Search Results (275)

Figure 1
<p>HSV-2 UL24 inhibits IFN-β production. (<b>A</b>) HSV-2 inhibits the activation of the IFN-β promoter. HEK 293T cells in 24-well plates were transfected with 500 ng HSV-2 UL24 or empty vector together with 250 ng p125-Luc. At 24 h post transfection, cells were stimulated with or without 100 HAU mL⁻¹ SeV for 24 h. (<b>B</b>) HSV-2 inhibits the production of IFN-β mRNA. HEK 293T cells in 6-well plates were transfected with 2 μg empty vector or plasmid expressing HSV-2 UL24 for 24 h, followed by stimulation with 100 HAU mL⁻¹ SeV for 24 h. (<b>C</b>) HSV-2 UL24 inhibits the production of IFN-β. HEK 293T cells in 6-well plates were transfected with 2 μg empty vector or HSV-2 UL24 expression plasmid for 24 h, followed by stimulation with or without 100 HAU mL⁻¹ SeV for 24 h. (<b>D</b>) The expression of HSV-2 UL24 was detected by Western blot using anti-Flag Ab. (<b>E</b>) Knockdown of HSV-2 UL24 impairs the capability of HSV-2 in inhibiting the production of IFN-β. HeLa cells in 6-well plates were transfected with 200 nM HSV-2 UL24 siRNA (siUL24) or negative control siRNA (N). At 10 h post transfection, cells were mock-infected or infected with HSV-2 at an MOI of 0.1. At 20 h post infection, cells were stimulated with or without 100 HAU mL⁻¹ SeV for 16 h, and the supernatants were harvested for ELISA. (<b>F</b>,<b>G</b>) The knockdown effect of HSV-2 UL24 siRNA. HeLa cells in 6-well plates were transfected with 200 nM HSV-2 UL24 siRNA (siUL24) or negative control siRNA (siNC) for 10 h, followed by transfection with 2 μg HSV-2 UL24 expression plasmid or empty vector. At 46 h post transfection of HSV-2 UL24 siRNA, cells were harvested for Western blot assay (<b>F</b>). HeLa cells in 6-well plates were transfected with 200 nM HSV-2 UL24 siRNA (siUL24) or negative control siRNA (N). At 10 h post transfection, cells were mock-infected or infected with HSV-2 at an MOI of 0.1. At 20 h post infection, cells were stimulated with or without 100 HAU mL⁻¹ SeV for 16 h, and the RNAs were exacted and detected by qPCR (<b>G</b>). Values for the samples were expressed as a percentage of the value induced in cells transfected with empty vector or mock-infected with DMEM (<b>A</b>–<b>C</b>,<b>E</b>,<b>G</b>). The data shown are representative of three independent experiments, with each condition performed in triplicate (mean ± SD). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001. ns, not significantly. N, negative. Rel, relative.</p> Full article ">Figure 2
<p>HSV-2 UL24 truncation mutants show no obvious cytotoxicity. (<b>A</b>,<b>B</b>) HSV-2 UL24 induces cell cycle arrest and cell apoptosis. (<b>C</b>) HSV-2 UL24 affects cell viability. (<b>D</b>) Schematic representation of HSV-2 UL24 truncation mutants. (<b>E</b>,<b>F</b>) HSV-2 UL24-N has no obvious cytotoxicity. (<b>G</b>) HSV-2 UL24-N has no obvious impact on cell viability. HEK 293T cells in 6-well plates were transfected with 2 μg plasmid expressing HSV-1 UL24 (<b>A</b>,<b>C</b>), HSV-2 UL24 (<b>A</b>,<b>C</b>,<b>E</b>,<b>G</b>,<b>H</b>), HSV-2 UL24 truncation mutant (<b>E</b>,<b>G</b>,<b>H</b>) or empty vector. HSV-1 UL24 (<b>A</b>,<b>C</b>) was used as a control for the arrest of cell cycle. At 48 h post transfection, cells were harvested and stained using a Cell Cycle and Apoptosis Analysis Kit for flow cytometry analysis (<b>A</b>,<b>E</b>) or lysed using a CellTiter-Lumi™ Plus II Luminescent Cell Viability Assay Kit for luciferase activity analysis (<b>C</b>,<b>G</b>). The results of A and E were displayed using the stacked bar chart (<b>B</b>,<b>F</b>). (<b>H</b>) The expression of HSV-1 and HSV-2 UL24s and HSV-2 UL24 truncation mutants was detected using anti-Flag or anti-HA Ab. A and E were presented separately, although both were part of the same experiment. Values for the samples were expressed as a percentage of the value in cells transfected with empty vector (<b>B</b>,<b>E</b>). The data shown are representative of three independent experiments, with each condition performed in triplicate (mean ± SD). * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001. ns, not significantly. Blue arrow indicates the apoptotic peak.</p> Full article ">Figure 3
<p>The N-terminal 1–202 AA domain of HSV-2 UL24 is the main functional region responsible for HSV-2 UL24-mediated inhibition of IFN-β production. (<b>A</b>) HSV-2 UL24-N inhibits the activation of the IFN-β promoter. HEK 293T cells in 24-well plates were transfected with 500 ng empty vector or plasmid expressing HSV-2 UL24 or its truncation mutant together with 250 ng p125-Luc. At 24 h post transfection, cells were stimulated with or without 100 HAU mL⁻¹ SeV for 24 h. (<b>B</b>,<b>C</b>) HSV-2 UL24-N inhibits the production of IFN-β. HEK 293T cells in 6-well plates were transfected with 2 μg empty vector or plasmid expressing HSV-2 UL24 or its truncation mutant for 24 h, followed by stimulation with or without 100 HAU mL⁻¹ SeV for 24 h. The mRNA and protein levels of IFN-β in cells were measured by qPCR (<b>B</b>) and ELISA (<b>C</b>). Values for the samples were expressed as a percentage of the value induced in cells transfected with empty vector. The data shown are representative of three independent experiments, with each condition performed in triplicate (mean ± SD). *** <span class="html-italic">p</span> &lt; 0.001. ns, not significantly. Rel, relative.</p> Full article ">Figure 4
<p>The N-terminal 1–202 AA domain of HSV-2 UL24 interferes with the IRF-3-mediated signaling pathway. (<b>A</b>) HSV-2 UL24-N inhibits the activation of the IRF-3-responsive IFN-β promoter. HEK 293T cells in 24-well plates were transfected with 500 ng empty vector or plasmid expressing HSV-2 UL24 or UL24 truncation mutant together with 250 ng PRD(III-I)₄–Luc. At 24 h post transfection, cells were stimulated with or without HAU mL⁻¹ SeV for 24 h. The expression of HSV-2 UL24 and UL24-N was detected by Western blot using anti-Flag Ab (<b>B</b>). (<b>C</b>–<b>G</b>) HSV-2 UL24-N inhibits the activation of the IRF-3 signaling pathway. HEK 293T cells in 24-well plates were cotransfected with 500 ng empty vector or plasmid expressing HSV-2 UL24 or its truncation mutant together with 250 ng p125-Luc and 50 ng plasmid expressing RIG-IN (<b>C</b>), MAVS (<b>D</b>), TBK-1 (<b>E</b>), IKK-ε (<b>F</b>), or IRF-3/5D (<b>G</b>). At 48 h post transfection, cells were harvested and luciferase activities were measured. Values for the samples were expressed as a percentage of the value induced in cells transfected with empty vector (<b>A</b>, <b>C</b>–<b>G</b>). Protein expression was detected by Western blot using anti-HA or anti-Flag Ab. The data shown are representative of three independent experiments, with each condition performed in triplicate (mean ± SD). *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 5
<p>The N-terminal 1-202 AA domain of HSV-2 UL24 inhibits the phosphorylation and nuclear translocation of IRF-3. (<b>A</b>,<b>B</b>) HSV-2 UL24-N does not affect the expression of IRF-3. (<b>C</b>,<b>D</b>) HSV-2 UL24-N suppresses the phosphorylation of IRF-3. (<b>E</b>,<b>F</b>) HSV-2 UL24-N induces the decrease in nuclear translocation of IRF-3. HEK 293T cells in 6-well plates were transfected with 2 μg empty vector or plasmid expressing HSV-2 UL24 or UL24 truncation mutant. At 24 h post transfection, cells were stimulated with or without 100 HAU mL⁻¹ SeV for 24 h. Cells were directly lysed to perform Western blot or collected to isolate the nuclear protein. Total cellular IRF-3 (<b>A</b>,<b>B</b>) and p-IRF-3 (<b>C</b>,<b>D</b>) or nuclear p-IRF-3 (<b>E</b>,<b>F</b>) were detected by Western blot using anti-IRF-3 or anti-p-IRF-3 Ab. β-actin and PCNA were used as loading controls. The relative intensities of IRF-3 and p-IRF-3 blots were quantified using Image J (<b>B</b>,<b>D</b>,<b>F</b>). One representative experiment out of three is shown. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, ns, not significantly.</p> Full article ">Figure 6
<p>The schematic model illustrates the mechanism by which HSV-2 UL24 blocks the IRF-3 signaling pathway. During HSV-2 infection, the viral dsRNA by-product is recognized by RIG-I. Subsequently, RIG-I binds to dsRNA and signals to the adaptor protein MAVS. Dimerized MAVS recruits and activates the downstream protein kinase complexes TBK-1/IKK-ε, leading to the phosphorylation and dimerization of IRF-3. The IRF-3 dimer translocates from the cytoplasm to the nucleus, where it binds to the IFN-β promoter to initiate the transcription of IFN-β. In the case of HSV-2 infection, the viral late protein UL24 interferes with IRF-3 phosphorylation, leading to the blockade of IFN-β production.</p> Full article ">

Figure 1
<p>In the selection of numerous feasible planning paths, cluttered environment and high risk always lead complex planning strategy and low flight speed, risk assessment and taking it in planning can achieve a balance between minimizing risk and optimizing paths, making MAVs have a tendency to fly safe and fast.</p> Full article ">Figure 2
<p>The process of DSP Map generation and the pedestrians motion representation of particle points. Green pyramid is the map space and blue view pyramid is the visible space, then scanning from the MAV on the top of pyramids, obstacles can be mapped into the map space. The red dashed boxes represent the mapped pedestrians.</p> Full article ">Figure 3
<p>Systerm overview. Our method includes three modules and performs each module in order: map construction, motion planning with risk assessment, and trajectory optimization.</p> Full article ">Figure 4
<p>(<b>a</b>) Motion snapshots of the MAV in simplified <math display="inline"><semantics> <msup> <mi mathvariant="double-struck">R</mi> <mn>2</mn> </msup> </semantics></math> environment with velocity direction indicated by purple lines. (<b>b</b>) Contours of occupancy risk cost <math display="inline"><semantics> <mi mathvariant="script">H</mi> </semantics></math> and the stretch direction is up to the velocity direction.</p> Full article ">Figure 5
<p>Simulation scenarios. Scenario A: A closed triangular area with dense cylindrical obstacles. Scenario B: A square with dense crowds walking in different directions. Scenario C: A park with oak trees and gazebos in addition to pedestrians. The walking speed in Scenario B and C ranges from 1.0 m/s to 1.5 m/s.</p> Full article ">Figure 6
<p>Traveled paths and in-process risk comparison in simulation scenarios. The x-axis represents flight time and the y-axis represents risk. EGO-Planner (yellow) [<a href="#B36-drones-08-00497" class="html-bibr">36</a>] always generates a efficient and short path but results in a higher likelihood of collision. RAST-Planner (blue) [<a href="#B20-drones-08-00497" class="html-bibr">20</a>] takes risk in consideration for reducing collision possibility finitely and sometimes makes the MAV stop moving and hover for re-planning. Our method (green) further reduces risk and makes the moving path more smooth and efficient.</p> Full article ">Figure 7
<p>MAV in our experiment. Hardware and sensors include a NVIDIA computing board and an Intel RealSense depth camera.</p> Full article ">Figure 8
<p>Real-world experiment compared with RAST-Planner [<a href="#B20-drones-08-00497" class="html-bibr">20</a>]. Subfigure (<b>a</b>,<b>b</b>): Overview snapshot and D435i view. The red dashed block reflects excellent mapping for dynamics. Subfigure (<b>c</b>): Risk flight corridor of RAST-Planner (red dashed block) and our method.</p> Full article ">Figure 9
<p>Risk comparison between RAST-Planner and ours in real-world experiment. The x-axis represents moving distance in start-to-goal direction and the y-axis represents risk. The dense lines zone like at <math display="inline"><semantics> <mrow> <mi>x</mi> <mo>∈</mo> <mo>(</mo> <mn>2</mn> <mo>,</mo> <mn>3</mn> <mo>)</mo> </mrow> </semantics></math> means the MAV stops moving and hovers for re-planning. Our method keeps lower risk most time and reduces frequency of re-planning efficiently.</p> Full article ">

Figure 1
<p>Details of MAV geometry and of six tested wing configurations.</p> Full article ">Figure 2
<p>PIV set-up with laser arm, camera and MAV model in wind tunnel (<b>top</b>). Example of PIV-acquired images at full scale (large field of view) and reduced scale (small field of view, in red) with reference system (<b>bottom</b>).</p> Full article ">Figure 3
<p>Lift and drag coefficients as functions of angle of attack for flat-plate MAV geometry. Present data are plotted for two Reynolds numbers and compared to data in [<a href="#B4-biomimetics-09-00553" class="html-bibr">4</a>,<a href="#B11-biomimetics-09-00553" class="html-bibr">11</a>].</p> Full article ">Figure 4
<p>Lift and drag coefficients as functions of angle of attack for the corrugated and profiled MAV wing geometry. Present data are plotted for two Reynolds numbers and compared to data in [<a href="#B4-biomimetics-09-00553" class="html-bibr">4</a>,<a href="#B11-biomimetics-09-00553" class="html-bibr">11</a>].</p> Full article ">Figure 5
<p>Lift and drag coefficients as functions of angle of attack for all tested MAV wing geometries for present large Reynolds number measurements.</p> Full article ">Figure 6
<p>Lift-to-drag ratio and polar curve for all tested MAV wing geometries.</p> Full article ">Figure 7
<p>The average absolute value of velocity and streamlines at an angle of attack of 15° for MAVs with the following wing configurations: flat (<b>a</b>), corrugated (<b>b</b>) and profiled (<b>c</b>). Large field of view. In the insets, detailed small-field-of-view plots are reported. Dashed lines indicate positions for velocity profiles.</p> Full article ">Figure 8
<p>Average absolute value of velocity and streamlines at angle of attack of 30° for flat (<b>a</b>), corrugated (<b>b</b>) and profiled (<b>c</b>) MAV wing configurations. Large field of view. In the insets, detailed small-field-of-view plots are reported. Dashed lines indicate positions for velocity profiles.</p> Full article ">Figure 9
<p>Average absolute value of velocity and streamlines at angle of attack of 36° for flat (<b>a</b>), corrugated (<b>b</b>) and profiled (<b>c</b>) MAV wing configurations. Large field of view. Dashed lines indicate positions for velocity profiles.</p> Full article ">Figure 10
<p>Average (on the left) and instantaneous (on the right) vorticity for an angle of attack equal to 15°: flat-plate (row <b>a</b>), corrugated (row <b>b</b>) and profiled (row <b>c</b>) MAV configurations from top to bottom. Large field of view.</p> Full article ">Figure 11
<p>Average (on the left) and instantaneous (on the right) vorticity for an angle of attack equal to 30°: flat-plate (row <b>a</b>), corrugated (row <b>b</b>) and profiled (row <b>c</b>) MAV configurations from top to bottom. Large field of view.</p> Full article ">Figure 12
<p>Average <span class="html-italic">rms</span> of streamwise velocity component for angle of attack equal to 15°: flat-plate (<b>a</b>), corrugated (<b>b</b>) and profiled (<b>c</b>) MAV configurations. Large field of view. In insets, detailed small-field-of-view plots are reported. Dashed lines indicate positions for <span class="html-italic">rms</span> profiles.</p> Full article ">Figure 13
<p>Average <span class="html-italic">rms</span> of streamwise velocity component for angle of attack equal to 30°: flat-plate (<b>a</b>), corrugated (<b>b</b>) and profiled (<b>c</b>) MAV configurations. Large field of view. In insets, detailed small-field-of-view plots are reported. Dashed lines indicate positions for <span class="html-italic">rms</span> profiles.</p> Full article ">Figure 14
<p>Average <span class="html-italic">rms</span> of streamwise velocity component for angle of attack equal to 36°: flat-plate (<b>a</b>), corrugated (<b>b</b>) and profiled (<b>c</b>) MAV configurations. Large field of view. Dashed lines indicate positions for <span class="html-italic">rms</span> profiles.</p> Full article ">Figure 15
<p>Vertical profiles of normalized streamwise velocity component at x/c = 0.3, for the three tested MAV configurations. Angles of incidence equal to 15° (<b>a</b>), 30° (<b>b</b>) and 36° (<b>c</b>).</p> Full article ">Figure 16
<p>Vertical profiles of normalized streamwise velocity component at x/c = 0.6, for the three tested MAV configurations. Angles of incidence equal to 15° (<b>a</b>), 30° (<b>b</b>) and 36° (<b>c</b>).</p> Full article ">Figure 17
<p>Vertical profiles of normalized <span class="html-italic">rms</span> streamwise component at x/c = 0.3, for the three tested MAV configurations. Angles of incidence equal to 15° (<b>a</b>), 30° (<b>b</b>) and 36° (<b>c</b>).</p> Full article ">Figure 18
<p>Vertical profiles of normalized <span class="html-italic">rms</span> streamwise component at x/c = 0.6 for the three tested MAV configurations. Angles of incidence equal to 15° (<b>a</b>), 30° (<b>b</b>) and 36° (<b>c</b>).</p> Full article ">

Figure 1
<p>Schematic representation of the exosome isolation protocol, exosome characterization procedure and co-culture with SCLC cell lines. Exosomes were isolated from PBMCs derived from SCLC patients by multiple ultracentrifugation steps. PBMC-EXs were characterized by SEM, nanoparticle tracking analysis was performed using a NanoSight Instrument and exosomal markers were determined by Western blot analysis. The graphical scheme was produced by the authors using the BioRender platform (<a href="https://www.biorender.com/" target="_blank">https://www.biorender.com/</a>) (basic license terms).</p> Full article ">Figure 2
<p>Isolation and characterization of exosomes from SCLC patients PBMC-EXs. (<b>A</b>) Representative FEG-SEM images of the isolated exosomes (scale bar = 300 nm). (<b>B</b>) Size distribution of the isolated PBMC-EXs. (<b>C</b>) Western blot analysis and its quantification of exosomal markers CD81, CD63, HSP70, DNA/RNA sensors of antitumor innate immune response (STING and MAVS) and EMT TFs Snail and c-Myc. The isolated exosomes were successfully isolated from the culture supernatants of PBMCs isolated from BR or NR SCLC patients. The data of PBMC-EX samples were expressed as the ratio of each PBMC-EX BR sample to the corresponding PBMC-EX NR sample to evaluate the relative fold-change induction. Red line indicated the band of each protein on the gel. Original western blots are presented in <a href="#app1-cancers-16-03151" class="html-app">File S1</a>.</p> Full article ">Figure 3
<p>Co-culture effect of PBMC-EXs with SCLC cell lines on cell viability. (<b>A</b>) Cell viability of H661 and H446 cells after co-culture for 24 h with PBMC-EXs from BR and NR SCLC patients. (<b>B</b>) Cell viability of H661 and H446 cells after co-culture for 72 h with PBMC-EXs from BR and NR SCLC patients. Data are expressed as the mean ± SD. Unpaired Student’s <span class="html-italic">t</span>-test with * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 4
<p>Co-culture effect of PBMC-EXs with SCLC cell lines on cell death. (<b>A</b>) Flow cytometry analysis of cell death by Annexin V/PI assay after co-culture for 72 h of PBMC-EXs from BR and NR donors. (<b>B</b>) Bar graph showing summary data of % Annexin V/PI positive cells; H661 (upper panel) and H446 (lower panel). Statistical significance: **** <span class="html-italic">p</span> &lt; 0.0001, *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, # = comparison between H661 and H446 apoptosis; #### <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 5
<p>Apoptotic markers, DNA damage/replication stress markers and EMT markers in co–cultures of SCLC cell lines with PBMC–EXs derived from BR and NR SCLC patients. Representative Western blotting of whole cell lysates from (<b>A</b>) H661 and (<b>B</b>) H446 cell lines showing levels of apoptotic markers (caspase 8, lamin A/C, BID/tBID), DNA damage, replication stress markers (Bcl–2, Bcl–xL, p–Chk2/Chk2, H2A.X) and EMT markers (e–cadherin, Snail, pMAPK/MAPK, TGFBR–I) after co-culture with BR or NR PBMC-EXs. GAPDH was used to ensure equal loading. At least three independent experiments were performed. Original western blots are presented in <a href="#app1-cancers-16-03151" class="html-app">File S1</a>.</p> Full article ">

Figure 1
<p>Ectromelia virus (ECTV) replication in L929 murine fibroblasts with modified mitochondrial network morphology. The plaque assay determined the ECTV titer in L929 cells at two time points: (<b>a</b>) 18 h post infection (hpi) under various treatments, including DMSO, Mdivi-1, or CCCP, with or without transfection of poly(I:C) (MHW) LyoVec (pIC LV); and (<b>b</b>) 10 hpi with transfection of control siRNA, Mfn1 siRNA, or Drp1 siRNA or treatment with DMSO, Mdivi-1, or CCCP. The data from both (<b>a</b>,<b>b</b>) are presented as mean ± standard deviation (SD), with statistical significance indicated as * <span class="html-italic">p</span> ≤ 0.05 and ** <span class="html-italic">p</span> ≤ 0.01. ECTV suspension was obtained from cell lysates in post-culture media. Western blot analysis was performed to confirm Mfn1 or Drp1 gene silencing in target cells (<b>c</b>) and to assess ECTV antigens expression in control and infected cells treated with DMSO, Mdivi-1, or CCCP at 2, 6, 10, and 18 hpi (<b>d</b>). (<b>e</b>) Fluorescence verification of mitochondrial network morphology in L929 fibroblast after treatment with DMSO (intact network), Mdivi-1 (elongated), and CCCP (fragmented) by using MitoRed fluorochrome (red). Scale bar: 20 µm.</p> Full article ">Figure 2
<p>ECTV replication in RAW 264.7 murine macrophages with modified mitochondrial network morphology. (<b>a</b>) Determination of ECTV titer by plaque assay in RAW 264.7 cells at 6 hpi treated with DMSO, Mdivi-1, or CCCP. ECTV suspension was obtained from cell lysates or post-culture media. (<b>b</b>) Fluorescence verification of mitochondrial network morphology in RAW 264.7 murine macrophages after treatment with DMSO (intact network), Mdivi-1 (elongated), and CCCP (fragmented). Macrophages were labeled using antibodies against MAVS (green) and DNA (blue). Scale bar: 20 µm.</p> Full article ">Figure 3
<p>Distribution of MAVS protein in L929 cells at 18 hpi with ECTV. Cells were treated with DMSO, Mdivi-1, or CCCP. (<b>a</b>) Magnifications indicate MAVS localization in the viral factories. Arrows show the direction of fluorescence intensity measurements. Fibroblasts are labeled using antibodies against MAVS (red), HSP60 (green), or ECTV (EV; green) and DNA (blue). Scale bar: 20 µm. (<b>b</b>) Percentage of MAVS and HSP60 colocalization (<span class="html-italic">n</span> = 20). Different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05) between groups.</p> Full article ">Figure 3 Cont.
<p>Distribution of MAVS protein in L929 cells at 18 hpi with ECTV. Cells were treated with DMSO, Mdivi-1, or CCCP. (<b>a</b>) Magnifications indicate MAVS localization in the viral factories. Arrows show the direction of fluorescence intensity measurements. Fibroblasts are labeled using antibodies against MAVS (red), HSP60 (green), or ECTV (EV; green) and DNA (blue). Scale bar: 20 µm. (<b>b</b>) Percentage of MAVS and HSP60 colocalization (<span class="html-italic">n</span> = 20). Different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05) between groups.</p> Full article ">Figure 4
<p>Flow cytometry analysis of MAVS protein level in L929 cells at 24 hpi with ECTV (EV). (<b>a</b>) Representative histograms indicate the fluorescence intensity of APC-labeled cells treated with DMSO, Mdivi-1, or CCCP and with or without transfection with poly(I:C)(HMW) LyoVec (pIC LV). A black line is drawn through the center of the histogram of uninfected DMSO-treated cells. (<b>b</b>) Dot plots of unlabeled and APC-labeled cells treated with DMSO. (<b>c</b>) Mean fluorescence intensity (MFI) of APC in cells treated with DMSO, Mdivi-1, or CCCP and with or without transfection with poly(I:C)(HMW) LyoVec.</p> Full article ">Figure 5
<p>Western blot analysis of proteins involved in MAVS-dependent immunity in L929 cells treated with DMSO, Mdivi-1, or CCCP during ECTV infection. (<b>a</b>) Representative Western blots of MDA-5, RIG-I, MAVS, pIRF3, and STING at 2, 10, 18, and 24 h post infection (hpi) with ECTV (EV). (<b>b</b>) Densitometry analysis of MDA-5, RIG-I, MAVS, pIRF3, and STING at 2, 10, 18, and 24 hpi with ECTV. The level of each protein was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The data ARE presented as mean ± standard deviation (SD), with statistical significance indicated as * <span class="html-italic">p</span> ≤ 0.05 AND ** <span class="html-italic">p</span> ≤ 0.01. pIC LV—cells transfected with poly(I:C)(HMW) LyoVec.</p> Full article ">Figure 6
<p>Oligomerization of MAVS protein in L929 cells transfected using poly(I:C)(HMW) LyoVec at 24 hpi with ECTV. (<b>a</b>,<b>b</b>) Uninfected control cells treated with Mdivi-1. Fibroblasts were labeled with specific antibodies against (<b>a</b>) MAVS (green) and pIRF3 (red) or (<b>b</b>) MAVS (red) and Hsp60 (green)—marker of mitochondria. (<b>c</b>) ECTV-infected cells treated with Mdivi-1 and labeled with specific antibodies against MAVS (red). DNA was labeled with DAPI (blue). White arrows indicate MAVS oligomers, the yellow arrow shows viral factory, and the white arrowhead indicates pIRF3 in the nucleus. Scale bar: 20 µm. (<b>d</b>) The bar chart presents the percentage of uninfected control cells with MAVS oligomers. Fibroblasts were treated with DMSO, Mdivi-1, or CCCP.</p> Full article ">Figure 7
<p>Extracellular level of IFN-α (<b>a</b>) and IFN-β (<b>b</b>) produced by L929 at 24 hpi with ECTV (EV). Cells were treated using DMSO, Mdivi-1, or CCCP and/or transfected with poly(I:C)(HMW) LyoVec (pIC LV). ND—non-detected. Different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05) between groups.</p> Full article ">Figure 8
<p>Colocalization of MAVS protein with RIG-I (<b>a</b>,<b>b</b>), MDA-5 (<b>c</b>,<b>d</b>), and STING (<b>e</b>,<b>f</b>) in L929 cells at 18 hpi with ECTV. Cells were treated using DMSO, Mdivi-1, or CCCP and/or transfected with poly(I:C)(HMW) LyoVec. Box plots (<b>a</b>,<b>c</b>,<b>e</b>) indicate the number of PLA dots per cell. Different letters indicate significant differences between groups (<span class="html-italic">p</span> ≤ 0.05). (<b>b</b>,<b>d</b>,<b>f</b>) Representative figures visualize the number of PLA dots per cell in each group. Scale bar: 20 µm.</p> Full article ">Figure 9
<p>Colocalization of MAVS with fission (<b>a</b>) and fusion (<b>b</b>) proteins in L929 at 18 hpi with ECTV. Cells were treated using DMSO, Mdivi-1, or CCCP and/or transfected with poly(I:C)(HMW) LyoVec. Box plots indicate the number of PLA dots per cell. Different letters indicate significant differences between groups (<span class="html-italic">p</span> ≤ 0.05).</p> Full article ">Figure 10
<p>Possible interaction between MAVS and selected proteins. E3—poxviral protein; ER—endoplasmic reticulum; Drp1—dynamin-related protein 1; Fis1—Fission 1 protein; IMM—inner mitochondrial membrane; MAVS—mitochondrial antiviral signaling protein; MDA5—melanoma differentiation-associated protein 5; Mdivi-1—mitochondrial division inhibitor; Mfn1/2—mitofusin 1/2; OMM—outer mitochondrial membrane; Opa1—optic atrophy 1; RIG-I—retinoic acid-inducible gene I; STING—stimulator of interferon genes.</p> Full article ">

Figure 1
<p>The yeast two-hybrid screen identifies PEX19 as an interacting partner of IAV M2. The yeast strain Y2H Gold was co-transformed with the bait plasmid pGBKT7-SC09(H1N1) M2, containing SC09(H1N1) M2 fused to the GAL4-binding domain (BD) in a pGBKT7 vector, together with the prey plasmid pGADT7-PEX19, which encodes PEX19 fused to the Gal4-activation domain (AD). Positive protein–protein interactions are indicated by the growth of blue colonies in the SD/–4/X/A plates in the presence of X-a-Gal. The co-transformation of pGBKT7-p53 encoding the Gal4-BD fused with murine p53 and pGADT7-T encoding the Gal4-AD fused with SV40 large T-antigen served as a positive control. The co-transformation of pGBKT7-Lamin, which encodes the Gal4-BD fused with Lamin and pGADT7-T, served as a negative control. SD/–2: SD/–Leu/–Trp; SD/–4: SD/–Ade/–His/–Leu/–Trp; and SD/–4/X/A: SD/–Ade/–His/–Leu/–Trp/X-a-Gal/AbA.</p> Full article ">Figure 2
<p>IAV M2 protein interacts with host factor PEX19. (<b>a</b>–<b>c</b>) HEK293T cells were co-transfected with the indicated combination of plasmids expressing Myc-tagged PEX19 and Flag-tagged SC09 (H1N1) M2 (<b>a</b>), WSN (H1N1) M2 (<b>b</b>), or AH05 (H5N1) M2 (<b>c</b>). Cell lysates were subjected to immunoprecipitation with mouse anti-Flag or anti-Myc mAb, and the bound proteins were detected by Western blotting. (<b>d</b>) Schematic representation of differences in amino acid sequences of M2. (<b>e</b>) A549 cells were infected with the WSN (H1N1) virus at an MOI of 2 for 24 h. Cell lysates were immunoprecipitated with a mouse anti-PEX19 mAb, and the bound proteins were detected by Western blotting. (<b>f</b>) HEK293T cells were co-transfected with plasmids expressing Myc-PEX19 and GFP or GFP-tagged BM2. Cell lysates were immunoprecipitated with a mouse anti-GFP mAb, and the bound proteins were detected by Western blotting. (<b>g</b>,<b>h</b>) A549 cells were transfected individually or in combination with plasmids expressing Flag-tagged WSN (H1N1) M2 and Myc-tagged PEX19 (<b>g</b>) or were infected with the WSN (H1N1) virus at an MOI of 1 (<b>h</b>). Cells were immunostained with anti-M2 and anti-PEX19 antibodies 24 h after transfection or the indicated timepoints p.i., and were visualized by confocal microscopy. The 3D analysis of images was performed using Image J.</p> Full article ">Figure 3
<p>The cytoplasmic tail domain of M2 and the C terminus of PEX19 are key regions of interactions. (<b>a</b>–<b>c</b>) HEK293T cells were co-transfected with the indicated combinations of plasmids. At 48 h post transfection, cell lysates were immunoprecipitated with a mouse anti-Myc mAb (<b>a</b>,<b>c</b>), or a mouse anti-Flag mAb (<b>b</b>), and the bound proteins were detected by Western blotting with a rabbit anti-GST pAb and a rabbit anti-Myc pAb (<b>a</b>) or with a rabbit anti-Flag pAb and a rabbit anti-Myc pAb (<b>b</b>,<b>c</b>).</p> Full article ">Figure 4
<p>PEX19 inhibits the replication of IAV. (<b>a</b>) A549 cells were transfected with siRNA targeting PEX19 or scrambled siRNA for 48 h. Whole-cell lysates were then analyzed by Western blotting with a mouse anti-PEX19 mAb. The band intensities of PEX19, quantified using ImageJ, were normalized to GAPDH and are expressed as relative ratios compared to RNAimax-treated cells. The cell viability of siRNA-treated A549 cells was measured using the CellTiter-Glo assay. The data are presented as means ± standard deviations for triplicate transfections. (<b>b</b>) PEX19 siRNA1- or scrambled siRNA-transfected A549 cells as in (<b>a</b>) were infected with the WSN/33 (H1N1), AH05 (H5N1), or AH13 (H7N9) virus. At 24 and 48 h p.i., supernatants were titrated for infectious viruses by plaque assays on MDCK cells. (<b>c</b>) The stable overexpression of PEX19 in PEX19-overexpressing cells was confirmed by Western blotting with a rabbit anti-PEX19 pAb. (<b>d</b>) The WSN (H1N1) virus was used to infect the PEX19-overexpressing or control A549 cells at an MOI of 0.01. Supernatants were collected at 24 and 48 h p.i., and virus titers were determined by plaque assays on MDCK cells. (<b>e</b>) A stable PEX19_KO A549 cell line was established by the CRISPR/Cas9 system, and the knockout of PEX19 was confirmed by Western blotting with a rabbit anti-PEX19 pAb. (<b>f</b>) PEX19_KO or control A549 cells were infected with the WSN/33 (H1N1) or AH05 (H5N1) virus. At 24 and 48 h p.i., supernatants were titrated for infectious viruses by plaque assays on MDCK cells. Three independent experiments were performed in (<b>b</b>,<b>d</b>,<b>f</b>), and data are shown as means ± standard deviations for triplicates from a representative experiment. *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01; and ***, <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 5
<p>PEX19 knockdown and IAV infection decrease the pool of peroxisomes in A549 cells. (<b>a</b>) A549 cells were transfected with PEX19 siRNA1 or scrambled siRNA for 24 h and were then subjected to immunofluorescence microscopy analysis using a mouse anti-PMP70 mAb. (<b>b</b>) A549 cells were infected with the WSN (H1N1) virus (MOI = 1) and were then subjected to immunofluorescence microscopy analysis using a mouse anti-PMP70 mAb and a rabbit anti-M2 pAb at 0 and 13 h p.i.</p> Full article ">Figure 6
<p>PEX19 deficiency promotes cell damage in IAV-infected cells by inducing ROS and compromising the ROS-processing function of peroxisomes. (<b>a</b>) HEK293T cells were treated with PEX19 siRNA1 or scrambled siRNA for 12 h, followed by mock infection or infection with the WSN (H1N1) virus (MOI = 1). Bright-field images were acquired under an inverted microscope at 24 h p.i. (<b>b</b>) The viability of HEK293T cells treated as in (<b>a</b>) was analyzed by a CellTiter-Glo assay at 24 h p.i. *, <span class="html-italic">p</span> &lt; 0.05 and ns, not significant. (<b>c</b>) PEX19_KO and control A549 cells were mock-infected or infected with the WSN (H1N1) virus (MOI = 1). Bright-field images were acquired under an inverted microscope at 36 h p.i. (<b>d</b>,<b>e</b>) A549 (<b>d</b>) and HEK293T (<b>e</b>) cells were left untreated or were treated with PEX19 siRNA1 for 12 h, followed by infection with the WSN (H1N1) virus (MOI = 1). The profiling of ROS formation was visualized by fluorescence microscopy with ROS detection reagents at 24 h p.i.</p> Full article ">Figure 7
<p>IAV infection and PEX19 knockout reduce the levels of type III interferons. (<b>a</b>,<b>b</b>) PEX19_KO and control A549 cells were infected with the WSN (H1N1) virus (MOI = 2) for 24 h. The mRNA levels of IFN-α and IFN-β (<b>a</b>) or type III IFNs (<b>b</b>) were determined by RT-qPCR (<span class="html-italic">n</span> = 3). *, <span class="html-italic">p</span> &lt; 0.05; ***, <span class="html-italic">p</span> &lt; 0.001; and ns, not significant.</p> Full article ">Figure 8
<p>The M2 protein disturbs the interactions between PEX19 and peroxisome-associated factors. (<b>a</b>,<b>b</b>) HEK293T cells were co-transfected with plasmids expressing Myc-PEX19, V5-PMP24 (<b>a</b>), or V5-PEX14 (<b>b</b>) and gradually increased amount of Flag-tagged WSN (H1N1) M2. At 24 h post transfection, cell lysates were immunoprecipitated with a mouse anti-Myc mAb, and the bound proteins were Western blotted with a rabbit anti-Flag, anti-Myc, or anti-V5 pAb.</p> Full article ">Figure 9
<p>Schematic diagram showing the role of PEX19 during IAV replication. IAV M2 interferes with the interactions between PEX19 and peroxisomal proteins, which may lead to a decrease in the peroxisome pool during IAV infection. The decrease in peroxisomes during IAV infection causes the accumulation of reactive oxygen species (ROS) and cell damage. Meanwhile, the IAV-induced reduction in peroxisome quantity also compromises the peroxisome MAVS-mediated type III IFN responses, which provides an environment that favors viral propagation.</p> Full article ">

Figure 1
<p>Conidia germination and reduction (%) of the eight strains of <span class="html-italic">Metarhizium anisopliae</span> irradiated with UV-A (<b>A</b>) or UV-A+B (<b>B</b>). ^a,b different literals in the same strain indicate significant differences (<span class="html-italic">p</span> &lt; 0.05) between the control and irradiation treatments.</p> Full article ">Figure 2
<p>Number of colony-forming units (CFU) and reduction (%) in the eight strains of <span class="html-italic">Metarhizium anisopliae</span> under UV-A and UV-A+B irradiation. Different literals in the same strain indicate significant differences (<span class="html-italic">p</span> &lt; 0.05) between the control and irradiation treatments.</p> Full article ">

Figure 1
<p>Regulation of TRIMs in innate immune signaling stimulated by viral PAMPs or mitochondrial DAMPs. When exposed to the cytosol, both viral PAMPs (e.g., viral DNA, dsRNA, etc.) and mitochondrial DAMPs can activate the same immune signal transduction pathways that are extensively regulated by TRIM proteins.</p> Full article ">Figure 2
<p>Regulation of MAVS signaling and stability by TRIM-mediated ubiquitination. MAVS contains 14 lysine residues distributed across its different domains, which include a single CARD domain, a proline-rich region (PRR), and a transmembrane domain (TM). Numbers in red indicate the target residues modified by TRIM-mediated ubiquitination. TRIM44 is a deubiquitinase that can stabilize MAVS by removing K48-linked polyubiquitin chains. TRIM7 is reported to induce K48-linked polyubiquitin chains, but the sites of TRIM7-mediated MAVS ubiquitination were not determined.</p> Full article ">Figure 3
<p>TRIM5-mediated mitophagy regulates immune activation. (<b>Top</b>), schematic of mitophagy pathway. Unwanted or damaged mitochondria and associated immunostimulatory molecules (e.g., mtDNA) or signaling factors (e.g., MAVS) are sequestered within an autophagosome and subsequently degraded following autophagosome/lysosome fusion. Elimination of mtDAMPs or signaling factors can prevent or attenuate mitochondria-based immune activation. (<b>Middle</b>), mechanism of TRIM5-mediated mitophagy. Mitochondrial damage activates TRIM5’s ubiquitin ligase activity. Active TRIM5 associates with TBK1 via shared interactions with Sequestosome-like receptors (SLRs, e.g., Optineurin, NDP52, TAX1BP1, p62, and NBR1). TRIM5 then ubiquitinates TBK1, allowing for the assembly of TRIM5-SLR-TBK1 complexes on damaged mitochondria in a feed-forward manner. TBK1 is concentrated in these complexes, enabling it to become activated by <span class="html-italic">trans</span>-autophosphorylation and carry out mitophagy functions. (<b>Bottom</b>), reported roles of TBK1 in mitophagy. TRIM5 promotes TBK1 activity in mitophagy. TBK1 can then phosphorylate SLRs, Beclin 1, and RAB7A. Phosphorylation of SLRs increases their ability to bind to ubiquitin chains on damaged mitochondria and increases the ability of SLRs to associate with other autophagy factors on mitochondria. Beclin 1 is a component of the class III phosphatidylinositol 3-kinase (PI3K) complex, and its phosphorylation at Ser15 enhances the complex’s activity to generate PI3P, a requirement for the recruitment of downstream autophagy factors to the autophagosome initiation site. RAB7A is essential for mitophagy. Its activation by TBK1 is important for the recruitment of the pro-mitophagy protein Pacer into Beclin 1 complexes, which takes the place of the autophagy-inhibitory Rubicon protein. RAB7A also recruits ATG9A vesicles to the autophagy initiation site in a manner requiring TBK1.</p> Full article ">

Figure 1
<p>CSFV infection significantly upregulated TRAF1 in TRAF family. (<b>A</b>) CSFV replication in PK-15 cells. RT-qPCR analyzed CSFV gRNA levels at 12, 24, 36, and 48 hpi in CSFV-infected (MOI = 1) PK-15 cells. (<b>B</b>) The mRNA levels of TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, and TRAF6 proteins were detected in CSFV-infected (MOI = 1) PK-15 cells by RT-qPCR analysis at 12, 24, 36, and 48 hpi. (<b>C</b>) The protein expression levels of CSFV E2 and TRAF1 in CSFV-infected (MOI = 1) PK-15 cells were assessed by WB analysis at 12, 24, 36, and 48 hpi. (<b>D</b>) The gray values of TRAF1 band from WB were measured by comparing them with β-actin using ImageJ software version 1.8.0. (<b>E</b>) Pearson analysis of the CSFV E2 and TRAF1 protein bands. All statistical analysis results were derived from comparisons between the experimental group and the Mock group (* <span class="html-italic">p</span> &lt; 0.05, **** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 2
<p>TRAF1 expression positively correlated with the RIG-I/MAVS pathway during CSFV infection. (<b>A</b>–<b>F</b>) The CSFV gRNA and the mRNA levels of TRAF1, RIG-I, MAVS, IRF1, and ISG15 proteins were detected in PK-15 cells infected with the CSFV Shimen strain (MOI = 1) at 3, 6, 9, 12, 24, 36, 48, and 72 hpi by RT-qPCR analysis. (<b>G</b>) The protein levels of CSFV E2, TRAF1, RIG-I, MAVS, IRF1, and ISG15 were detected in PK-15 cells infected with the CSFV Shimen strain (MOI = 1) at 3, 6, 9, 12, 24, 36, 48, and 72 hpi by WB analysis. (<b>H</b>–<b>M</b>). (<b>N</b>) Pearson analysis of the CSFV E2, TRAF1, RIG-I, MAVS, IRF1, and ISG15 protein bands. The gray value of each target band from WB was measured by comparing them to β-actin using ImageJ software version 1.8.0. All statistical analysis results were derived from comparisons between the experimental group and the Mock group (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 3
<p>TRAF1 interacts with MAVS. (<b>A</b>) The expression of TRAF1 and MAVS proteins was analyzed in HEK 293T cells through co-transfection of 1 µg of pcDNA3.0-TRAF1^-Flag and pCMV-MAVS^-Myc vectors, followed by WB analysis at 36 hpi. A portion of the cell extract was utilized for assessing TRAF1 and MAVS protein expression in total cells (lane 3), while the remainder was subjected to Co-IP analysis using anti-TRAF1 antibody (lane 2) or normal mouse IgG (lane 1). (<b>B</b>) Most experimental procedures were the same as above, except that the remainder was subjected to Co-IP analysis using anti-MAVS antibody (lane 2) or normal mouse IgG (lane 1). (<b>C</b>) Purified GST and GST-MAVS fusion proteins from the recombinant prokaryotic expression systems were incubated with pcDNA3.0-TRAF1^-Flag transfected HEK-293T cell lysate, TRAF1, MAVS, and GST control in Pull-down samples were detected by WB analysis using specific antibodies. (<b>D</b>) The interaction between TRAF1 and MAVS was observed in the cytoplasm. The co-transfection of 1 µg of pcDNA3.0-TRAF1^-Flag and pCMV-MAVS^-Myc vectors into PK-15 cells was analyzed using indirect immunofluorescence at 36 hpi.</p> Full article ">Figure 4
<p>TRAF1 downregulation by siRNA-enhanced CSFV replication. (<b>A</b>–<b>F</b>) The CSFV gRNA and the mRNA levels of TRAF1, RIG-I, MAVS, IRF1, and ISG15 proteins were detected in different kinds of PK-15 cells, such as infection of CSFV Shimen strain at an MOI of 1.0 as a positive control, temporary transfection of 100 nM TRAF1 siRNA 6 h then infection of CSFV Shimen strain, temporary transfection of 100 nM control siRNA 6 h then infection of CSFV Shimen strain as a negative control by RT-qPCR analysis at 24, 36, and 48 hpi. (<b>G</b>) The experimental treatment was consistent with the (<b>A</b>) and WB analysis at 24, 36, and 48 hpi. (<b>H</b>–<b>M</b>) The gray value of each target band from WB was measured by comparing them to β-actin using ImageJ software version 1.8.0. (<b>N</b>) The experimental treatment was consistent with (<b>A</b>). Culture supernatant from PK-15 cells subjected to different treatments was collected, and the viral titer was tested by IFA at 12, 24, 36, 48, and 60 hpi. All statistical analysis results were derived from comparisons between the experimental group and the Mock group (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 5
<p>TRAF1 overexpression inhibited CSFV replication by positive regulation of IRF1 and ISG15. (<b>A</b>–<b>F</b>) The CSFV gRNA and mRNA levels of TRAF1, RIG-I, MAVS, IRF1, and ISG15 proteins were detected in different kinds of PK-15 cells, such as infection of CSFV Shimen strain at an MOI of 1.0 as a positive control, temporary transfection of 1.0 µg pcDNA3.0-TRAF1^-Flag vector 12 h then infection of CSFV Shimen strain, temporary transfection of 1.0 µg empty vector 12 h then infection of CSFV Shimen strain as negative control by RT-qPCR analysis at 24, 36, and 48 hpi. (<b>G</b>) The experimental treatment was consistent with (<b>A</b>) and the WB analysis at 24, 36, and 48 hpi. (<b>H</b>–<b>M</b>) The gray value of each target band from WB was measured by comparing them to β-actin using ImageJ software version 1.8.0. (<b>N</b>) The experimental treatment was consistent with (<b>A</b>). Culture supernatant from PK-15 cells subjected to different treatments was collected, and the viral titer was tested by IFA at 12, 24, 36, 48, and 60 hpi. All statistical analysis results were derived from comparisons between the experimental group and the Mock group (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 6
<p>TRAF1 participated in the RIG-I/MAVS pathway against CSFV infection. RIG-I identifies short plus-stranded RNA of CSFV. After activation of RIG-I, its CARD region merges with the CARD region of MAVS to activate downstream signal molecules. TRAF1 binds to the PRR domain of MAVS to promote IRF1 activation and subsequent stimulation of ISG15 expression.</p> Full article ">

Figure 1
<p>Overall process diagram for the approach of the research.</p> Full article ">Figure 2
<p>EMG-USB2+ multichannel amplifier by OTBioelecttronica [<a href="#B12-applsci-14-05784" class="html-bibr">12</a>].</p> Full article ">Figure 3
<p>GRABMyo dataset setup for electrodes on the forearm [<a href="#B12-applsci-14-05784" class="html-bibr">12</a>].</p> Full article ">Figure 4
<p>GRABMyo dataset folder and file exploration.</p> Full article ">Figure 5
<p>GRABMyo dataset flowchart for signal acquisition.</p> Full article ">Figure 6
<p>Available gesture list and selected research gestures.</p> Full article ">Figure 7
<p>Raw EMG signals captured from the wrist in the time domain.</p> Full article ">Figure 8
<p>Raw EMG signals captured from the forearm in the time domain.</p> Full article ">Figure 9
<p>Power Spectrum Density (PSD) analysis of raw EMG signals captured from the wrist in the frequency domain.</p> Full article ">Figure 10
<p>Power Spectrum Density (PSD) analysis of raw EMG signals captured from the forearm in the frequency domain.</p> Full article ">Figure 11
<p>PSD analysis of EMG signals captured from the wrist after average referencing.</p> Full article ">Figure 12
<p>PSD analysis of EMG signals captured from the forearm after average referencing.</p> Full article ">Figure 13
<p>PSD analysis of EMG signals captured from the wrist after the application of a Band-pass filter with a range of 10 Hz to 450 Hz.</p> Full article ">Figure 14
<p>PSD analysis of EMG signals captured from the wrist after the application of the 60 Hz Notch filter.</p> Full article ">Figure 15
<p>PSD analysis post filtering of EMG signals captured from the wrist.</p> Full article ">Figure 16
<p>PSD analysis post filtering of EMG signals captured from the forearm.</p> Full article ">Figure 17
<p>Discrete Wavelet Transform decomposition, block diagram form, with 4 levels of decomposition.</p> Full article ">Figure 18
<p>Discrete Wavelet Transform decomposition with 6 levels of decomposition.</p> Full article ">Figure 19
<p>Discrete Wavelet Transform decomposition, analysis form, with 4 levels of decomposition.</p> Full article ">Figure 20
<p>Comparison of signal distribution before and after Linear Discriminant Analysis (LDA) for 2 classes.</p> Full article ">Figure 21
<p>Visualization of K-Nearest Neighbor classes and classification method.</p> Full article ">Figure 22
<p>Visualization of Support Vector Machine classification methods [<a href="#B11-applsci-14-05784" class="html-bibr">11</a>].</p> Full article ">Figure 23
<p>Final process diagram illustrating the results of the research.</p> Full article ">Figure 24
<p>Scatter plot illustrating the classification of 215 EMG samples using the PCA–SVM model.</p> Full article ">Figure 25
<p>Scatter plot illustrating the classification of 215 EMG samples using the LDA–SVM model.</p> Full article ">Figure 26
<p>Methodology for processing the three sessions from the GRABMyo dataset.</p> Full article ">Figure 27
<p>Comparison of scatter plots between one session and the complete dataset of three sessions of data.</p> Full article ">Figure 28
<p>Confusion matrix for the SVM model using data from the complete dataset of three sessions of data.</p> Full article ">Figure 29
<p>Performance metrics for KNN, NB, and SVM classifiers using data from one session.</p> Full article ">Figure 30
<p>Comparing performance metrics for SVM classifiers utilizing data from one session and the complete dataset of three sessions of data.</p> Full article ">Figure 31
<p>Diagram depicting the schematic pathway of nerves extending from the brain to the hand [<a href="#B30-applsci-14-05784" class="html-bibr">30</a>,<a href="#B31-applsci-14-05784" class="html-bibr">31</a>,<a href="#B32-applsci-14-05784" class="html-bibr">32</a>,<a href="#B33-applsci-14-05784" class="html-bibr">33</a>].</p> Full article ">

Figure 1
<p>Auxiliary vehicle component module.</p> Full article ">Figure 2
<p>Accelerometer peak point extraction.</p> Full article ">Figure 3
<p>Key point recognition of motion capture system based on OpenPose.</p> Full article ">Figure 4
<p>Six-axis force sensor.</p> Full article ">Figure 5
<p>Knee immobilization device.</p> Full article ">Figure 6
<p>Schematic of CNN model.</p> Full article ">Figure 7
<p>Normal and abnormal walking gait cycles. ** (0.001 ≤ <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 8
<p>Mean and peak values of left and right leg accelerometers. (<b>a</b>) Average acceleration. (<b>b</b>) Peak acceleration. * (0.01 ≤ <span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 9
<p>Scatterplot of left- and right-hand forces. (<b>a</b>) Scatter plot of left-hand force. (<b>b</b>) Scatter plot of right-hand force.</p> Full article ">Figure 10
<p>Model accuracy.</p> Full article ">Figure 11
<p>Weight of important features in the classification task.</p> Full article ">

Figure 1
<p>Schematic representation of the proposed integrated system.</p> Full article ">Figure 2
<p>Thermo-environ-economic modeling framework.</p> Full article ">Figure 3
<p>Computational flowchart of the objective function.</p> Full article ">Figure 4
<p>Geographical location of Kish Island.</p> Full article ">Figure 5
<p>The average monthly solar radiation in Kish Island.</p> Full article ">Figure 6
<p>The average monthly wind speed data in Kish Island.</p> Full article ">Figure 7
<p>The average monthly ambient temperature data in Kish Island.</p> Full article ">Figure 8
<p>Exergy destruction of the subsystems.</p> Full article ">Figure 9
<p>Effects of the subsystems on MTAC of the integrated system.</p> Full article ">Figure 10
<p>Normalized cost rates of each subsystem.</p> Full article ">Figure 11
<p>Normalize total annual cost, exergy destruction cost rate, and environmental cost rate.</p> Full article ">Figure 12
<p>The impact of the unit price of fuel on MTAC in the integrated system.</p> Full article ">Figure 13
<p>Effect of solar radiation on MTAC in the integrated system.</p> Full article ">Figure 14
<p>Effect of the wind speed on MTAC in the integrated system.</p> Full article ">

Figure 1
<p>Artist’s rendition of the Mars Sample Return (MSR) campaign’s major components. In the lower left is the Perseverance Mars rover that successfully landed on Mars on 18 February 2021 and is currently acquiring up to 38 samples from the Mars surface and atmosphere. In the lower right is shown the Sample Retrieval Lander (SRL) which delivers to the Martian surface the Sample Transfer Arm and the Mars Ascent Vehicle (MAV) which is shown launching the Orbiting Sample (OS) container into Mars orbit. In the far left one of two SRL helicopters is shown which will be used as backup to retrieve cached sample tubes in case Perseverance experiences a failure prior to the arrival of the SRL. At the top is shown the Earth Return Orbiter (ERO) which will rendezvous with the OS container and capture it using the Capture, Containment and Return System (CCRS) that is included as part of its payload. After successfully capturing and processing the OS for Earth return, the ERO will leave Mars orbit and deliver the samples back to Earth.</p> Full article ">Figure 2
<p>Conceptual computer aided design (CAD) views of the Orbiting Sample (OS) container as it appears in Mars orbit after release from the MAV (Mars Ascent Vehicle) showing an oblique view of the base endcap (<b>left</b>) and the lid endcap (<b>right</b>).</p> Full article ">Figure 3
<p>Conceptual computer aided design (CAD) views of the ERO (Earth Return Orbiter) Capture, Containment and Return System (CCRS) payload. The coordinate system axes shown in the lower right have their origins offset from the actual (0, 0, 0) vertex for clarity.</p> Full article ">Figure 4
<p>Simplified internal view of a portion of the CCRS (capture enclosure) showing the location of the vision system with respect to the capture cone (pale green) and the post-capture OS (off white). Brackets mount the two vision system cameras and illumination modules to two different CCRS bulkheads. Views of the OS and apertures for illumination are provided by three portholes in the capture cone cylinder. Each camera has a dedicated porthole while the two illumination modules share one large porthole. The colors (black) shown for the vision system components are true-to-life whereas the colors applied to all the other components are for figure clarity. The orientation mechanism is not shown to make the post-capture OS position clearly visible.</p> Full article ">Figure 5
<p>Electrical block diagram of the vision system camera head.</p> Full article ">Figure 6
<p>Diagram of the OS depth-of-field volume with respect to the nominal camera locations and volume allocations. The 461 mm distance shown is from the vertex of the first lens surface to the first plane in the depth-of-field cylindrical volume. The best focus is set for a position 493 mm from the first lens vertex. MTF curves at the field positions illustrated above are provided in <a href="#aerospace-11-00456-f008" class="html-fig">Figure 8</a>.</p> Full article ">Figure 7
<p>Layout and ray trace of the vision system camera lens.</p> Full article ">Figure 8
<p>The minimum required and predicted camera MTF performance at the center and corners of the camera detector active imaging area for flat planes located at the near and far planes of the depth-of-field requirement volume, as depicted in <a href="#aerospace-11-00456-f006" class="html-fig">Figure 6</a>. The plot ends at a spatial frequency of 454.5 mm⁻¹ which is equivalent to the highest sampling frequency of the detector. The prediction includes the optical as well as the detector MTF.</p> Full article ">Figure 9
<p>Ray trace model calculations of the detector sampling (<b>top row</b>) and the MTF values corresponding to two cycles across 3 mm (<b>bottom row</b>) at the two extreme depth-of-field planes for the selected vison system cameras. The calculations include optical as well as detector MTF effects. The results indicate that the cameras will have sufficient resolution and contrast to discriminate between features on the OS endcaps.</p> Full article ">Figure 10
<p>Image of a previously constructed Malin Space Science Systems (MSSS) ECAM camera that is identical to the CCRS vision system camera design.</p> Full article ">Figure 11
<p>Simplified block diagram of the interface between the CCRS avionics assembly and the capture enclosure camera head. The key ground path is shown, including isolation of the camera electronics from chassis ground, which is connected to both the CCRS structure and the internal SpaceWire shields. The SpaceWire strobe signals are not explicitly shown (Acronyms: 1. TSP Twisted, shielded pair; 2. LVPC Low-Voltage Power Converter; 3. RTN Return; 4. JA Jettison Avionics; 5. CCRS Capture, Containment Return System; 6. SpW Spacewire; 7. AD590 Analog Devices 590 Temperature Sensor).</p> Full article ">Figure 12
<p>The camera head ground plane connected to a +5 V return through a Ferrite bead and isolated from the chassis ground by 11 MΩ resistance (dual 22 MΩ resistors in parallel). Chassis grounded to the CCRS structure. Chassis connected to SpaceWire outer shield and power inner shield.</p> Full article ">Figure 13
<p>The camera 15-pin Micro-D Socket (M83513/01-BN) pinout shows the chassis ground connected to the power twisted pair harness internal shields; the chassis ground connected to the SpaceWire bundle shield and to strobe input and output twisted pair shields and data output twisted pair shield; data input twisted pair shield left isolated; Pin 3 not connected through harness following Spacewire Type A standard and temperature sensor also not connected through harness.</p> Full article ">Figure 14
<p>LED variation of voltage with temperature from −70 °C to 65 °C at a constant current of 300 mA for LXZ1-4070 LED manufactured by Lumileds (San Jose, CA, USA).</p> Full article ">Figure 15
<p>Changes in LED illuminance with current sweeps ranging from 10 mA to either 500 mA or 1000 mA across a temperature range from −70 °C to +65 °C.</p> Full article ">Figure 16
<p>Variation of LED light efficiency versus current at 55 °C for the least efficient LED among the six LEDs tested. This represents a worst-case scenario estimation of light power, thereby providing insight into the maximum potential thermal dissipation for our illumination system.</p> Full article ">Figure 17
<p>Pre- and post-radiation test optical characterization for LEDs exposed to various radiation dosages. No discernible changes were observed in the measured LED illumination patterns (<b>a</b>). The illuminance percent change data (<b>b</b>) indicate that optical degradation due to radiation exposure was within the measurement noise—not exceeding 3% even under the most pessimistic assumptions.</p> Full article ">Figure 18
<p>Electrical diagram for a single illumination module, constructed from two independent circuits. Each circuit contains seven LEDs (LXZ1-4070) and three resistors (2010 size, 800 mW Power Rating, sourced from Vishay Intertechnology).</p> Full article ">Figure 19
<p>Front and back surfaces of the illumination system PCB design.</p> Full article ">Figure 20
<p>Illustration of the illumination module illumination areas over which the illuminance and uniformity requirements are to be met to ensure visibility of the OS endcaps (bottom). The illumination module baffle was designed using the near requirement area (located 677 mm away from the LED emission area) due to its being the most constraining. An oversized rectangle surrounding the 14 LEDs was used to represent the LED emission area in the design. Additional clearance to the LED emission area was added to one side of the rectangle to eliminate mechanical interference between one of the illumination modules’ baffle petals and the CCRS capture cone.</p> Full article ">Figure 21
<p>Illumination module baffle designs for the −Z (<b>left</b>) and +Z (<b>right</b>) illumination modules that maximize the light uniformity in the required illumination areas but minimize the light that strikes other areas. Dimensions indicate the distances between the LED emission plane and the baffle aperture minima and maxima dimensions.</p> Full article ">Figure 22
<p>Mechanical design with coating specifications, showing two unique baffle designs and one common base design for each module.</p> Full article ">Figure 23
<p>Overall dimensions of the illumination modules.</p> Full article ">Figure 24
<p>Thermal model simulation results showing the LED junction and resistor temperatures meet the component specifications with margin.</p> Full article ">Figure 25
<p>Graphical representation of the EDU illumination module thermal vacuum test temperature profile. Section A is the initial ambient condition. Section B is the ramp up to the hot survival temperature. Section C is the hot survival test. Sections D and E are the hot operational and ramp-down operational tests. Section F is the cold survivability test. Sections G and H are the cold operational and ramp-up tests and Section I is the final ambient test condition.</p> Full article ">Figure 26
<p>EDU illumination module thermal vacuum test set-up prior to chamber door closure. Six test articles are visible with views of the PCBs looking along the baffle interiors.</p> Full article ">Figure 27
<p>EDU illumination module vibration test arrangement. This set up allowed two illumination modules to be tested at the same time.</p> Full article ">Figure 28
<p>Illustration of the changes made to the initial illumination module design based on the lessons learned from the EDU fabrication and test activities.</p> Full article ">Figure 29
<p>Image of two of the EDU illumination modules constructed from the flight model design. Although the −Z module (<b>left</b>) and the +Z module (<b>right</b>) baffles appear to be mirror copies, they are not exact mirror matches due to the use of a common module base. The small hole visible near the PCB in each module is for EDU test instrumentation and will not be present in the flight models (Photo Credit: Katherine M. Mellos).</p> Full article ">Figure 30
<p>Illustration of the EDU illumination module optical test set-up. The diagram on the left shows the alignment approach used to minimize the tip/tilt between the illumination module LED emission plane surface normal and the optical measurement plane. The diagram on the right shows the optical test dimensions. Note the test distance is 1 mm different from the distance originally used to design the baffle due to late changes in the illumination module design and CCRS accommodations.</p> Full article ">Figure 31
<p>EDU illumination module optical test results at both the near (<b>left</b>) and far (<b>right</b>) evaluation planes for the two +Z illumination modules and two −Z illumination modules. In all eight cases, the maximum and minimum illuminance requirements are met over the required area (red circle). The illuminance variation requirement is also met over most of the required area (red circle) for all eight EDU modules except for areas ~5 mm × 12 mm in size near the edge of the requirement zone where the variation is 5% or more. A subsequent investigation has found that this non-compliance was mistakenly designed into the baffles due to a misunderstanding of the SolidWorks “Lofted Cut” feature. Note that the low-level periodic variation running left to right in the illuminance uniformity results are not present in the optical patterns but are caused by periodic noise in the photometer.</p> Full article ">Figure 32
<p>EDU illuminance results over the required area (red circle) when the primary 7 LEDs in each illumination module are powered on (<b>left</b>) and all 14 LEDs in each illumination module are powered on (<b>right</b>). With 7 LEDs in each illumination module powered on, the illuminance doubles when compared to only one module being powered on (see <a href="#aerospace-11-00456-f031" class="html-fig">Figure 31</a>). With 14 LEDs in each illumination module powered on, the illuminance doubles when compared to 7 LEDs being powered in each illumination module. For the case with all LEDs powered on, the small areas at the near plane where the illuminance uniformity is &gt;5% appear on both sides of the required area while the individual areas of non-uniformity shrink. Although the nominal vision system operations concept assumes only 7 LEDs in each illumination module are powered on, for operational efficiency the CCRS operations concept currently assumes all LEDs (28) will be powered on during imaging.</p> Full article ">Figure 33
<p>BRDF measurements of CCRS surface treatments relevant to the vision system at 440 nm (<b>left column</b>), 550 nm (<b>middle column</b>) and 700 nm (<b>right column</b>) for (<b>a</b>) Ceranovis pre-friction test, (<b>b</b>) Ceranovis post-friction test, (<b>c</b>) aluminum 6061 with Teflon coating and (<b>d</b>) aluminum 7075 with Teflon coating.</p> Full article ">Figure 34
<p>Vison system surface treatment BRDF measurements of (<b>a</b>) 3D-printed material at 440 nm (<b>left</b>), 550 nm (<b>center</b>) and 700 nm (<b>right</b>) for the OS endcap surrogates, (<b>b</b>) 550 nm measurements of bead-blasted aluminum with clear anodize at 20 psi (<b>left</b>), 30 psi (<b>center</b>) and 40 psi (<b>right</b>), (<b>c</b>) 30 psi bead-blasted aluminum with clear anodize at 440 nm (<b>left</b>), 550 nm (<b>center</b>) and 700 nm (<b>right</b>) and (<b>d</b>) black anodized aluminum at 440 nm (<b>left</b>), 550 nm (<b>center</b>) and 700 nm (<b>right</b>).</p> Full article ">Figure 35
<p>Vision system laboratory testbed utilizing commercially available off-the-shelf (COTS) cameras, engineering development unit (EDU) illumination modules and 3D-printed representations of the CCRS capture cone, orientation mechanism and both OS container endcaps (Photo Credit: Katherine M. Mellos).</p> Full article ">Figure 36
<p>MTF of the lab testbed lens (orange), the flight camera lens (blue) and the minimum allowable vision system camera optics (red).</p> Full article ">Figure 37
<p>BRDF comparison for a variety of incidence angles (AOI) of the two laboratory testbed OS surface finishes (3D resin and 3D metallic paint) to the two OS surface finishes (CN145 Li-Doped and Req. limit) used in the non-sequential ray trace model. The 3D resin (<b>a</b>) represents the current OS surface finish in the testbed and matches well to the Ceranovis BRDF modeled in the non-sequential model except for the specular peak caused via friction testing. The metallic paint (<b>b</b>) represents the worst-case OS surface finish currently allowed by OS requirements. It agrees well with the OS BRDF requirement limit, particularly near the specular peaks.</p> Full article ">Figure 38
<p>System-level images from the −Z camera position in the laboratory testbed, showing the vision system baseline performance for the lab surrogate of the current OS surface finish. The right column shows the same images reporting calibrated luminance values. Red indicates the areas in the image below the 7.8 candela/m2 OS luminance requirement. No areas on the OS are below the luminance requirement.</p> Full article ">Figure 39
<p>System-level images from the −Z camera position laboratory testbed, showing the baseline vision system performance for an OS with the most specularly reflective surface finish allowed by requirements. The right column shows the same images reporting calibrated luminance values. Red indicates the areas in the image below the 7.8 candela/m2 OS luminance requirement. Except for a few small points, the majority of the OS surface meets the luminance requirement.</p> Full article ">Figure 40
<p>FRED BRDF model fit at various angles of incidence (AOI) to the measured data for the Ceranovis-145 with Li-Doped sealant after going through surface friction testing.</p> Full article ">Figure 41
<p>View of the CCRS vision system non-sequential ray trace model components. It includes both cameras, both illumination modules, the capture cone with porthole strengthening members, the major portions of the orientation mechanism and the OS.</p> Full article ">Figure 42
<p>View of the CCRS vision system non-sequential ray trace model components looking from the capture side along the CCRS Y-axis. Viewable items include both cameras, both illumination modules, the capture cone interior, one OS endcap and small portions of the orientation mechanism.</p> Full article ">Figure 43
<p>Comparison of the OS base luminance results from the laboratory measurements and the FRED non-sequential ray trace model prediction. Agreement between the two results is typically ~10%, consistent with our photometer calibration uncertainty and our ability to measure and model the OS BRDF. Luminance values shown in the table above are region averages calculated within square windows equivalent to a 28 × 28 pixel area on the flight vision system camera detector.</p> Full article ">Figure 44
<p>Four nominal vision system imaging performance predictions for the OS lid (<b>top row</b>) and OS base (<b>bottom row</b>) based on the laboratory testbed results for the +Z camera (<b>left column</b>) and −Z camera (<b>right column</b>) with the primary LED circuits in both illumination modules providing illumination.</p> Full article ">Figure 45
<p>Eight nominal vision system performance predictions for the OS lid (<b>top row</b>) and OS base (<b>bottom row</b>) based on non-sequential ray trace modeling in FRED. Left to right: +Z camera, primary circuit LEDs; +Z camera, redundant circuit LEDs; −Z camera, primary circuit LEDs and −Z camera, redundant circuit LEDs.</p> Full article ">Figure 46
<p>Eight worst-case vision system performance predictions for the OS lid (<b>top row</b>) and OS base (<b>bottom row</b>) based on non-sequential ray trace modeling in FRED. The worst-case lens contamination; OS surface treatment; OS position and orientation; lens performance and detector noise are added to the nominal conditions to create the images. Left to right: +Z camera, primary circuit LEDs; +Z camera, redundant circuit LEDs; −Z camera, primary circuit LEDs; −Z camera, redundant circuit LEDs.</p> Full article ">Figure 47
<p>Four nominal vision system performance predictions for the OS lid (<b>top row</b>) and OS base (<b>bottom row</b>) based on non-sequential ray trace modeling in FRED when all 28 vision system LEDs are powered on. +Z camera images are shown in the left column. −Z camera images are shown in the right column.</p> Full article ">Figure 48
<p>The receiver operating characteristic (ROC) curves for the trained fully connected network (FCN) and convolutional neural network (CNN) models, which illustrates the false positive rate at different decision thresholds. ROC curves that stay close to the top left corner indicate better performance while the dashed line corresponds to random (50/50) classification.</p> Full article ">Figure 49
<p>Examples of misidentified images and the corresponding probabilities calculated by each neural network model for a decision (identification) threshold set to <span class="html-italic">p</span> &gt; 0.5. (<b>Left</b>) A random sample of 9 test images from the 38 that were misidentified via the FCN model. Several images that are easily identified by humans are confidently assigned the incorrect label via the model; for example, the bottom middle image is identified as a base endcap with an 89% probability. (<b>Right</b>) The three test images that were misidentified via the CNN model. These would also be challenging for a human observer to identify.</p> Full article ">Figure 50
<p>The impact of increasing Gaussian blur widths on the confidence of the CNN model. Different levels of blurring were applied to an image of the lid endcap, which is outlined in green on the rightmost panel. All units are in image space. (<b>Left</b>) Probabilities generated via the CNN for different Gaussian convolution kernel widths. Images that fall below the decision threshold of 0.5 are colored pink. Bluer points correspond to a higher confidence in identification. (<b>Middle top</b>) The Gaussian convolution kernels, which are effectively PSFs, next to the expected PSF of the vision system cameras (labeled “Malin PSF”). The curves are color-coded to match the points—or probabilities—shown to the left. (<b>Middle bottom</b>) The MTF for each of the blurring cases, which are the modulus of the Fourier transform of the PSFs. (<b>Right</b>) Five levels of blurring applied to the images of the lid and the base. The top row corresponds to the nominal vision system camera PSF.</p> Full article ">Figure 51
<p>Summary of the vision system camera PCB pre-population inspection process.</p> Full article ">Figure 52
<p>Summary flow diagram of the illumination system printed circuit board (PCB) flight screening and illumination module qualification plans.</p> Full article ">