Denatured-State Conformation As Regulator of Amyloid Assembly Pathways?

Additional file 1: Figure S1. Disease incidence of ginger bacterial wilt. Figure S2. The rarefaction curve of samples. Table S1. Soil Physicochemical Data. Table S2. The top ten Phyla of samples. Dataset S1. Discriminative taxa analyzed by LEfSe in all samples

Video3_Steps of actin filament branch formation by Arp2/3 complex investigated with coarse-grained molecular dynamics.mp4

The nucleation of actin filament branches by the Arp2/3 complex involves activation through nucleation promotion factors (NPFs), recruitment of actin monomers, and binding of the complex to the side of actin filaments. Because of the large system size and processes that involve flexible regions and diffuse components, simulations of branch formation using all-atom molecular dynamics are challenging. We applied a coarse-grained model that retains amino-acid level information and allows molecular dynamics simulations in implicit solvent, with globular domains represented as rigid bodies and flexible regions allowed to fluctuate. We used recent electron microscopy structures of the inactive Arp2/3 complex bound to NPF domains and to mother actin filament for the activated Arp2/3 complex. We studied interactions of Arp2/3 complex with the activating VCA domain of the NPF Wiskott-Aldrich syndrome protein, actin monomers, and actin filament. We found stable configurations with one or two actin monomers bound along the branch filament direction and with CA domain of VCA associated to the strong and weak binding sites of the Arp2/3 complex, supporting prior structural studies and validating our approach. We reproduced delivery of actin monomers and CA to the Arp2/3 complex under different conditions, providing insight into mechanisms proposed in previous studies. Simulations of active Arp2/3 complex bound to a mother actin filament indicate the contribution of each subunit to the binding. Addition of the C-terminal tail of Arp2/3 complex subunit ArpC2, which is missing in the cryo-EM structure, increased binding affinity, indicating a possible stabilizing role of this tail.</p

Video8_Steps of actin filament branch formation by Arp2/3 complex investigated with coarse-grained molecular dynamics.mp4

The nucleation of actin filament branches by the Arp2/3 complex involves activation through nucleation promotion factors (NPFs), recruitment of actin monomers, and binding of the complex to the side of actin filaments. Because of the large system size and processes that involve flexible regions and diffuse components, simulations of branch formation using all-atom molecular dynamics are challenging. We applied a coarse-grained model that retains amino-acid level information and allows molecular dynamics simulations in implicit solvent, with globular domains represented as rigid bodies and flexible regions allowed to fluctuate. We used recent electron microscopy structures of the inactive Arp2/3 complex bound to NPF domains and to mother actin filament for the activated Arp2/3 complex. We studied interactions of Arp2/3 complex with the activating VCA domain of the NPF Wiskott-Aldrich syndrome protein, actin monomers, and actin filament. We found stable configurations with one or two actin monomers bound along the branch filament direction and with CA domain of VCA associated to the strong and weak binding sites of the Arp2/3 complex, supporting prior structural studies and validating our approach. We reproduced delivery of actin monomers and CA to the Arp2/3 complex under different conditions, providing insight into mechanisms proposed in previous studies. Simulations of active Arp2/3 complex bound to a mother actin filament indicate the contribution of each subunit to the binding. Addition of the C-terminal tail of Arp2/3 complex subunit ArpC2, which is missing in the cryo-EM structure, increased binding affinity, indicating a possible stabilizing role of this tail.</p

Video5_Steps of actin filament branch formation by Arp2/3 complex investigated with coarse-grained molecular dynamics.mp4

The nucleation of actin filament branches by the Arp2/3 complex involves activation through nucleation promotion factors (NPFs), recruitment of actin monomers, and binding of the complex to the side of actin filaments. Because of the large system size and processes that involve flexible regions and diffuse components, simulations of branch formation using all-atom molecular dynamics are challenging. We applied a coarse-grained model that retains amino-acid level information and allows molecular dynamics simulations in implicit solvent, with globular domains represented as rigid bodies and flexible regions allowed to fluctuate. We used recent electron microscopy structures of the inactive Arp2/3 complex bound to NPF domains and to mother actin filament for the activated Arp2/3 complex. We studied interactions of Arp2/3 complex with the activating VCA domain of the NPF Wiskott-Aldrich syndrome protein, actin monomers, and actin filament. We found stable configurations with one or two actin monomers bound along the branch filament direction and with CA domain of VCA associated to the strong and weak binding sites of the Arp2/3 complex, supporting prior structural studies and validating our approach. We reproduced delivery of actin monomers and CA to the Arp2/3 complex under different conditions, providing insight into mechanisms proposed in previous studies. Simulations of active Arp2/3 complex bound to a mother actin filament indicate the contribution of each subunit to the binding. Addition of the C-terminal tail of Arp2/3 complex subunit ArpC2, which is missing in the cryo-EM structure, increased binding affinity, indicating a possible stabilizing role of this tail.</p

High glucose treatment increases full-length APP protein level.

<p>SH-SY5Y cells were treated with different concentration of glucose for 24 hours (<b>A</b>), and 48 hours(<b>C</b>) with2.5 mM glucose treatment serving as control. The cell lysates were analyzed by Western blot. Full-length APP was detected by C20 antibody. β-actin, serving as internal control, was detected by AC-15 antibody. 24-hour and 48-hour of high glucose treatment significantly increased full-length APP protein level.Quantification of full-length APP after 24-hour treatment of high glucose in SH-SY5Y cells (<b>B</b>). The values are expressed as mean±S.E.M, n = 4,*p<0.05 by ANOVA. Quantification of full-length APP after 48-hour treatment of high glucose in SH-SY5Y cells (<b>D</b>) The values are expressed as mean±S.E.M, n = 3,*p<0.05 by ANOVA.</p

High glucose treatment increases C99 and Aβ₄₀ production.

<p>The 20E2 cells were cultured and treated with different concentrations of glucose for 24 hours. Media containing 5.5 mM glucose served as control. The cell lysates were analyzed by western blot (<b>A</b>).C99 was detected by C20 antibody. β-actin, serving as internal control, was detected by AC-15 antibody. Quantification of C99 after 24-hour treatment of high glucose in 20E2 cells (<b>B</b>) The values are expressed as mean±S.E.M, n = 3,*p<0.001 by ANOVA The level of Aβ₄₀ in conditioned media of 20E2 cells was measured by ELISA(<b>C</b>). The values are expressed as mean±S.E.M, n = 4, *p<0.001, by ANOVA.</p

High glucose treatment inhibits APP protein degradation.

<p>20E2 cells were treated with 5.5 mM, 10 mM or 25 mM glucose for 24 hours, and the cell lysates were analyzed by Western blot (<b>A</b>). Full-length APP was detected by C20 antibody. β-actin, serving as internal control, was detected by AC-15 antibody. The level of APP protein was quantified by Image J (<b>B</b>). The values are expressed as mean±S.E.M. n = 3,*p<0.0001, by ANOVA<b>.</b> For APP degradation experiment, 20E2 cells were treated with culturing media containing 100 ug/ml CHX along with 5.5 mM or 10 mM glucose. The cell lysates were harvested at 0, 15, 30 or 60 minutes after treatment and analyzed by Western blot (<b>C, D</b>).Quantification of APP protein by Image J (<b>E</b>). APP protein level was plotted as a percentage of the amount at 0 minute. The values are expressed as mean±S.E.M. n = 3,*p<0.001, by two-way ANOVA. The APP degradation experiment was also conducted using SH-SY5Y cells treated with 100 ug/ml CHX along with 5.5 mM, 10 mM or 25 mM glucose for 30 minutes. The cell lysates were analyzed by Western blot (<b>F</b>).The level of APP protein was quantified by Image J(<b>G</b>) andwas plotted as a percentage of the amount at 0 minute. The values are expressed as mean±S.E.M. n = 3,*p<0.05, by ANOVA.</p

High glucose treatment does not affect APP transcription.

<p>The 2.94 kb human APP promoter was transfected into SH-SY5Ycells and treated with high glucose for 24 hours (<b>A</b>) and 48 hours (<b>B</b>). 2.5 mM glucose serves as control. Luciferase assasy was performed.High glucose treatment did not affect APP promoter activity. All the promoter data shown are results of 4 independent experiments, with each condition performed in triplicates. The values are expressed as mean±S.E.M. n = 4, by ANOVA. SH-SY5Y cells were treated with different concentration of glucose for 24 hours(<b>C</b>) and 48 hours (<b>E</b>). RNA was extracted and APP mRNA level was measured by semi-quantitative PCR with specific primers. β-actin served as an internal control. 24-hourand 48-hourtreatment of high glucose did not significantly affect APP mRNA. Quantification of full-length APP after 24-hour treatment of high glucose (<b>D</b>)The values are expressed as mean±S.E.M, n = 7, by ANOVA. Quantification of full-length APP after 48-hour treatment of high glucose (<b>F</b>)The values are expressed as mean±S.E.M, n = 5, by ANOVA.</p

Synthesis of Benzidine Derivatives via FeCl₃·6H₂O‑Promoted Oxidative Coupling of Anilines

Under open-flask conditions in the presence of commercially available FeCl₃·6H₂O, N,N-disubstituted anilines can be converted into diversely functionalized benzidines with yields of up to 99%. Oxidative coupling was extended to N-monosubstituted anilines, and the method was applied to the efficient preparation of 6,6′-biquinoline. Mechanistic investigations have also been performed to explain the observed reactivities