Calcineurin activation and 14-3-3ε suppression together contribute to Hcy-induced apoptosis.

<p>Hippocampal neurons transduced with lentiviral 14-3-3ε for 48 h were then pretreated with 1 µM CsA for 30 min before treatment with 500 µM Hcy (<i>Hcy</i>) for 24 h. A, Total Bad and phospho-Bad levels were assessed by western blotting and densitometric analysis. B, Western blot analysis of Bad in mitochondrial (Mit) and cytoplasmic (Cyto) fractions from hippocampal neurons. C, Percentage of total Bad (cytoplasmic+mitochondrial fractions) localized in the mitochondrial fraction as determined by densitometric analysis of the data from B. D, The level of apoptosis was determined by counting the number of neurons with apoptotic nuclei. Four randomly selected fields of Hoechst-stained cells were counted and the average percentage of apoptotic cells per total number of cells was determined. E, Cleaved PARP was measured using western blotting. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group. ^Δ<i>P</i><0.05 vs. CsA group.</p

Inhibition of Hcy-induced apoptosis by a calcineurin inhibitor or intracellular Ca²⁺ chelator.

<p>Cells were pretreated with 1 µM CsA or 10 µM BAPTA-AM for 30 min before treatment with 500 µM Hcy (<i>Hcy</i>) for 24 h. A, The level of apoptosis was determined by counting the number of neurons with apoptotic nuclei. Four randomly selected fields of Hoechst-stained cells were counted and the average percentage of apoptotic cells per total number of cells was determined. B, Cleaved PARP was studied by western blotting. Results are expressed as the mean ± S.E.M. of three experiments. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group.</p

DataSheet1_Optimal scheduling of electric-gas-heat system considering dual virtual energy storage of gas network management and heat network storage.pdf

Aiming at the problem of wind curtailment caused by the lack of system flexibility, an optimal scheduling strategy for improving the flexibility of the electricity-gas-heat interconnection system by the coordinated operation of the gas network management and storage characteristics and the heat storage characteristics of the heat network is proposed. Firstly, the influence of gas network storage and heat network storage on improving flexibility is analyzed respectively. Then, an electricity-gas-heat interconnected system scheduling model considering the dynamic characteristics of gas network management and t the delay characteristics of heat storage in heat network, which greatly improves the imbalance between supply and demand of flexibility in space and time brought by the anti-peak regulation characteristics of wind power to the system. Finally, the IEEE-24 nodes power system, Belgium’s 20-node natural gas network and 6-node thermal network are used, for example, analysis. The results show that the proposed scheduling scheme improves the flexibility of the system while its operating cost and wind curtailment cost are the lowest, which promotes the consumption of wind power.</p

Calcineurin activation contributes to Hcy-induced dephosphorylation and mitochondrial translocation of Bad.

<p>Cells were pretreated with 1 µM CsA or 10 µM BAPTA-AM for 30 min before treatment with 500 µM Hcy (<i>Hcy</i>) for 24 h. A, Total Bad and phospho-Bad levels were assessed by western blotting and densitometric analysis. B, Western blot analysis of Bad in mitochondrial (Mit) and cytoplasmic (Cyto) fractions from hippocampal neurons. C, Percentage of total Bad (cytoplasmic +mitochondrial fractions) localized to the mitochondrial fraction was determined by densitometric analysis of the data from B. Results are expressed as the mean ± S.E.M. of three experiments. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group.</p

Hcy induces apoptosis and suppresses 14-3-3ε expression in cultured hippocampal neurons.

<p>A, Cultures were exposed for 24 h to either saline (<i>Con</i>) or 500 µM Hcy (<i>Hcy</i>) and were subsequently stained with the fluorescent DNA-binding dye Hoechst 33342 (<i>top</i>) or photographed under phase-contrast optics (<i>bottom</i>). Note the nuclear DNA condensation and fragmentation in neurons exposed to Hcy (arrow). Scale bar: 100 µm. B, Cultures were exposed to the indicated concentrations of Hcy for 24 h, and the percentages of neurons with apoptotic nuclei were quantified. Values are the mean ±S.E.M. of counts made in four to six cultures. C, Cells were treated as in B, and the levels of cleaved PARP were determined by western blotting. Top, representative western blots. Bottom, densitometric analysis of cleaved PARP normalized by β-actin (<i>n</i> = 3). D, 14-3-3ε mRNA and protein was measured by real-time quantitative PCR (<i>top</i>) and western blotting (<i>mid and bottom</i>), respectively. *<i>P</i><0.05 vs. control.</p

The effect of lentiviral 14-3-3ε transduction on apoptosis in hippocampal neurons.

<p>Hippocampal neurons transduced with lentiviral 14-3-3ε for 48 h were treated with Hcy for 24 h. A, 14-3-3ε mRNA and proteins were analyzed by real-time quantitative PCR or western blotting, respectively. B, The percentages of neurons with apoptotic nuclei (Hoechst staining) were quantified. C, Cleaved PARP was determined by western blotting. D, Total Bad and phospho-Bad levels were assessed by western blotting and densitometric analysis. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group.</p

Activation of calcineurin by Hcy and inhibition by specific blockers.

<p>A, Cultures were exposed to the indicated concentrations of Hcy (<i>Hcy</i>) for 24 h, and cellular calcineurin activity was then determined. B, Cells were pretreated with 1 µM CsA or 10 µM BAPTA-AM for 30 min before exposed to 500 µM Hcy (<i>Hcy</i>). Cellular calcineurin activity was measured at the indicated time periods after the addition of Hcy to the cells. Values represents the mean ± S.E.M. of four or more assays. C, Cells were pretreated with 1 µM CsA or 10 µM BAPTA-AM for 30 min before exposed to 500 µM Hcy (<i>Hcy</i>) for 24h. Calcineurin A (Calcineurin) protein level was determined by western blotting. Top, representative western blots. Bottom, densitometric analysis. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group.</p

Metal-Free DNA Linearized Nuclease Based on PASP–Polyamine Conjugates

Genome manipulation controlled by small metal complexes has attracted extensive interest because of their potential application in the fields of molecular biotechnology and drug development. However, their medicinal application is still limited due to the distinct toxicity of the free radicals generated by partial metal complexes based on oxidative cleaving processes. Thus, it is still a challenge for us to use metal free agent to cleave DNA. In this work, we showed that a family of polyamine-grafted PASP (poly­(aspartic acid)) conjugates is able to rapidly induce DNA cleavage in the absence of metal ions, and obtain a high-yield linearization product via a hydrolytic path. From the results of detailed control experiments, it was revealed that the formation of polyamine cation/phosphate anion pair and free ungrafted nucleophilic groups would be the key factors to improve DNA linearization. Constructing polyamine conjugates based on short peptide such as polyamine-grafted PASP, as achieved here, could provide an attractive strategy for developing mild and efficient artificial nucleases as well as researching catalytic mechanisms on DNA chemistry

E3d HPC lead to the loss of HPC-induced neuroprotection.

<p>A schematic diagram shows the experimental protocols for HPC, E3dHPC and E6dHPC (A). Each black box represents an episode of hypoxia (8% O₂ for 5 hours). Bar charts shows the stroke volume in mice following a single episode of HPC (C), E3d HPC (C) and E6d HPC (D). Stroke volume was evaluated 24 hours after MCAO. A significantly reduced stroke volume was induced by a single episode of HPC and E6d HPC, 3 days after the last exposure, although no effects were observed following E3d HPC. Data are shown as mean ± SD; <i>n</i> = 6–8 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.01 compared to the control.</p

CD39 and CD73 were highly upregulated after HPC and were mainly distributed in neurons.

<p>Immunofluorescence assay was used to show the expression of CD39 (A, B, C) and CD73 (D, E, F) in neurons and astrocytes in different groups. Neuron and astrocyte cytoskeletal proteins, MAP-2 and GFAP, were labeled with green fluorescence, while CD39 or CD73 were labeled with red fluorescence. CD39 and CD73 were highly upregulated after HPC but not after E3d HPC (A, B, D, E). Quantitative analysis of receptor expression showed that both CD39 and CD73 were mainly localized in neurons (C, F). Data are shown as mean ± SD; <i>n</i> = 4 mice in each group.</p