combined cpDNA sequences_aligned

an aligned sequence file of combined cpDNA sequences from 100 individual sample

Permeability Transition Pore-Mediated Mitochondrial Superoxide Flashes Regulate Cortical Neural Progenitor Differentiation

<div><p>In the process of neurogenesis, neural progenitor cells (NPCs) cease dividing and differentiate into postmitotic neurons that grow dendrites and an axon, become excitable, and establish synapses with other neurons. Mitochondrial biogenesis and aerobic metabolism provide energy substrates required to support the differentiation, growth and synaptic activity of neurons. Mitochondria may also serve signaling functions and, in this regard, it was recently reported that mitochondria can generate rapid bursts of superoxide (superoxide flashes), the frequency of which changes in response to environmental conditions and signals including oxygen levels and Ca²⁺ fluxes. Here we show that the frequency of mitochondrial superoxide flashes increases as embryonic cerebral cortical neurons differentiate from NPCs, and provide evidence that the superoxide flashes serve a signaling function that is critical for the differentiation process. The superoxide flashes are mediated by mitochondrial permeability transition pore (mPTP) opening, and pharmacological inhibition of the mPTP suppresses neuronal differentiation. Moreover, superoxide flashes and neuronal differentiation are inhibited by scavenging of mitochondrial superoxide. Conversely, manipulations that increase superoxide flash frequency accelerate neuronal differentiation. Our findings reveal a regulatory role for mitochondrial superoxide flashes, mediated by mPTP opening, in neuronal differentiation.</p> </div

Expression of mitochondrial proteins in NPCs during differentiation.

<p>(A, B and C) Immunoblot analysis was performed by using antibodies that selectively recognize cytochrome c, MnSOD and cyclophylin D. Blots were reprobed with antibodies against Actin. Panel A shows representative blots; panels B and C show results of densitometric analysis of cyclophylin D and MnSOD, which were normalized to the corresponding mitochondrial cytochrome c level on each differentiation day. Values are expressed as a percentage of the mean of NPCs at day 0 (n = 3-4 separate experiments performed on cells cultured from 3-4 pregnant mice); *p<0.05 compared to the values of NPCs at day 0.</p

Characterization of mitochondrial SO flashes in NPCs during differentiation.

<p>(A) Distribution of mitochondrial SO flashes in NPCs at different days of differentiation. Left, middle and right panels show typical flashes (high intensity fluorescence) in mitochondria of cells on differentiation days 1, 2 and 4. See Movies S1 and S2 for a dynamic view of the superoxide flashes. Bar = 5 µm. (B) Flash pattern changes during NPC differentiation indicated by percentages of flashes occurring in mitochondria throughout the whole cell, in perinuclear regions or in processes/neurites, among all flashes detected. n=60-100 flashes (from four independent cultures) analyzed on each individual day of differentiation. (C) Flash incidence in NPCs during cell differentiation. Flash incidence refers to percentage of cells exhibiting mitochondrial superoxide flashes among all cells scanned during the imaging period (n=4 independent experiments and approximately 150-210 cells scanned per experiment on each individual day of differentiation). Values are expressed as mean ± SD. *p<0.05 vs. total flash incidence of NPCs at day 0; #p<0.05 vs. incidence of flashes in whole cell or perinuclear region at day 0. (D) Flash amplitude (ΔF/F0) in NPCs during differentiation. Flash amplitude was calculated as the ratio ΔF /F0. ΔF indicates difference between peak fluorescence intensity and basal fluorescence intensity. F0 refers to basal fluorescence intensity. Values are expressed as mean ± SD. 60-100 SO flashes (from 4 independent experiments) analyzed on each individual day of differentiation.</p

Characterization of mitochondrial SO flashes in NPCs.

<p>(A) Cell differentiation was induced by growth factor removal. NPCs and neurons were identified by antibodies against Sox2 (red) and Tuj1 (green), respectively, at different days of differentiation. The nuclei of NPCs were counterstained with DAPI (blue). Bar = 20 µm. (B-C) Characteristics of typical mitochondrial SO flashes in NPCs (day 0). The area circumscribed with the red dashed line is a perinuclear region where SO flashes occurred, and the area circumscribed with the white dashed line is the nucleus. Bar = 5µm (B). Change of mt-cpYFP fluorescence intensity during time course of a typical SO flash (C).</p

Mitochondrial mass and function changes in NPCs during differentiation.

<p>(A, B and C) Mitochondrial mass, measured by Mitotracker green loading (A), mitochondrial ATP content (B) and mitochondrial superoxide production, evaluated by MitoSOXred loading (C) are normalized by protein concentration in differentiated cells. (D) Mitochondrial membrane potential (∆Ψm) was imaged by mitoCMXros loading in NPCs on differentiation days 0, 2 and 4. Bar = 20 µm. (E) Mitochondrial membrane potential (∆Ψm) was determined by the ratio of MitoCMXros to Mitotracker green intensity. Values are expressed as a percentage of the mean of NPCs at day 0 (n = 4 separate experiments performed on NPCs cultured from 4 pregnant mice) *p<0.05, **p<0.001 compared to the values of NPCs at day 0.</p

Mitochondrial superoxide flashes promote NPC differentiation.

<p>(A) Mitochondrial membrane potential in differentiated cells at day 2. Cells were treated with 100 nM CsA, 1µM ATR or vehicle at the time of growth factor removal. Values are expressed as a percentage of the mean of the control condition in NPCs (day 0). *p<0.05 compared to the control value in cells (day 2). (B) Mitochondrial superoxide in differentiated cells at day 2. Cells were treated with 1µM Mito-Tempol (MitoTEMO), 1µM PQ or vehicle at the time of growth factor removal. Values are expressed as a percentage of the mean of the control condition in NPCs (day 0), *p<0.05 compared to the control value in cells (day 2). (C) Superoxide flash incidence in differentiated cells at day2. Cells were treated with CsA, ATR, Mito-Tempol and PQ as indicated in A and B. Values are expressed as a percentage of the mean of the control condition in NPCs (day 0); Perinuclear or whole cell flash: *p<0.05 compared to the control value in cells (day 2); Flash at process: #p<0.05 compared to the control value in cells (day 2). (D, E and F) Percentage of NPCs, neurons and astrocytes at different days of differentiation. NPCs, neurons and astrocytes were identified by antibodies against Sox2, Tuj1 and GFAP at different days after differentiation. Total number of cells was counted by counterstaining nuclei with DAPI. n = 4 separate experiments performed on NPCs cultured from 4 pregnant mice. *p<0.05 compared to the control value of each individual day.</p

Total geographical ranges (lines) and sampling sites (dots) of <i>Diapensia</i>.

<p>HHM: Himalayan-Hengduan Mountains. Sample IDs refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140175#pone.0140175.t001" target="_blank">Table 1</a>. The ranges of <i>D</i>. <i>lapponica</i> and <i>D</i>. <i>obovata</i> are redrawn after Hultén & Fries [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140175#pone.0140175.ref044" target="_blank">44</a>]. The photo of <i>D</i>. <i>lapponica</i> was attributed by Alinja (<a href="https://commons.wikimedia.org/wiki/File:Diapensia_lapponica_Kilpisj%C3%A4rvi_2012-07.jpg#/media/File:Diapensia_lapponica_Kilpisj%C3%A4rvi_2012-07.jpg" target="_blank">https://commons.wikimedia.org/wiki/File:Diapensia_lapponica_Kilpisj%C3%A4rvi_2012-07.jpg#/media/File:Diapensia_lapponica_Kilpisj%C3%A4rvi_2012-07.jpg</a>)</p

Polyoxometalate Metal–Organic Frameworks: Keggin Clusters Encapsulated into Silver-Triazole Nanocages and Open Frameworks with Supercapacitor Performance

To investigate the relationship between the structures of polyoxometalate host–guest materials and their energy-storage performance, three novel polyoxometalate-based metal–organic compounds, [Ag10(C2H2N3)8]­[HVW12O40], [Ag10(C2H2N3)6]­[SiW12O40], and [Ag­(C2H2N3)]­[Ag12(C2H2N3)9]­[H2BW12O40] are synthesized by a one-step hydrothermal method and further confirmed by single-crystal X-ray diffraction analyses and other numerous characterization techniques. In compound [Ag10(C2H2N3)8]­[HVW12O40], the Keggin clusters are intersected into channels formed by a 3D open metal–organic framework. In contrast, in compounds [Ag10(C2H2N3)6]­[SiW12O40] and [Ag­(C2H2N3)]­[Ag12(C2H2N3)9]­[H2BW12O40], the Keggin clusters are encapsulated into silver-triazole metal–organic nanocages to construct core–shell structures, which are further fused together by covalent bonds to form 3D polyoxometalate-based metal–organic frameworks. The electrochemical properties of three compound-based electrodes are estimated by cyclic voltammetry, galvanostatic charge–discharge, electrochemically active surface area, and electrochemical impedance spectroscopy. The results of the electrochemical performance tests indicate that these compounds possess high specific capacitance and cycling stability, especially [Ag10(C2H2N3)8]­[HVW12O40], showing a specific capacitance of 93.5 F g–1, which is higher than that of many other polyoxometalate-based electrode materials. A possible mechanism of the electrochemical performance is explored, which is mainly related to the redox capacity of polyoxometalate, the electrochemically active surface area, the electrochemical impedance spectroscopy, and the microstructures of polyoxometalate-based metal–organic frameworks