Probing the influence of SIBLING proteins on collagen-I fibrillogenesis and denaturation

<p>Bone tissue is comprised of collagen, non-collagenous proteins, and hydroxyapatite and the SIBLING (small integrin binding, N-linked glycoprotein) family of proteins is the primary group of non-collagenous proteins. By replicating the native interactions between collagen and the SIBLING proteins at the interface of an implant, it is believed that a bone scaffold will more easily integrate with the surrounding tissue. In this work, bone sialoprotein, osteopontin (OPN), dentin sialoprotein (DSP), dentin phosphoprotein (DPP), C-terminal fragment of dentin matrix protein 1 (DMP1-C), and proteoglycan versions of DSP (DSP-PG) and DMP1 (DMP1-PG) were tested individually to determine their roles in collagen fibrillogenesis and the prevention of denaturation. It was shown that DSP and DPP slowed down fibrillogenesis, while other SIBLINGs had limited impact. In addition, the denaturation time was faster in the presence of DSP and OPN, indicating a negative impact. The role of calcium ions in these processes was also investigated. The presence of calcium ions sped up fibrillogenesis in all scenarios tested, but it had a negative impact by reducing the extent. Calcium also sped up the denaturation in most cases, with the exception of DMP1-C and DSP where the opposite was seen. Calcium had a similar effect on the proteoglycan variants in the fibrillogenesis process, but had no impact on the denaturation process in the presence of these two. It is believed that incorporating DMP1-C or DSP on the surface of a bone implant may improve the collagen interactions with the implant, thereby facilitating improved osteointegration.</p

Copper oxide nanoparticles induce autophagy in A549 cells.

<p>(A) Western blot analysis of LC3-I and LC3-II in A549 cells treated with CuO or SiO2 NPs (30 µg/ml) for 12 h, bar graph indicates the LC3 II/β-actin relative ratio (B) EM images of A549 cells treated with control, rapamycin (5 µM), CuO (30 µg/ml) or SiO₂ (30 µg/ml); bar graph indicates the percentage of autophagy-positive cells (C) Confocal images of A549 cells transfected with LC3-EGFP plasmid and treated with control, rapamycin (5 µM), CuO (30 µg/ml) or SiO₂ (30 µg/ml); bar graph indicates the percentage of LC3-positive cells (D) Western blot analysis of LC3-I and LC3-II in A549 cells treated with bafilomycin A1 (50 nm) 1 h before exposure to CuO NPs (30 µg/ml) for 3 h, bar graph indicates the LC3 II/LC3 I relative ratio (E) MTS assay results for A549 cells treated with bafilomycin A1 (50 nm) 1 h before exposure to CuO NPs (30 µg/ml) for 18 h (F) Western blotting analysis of the efficiency of ATG5-knockdown efficiency by ATG5 siRNA, bar graph indicates the Atg5/β-actin relative ratio (G) Western blotting analysis of the expression of LC3-I and LC3-II in ATG5 siRNA treated cells or control siRNA treated cells, bar graph indicates LC3 II/LC3 I relative ratio (H) Cell viability of A549 cells upon treatment of CuO NPs (30 µg/ml) for 24 h in ATG5 siRNA treated group and control siRNA treated group (I) MTS assay results for A549 cells treated with 30 µg/ml CuO NPs and cell death inhibitors for 24 h; **<i>p</i><0.01 versus control and *<i>p</i><0.05 versus control.</p

Copper oxide nanoparticles induce cell death through a non-apoptotic pathway in A549 cells.

<p>(A) TUNEL assay results for A549 cells treated with CuO or SiO₂ NPs (30 µg/ml) for 8 h. The magnitude = 400×(B) Western blot analysis of cleaved caspase 3 in A549 cells treated with 30 µg/ml CuO NPs for 12 h. 4% DMSO was used as a positive control.</p

Copper oxide nanoparticles cause cell death in respiratory cell lines.

<p>(A) MTS assay results for A549 cells treated with CuO, SiO₂, TiO₂ (10 nm, 32 nm), Fe₂O₃ or Fe₃O₄ NPs (30 µg/ml) for 24 h (B) MTS assay results for A549 cells treated with CuO NPs (0.1, 0.3, 1, 3, 10 and 30 µg/ml) for 24 h (C) MTS assay results for H1650 cells treated with NPs (30 µg/ml) for 24 h (D) MTS assay results for CNE-2Z cells treated with NPs (30 µg/ml) for 24 h. ***<i>p</i><0.001 versus control and **<i>p</i><0.01 versus control.</p

In Situ Observation of Dynamic Wetting Transition in Re-Entrant Microstructures

Re-entrant microstructures exhibit excellent wetting stability under different pressure levels, but the underlying mechanism determined by wetting transition behavior at the microscale level remains unclear. We propose the “wetting chip” method for in situ assessment of the dynamic behavior of wetting transition in re-entrant microstructures. High sag and transverse depinning were observed in re-entrant microstructures. Analysis indicated that high sag and transverse depinning mainly influenced the stability of the structures. The threshold pressure and longevity of wetting transition were predicted and experimentally verified. The design criteria of wetting stability, including small geometry design, hydrophobic material selection, and sidewall condition, were also presented. The proposed method and model can be applied to different shapes and geometry microstructures to elucidate wetting stability

Incubation with heparin and degradation of HS polysaccharides on the cell surface by heparinase inhibits SARS pseudovirus infection of HEK293E/ACE2-Myc cells.

<p>(<b>A</b>) Heparin inhibits SARS pseudovirus entry into HEK293E/ACE2-Myc cells. Before incubation with SARS pseudovirus at 37°C for 4 h, HEK293E/ACE2-Myc cells were treated with heparin for 10 min at the concentration of 0.625 µM, 1.25 µM, 2.5 µM, 5 µM or 10 µM. GFP-expressing HEK293E/ACE2-Myc cells in the total population were analyzed by flow cytometry. The relative viral infection ratio was measured by comparing the percentage of GFP expressing cells of each group to that of the BSA control. Error bars represent the SD of three independent experiments. (<b>B</b>) Lysis of cell-surface HS by heparinase blocks the infection of HEK293E/ACE2-Myc cells by SARS pseudovirus. After incubation with 10 U of heparinase I or chondroitinase ABC for 1 h at 37°C, the cells were treated with SARS pseudovirus as described above. The relative viral infection ratio was calculated by the same method. *P<0.05.</p

Lactoferrin localizes to the cell membrane by targeting HSPGs.

<p>(<b>A</b>) LF is present on the cell surface. Oregon Green-labeled LF localization was observed under confocal microscopy. LF untreated cells were used as control. Cell membrane and nuclei were stained with DiI and Hoechst33342, respectively. Scale bar, 8 µm. (<b>B</b>) Heparin inhibits LF binding to HEK293E/ACE2-Myc cells. HEK293E/ACE2-Myc cells were incubated with 0.5 µM Oregon Green-labeled LF at 4°C for 1 h after pretreatment with heparin at the concentration of 3 µM, 10 µM or 30 µM. The MFI was measured for each group by flow cytometry as described above. Error bars represent the SD of three independent experiments. **P<0.01.</p

Lactoferrin blocks the interaction between spike protein and HEK293E/ACE2-Myc cells in an ACE2-independent fashion.

<p>(<b>A</b>) LF inhibits the binding of S1190-Fc to HEK293E/ACE2-Myc cells. Before incubation of S1190-Fc with HEK293E/ACE2-Myc cells at 4°C for 1 h, the cells were treated with LF at 37°C for 1 h at concentrations of 1 µM, 3 µM and 10 µM. Fc protein was used as a control. S1190-Fc binding to the cells was detected by flow cytometry as described in Methods. Error bars represent the SD of three independent experiments. ***P<0.001, **P<0.01 and *P<0.05. (<b>B</b>) LF does not disrupt the binding of S1190-Fc to ACE2-Myc. Error bars represent the SD of three independent experiments. ** P<0.01.</p

LF, heparin or enzymatic removal of cell surface HSPGs can prevent SARS pseudovirus entry into Vero E6 or Caco-2 cells.

<p>(<b>A</b>) Interference of the interaction between Vero E6 and SARS pseudovirus by LF, heparin and heparinase leads to reduction of viral infection. Vero E6 cells were treated by the same methods above with 10 µM LF, 10 µM heparin or 10 U of heparinase I, respectively. Then, the cells were incubated with SARS pseudovirus as described in Methods. GFP-expressing cells in the total population were analyzed by flow cytometry. The relative viral infection ratio was measured by comparing the percentage of GFP expressing cells of each group to that of the control. Error bars represent the SD of three independent experiments. *** P<0.001 and **P<0.01. (<b>B</b>) Incubation with LF or heparin, or degradation of HSPGs by heparinase inhibits SARS pseudovirus infection of Caco-2 cells. The Caco-2 cells were treated by same methods as described above. *** P<0.001, **P<0.01 and *P<0.05.</p

Addition of exogenous heparin and enzymatic removal of HS chains by heparinase reduce spike protein binding to HEK293E/ACE2-Myc cells.

<p>(<b>A</b>) Heparin blocks the binding of S1190-Fc to HEK293E/ACE2-Myc cells. HEK293E/ACE2-Myc cells were incubated with heparin for 10 min at the concentration of 10 µM, 30 µM or 100 µM. S1190-Fc was added to each group and incubated at 4°C for 1 h. The MFI of each group was measured as described above. Error bars represent the SD of three independent experiments. *** P<0.001, **P<0.01 and *P<0.05. (<b>B</b>) Enzymatic degradation of cell-surface heparan sulfate (HS) chains reduces S1190-Fc binding. After treatment with 10 U of heparinase I, the cells were incubated with S1190-Fc at 4°C for 1 h. The MFI test was performed using the same method as above.</p