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Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and ‘cleaner’ knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.

Aberrant mitophagy has been implicated in a broad spectrum of disorders. PINK1, Parkin, and ubiquitin have pivotal roles in priming mitophagy. However, the entire regulatory landscape and the precise control mechanisms of mitophagy remain to be elucidated. Here, we uncover fundamental mitophagy regulation involving PINK1 and a non-canonical role of the mitochondrial Tu translation elongation factor (TUFm). The mitochondrion-cytosol dual-localized TUFm interacts with PINK1 biochemically and genetically, which is an evolutionarily conserved Parkin-independent route toward mitophagy. A PINK1-dependent TUFm phosphoswitch at Ser222 determines conversion from activating to suppressing mitophagy. PINK1 modulates differential translocation of TUFm because p-S222-TUFm is restricted predominantly to the cytosol, where it inhibits mitophagy by impeding Atg5-Atg12 formation. The self-antagonizing feature of PINK1/TUFm is critical for the robustness of mitophagy regulation, achieved by the unique kinetic parameters of p-S222-TUFm, p-S65-ubiquitin, and their common kinase PINK1. Our findings provide new mechanistic insights into mitophagy and mitophagy-associated disorders.

Based on 3D fractal theory the rough surface is characterized. And its structural parameters are determined by measurement of the roughness of a real frictional surface. A thermo-mechanical coupling model for the rough surface sliding friction is established. The model integrates the heat flux coupling between the sliding surfaces and allows the analysis of the effects of elastic deformation of contact subjects and all of the correlation between asperities. To obtain the transient contact process of the rough surfaces during frictional sliding, the thermoelastic problem under this three-dimensional model is solved by the nonlinear finite element multiphysical methods in ANSYS8.1 software. The effects of the different external applied pressure on the highest contact temperature, the maximum surface contact pressure and the biggest equivalent VonMises stress during frictional sliding are analyzed. The numerical results from the analysis and simulation show the highest contact temperature is approximate proportion to the external applied pressure. But some differences exist. The difference becomes more distinctness with the increase of the sliding distance and the temperature rise. The synthetical function of multiple factors such as the elastic deformation, the rising temperature of contact subject, the variation of the number of the contact asperities and the interaction between asperities make the instantaneous maximum contact pressure and the biggest VonMises equivalent stress do not increase linearly with the increase of the external applied pressure.