Biology subjects

Wu, C.-C.

Publications and source records attributed to Wu, C.-C..

29 records · Page 2Linked to original sources

bioRxiv2020

Is zebrafish heart regeneration complete? Lineage-restricted cardiomyocytes proliferate to pre-injury numbers but some fail to differentiate in fibrotic hearts

Adult zebrafish are frequently described to be able to "completely" regenerate the heart. Yet, the extent to which cardiomyocytes lost to injury are replaced is unknown, since only indirect or non-quantitative evidence for cardiomyocyte proliferation exists. We established stereological methods to quantify the number of cardiomyocytes at several time-points post cryoinjury. Intriguingly, after cryoinjuries that killed about 1/3 of the ventricular cardiomyocytes, pre-injury cardiomyocyte numbers were restored already within 30 days. Yet, many hearts retained small residual scars, and a subset of cardiomyocytes bordering these fibrotic areas remained smaller, lacked differentiated sarcomeric structures, and displayed defective calcium signaling. Thus, a subset of regenerated cardiomyocytes failed to fully mature. While lineage-tracing experiments have shown that regenerating cardiomyocytes are derived from differentiated cardiomyocytes, technical limitations have previously made it impossible to test whether cardiomyocyte trans-differentiation contributes to regeneration of non-myocyte cell lineages. Using Cre responder lines that are expressed in all major cell types of the heart, we found no evidence for cardiomyocyte transdifferentiation into endothelial, epicardial, fibroblast or immune cell lineages. Overall, our results imply a refined answer to the question whether zebrafish can completely regenerate the heart: in response to cryoinjury, preinjury cardiomyocyte numbers are indeed completely regenerated, while restoration of cardiomyocyte differentiation and function, as well as resorption of scar tissue, is less robustly achieved.

developmental biology↗

bioRxiv2020

Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 is a worldwide health emergency. The immense damage done to public health and economies has prompted a global race for cures and vaccines. In developing a COVID-19 vaccine, we applied technology previously used for MERS-CoV to produce a prefusion-stabilized SARS-CoV-2 spike protein by adding two proline substitutions at the top of the central helix (S-2P). To enhance immunogenicity and mitigate the potential vaccine-induced immunopathology, CpG 1018, a Th1-biasing synthetic toll-like receptor 9 (TLR9) agonist was selected as an adjuvant candidate. S-2P was combined with various adjuvants, including CpG 1018, and administered to mice to test its effectiveness in eliciting anti-SARS-CoV-2 neutralizing antibodies. S-2P in combination with CpG 1018 and aluminum hydroxide (alum) was found to be the most potent immunogen and induced high titer of spike-specific antibodies in sera of immunized mice. The neutralizing abilities in pseudotyped lentivirus reporter or live wild-type SARS-CoV-2 were measured with reciprocal inhibiting dilution (ID50) titers of 5120 and 2560, respectively. In addition, the antibodies elicited were able to cross-neutralize pseudovirus containing the spike protein of the D614G variant, indicating the potential for broad spectrum protection. A marked Th-1 dominant response was noted from cytokines secreted by splenocytes of mice immunized with CpG 1018 and alum. No vaccine-related serious adverse effects were found in the dose-ranging study in rats administered single- or two-dose regimens with up to 50 g of S-2P combined with CpG 1018 alone or CpG 1018 with alum. These data support continued development of CHO-derived S-2P formulated with CpG 1018/alum as a candidate vaccine to prevent COVID-19 disease.

immunology↗

bioRxiv2020

Fibroblast growth factor 9 stimulates neurite outgrowth through NF-kB signaling in striatal cell Huntington's disease models

Proper development of neuronal cells is important for brain functions, and impairment of neuronal development may lead to neuronal disorders, implying that improvement in neuronal development may be a therapeutic direction for these diseases. Huntingtons disease (HD) is a neurodegenerative disease characterized by impairment of neuronal structures, ultimately leading to neuronal death and dysfunctions of the central nervous system. Based on previous studies, fibroblast growth factor 9 (FGF9) may provide neuroprotective functions in HD, and FGFs may enhance neuronal development and neurite outgrowth. However, whether FGF9 can provide neuronal protective functions through improvement of neuronal morphology in HD is still unclear. Here, we study the effects of FGF9 on neuronal morphology in HD and attempt to understand the related working mechanisms. Taking advantage of striatal cell lines from HD knock-in mice, we found that FGF9 increases neurite outgrowth and upregulates several structural and synaptic proteins under HD conditions. In addition, activation of nuclear factor kappa B (NF-kB) signaling by FGF9 was observed to be significant in HD cells, and blockage of NF-kB leads to suppression of these structural and synaptic proteins induced by FGF9, suggesting the involvement of NF-kB signaling in these effects of FGF9. Taken these results together, FGF9 may enhance neurite outgrowth through upregulation of NF-kB signaling, and this mechanism could serve as an important mechanism for neuroprotective functions of FGF9 in HD.

neuroscience↗

bioRxiv2020

Toward the pathogenicity of the SLC26A4 p.C565Y variant using a genetically driven mouse model

Recessive variants of the SLC26A4 gene are a common cause of hearing impairment worldwide. In the past, cell lines and transgenic mice have been widely used to investigate the pathogenicity associated with the SLC26A4 variants. However, discrepancies in the pathogenicity between humans and cell lines or transgenic mice have been documented for some of the SLC26A4 variants. For instance, the p.C565Y variant, which has been reported to be pathogenic in humans, did not exhibit functional pathogenic consequences in cell lines. To address the pathogenicity of p.C565Y, we used a genotype-based approach in which we generated knock-in mice heterozygous (Slc26a4+/C565Y), homozygous (Slc26a4C565Y/C565Y), and compound heterozygous (Slc26a4919-2A>G/C565Y) for this variant. Subsequent phenotypic characterization revealed that mice segregating these genotypes demonstrated normal auditory and vestibular functions and normal inner ear morphology and expression of pendrin. These findings indicate that the p.C565Y variant is non-pathogenic for mice and that a single p.C565Y allele is sufficient to maintain normal inner ear physiology in mice. Our results highlight the differences in the pathogenicity associated with certain SLC26A4 variants between transgenic mice and humans, which should be taken into consideration while interpreting the results of animal studies for SLC26A4-related deafness.

genetics↗

bioRxiv2020

A computational network approach to identify predictive biomarkers and therapeutic combinations for anti-PD-1 immunotherapy in cancer

BackgroundDespite remarkable success, only a subset of cancer patients have shown benefit from the anti-PD1 therapy. Therefore, there is a growing need to identify predictive biomarkers and therapeutic combinations for improving the clinical efficacy. ResultsBased upon the hypothesis that aberrations of any gene that are close to MHC class I genes in the gene network are likely to deregulate MHC I pathway and affect tumor response to anti-PD1, we developed a network approach to infer genes, pathway, and potential therapeutic target genes associated with response to PD-1/PD-L1 checkpoint immunotherapies in cancer. Our approach successfully identified genes (e.g. B2M and PTEN) and pathways (e.g. JAK/STAT and WNT) known to be associated with anti-PD1 response. Our prediction was further validated by 5 CRISPR gene sets associated with tumor resistance to cytotoxic T cells. Our results also showed that many cancer genes that act as hubs in the gene network may drive immune evasion through indirectly deregulating the MHC I pathway. The integration analysis of transcriptomic data of the 34 TCGA cancer types and our prediction reveals that MHC I-immunoregulations may be tissue-specific. The signature-based score, the MHC I association immunoscore (MIAS), calculated by integration of our prediction and TCGA melanoma transcriptomic data also showed a good correlation with patient response to anti-PD1 for 354 melanoma samples complied from 5 cohorts. In addition, most targets of the 36 compounds that have been tested in clinical trials or used for combination treatments with anti-PD1 are in the top list of our prediction (AUC=0.833). Integration of drug target data with our top prediction further identified compounds that were recently shown to enhance tumor response to anti-PD1, such as inhibitors of GSK3B, CDK, and PTK2. ConclusionOur approach is effective to identify candidate genes and pathways associated with response to anti-PD-1 therapy, and can also be employed for in silico screening of potential compounds to enhances the efficacy of anti-PD1 agents against cancer.

bioinformatics↗

bioRxiv2020

Correlation between cancer stem cells, inflammation and malignant transformation in a DEN-induced model of hepatic carcinogenesis

AimsChronic inflammation and cancer stem cells are known risk factors for tumorigenesis. The aetiology of hepatocellular carcinoma (HCC) involves a multistep pathological process characterised by chronic inflammation and hepatocyte damage but the correlation between HCC, inflammation and cancer stem cell remains unclear. In this study, we examined the role of hepatic progenitor cells in a mouse model of chemical-induced hepatocarcinogenesis to elucidate the relationship between inflammation, malignant transformation and cancer stem cells. Methods and resultsWe used diethylnitrosamine (DEN) to induce liver tumour and scored for H&E and reticulin staining, and also immunohistochemistry staining for OV-6 expression and analysed the statistical correlation between each other. DEN progressively induced inflammation at 7 week 7 (40%, 2/5), week 27 (75%, 6/8), week 33 (62.5%, 5/8) and week 50 (100%, 12/12). DEN progressively induced malignant transformation at week 7 (0%, 0/5), week 27 (87.5%, 7/8), week 33 (100%, 8/8) and week 50 (100%, 12/12). Data obtained showed that DEN progressively induced high-levels of OV-6 expression at week 7 (20%, 1/5), week 27 (37.5%, 3/8), week 33 (50%, 4/8) and week 50 (100%, 12/12). DEN-induced inflammation, malignant transformation and high-level OV-6 expression in hamster liver as shown above and applying Spearmans correlation to the data showed that expression of OV-6 was significantly correlated to inflammation (p = 0.001) and malignant transformation (p < 0.001) ConclusionsThere was a significant correlation between number of cancer stem cells, inflammation and malignant transformation in a DEN-induced model of hepatic carcinogenesis in the hamster.

cancer biology↗

bioRxiv2019

Enhancer regulation for induced WNT3A expression during neuronal regeneration

The treatment of traumatic brain injury (TBI) is limited by a lack of knowledge about the mechanisms underlying neuronal regeneration. WNT family members have been implicated in neurogenesis and aberrant WNT signaling has been associated with neurodegenerative diseases. The current study compared the expression of WNT genes during regeneration of injured cortical neurons. Recombinant WNT3A showed positive effect in promoting neuronal regeneration via in vitro and in vivo TBI models. Intranasal administration of WNT3A protein to TBI mice increased NeuN+ cells compared to control mice as well as retained motor function based on behavior analysis. Since TBI is known to reprogram the epigenome, chromatin immunoprecipitation-sequencing of histone H3K27ac and H3K4me3 was performed to address the transcriptional regulation of WNT3A during neuronal regeneration. We predicted, characterized and proposed that a histone H3K4me1-marked enhancer may undergo topological transformation to regulate the WNT3A gene expression.

molecular biology↗

bioRxiv2018

In situ quantification of individual mRNA transcripts in melanocytes discloses gene regulation of relevance to speciation

Functional validation of candidate genes for adaptation and speciation remains challenging. We here exemplify the utility of a method quantifying individual mRNA transcripts in revealing the molecular basis of divergence in feather pigment synthesis during early-stage speciation in crows. Using a padlock probe assay combined with rolling circle amplification we quantified cell-type specific gene expression in the native, histological context of growing feather follicles. Expression of Tyrosinase related protein 1 (TYRP1), Solute carrier family 45 member 2 (SLC45A2) and Hematopoietic prostaglandin D synthase (HPGDS) was melanocyte-limited and significantly reduced in follicles from hooded crow explaining the substantially lower melanin content in grey vs. black feathers. The central upstream transcription factor Microphthalmia-associated transcription factor (MITF) only showed differential expression specific to melanocytes - a feature not captured by bulk RNA-seq. Overall, this study provides insight into the molecular basis of an evolutionary young transition in pigment synthesis, and demonstrates the power of histologically explicit, statistically substantiated single-cell gene expression quantification for functional genetic inference in natural populations.

evolutionary biology↗

bioRxiv2018

Human Cytomegalovirus Immediate-Early 1 Protein Causes Loss of SOX2 from Neural Progenitor Cells by Trapping Unphosphorylated STAT3 in the Nucleus

The mechanisms underlying neurodevelopmental damage caused by virus infections remain poorly defined. Congenital human cytomegalovirus (HCMV) infection is the leading cause of fetal brain development disorders. Previous work has linked HCMV to perturbations of neural cell fate, including premature differentiation of neural progenitor cells (NPCs). Here we show that HCMV infection of NPCs results in the loss of the SOX2 protein, a key pluripotency-associated transcription factor. SOX2 depletion maps to the HCMV major immediate-early (IE) transcription unit and is individually mediated by the IE1 and IE2 proteins. IE1 causes SOX2 down-regulation by promoting the nuclear accumulation and inhibiting the phosphorylation of STAT3, a transcriptional activator of SOX2 expression. Deranged signaling resulting in depletion of a critical stem cell protein is an unanticipated mechanism by which the viral major IE proteins may contribute to brain development disorders caused by congenital HCMV infection.\n\nIMPORTANCEHuman cytomegalovirus (HCMV) infections are a leading cause of brain damage, hearing loss and other neurological disabilities in children. We report that the HCMV proteins known as IE1 and IE2 target expression of human SOX2, a central pluripotency-associated transcription factor that governs neural progenitor cell (NPC) fate and is required for normal brain development. Both during HCMV infection and when expressed alone, IE1 causes the loss of SOX2 from NPCs. IE1 mediates SOX2 depletion by targeting STAT3, a critical upstream regulator of SOX2 expression. Our findings reveal an unanticipated mechanism by which a common virus may cause damage to the developing nervous system and suggest novel targets for medical intervention.

microbiology↗

bioRxiv2018

Targeting redox regulatory site of protein kinase B impedes neutrophilic inflammation

Neutrophil activation has a pathogenic effect in inflammatory diseases. Protein kinase B (PKB)/AKT regulates diverse cellular responses. However, the significance of AKT in neutrophilic inflammation is still not well understood. Here, we identified CLLV-1 as a novel AKT inhibitor. CLLV-1 inhibited respiratory burst, degranulation, chemotaxis, and AKT phosphorylation in activated human neutrophils and dHL-60 cells. Significantly, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro. The AKT309-313 peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Additionally, CLLV-1 ameliorated lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. CLLV-1 acts as a covalent allosteric AKT inhibitor by targeting AKT Cys310 to restrain inflammatory responses in human neutrophils and LPS-induced ALI in vivo. Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation.

pharmacology and toxicology↗

bioRxiv2017

mTORC1-mediated inhibition of 4EBP1 is essential for Hedgehog (HH) signaling and can be targeted to suppress HH-driven medulloblastoma

Mechanistic target of rapamycin (MTOR) cooperates with Hedgehog (HH) signaling, but the underlying mechanisms are incompletely understood. Here, we provide genetic, biochemical, and pharmacologic evidence that MTOR complex 1 (mTORC1)-dependent translation is a prerequisite for HH signaling. The genetic loss of mTORC1 function inhibited HH signaling- driven growth of the cerebellum and medulloblastoma. Inhibiting translation or mTORC1 blocked HH signaling. Depleting 4EBP1, an mTORC1 target that inhibits translation, alleviated the dependence of HH signaling on mTORC1. Consistent with this, phosphorylated 4EBP1 levels were elevated in HH signaling-driven medulloblastomas in mice and humans. In mice, an mTORC1 inhibitor suppressed medulloblastoma driven by a mutant SMO that is resistant to an SMO inhibitor in the clinic, prolonging the survival of the mice. Our study reveals mTORC1-mediated translation to be a key component of HH signaling and an important target for treating medulloblastoma and other cancers driven by HH signaling.

developmental biology↗