210 research outputs found
Effects of eliminating N-glycosylation sequons on WHc functions.
No full textThe WHV replicon construct expressing the T77E, T92N, or T77E/T92N WHc mutant, or WT WHc was transfected into WC3 or Huh7 cells. (A) The assembled capsids (top) and packaged pgRNA (middle) were detected by the C33 anti-HBc/WHc mAb and anti-sense WHV RNA probe, respectively, following the resolution of cytoplasmic lysates from the transfected WC3 cells by NAGE and transfer to nitrocellulose membrane. Levels of WHc proteins (bottom) were measured by western blot using 19C18 anti-HBc/WHc mAb after SDS-PAGE. Capsid assembly efficiency (B) was determined by normalizing the levels of capsids to those of total WHc protein, and pgRNA packaging efficiency (C) was determined by normalizing the levels of pgRNA to those of capsids, with the efficiencies of WT WHc set to 1.0. (D)-(F) Cytoplasmic lysates from WHV replicon transfected Huh7 cells were analyzed for capsid assembly and pgRNA packaging, as described for WC3 cells. (G) WHV core DNA was released from the NCs of cytoplasmic lysate by SDS-proteinase K treatment and detected by Southern blot analysis. Due to the non-detectable WHV rcDNA in transfected WC3 cells, (rcDNA) denotes for the expected position of WHV rcDNA. WHV PF-DNA was extracted from the transfected WC3 cells by Hirt extraction. The extracted DNA was treated with Dpn I plus the exonucleases I and III (Exo I & III) to remove all DNA with free 3’ ends. The ssDNA synthesis efficiency (H) was determined by normalizing the levels of ssDNA to those of pgRNA in (A) with the efficiency of WT WHc set to 1.0. Similarly, core DNA and PF-DNA from WHV replicon transfected Huh7 cells were analyzed (I) and the ssDNA synthesis efficiency (J) was determined by normalizing the levels of ssDNA to those of pgRNA in (D) with the efficiency of WT WHc set to 1.0. The cccDNA formation efficiency (K) was determined by normalizing the levels of cccDNA in (I) to those of rcDNA with the efficiency from WT WHc set to 1.0. Data is shown as mean ± SD. Two-tailed unpaired Student’s t test was used to compare the difference of each data set versus WT WHc (*, p p p (TIF)</p
Effects of eliminating N-glycosylation sequons on WHc functions.
No full textThe WHV replicon construct expressing the T77E, T92N, or T77E/T92N WHc mutants, or WT WHc was transfected into HpeG2 or WCH-17 cells. (A) The assembled capsids (top) and packaged pgRNA (middle) were detected by the C33 anti-HBc/WHc mAb and anti-sense WHV RNA probe, respectively, following the resolution of cytoplasmic lysates from the transfected HepG2 cells by native agarose gel electrophoresis (NAGE) and transfer to nitrocellulose membrane. Levels of WHc proteins (bottom) were measured by western blot using 19C18 anti-HBc/WHc mAb after SDS-PAGE. Capsid assembly efficiency (B) was determined by normalizing the levels of capsids to those of total WHc protein, and pgRNA packaging efficiency (C) was determined by normalizing the levels of pgRNA to those of capsids, with the efficiencies of WT WHc set to 1.0. (D)-(F) Cytoplasmic lysates from WHV replicon transfected WCH-17 cells were analyzed for capsid assembly and pgRNA packaging, as described for HepG2 cells. (G) WHV core DNA was released from the NCs of cytoplasmic lysate by SDS-proteinase K treatment and detected by Southern blot analysis. WHV PF-DNA was extracted from the transfected cells by the Hirt extraction method. The extracted DNA was treated with Dpn I plus the exonucleases I and III (Exo I & III) to remove all DNA with free 3’ ends. The ssDNA synthesis efficiency (H) was determined by normalizing the levels of ssDNA to those of pgRNA in (A), and the cccDNA formation efficiency (I) was determined by normalizing the levels of cccDNA to those of rcDNA in (G), with the efficiencies of WT WHc set to 1.0. Similarly, core DNA and PF-DNA from WHV replicon transfected WCH-17 cells were analyzed (J)-(L). Data is shown as mean ± SD. Two-tailed unpaired Student’s t test was used to compare the difference of each dataset versus WT WHc (*, p p p < 0.001). Ca, capsid; pgRNA, pregenomic RNA; WHc, WHV core protein; ssDNA, single-strand DNA; rcDNA, relaxed circular DNA; cccDNA, covalently closed circular DNA; cM-DNA, closed minus strand DNA.</p
Effects of HBc or WHc on supporting HBV virion secretion.
No full text(A) concentrated culture supernatant of HepG2 cells co-transfected with WT HBc or WHc and HBV-C(-) (genotype A or D) was analyzed by agarose gel electrophoresis. HBV DNA in virions or naked capsids was detected by a HBV DNA probe. Concentrated culture supernatant of HepG2 cells co-transfected with WT HBc or WHc along with HBV-C(-) (genotype D) was fractionated by CsCl density gradient ultracentrifugation. The fractions from WT HBc (B) or WT WHc (C) co-transfected culture supernatant was analyzed by agarose gel electrophoresis. HBV DNA in virions or naked capsids was detected by a HBV DNA probe. The density profile of each fraction is indicated at the bottom. The density of HBV virions (1.250 g/cm3) is highlighted in bold. (D) concentrated cell culture supernatant of HepG2 cells co-transfected with HBV-C(-) (genotype D) and WT or mutant HBc or WHc was analyzed by NAGE and DNA in virions or naked capsids was detected by a HBV DNA probe. NC, nucleocapsid; V, virion.</p
Effects of WT and mutant HBc or WHc on supporting the replication of HBV and WHV.
No full textThe HBV replicon construct that is defective in HBc expression was co-transfected with WT and mutant HBc or WHc expression constructs in HepG2 cells. (A) The assembled capsids (top) and packaged pgRNA (middle) were detected by the C33 anti-HBc/WHc mAb and anti-sense HBV RNA probe, respectively, following the resolution of cytoplasmic lysates by NAGE and transfer to nitrocellulose membrane. Levels of HBc or WHc proteins (bottom) were measured by western blot using 19C18 anti-HBc/WHc mAb after SDS-PAGE. Quantitative results of capsid assembly efficiency (B) and pgRNA packaging efficiency (C) were obtained as described in Fig 4 and are shown. (D) core DNA was released from the NCs of cytoplasmic lysate by SDS-proteinase K treatment and detected by Southern blot analysis. (E) PF-DNA was extracted by the Hirt extraction method. The extracted DNA was treated with DpnI plus Exo I & III to remove all DNA with free 3’ ends. The cccDNA formation efficiency (F) was determined by normalizing the levels of cccDNA to those of rcDNA, with the efficiency from WT HBc set to 1.0. Similarly, a WHV replicon construct that is defective in WHc expression was co-transfected with WT and mutant HBc or WHc expression constructs in WCH-17 cells. (G) The assembled capsids (top) and packaged pgRNA (middle) were detected by the C33 anti-HBc/WHc mAb and anti-sense WHV RNA probe, respectively. Levels of HBc or WHc proteins (bottom) were measured by western blot using 19C18 anti-HBc/WHc mAb after SDS-PAGE. Capsid assembly efficiency (H) and pgRNA packaging efficiency (I) were determined as described above and are shown. (J) core DNA and (K) PF-DNA from the transfected cells was analyzed by Southern blot analysis. (L) cccDNA formation efficiency of WHV was determined by normalizing the levels of cccDNA to those of rcDNA, with the efficiency from WT HBc set to 1.0. Data is shown as mean ± SD. Two-tailed unpaired Student’s t test was used to compare the difference of each dataset versus WT HBc (*, p p p < 0.001). Ca, capsid; pgRNA, pregenomic RNA; HBc, HBV core protein; WHc, WHV core protein; ssDNA, single-strand DNA; rcDNA, relaxed circular DNA; cccDNA, covalently closed circular DNA; cM-DNA, closed minus strand DNA.</p
Analysis of WHV virion secretion from the WHc N-glycosylation mutants.
No full textThe WHV replicon construct expressing the WT WHc (A) or the T77E (B), T92N (C), or T77E/T92N (D) WHc mutants was transfected into HepG2 cells. Cell culture supernatant from WHV-transfected HepG2 cells were collected at day 14 post-transfection and fractionated by CsCl gradient ultracentrifugation. Indicated fractions (fractions 14 to 22) were resolved by NAGE and detected with a WHV DNA probe. Fraction 17 is known to contain the WHV virion peak at a density of 1.258 g/cm3. NC, nucleocapsids. (TIF)</p
Ability of WT and mutant HBc or WHc to support WHV replication in the <i>trans</i>-complementation assay.
No full textThe WHV replicon construct that is defective in WHc expression was co-transfected with WT or mutant HBc or WHc expression construct into HepG2 cells. (A) The assembled capsids (top) and packaged pgRNA (middle) were detected by the C33 anti-HBc/WHc mAb and anti-sense HBV RNA probe, respectively, following the resolution of cytoplasmic lysates by NAGE and transfer to nitrocellulose membrane. Levels of HBc or WHc proteins (bottom) were measured by western blot using 19C18 anti-HBc/WHc mAb after SDS-PAGE. Capsid assembly efficiency (B) was determined by normalizing the levels of capsids to those of total HBc/WHc protein, and pgRNA packaging efficiency (C) was determined by normalizing the levels of pgRNA to those of capsids, with the efficiencies of WT HBc set to 1.0. (D) core DNA was released from the NCs of cytoplasmic lysate by SDS-proteinase K treatment and detected by Southern blot analysis. (E) HBV PF-DNA was isolated from the transfected cells by the Hirt extraction method. The extracted DNA was treated with Dpn I plus Exo I & III to remove all DNA with free 3’ ends. (F) The cccDNA formation efficiency was calculated by normalizing the levels of cccDNA to those of rcDNA, with the efficiency from WT HBc set to 1.0. (G) Summary of all parameters of WHV replication in HepG2 and WCH-17 cells (shown in Fig 6). Increased parameters compared to HBc-WT were marked in red while decreased parameters compared to HBc-WT were marked in blue. Data is shown as mean ± SD. Two-tailed unpaired Student’s t test was used to compare the difference of each data set versus WT HBc (*, p p p (TIF)</p
Ability of WT and mutant HBc or WHc to support HBV replication in the <i>trans</i>-complementation assay.
No full textThe HBV replicon construct that is defective in HBc expression was co-transfected with WT or mutant HBc or WHc expression construct into WCH-17 cells. (A) The assembled capsids (top) and packaged pgRNA (middle) were detected by the C33 anti-HBc/WHc mAb and anti-sense HBV RNA probe, respectively, following the resolution of cytoplasmic lysates by NAGE and transfer to nitrocellulose membrane. Levels of HBc or WHc proteins (bottom) were measured by western blot using the 19C18 anti-HBc/WHc mAb after SDS-PAGE. Capsid assembly efficiency (B) was determined by normalizing the levels of capsids to those of total HBc/WHc protein, and pgRNA packaging efficiency (C) was determined by normalizing the levels of pgRNA to those of capsids, with the efficiencies of WT HBc set to 1.0. (D) core DNA was released from the NCs of cytoplasmic lysate by SDS-proteinase K treatment and detected by Southern blot analysis. (E) HBV PF-DNA was isolated from the transfected cells by the Hirt extraction method. The extracted DNA was treated with Dpn I plus Exo I & III to remove all DNA with free 3’ ends. (F) The cccDNA formation efficiency was calculated by normalizing the levels of cccDNA to those of rcDNA, with the efficiency from WT HBc set to 1.0. (G) Summary of all parameters of HBV replication in HepG2 (shown in Fig 6) and WCH-17 cells. Increased parameters compared to HBc-WT were marked in red while decreased parameters compared to HBc-WT were marked in blue. Data is shown as mean ± SD. Two-tailed unpaired Student’s t test was used to compare the difference of each dataset versus WT HBc (*, p p p (TIF)</p
Effects of HBc or WHc on protecting the rcDNA content inside NCs.
No full textCore DNA released from the cytoplasmic lysate of WT or mutant HBc or WHc co-transfected with HBV-C(-) from WCH-17 cells (A) or with WHV-C(-) from HepG2 cells (B), with or without prior Turbo DNase digestion, was detected by Southern blot analysis. The rcDNA signals obtained after Turbo DNase treatment is indicated by the dashed, red box for comparison to rcDNA signals present without nuclease treatment. ssDNA, single strand DNA; rcDNA, relaxed circular DNA. (TIF)</p
Effects of HBc or WHc on protecting the rcDNA content inside NCs.
No full text(A) schematic diagram of nuclease treatment of HBV or WHV NCs. Turbo DNase was used to degrade viral DNA inside unstable NCs. Following inactivation of the nuclease, NC-protected DNA was isolated and detected by Southern blot analysis. Core DNA released from the cytoplasmic lysate of WT and mutant HBc or WHc co-transfected with HBV-C(-) from HepG2 cells (B) or with WHV-C(-) from WCH-17 cells (C), with or without prior Turbo DNase digestion, was detected by Southern blot analysis. The rcDNA signals obtained after Turbo DNase treatment is indicated by the dashed, red box for comparison to rcDNA signals present without nuclease treatment. ssDNA, single strand DNA; rcDNA, relaxed circular DNA.</p
Towards teleoperation with human-like dynamics: Human use of elastic tools
No full textVariable stiffness actuators undergo lower peak force in contacts compared to their rigid counterparts, and are thus safer for human-robot interaction. Furthermore, they can store energy in their elastic element and can release it later to achieve human-like dynamic movements. However, it is not clear how to integrate them in teleoperator systems so that they can be controlled intuitively by a human. We performed an experiment to study human use of elastic tools to determine how a teleoperator system with an elastic slave would need to be designed. For this, we had 13 untrained participants hammer with an elastic tool under different stiffness conditions, asking them to try to find the best timing for a backward-forward swing motion in order to achieve the strongest impact. We found that the participants generally executed the task efficiently after a few trials and they converged to very similar solutions. The stiffness influenced the performance slightly, a stiffness between 2.3 Nm/rad and 4.1 Nm/rad showing the best results. We conclude that humans intuitively know how to efficiently use elastic tools for hammering type tasks. This could facilitate the control of teleoperator systems with elastic slave manipulators for tasks requiring explosive movements like hammering.Accepted Author ManuscriptBiomechatronics & Human-Machine Contro
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