Divergent roles of BECN1 in LC3 lipidation and autophagosomal function

<div><p>BECN1/Beclin 1 is regarded as a critical component in the class III phosphatidylinositol 3-kinase (PtdIns3K) complex to trigger autophagy in mammalian cells. Despite its significant role in a number of cellular and physiological processes, the exact function of BECN1 in autophagy remains controversial. Here we created a <i>BECN1</i> knockout human cell line using the TALEN technique. Surprisingly, the complete loss of BECN1 had little effect on LC3 (MAP1LC3B/LC3B) lipidation, and LC3B puncta resembling autophagosomes by fluorescence microscopy were still evident albeit significantly smaller than those in the wild-type cells. Electron microscopy (EM) analysis revealed that BECN1 deficiency led to malformed autophagosome-like structures containing multiple layers of membranes under amino acid starvation. We further confirmed that the PtdIns3K complex activity and autophagy flux were disrupted in <i>BECN1</i>^−/− cells. Our results demonstrate the essential role of BECN1 in the functional formation of autophagosomes, but not in LC3B lipidation.</p></div

Divergent roles of BECN1 in LC3 lipidation and autophagosomal function

<div><p>BECN1/Beclin 1 is regarded as a critical component in the class III phosphatidylinositol 3-kinase (PtdIns3K) complex to trigger autophagy in mammalian cells. Despite its significant role in a number of cellular and physiological processes, the exact function of BECN1 in autophagy remains controversial. Here we created a <i>BECN1</i> knockout human cell line using the TALEN technique. Surprisingly, the complete loss of BECN1 had little effect on LC3 (MAP1LC3B/LC3B) lipidation, and LC3B puncta resembling autophagosomes by fluorescence microscopy were still evident albeit significantly smaller than those in the wild-type cells. Electron microscopy (EM) analysis revealed that BECN1 deficiency led to malformed autophagosome-like structures containing multiple layers of membranes under amino acid starvation. We further confirmed that the PtdIns3K complex activity and autophagy flux were disrupted in <i>BECN1</i>^−/− cells. Our results demonstrate the essential role of BECN1 in the functional formation of autophagosomes, but not in LC3B lipidation.</p></div

The Special Activity of Stone–Wales Defect Graphene for the Oxygen Reduction Reaction: A Comparison Study between the Charge-Neutral Model and the Constant Potential Model Calculated by Density Functional Theory

This study investigates the difference of Stone–Wales defect graphene on the oxygen reduction reaction (ORR) under the charge-neutral model (CNM) and the constant potential model (CPM) using density functional theory (DFT). Under the CPM, two methods, the double reference method (DRM) and grand-canonical density functional theory (GC-DFT), are used. The calculated limiting potential is 0.854 V under the CNM. The limiting potential calculated by the DRM decreases first and then increases with an increase in pH. The limiting potential calculated by GC-DFT increases with pH increasing, and another limiting potential appears at pH ≥ 5, which means that the Gibbs free energies for each step are all smaller than 0 only at a certain potential range (between the two limiting potentials). These results broke the scaling relation and volcano plot obtained under the CNM. The different limiting potential or activity under the CNM and the CPM can be primarily attributed to the position of the surface Van Hove singularity (SVHS); the farther away it is from the Fermi level, the lower is the limiting potential. Microkinetic simulation was performed to obtain the turnover frequency (TOF) for H2O and the coverage of *O and *OH changed with the applied potential. The half-wave potential calculated under the CNM or GC-DFT is equal to the corresponding limiting potential. However, the half-wave potential calculated by the DRM at pH = 0 is larger than the calculated limiting potential, which indicates an unneglected kinetic effect

Examples of disruption of genes in human cell lines by ULtiMATE-engineered TALENs.

<p>(<b>A</b>, <b>D</b> and <b>G</b>) Partial sequences of <i>HBEGF</i>, <i>ANTXR1</i>, and <i>LRP1</i> genes in genome containing TALENs binding regions (overlined for TALEN^L and underlined for TALEN^R). Restriction enzyme cutting sites are highlighted in yellow. (<b>B</b>, <b>E</b> and <b>H</b>) Measurement of indel rates in TALENs-treated HeLa and HEK293T cells by restriction enzyme digestion. The uncleaved bands indicate potential indels. Both wild type and cells treated by TALENs targeting <i>ANTXR2</i> gene are used as controls. The percentage of uncleaved band (indicated by red arrow) was measured using ImageJ (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>). (<b>C</b>, <b>F</b> and <b>I</b>) Sequencing analysis of mutated alleles from 4-6 randomly selected TALENs clones (in HeLa cells). The TALENs binding sites (underlined) and restriction enzyme cutting sites (in yellow) are highlighted. Dashes and red letters indicate deletions and insertions, respectively.</p

Carbon Nanosheets Containing Discrete Co‑N_<i>x</i>‑B_<i>y</i>‑C Active Sites for Efficient Oxygen Electrocatalysis and Rechargeable Zn–Air Batteries

Structural and compositional engineering of atomic-scaled metal−N−C catalysts is important yet challenging in boosting their performance for the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER). Here, boron (B)-doped Co−N−C active sites confined in hierarchical porous carbon sheets (denoted as Co-N,B-CSs) were obtained by a soft template self-assembly pyrolysis method. Significantly, the introduced B element gives an electron-deficient site that can activate the electron transfer around the Co−N−C sites, strengthen the interaction with oxygenated species, and thus accelerate reaction kinetics in the 4e⁻ processed ORR and OER. As a result, the catalyst showed Pt-like ORR performance with a half-wave potential (<i>E</i>_1/2) of 0.83 V <i>versus</i> (<i>vs</i>) RHE, a limiting current density of about 5.66 mA cm⁻², and higher durability (almost no decay after 5000 cycles) than Pt/C catalysts. Moreover, a rechargeable Zn–air battery device comprising this Co-N,B-CSs catalyst shows superior performance with an open-circuit potential of ∼1.4 V, a peak power density of ∼100.4 mW cm⁻², as well as excellent durability (128 cycles for 14 h of operation). DFT calculations further demonstrated that the coupling of Co-N<i>_x</i> active sites with B atoms prefers to adsorb an O₂ molecule in side-on mode and accelerates ORR kinetics