, both in vitro and ex vivo. In vivo, oral gavage of GSK2606414 preserved ~50% of the normal blood flow 60 min after FeCl3-induced vascular injury.\n\nConclusionsPERK inhibition is dual beneficial in mitigating restenosis and thrombosis, thus implicating a potential design for anti-restenotic intervention to overcome the thrombogenicity of DES.

pathology↗

bioRxiv2019

LonP1 Orchestrates UPRmt and UPRER and Mitochondrial Dynamics to Regulate Heart Function

Protein quality control is pivotal to cellular homeostasis and integrity of cardiomyocytes for maintenance of normal heart function. The unfolded protein response (UPR) is an adaptive process to modulate protein quality control in the endoplasmic reticulum (ER) and mitochondria, and is accordingly termed UPRER and UPRmt, respectively. Lon protease (LonP1) is a highly conserved mitochondrial protease to modulate UPRmt, which is involved in regulating metabolism, mitophagy, and stress response. However, whether LonP1 regulates UPRER remains elusive. To investigate the regulation of protein quality control in cardiomyocytes, we generated cardiac-specific LonP1 deletion mice. Our findings show that LonP1 deficiency caused impaired mitochondrial respiratory function and fragmentation. Surprisingly, both UPRER and UPRmt is substantially induced in LonP1-deletion heart suggesting of LonP1 as a novel regulator of UPRER; however, the activation of UPRER occurs earlier than UPRmt in response to LonP1 deletion. Consequently, cardiac-specific LonP1 deficiency causes aberrant metabolic reprogramming of cardiomyocytes, pathological heart remodeling, as well as impeded heart function. Thus, we uncovered the novel function of LonP1 as an UPRmt mediator, and reciprocal orchestration of UPRmt and UPRER and mitochondrial dynamics regulated by LonP1 in the cardiomyocytes that is critical to maintain heart function, which offers exciting new insights into the potential therapeutic strategy for heart failure.

cell biology↗

bioRxiv2018

Garcinia cambogia extract removes calcium oxalate kidney stones in both genetic and non-genetic Drosophila models of nephrolithiasis

Kidney stone formers with family history have a high rate of stone recurrence after kidney stone removal surgery and there is no effective medication available for treatment. Here, we show that Garcinia cambogia extract (GCE) efficiently removes calcium oxalate kidney stones from Malpighian tubules in both genetic and non-genetic Drosophila models of nephrolithiasis, and hydroxycitrate -a major component of GCE, directly dissolves calcium oxalate stones in Drosophila Malpighian tubules ex vivo. Our study discovers a potential novel therapeutic strategy for the clinical treatment of nephrolithiasis and suggests that clinical-grade Garcinia cambogia extract could be used to treat patients with nephrolithiasis in the future.

pharmacology and toxicology↗

bioRxiv2017

DNA unwinding is the primary determinant of CRISPR-Cas9 activity

Bacterial adaptive immunity utilizes RNA-guided surveillance complexes composed of CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target foreign nucleic acids for destruction. Cas9, a type II CRISPR-Cas effector complex, can be programed with a single guide RNA that base-pairs with the target strand of dsDNA, displacing the non-target strand to create an R-loop, where the HNH and RuvC nuclease domains can cleave opposing strands. Cas9 has been repurposed for a variety of important genome engineering applications. While many structural and biochemical studies have shed light on the mechanism of Cas9 cleavage, a clear unifying model has yet to emerge. Our detailed kinetic characterization of the enzyme reveals that DNA binding is reversible, R-loop formation is rate-limiting, occurring in two steps, one for each of the nuclease domains. Although the HNH nuclease activity is stimulated by Mg2+ with a single measureable Kd, the RuvC activity requires two distinct Mg2+ binding events. The specificity constant for cleavage is determined through an induced-fit mechanism as the product of the equilibrium binding affinity for DNA and the rate of R-loop formation.

biochemistry↗

bioRxiv2017

Senkyunolide A Protects Corticosterone-Induced Cell Apoptosis Via Modulating Protein Phosphatase 2A And α-Synuclein

Dan-zhi-xiao-yao-san is a Traditional Chinese Medicine (TCM) formula that is widely used to treat depression related neurological disorders, however, the active compound(s) and underlying mechanisms are unclear. In the present study, we found that senkyunolide A (SenA) has neuroprotective effects in corticosterone (Cort)-induced depression cell model in PC12 cells. Firstly, we found that SenA protects Cort-induced cell injury in PC12 cells. In addition, SenA attenuates Cort-induced the reduction of phosphatase 2A (PP2A) activities, and the increase of p-PP2A, -syn and p--syn-Ser129 levels. Furthermore, PP2A inhibitor okadaic acid (OA) decreased, and PP2A activator D-erythro-Sphingosine (SPH) increased Cort-induced cell apoptosis. Importantly, we also found that the neuroprotective effects of SenA in Cort-induced cell injury via modulating -syn levels. Collectively, our results suggest that the neuroprotective effects of SenA in Cort-induced depression cell model via modulating PP2A activities and -syn levels, and bring a breakthrough to the anti-depression mechanisms for natural compound SenA.

cell biology↗