Visualization 2: Complex amplitude reconstruction for dynamic beam quality M² factor measurement with self-referencing interferometer wavefront sensor

Reconstructed phase and intensity of the aberration beam. Originally published in Applied Optics on 20 December 2016 (ao-55-36-10180

Variable Valence State of Trace Elements Regulating Toxic Potencies of Inorganic Particulate Matter

Trace element is known to be one major component in determining particulate matter (PM) toxicities. However, there is still no accurate assessment of the toxic potency of the mixed valences. Here, we reported the oxidative stress and cytotoxicity potencies of 14 trace elements in their various valence states and estimated the toxic gaps of inorganic PM resulting from variations in element valences. Substantial discrepancies of up to 3 orders of magnitude in toxic potencies were observed among their different valences. When considering their abundance in PM, the toxicity gaps are estimated to range from 5 to 6 times between the greatest and weakest toxic valence states in the inorganic PM emitted from industrial sources, with iron contributing to 65.5%–91.0% of the overall gaps. Furthermore, the relative toxic variation of inorganic PM shows a significant correlation with the additive toxicities of Fe(II) and Fe(III) ions during aging process. The finding highlights that the multiple coexisting valence states of trace elements in PM need to be taken into account when estimating their toxic potencies

X-ray Absorption Spectral Signature of Quantum Nuclei in Liquid Water

<div> <div> <div> <p>Based on electron-hole excitation theory, we investigate the X-ray absorption spectral signature of nuclear quantum effect in liquid water, whose molecular structure is simulated by path-integral molecular dynamics using the MB-pol model. Compared to spectra generated from classically modeled water structure, quantum nuclei has important effect on spectra in terms of both the spectral energies and their line shapes. At the short-range ordering of H-bond network, the delocalized protons increase the fluctuations on the intramolecular covalency and broaden the pre-edge of the spectra. For intermediate-range and long-range orderings, the observed red and blue shifts of the main-edge and post-edge are attributed to the so-called competing quantum effects, under which both the weak and well-formed H-bonds are promoted. The theoretical spectra are in nearly quantitative agreement with the available experimental data. </p> </div> </div> </div

Mitochondrial Protein PGAM5 Regulates Mitophagic Protection against Cell Necroptosis

<div><p>Necroptosis as a molecular program, rather than simply incidental cell death, was established by elucidating the roles of receptor interacting protein (RIP) kinases 1 and 3, along with their downstream partner, mixed lineage kinase-like domain protein (MLKL). Previous studies suggested that phosphoglycerate mutase family member 5 (PGAM5), a mitochondrial protein that associates with RIP1/RIP3/MLKL complex, promotes necroptosis. We have generated mice deficient in the pgam5 gene and surprisingly found PGAM5-deficiency exacerbated rather than reduced necroptosis in response to multiple <i>in vitro and in vivo</i> necroptotic stimuli, including ischemic reperfusion injury (I/R) in the heart and brain. Electron microscopy, biochemical, and confocal analysis revealed that PGAM5 is indispensable for the process of PINK1 dependent mitophagy which antagonizes necroptosis. The loss of PGAM5/PINK1 mediated mitophagy causes the accumulation of abnormal mitochondria, leading to the overproduction of reactive oxygen species (ROS) that worsen necroptosis. Our results revise the former proposal that PGAM5 acts downstream of RIP1/RIP3 to mediate necroptosis. Instead, PGAM5 protects cells from necroptosis by independently promoting mitophagy. PGAM5 promotion of mitophagy may represent a therapeutic target for stroke, myocardial infarction and other diseases caused by oxidative damage and necroptosis.</p></div

PGAM5 fails to stabilize PINK1 in HT-29 cells.

<p>(A) HT-29 cells transduced with shRNA against PGAM5 were treated with TCZ to induce necroptosis. Cell viability was evaluated by the MTT assay. (B) Mitochondria in HeLa cells and HT-29 cells were stained with anti-Tomm20 and mitochondrial morphology was evaluated by confocal. (C) PGAM5 was knocked down by lentiviral shRNA in HeLa, HT-29 and SY5Y cell lines. Then PINK1 mRNA in the control (NS) and Knock-down cells (PGsh) were quantified by RT-PCR. (D) HT-29 and HeLa cells were treated with CCCP for 3 hours and PINK1 was detected by western blot. * indicated full length PINK1 and Δ indicated cleaved PINK1.(E)HT-29 control (NS) and PGAM5 knock-down cells (PGsh) were treated with CCCP for 3 hours followed by western blot for detecting indicated proteins.</p

PGAM5 deficiency exacerbates necroptosis.

<p>(A) Phase contrast photomicrographs of WT or KO MEFs treated with DMSO (a, b) or TCZ (c, d) for 24 hr. Cell viability was evaluated by the MTT assay, and propidium iodide staining (inset c, d). (B) Cell viability results for WT (black) and KO (white) MEFs, which were treated with TCZ with or without 50 μM Nec1 or 100 μM BHA; (C) WT and Pgam5 KO MEFs were treated with DMSO or TCZ for 6 hr and then stained with the mitochondria membrane potential dependent dye DAPI (blue), anti-HMGB1 (green) and Mitotracker (red); (D) WT and Pgam5 KO MEFs were treated with TCZ for 3 hrs, and followed by purification of mitochondria. Western blots with the indicated antibodies are shown. Tubulin and VDAC are loading controls. (E) Representative TEM micrographs of necrotic WT and Pgam5 KO MEFs treated with TCZ or DMSO vehicle for 12 hrs. (F) Jurkat I2.1 (Caspase 8 deficient) cells transfected with nonspecific (NS) or PGAM5 (siPG) siRNAs were treated with 0.5 ug/ml human TNF-α for 24 hrs with or without necrostatin (Nec1), then cell death was quantified by taking the percentage outside of the life gate (indicated by the box using dot plots with PI staining on the y-axis and forward scatter on the x-axis (left panels). Quantification is shown in a bar graph (right upper panel) and the quality of the knockdown shown by Western blot (right lower panel).</p

PGAM5 stabilizes PINK1 to protect cell from necroptosis.

<p>(A) Mitochondrial extracts of WT and Pgam5 KO MEFs treated with DMSO (D), CCCP(C), or TCZ (T) for 3 hours were analyzed by immunoblot as above. (B) PINK1 immunoblot of mitochondrial fractions from WT and KO hearts subjected to I/R (+) or perfusion only (-). (C) Cell viability of Pink1 WT/KO MEFs was quantified by MTT assay. Black bars represent WT controls and white bars the respective KO. Cells were treated with TCZ, TCZ plus Nec1, or TCZ plus BHA for 12 hours. (D) Cell viability of WT and Pink1 KO MEF cells was transduced with scrambled (NC) or Pgam5-specific shRNA (PG) lentiviruses, treated with TCZ, and then tested for viability by the MTT assay at indicated treatments. <i>p</i> <0.01 by Student <i>t</i>-test.</p

MHV-3 fails to induce FGL2 production and neutrophil infiltration in the livers of <i>IL-1R1</i>^<i>-/-</i> mice.

<p><i>IL-1R1</i>^<i>-/-</i> mice and their C57BL/6 WT littermates were infected with MHV-3 (100 PFU). <b>(A)</b> Peritoneal exudative macrophages (PEMs) were isolated and the expression of FGL2 was detected by western-blotting. <b>(B)</b> The expression of FGL2 in liver at 48h and 72h post-infection was analyzed by western-blotting. Four representative samples <i>per</i> group are shown. <b>(C)</b> Serum FGL2 levels in virus infected mice were measured by ELISA.*<i>p</i><0.05 and **<i>p</i><0.0001, NS: no significant difference, n = 5 <i>per</i> group. <b>(D)</b> The liver fibrinogen deposition post-infection was analyzed by immunohistochemistry. Scale bar 20 μm, n = 6~8 <i>per</i> group. <b>(E)</b> Liver recruitment of CD45⁺Gr-1^high neutrophils after MHV-3 infection was measured by flow cytometry. The left panels are gate strategies, and number indicates the percentage of positive cells in the gate. One representative sample from five mice <i>per</i> group is showed. <b>(F)</b> Statistical analysis of liver CD45⁺Gr-1^high neutrophil infiltration. *<i>p</i><0.05 compared to WT littermates in each group, n = 5 <i>per</i> group.</p

<i>IL-18</i>^<i>-/-</i> mice are susceptible to MHV-3-mediated hepatitis.

<p><i>IL-18</i>^<i>-/-</i> mice and their C57BL/6 WT littermates were treated with MHV-3 (100PFU). <b>(A)</b> Peritoneal exudative macrophages (PEMs) and liver tissues were isolated from C57BL/6 WT mice, and the transcription of <i>proIL-18</i> mRNA was measured by qPCR. *<i>p</i> < 0.05. <b>(B)</b> Serum IL-18 levels in virus-infected WT mice at the indicated time points were measured by ELISA. **<i>p</i> < 0.001, n = 5~8 <i>per</i> group, NS: no significant difference. <b>(C)</b> RAW264.7 cells and SVE-10 endothelial cells were treated with IFN-γ (50 ng/ml), TNF-α (100 ng/ml) and IL-18 (20 ng/ml) alone or in combination, and <i>fgl2</i> mRNA transcription was detected by qPCR at 24h. *<i>p</i> < 0.05, NS: no significant difference. <b>(D)</b> The expression of FGL2 in MHV-3 infected livers was compared by western-blotting. Three representative samples <i>per</i> group are shown. <b>(E)</b> Liver fibrinogen deposition was analyzed by immunohistochemistry, architecture was detected by H&E staining, and cell apoptosis by TUNEL staining. Scale bar 20 μm, arrows indicates TUNEL-positive cells, blue color indicates nuclear staining with 4',6-diamidino-2-phenylindole (DAPI), n = 5 <i>per</i> group. <b>(F)</b> Liver expression of Bgp1 was detected by western-blot (up) and the virus titers in livers at 72h post-infection were analyzed by plaque assay, and their levels were compared by statistical analysis (down). *<i>p</i><0.05, n = 5 <i>per</i> group. <b>(G)</b> The survival rate of virus-infected mice was monitored for 20 days. One of three experiments with similar results is shown. NS: no significant difference.</p

Enhanced ROS in macrophages following MHV-3 infection.

<p>PEMs and RAW264.7 cells were infected with MHV-3 (MOI = 1) <i>in vitro</i>, the NOX-derived ROS (DCFH), mitochondrial damage (stained with MitoTracker Red FM/MitoTracker Green FM) and the secretion of mitochondrial ROS (MitoSOX) were detected by flow cytometry <b>(A).</b> The gate strategies were similar to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005155#ppat.1005155.g001" target="_blank">Fig 1D</a>, and number indicates the percentage of positive cells in the gate. One of three experiments with similar results was shown. <b>(B)</b> The expression of NADPH oxidase subunits including gp91^phox, p47^phox and p67^phox and NOX-4 in virus infected Raw264.7 cells was measured by western-blot. <b>(C)</b> Transmission electron microscopy analysis of mitochondrial morphology in virus infected cells. Arrows indicate the damaged mitochondria, whereas arrow heads indicate the normal mitochondria. (<b>D</b>) RAW264.7 cells were infected with MHV-3 for a total 24h, and cells were incubated with different doses of DPI in the last 4h. The secretion of DCFH and MitoSOX was measured by flow cytometry. Data were normalized to the increase in fluorescence of the MHV-3 infected alone sample without DPI treatment for each experiment (n = 3 independent experiments). <b>(E)</b> The expression of Caspase-1 p20 and IL-1β p17 in DPI treated RAW264.7 cells as well as PEMs was detected by western-blotting. One representative of three experiments with similar results is shown.</p