Global Embodied Energy Flow and Stock Analysis with Endogeneous Fixed Capital

Fixed capital stock functions as an embodied energy storage system that connects economic activities which do not happen simultaneously. This paper constructs a dynamic energy input–output model to analyze embodied energy flows and stocks along both temporal and spatial dimensions from 2000 to 2014. The results show that 2043 exajoule of embodied energy was stored in the global fixed capital stock in 2014, which was about three times the world’s direct energy use. Compared with those in developed countries, the gaps between the dynamic energy footprints and the traditional ones were larger in fast-developing countries. Net embodied energy usually flowed from high-intensity economies to lower-intensity economies, and around 10% of the energy embodied in trade came from depreciation. The dynamic embodied energy indicators provide information for improving energy efficiency and mitigating corresponding problems from the perspective of consumption

Organic Photovoltaics and Bioelectrodes Providing Electrical Stimulation for PC12 Cell Differentiation and Neurite Outgrowth

Current bioelectronic medicines for neurological therapies generally involve treatment with a bioelectronic system comprising a power supply unit and a bioelectrode device. Further integration of wireless and self-powered units is of practical importance for implantable bioelectronics. In this study, we developed biocompatible organic photovoltaics (OPVs) for serving as wireless electrical power supply units that can be operated under illumination with near-infrared (NIR) light, and organic bioelectronic interface (OBEI) electrode devices as neural stimulation electrodes. The OPV/OBEI integrated system is capable to provide electrical stimulation (ES) as a means of enhancing neuron-like PC12 cell differentiation and neurite outgrowth. For the OPV design, we prepared devices incorporating two photoactive material systemsβ-carotene/<i>N</i>,<i>N</i>′-dioctyl-3,4,9,10-perylenedicarboximide (β-carotene/PTCDI-C8) and poly­(3-hexylthiophene)/phenyl-C₆₁-butyric acid methyl ester (P3HT/PCBM)that exhibited open circuit voltages of 0.11 and 0.49 V, respectively, under NIR light LED (NLED) illumination. Then, we connected OBEI devices with different electrode gaps, incorporating biocompatible poly­(hydroxymethylated-3,4-ethylenedioxythiophene), to OPVs to precisely tailor the direct current electric field conditions during the culturing of PC12 cells. This NIR light-driven OPV/OBEI system could be engineered to provide tunable control over the electric field (from 220 to 980 mV mm^–1) to promote 64% enhancement in the neurite length, direct the neurite orientation on chips, or both. The OPV/OBEI integrated systems under NIR illumination appear to function as effective power delivery platforms that should meet the requirements for wirelessly offering medical ES to a portion of the nervous system; they might also be a key technology for the development of next-generation implantable bioelectronics

Consecutive Gated Injection-Based Microchip Electrophoresis for Simultaneous Quantitation of Superoxide Anion and Nitric Oxide in Single PC-12 Cells

As important reactive oxygen species (ROS) and reactive nitrogen species (RNS), cellular superoxide anion (O₂^•–) and nitric oxide (NO) play significant roles in numerous physiological and pathological processes. Cellular O₂^•– and NO also have a close relationship and always interact with each other. Thus, the simultaneous detection of intracellular O₂^•– and NO, especially at the single-cell level, is important. In this paper, we present a novel method to simultaneously detect and quantify O₂^•– and NO in single cells using microchip electrophoresis based on a new consecutive gated injection method. This novel injection method achieved consecutive manipulation of single cells, guaranteeing an almost constant volumetric flow rate and thus good quantitative reproducibility. After cellular content separation by microchip electrophoresis and detection by laser-induced fluorescence (MCE–LIF), O₂^•– and NO in single PC-12 cells were simultaneously quantified in an automated fashion. This is the first report of consecutive absolute quantitation at the single-cell level. The quantitative results obtained from single cells is beneficial for deep understanding of the biological roles of cellular O₂^•– and NO. This new method constitutes a consecutive, accurate way to study the synergistic function of O₂^•– and NO and other biomolecules in various biological events at the single-cell level

Mouse <i>Wuho</i> (m<i>Wh</i>) is an essential gene.

<p>(A) Gene targeting strategy. The gene targeting vector is a construct harboring positive (neomycin resistant gene [Neo], <i>red</i>) and negative (diphtheria toxin gene [DT], <i>blue</i>) selection markers. Through homologous recombination, the genomic region with exons 2 (E2) and 3 (E3) of m<i>Wh</i> was replaced and resulted in the introduction of one loxP site before E2, and the Neo cassette flanked by two loxP sites in Intron 3. Subsequently, E2 and E3 were deleted by Cre recombinase through recombination between two distal loxP’s. The symbols “>” and “<” represent the locations of forward and reverse primers used in PCR for genotyping. The PCR products are 3.6 kb from in wild type allele and 0.5 kb from knockout allele. (B) A representative genotyping experiment for embryos of E9.5 from heterozygous parents. PCR products can distinguish among genotypes of +/+, +/-, and -/-. (C) Knockout of m<i>Wh</i> results in DNA damage and apoptosis. The embryos genotyped in (B) were processed for western blot to examine expression of mWH, appearance of γ-H2AX for DNA damage, Cleaved PARP for apoptosis, and actin as loading controls. (D) Development of -/- embryos at E9.5 (left panel) and 10.5 (right panel) is abnormal. In E10.5 -/- embryos, they show varying morphology, potentially due to different degrees of resorption. Severe extents of resorption and are small (the left one of -/-). The other null embryo (the right one of -/-) show minor morphological defects and has brain development defects and internal bleeding. Scale bar is 1 mm.</p

FEN1 plays a vital role in DNA damage and cell death induced by knockdown of hWH.

<p>(A) Depletion of FEN1 mitigates DNA damage and cell death resulting from hWH knockdown in human cells. Double knockdowns of hWH and FEN1 were conducted in HCT116 p53^+/+ cells by siRNAs. We utilized the siRNA pool to knockdown hWH, and three different single siRNAs for FEN1 knockdown. The depletion efficiency of hWH and FEN1, and the DNA damage level revealed by γ-H2AX were determined by western blot. Viability of each treatment was measured by MTT assay. Two of the FEN1 siRNA showing better FEN1 knockdown efficiency (sihFEN1-1 and -2, lanes 2 and 3) were able to better rescue DNA damage and cell death induced by hWH knockdown (lanes 6 and 7). (B) Ectopic expression of FEN1 reverses the rescuing effect by FEN1 knockdown and makes hWH knockdown more toxic. Transfection of vector for ectopic expression of FEN1 tagged with myc and DDK was conducted with pCMV6/FEN1-myc-DDK construct (0.2 μg/ml for 10⁵ cells). Knockdowns of hWH and FEN1 were carried out with siRNA pools. While knockdown of FEN1 can rescue cytotoxicity induced by hWH knockdown (lanes 4 versus 6), ectopic expression of FEN1 can restore the cytotoxicity of hWH knockdown (lanes 6 versus 8). Interestingly, combined expression of FEN1 from both endogenous and ectopic sources exacerbates cytotoxic effect of hWH depletion (lanes 4 versus 7).</p

p53 plays a vital role in cell death following hWH depletion.

<p>(A) Depletion of hWH induces DNA damage (appearance of γ-H2AX) in both HCT116 p53^+/+ and HCT116 p53^-/- cells, but only causes loss of viability in HCT116 p53^+/+ cells. (B) Depletion of hWH causes apoptosis in HCT116 p53^+/+ cells (revealed by the cleavage of Procaspase-3 and PARP), but not in HCT116 p53^-/- cells. (C) Knockdown of p53 mitigates apoptosis and improves viability after depleting hWH in HCT116 p53^+/+ cells. Simultaneously knocking down hWH and p53 (by p53 siRNA, sip53) reduces PARP cleavage and increases cell viability in comparison with single knockdown of hWH. (D) Transfection of ectopic p53 in HCT116 p53^-/- cells promotes cell death resulting from depletion of hWH. Reversion of p53 status in HCT116 p53^-/- cells was carried out by transfection of pcDNA3.1/p53 construct (p[p53]). The expression of p53 protein was observed in p[p53] cells but not in vector control (p[V]). Single asterisk indicates significant difference at <i>p</i> < 0.05, according to Student’s <i>t</i> test.</p

Wuho Is a New Member in Maintaining Genome Stability through its Interaction with Flap Endonuclease 1

<div><p>Replication forks are vulnerable to wayward nuclease activities. We report here our discovery of a new member in guarding genome stability at replication forks. We previously isolated a <i>Drosophila</i> mutation, <i>wuho</i> (<i>wh</i>, no progeny), characterized by a severe fertility defect and affecting expression of a protein (WH) in a family of conserved proteins with multiple WD40 repeats. Knockdown of WH by siRNA in <i>Drosophila</i>, mouse, and human cultured cells results in DNA damage with strand breaks and apoptosis through ATM/Chk2/p53 signaling pathway. Mice with m<i>Wh</i> knockout are early embryonic lethal and display DNA damage. We identify that the flap endonuclease 1 (FEN1) is one of the interacting proteins. Fluorescence microscopy showed the localization of WH at the site of nascent DNA synthesis along with other replication proteins, including FEN1 and PCNA. We show that WH is able to modulate FEN1’s endonucleolytic activities depending on the substrate DNA structure. The stimulatory or inhibitory effects of WH on FEN1’s flap versus gap endonuclease activities are consistent with the proposed WH’s functions in protecting the integrity of replication fork. These results suggest that <i>wh</i> is a new member of the guardians of genome stability because it regulates FEN1’s potential DNA cleavage threat near the site of replication.</p></div

hWH knockdown results in DNA damage at the replication sites.

<p>(A) HCT116 p53^+/+ cells were treated with either hWH or control siRNA for 24, 48, and 72 h. Afterward cells are harvested and stained with DAPI (blue), EdU (green), and γ-H2AX (red). (B) After siRNA treatment for 48h, γ-H2AX positive cells percentage in sihWH is significantly higher than that of siControl. Double asterisks indicate significant differences at <i>p</i> < 0.01, according to Student’s <i>t</i> test.</p

DNA damage precedes apoptosis after mWH depletion in mouse JB6 cells.

<p>(A) Time course of DNA damage and apoptosis following mWH depletion. Samples were collected for analysis following mWH knockdown at 0, 24, 48, and 72 h. DNA damage (γ-H2AX signals) was detected at 48 h after siRNA treatment. Apoptotic signals including cleavage of Procaspase-9, Procaspase-3, and PARP, were detected only after 72 h of treatment. (B) Inhibitors of Caspases-9 and -3, but not for Caspase-8, block apoptosis ensuing depletion of mWH. However, DNA damage signals were unaffected and present under these conditions. Cells were co-cultured with simWH and different Caspase-specific inhibitors (final concentrations of 2 μM in the culture medium) including Z-DEVD-FMK (Caspase-3), Z-IETD-FMK (Caspase-8), and Z-LEHD-FMK (Caspase-9). After 72 h of treatment, samples were collected and processed for western blot.</p

Multicolor Fluorescence Detection-Based Microfluidic Device for Single-Cell Metabolomics: Simultaneous Quantitation of Multiple Small Molecules in Primary Liver Cells

Single-cell metabolomics can be used to study cell diversity and how cells respond to environment. There is an urgent need to develop effective detection methods for single-cell metabolomics. Microchip electrophoresis with laser-induced fluorescence detection (MCE-LIFD) is a powerful tool to detect metabolites at the single-cell level. However, the existing one-laser excitation and one-color fluorescence collection in MCE-LIFD is not sufficient for the simultaneous detection of multiple small molecules with wide variations in their fluorescence excitation and emission spectra. In this manuscript, we describe a multicolor fluorescence detection-based microfluidic device (MFD-MD) for single-cell metabolomics research. We selected primary liver cells from acute ethanol-stimulated mice as the model cells and hydrogen peroxide (H₂O₂), glutathione (GSH), and cysteine (Cys) as representative small-molecule metabolites for single-cell analysis. The microfluidic chip enabled accurate single-cell manipulation and effective electrophoresis separation. The new multicolor fluorescence detection permitted simultaneous analysis of H₂O₂, GSH, and Cys. Ethanol exposure induced an increase in H₂O₂ and a decrease in GSH and Cys. Obvious cell heterogeneity was observed. These results provide insights regarding the intracellular oxidative/antioxidative molecular mechanism in response to external stimuli. The MFD-MD provides a new opportunity for simultaneous single-cell analysis of multiple metabolites