Le Sun

A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

WDR45/WIPI4, encoding a WD40 repeat-containing PtdIns(3)P binding protein, is essential for the basal autophagy pathway. Mutations in WDR45 cause the neurodegenerative disease β-propeller protein-associated neurodegeneration (BPAN), a subtype of NBIA. We generated CNS-specific Wdr45 knockout mice, which exhibit poor motor coordination, greatly impaired learning and memory, and extensive axon swelling with numerous axon spheroids. Autophagic flux is defective and SQSTM1 (sequestosome-1)/p62 and ubiquitin-positive protein aggregates accumulate in neurons and swollen axons. Nes-Wdr45(fl/Y) mice recapitulate some hallmarks of BPAN, including cognitive impairment and defective axonal homeostasis, providing a model for revealing the disease pathogenesis of BPAN and also for investigating the possible role of autophagy in axon maintenance.

Improving the degree of vascularization through the regulation of wound microenvironment is crucial for wound repair. Gene activated matrix (GAM) technology provides a new approach for skin regeneration. It is a local gene delivery system that can not only maintain a moist environment, but also increase the concentration of local active factors. For this purpose, we fabricated the mVEGF165/TGF-β1 gene-loaded N-carboxymethyl chitosan/sodium alginate hydrogel and studied its effect on promoting deep second degree burn wound repair. The average diameter of the hydrogel pores was 100 μm and the porosity was calculated as 50.9%. SEM and CLSM images showed that the hydrogel was suitable for cell adhesion and growth. The NS-GAM could maintain continuous expression for at least 9 days in vitro, showing long-term gene release and expression effect. Deep second-degree burn wound model was made on the backs of Wistar rats to evaluate the healing effect. The wounds were healed by day 22 in NS-GAM group with the prolonged high expression of VEGF and TGF-β1 protein. A high degree of neovascularization and high expression level of CD34 were observed in NS-GAM group in 21 days. The histological results showed that NS-GAM had good tissue safety and could effectively promote epithelialization and collagen regeneration. These results indicated that the NS-GAM could be applied as a promising local gene delivery system for the repair of deep second-degree burn wounds.

Abstract The low-voltage activated T-type calcium channels regulate cellular excitability and oscillatory behavior of resting membrane potential which trigger many physiological events and have been implicated with many diseases. Here, we determine structures of the human T-type Ca V 3.3 channel, in the absence and presence of antihypertensive drug mibefradil, antispasmodic drug otilonium bromide and antipsychotic drug pimozide. Ca V 3.3 contains a long bended S6 helix from domain III, with a positive charged region protruding into the cytosol, which is critical for T-type Ca V channel activation at low voltage. The drug-bound structures clearly illustrate how these structurally different compounds bind to the same central cavity inside the Ca V 3.3 channel, but are mediated by significantly distinct interactions between drugs and their surrounding residues. Phospholipid molecules penetrate into the central cavity in various extent to shape the binding pocket and play important roles in stabilizing the inhibitor. These structures elucidate mechanisms of channel gating, drug recognition, and actions, thus pointing the way to developing potent and subtype-specific drug for therapeutic treatments of related disorders.