Biology subjects

Fu, W.

Publications and source records attributed to Fu, W..

4 recordsLinked to original sources

bioRxiv2018

Targeting RyR2 with a phosphorylation site-specific nanobody Reverses Dysfunction of Failing Cardiomyocytes in Rat

Chronic PKA phosphorylation of RyR2 has been shown to increased diastolic SR Ca2+ leak and lead to cardiac dysfunction. Since the change of phosphorylation level of RyR2 is a biomarker of failing heart, we attempted to verify the hypothesis that intracellular gene delivery of a RyR2 targeting phosphorylation site-specific nanobody could preserve contractility of failing myocardium. In present study, we acquired the RyR2-specific nanobodies from a phage display library which are variable domains of camellidae heavy chain-only antibodies (VHH). One of the monoclonal nanobodies, AR185, inhibiting RyR2 phosphorylation in an in vitro assay was then chosen for further investigation. We investigated the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of AR185 against post-ischemic heart failure. Adeno-associated virus gene delivery elevated the intracellular expression AR185 protein in the ischemic heart failure model of rats, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo and in vitro by reversing myocardial Ca2+ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of sarcoplasmic reticulum (SR) calcium leak, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited PKA phosphorylation of RyR2 in failing cardiomyocytes. Our results provided strong pre-clinical experimental evidence of the cardiac expression of RyR2 nanobody with AAV9 vectors as a promising therapeutic strategy for ischemic heart failure.

pathology↗

bioRxiv2018

A familial case of Syndactyly type IV due to a novel duplication of ~222.23 kb covering exons 2-17 of the LMBR1 gene: a case report

Syndactyly is one of the most frequent hereditary limb malformations with clinical and genetical complexity. Autosomal dominant Syndactyly type IV (SD4) is a very rare form of syndactyly, occurring as a result of heterozygous mutation in an SHH regulatory element (ZRS) that resides in intron 5 of the LMBR1 gene on chromosome 7q36.3. The SD4 is characterized by complete cutaneous syndactyly of all fingers, cup-shaped hands due to flexion of the fingers and accompanied by polydactyly. Here, we firstly reported a big Chinese family, manifesting cup-shaped hands consistent with SD4 and intrafamilial heterogeneity in clinical phenotype of tibial and fibulal shortening, triphalangeal thumb-polysyndactyly syndrome (TPTPS). Genetically, we identified a novel duplication of [~]222.23 kb covering exons 2-17 of the LMBR1 gene in this family by next generation sequencing. This case expands our new clinical understanding of SD4 phenotype.

genetics↗

bioRxiv2018

New human chromosomal safe harbor sites for genome engineering with CRISPR/Cas9, TAL effector and homing endonucleases

Safe Harbor Sites (SHS) are genomic locations where new genes or genetic elements can be introduced without disrupting the expression or regulation of adjacent genes. We have identified 35 potential new human SHS in order to substantially expand SHS options beyond the three widely used canonical human SHS, AAVS1, CCR5 and hROSA26. All 35 potential new human SHS and the three canonical sites were assessed for SHS potential using 9 different criteria weighted to emphasize safety that were broader and more genomics-based than previous efforts to assess SHS potential. We then systematically compared and rank-ordered our 35 new sites and the widely used human AAVS1, hROSA26 and CCR5 sites, then experimentally validated a subset of the highly ranked new SHS together versus the canonical AAVS1 site. These characterizations included in vitro and in vivo cleavage-sensitivity tests; the assessment of population-level sequence variants that might confound SHS targeting or use for genome engineering; homology-dependent and -independent, SHS-targeted transgene integration in different human cell lines; and comparative transgene integration efficiencies at two new SHS versus the canonical AAVS1 site. Stable expression and function of new SHS-integrated transgenes were demonstrated for transgene-encoded fluorescent proteins, selection cassettes and Cas9 variants including a transcription transactivator protein that were shown to drive large deletions in a PAX3/FOXO1 fusion oncogene and induce expression of the MYF5 gene that is normally silent in human rhabdomyosarcoma cells. We also developed a SHS genome engineering toolkit to enable facile use of the most extensively characterized of our new human SHS located on chromosome 4p. We anticipate our newly identified human SHS, located on 16 chromosomes including both arms of the human X chromosome, will be useful in enabling a wide range of basic and more clinically-oriented human gene editing and engineering.

genomics↗

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↗