Additional file 1 of Physiology education in China: the current situation and changes over the past 3 decades

Supplementary Material 1

Unlocking DCAFs To Catalyze Degrader Development: An Arena for Innovative Approaches

Chemically induced proximity-based targeted protein degradation (TPD) has become a prominent paradigm in drug discovery. With the clinical benefit demonstrated by certain small-molecule protein degraders that target the cullin-RING E3 ubiquitin ligases (CRLs), the field has proactively strategized to tackle anticipated drug resistance by harnessing additional E3 ubiquitin ligases to enrich the arsenal of this therapeutic approach. Here, we endeavor to explore the collaborative efforts involved in unlocking a broad range of CRL4DCAF for degrader drug development. Throughout the discussion, we also highlight how both conventional and innovative approaches in drug discovery can be taken to realize this objective. Moving ahead, we expect a greater allocation of resources in TPD to pursue these high-hanging fruits

Estrogen signaling to ezrin increases T47-D cell migration and invasion.

<p>(A) T47-D cells were transfected with scrambled siRNA or ezrin targeted siRNA 1 and 2 for 48 h. Ezrin protein expression was detected by western blot and β-actin intensity was used as the loading control as indicated. (B) Cells were stained with an Ab vs. ezrin (FITC; green staining) as well as with Texas Red-phalloidin (in red). Nuclei are counterstained in blue. All the experiments were repeated three times with consistent results, and a representative result is shown. (C) Cells were transfected with 100 nM target siRNAs for ezrin or scrambled siRNA for 48 h and then treated with E2 (10 nM) for 48 h. Cell migration distances were measured and values are presented within brackets as mean migration distance (mm) ± SD. The black line indicates the starting line. * = P<0.01 vs. control. (D) Cells were treated with E2 (10 nM) for 48 h, in the presence or absence of ICI 182,780 (ICI - 1 µM), of wortmannin (WM - 30 nM), of Y-27632 (Y - 10 µM) or of PP2 (10 µM). * = P<0.01 vs. control; # = P<0.01 vs. E2. The experiments were performed in triplicates and representative images are shown. (E) T47-D cells were treated with 10 nM E2 for 24 h, in the presence or absence of ICI 182,780 (ICI - 1 µM), of wortmannin (WM - 30 nM), of Y-27632 (Y - 10 µM), of PP2 (10 µM) or after transfection with 100 nM siRNAs toward ezrin or control scrambled siRNAs for 48 h. Cell invasion was assayed using invasion chambers. Invading cells were counted in three different central fields of triplicate membranes. Statistics for invasion indexes and representative images are shown. * = P<0.01 vs. control, # = P<0.01 vs E2 without transfection. The experiments were performed in triplicates and representative images are shown.</p

ERα supports the extra-nuclear signaling of E2 to ezrin.

<p>(A) T47-D cells were treated with E2 (10⁻⁸ M) or E-BSA (10⁻⁸ M) for 15 min, in the presence or absence of actinomycin D (Act D - 10 µM) or cycloheximide (CHX - 200 µM). Ezrin and phosphorylated ezrin are shown. ** = P<0.01 vs control. (B) T47-D cells were treated with E2 (10⁻⁸ M), PPT (10⁻⁹ M) or DPN (10⁻⁹ M) for 15 min, in the presence or absence of ICI 182,780 (ICI - 1 µM). Ezrin and phosphorylated ezrin are shown. ** = P<0.01 vs. control. # = P<0.01 vs. E2 or PPT, respectively. All the experiments were repeated three times with consistent results, and the representative images are shown.</p

E2 activates ezrin and induces rapid actin cytoskeleton rearrangement in T47-D cells.

<p>(A) and (B) show the time- and dose-dependent ezrin activation in T47-D breast cancer cells after treatment with E2. Total cell amount of wild-type (ezrin) or Thr⁵⁶⁷-phosphorylated ezrin (P-ezrin) are shown with western blot. P-ezrin densitometry values were adjusted to ezrin intensity, then normalized to expression from the control sample. * = P<0.05 vs. corresponding control; ** = P<0.01 vs. corresponding control. (C) shows dose-dependent ezrin phosphorylation at Tyr³⁵³ after treatment with E2 for 15 minutes in T47-D cells. Tyr³⁵³-phosphorylated ezrin (P-ezrin) are shown with western blot and actin is taken as the loading control. (D) In MCF-7 breast cancer cells, E2 induced ezrin phosphorylation at Thr⁵⁶⁷ in a time-dependent manner. (E) T47-D cells were treated with E2 (10⁻⁸ M) for the indicated time. Then the cells were stained with anti-phospho-Thr⁵⁶⁷ ezrin (P-ezrin) linked to FITC. Actin was stained with phalloidin linked to Texas Red and nuclei were counterstained with DAPI. White arrows indicate the lamellipodia. All the experiments were repeated three times with consistent results, and the representative images are shown.</p

c-Src and phosphatidylinositol-3-kinase (PI3K)/Akt are implicated in E2-induced ezrin phosphorylation.

<p>(A) T47-D cells were exposed to 10⁻⁸ M E2 for 15 min, in the presence or absence of the pure ER antagonist ICI 182,780 (ICI - 1 µM), of the MEK inhibitor PD98059 (PD - 5 µM), of the G protein inhibitor, PTX (100 ng/mL), of the PI3K inhibitor wortmannin (WM - 30 nM), of the ROCK-2 inhibitor, Y-27632 (Y - 10 µM) or of the c-Src kinase inhibitor, PP2 (10 µM). Cell content of wild-type or phosphorylated ezrin are shown. P-ezrin densitometry values were adjusted to ezrin intensity and then normalized to expression from the control sample. ** = P<0.01 vs. control, # = P<0.01 vs. E2. (B) and (C) show the time- and dose-dependent c-Src activation in T47-D breast cancer cells after treatment with E2. Total cell amount of wild-type (c-Src) or Tyr⁴¹⁶-phosphorylated c-Src (P-c-Src) are shown with western blot. P-c-Src densitometry values were adjusted to c-Src intensity, then normalized to expression from the control sample. (D) T47-D cells were exposed to 10⁻⁸ M E2 for 15 min after transfection with 100 nM c-Src siRNAs or control scrambled siRNAs for 48 h. Total actin, c-Src, ezrin, P-ezrin, Akt and P-Akt amounts and statistics for densitometry are shown. ** = P<0.01 vs. corresponding control. (E) T47-D cells were treated with 10⁻⁸ M E2 for 15 min, with or without the ER antagonist ICI 182,780 (ICI - 1 µM), PI3K inhibitor wortmannin (WM - 30 nM) or c-Src kinase inhibitor, PP2 (10 µM). Active Akt (P-Akt) densitometry values were adjusted to wild-type Akt intensity, then normalized to expression from the control sample. ** = P<0.01 vs. control. # = P<0.01 vs. P. (F) Cells were exposed to 10 nM E2 for 15 min after transfection with wild type p85α (WT p85α, 1.5 µg) or dominant-negative p85α (Δp85α, 1.5 µg) for 48 h. Cell contents of actin, p85 protein, wild-type and phosphorylated ezrin and statistics for densitometry are shown. ** = P<0.01 vs. corresponding control without transfection. All these experiments were performed in triplicates and representative images are shown.</p

RhoA and ROCK-2 are activated during ER signaling to ezrin.

<p>(A) RhoA activity was assayed in cells treated with E2 (10⁻⁸ M) for 15 min in the presence or absence of the ICI 182,780 (ICI - 1 µM), of the PI3K inhibitor wortmannin (WM - 30 nM), or of the c-Src kinase inhibitor, PP2 (10 µM). Active, GTP-bound RhoA was immunoprecipitated with Rhotekin and subsequently assayed with western analysis with an anti-RhoA Ab (lower boxes). The upper blot shows total RhoA content in the input. RhoA-GTP densitometry values were adjusted to total RhoA intensity, then normalized to expression from the control sample. ** = P<0.01 vs. control. # = P<0.01 vs. E2. (B) Cells were treated with 10⁻⁸ M E2 for 15 min in the presence or absence of ICI 182,780 (ICI - 1 µM), of wortmannin (WM - 30 nM) or of PP2 (10 µM). ROCK-2 was immunoprecipitated with a specific Ab and the IPs were used to phosphorylate the bait protein, myelin basic protein (MBP). ROCK-2 kinase activity is shown as the amount of phosphorylated MBP (P-MBP). P-MBP densitometry values were adjusted to ROCK-2 intensity, then normalized to expression from the control sample. ** = P<0.01 vs. control. # = P<0.01 vs. E2. (C) T47-D cells were either mock-transfected or exposed to dominant-negative RhoA or constitutively active (RhoA DN or RhoA CA). Cells were then treated with E2 (10 nM) for 15 min and wild type or phosphorylated ezrin were analyzed. P-ezrin densitometry values were adjusted to actin intensity, then normalized to expression from the corresponding control sample. ** = P<0.01 vs. corresponding control without transfection. (D) Cells were exposed to 10⁻⁸ M E2 for 15 min after transfection with 100 nM siRNAs towards ROCK-2 or control scrambled siRNAs for 48 h. P-ezrin densitometry values were adjusted to ezrin intensity, then normalized to expression from the corresponding control sample. ** = P<0.01 vs. corresponding scrambled control. (E) Cells were exposed to 10⁻⁸ M E2 for 15 min after transfection with c-Src siRNAs for 48 h. Active, GTP-bound RhoA and ROCK-2 activity were assayed RhoA-GTP and P-MBP densitometry values were adjusted to total RhoA and ROCK-2 intensities respectively, then normalized to expression from the control sample. ** = P<0.01 vs. control. All these experiments were performed in triplicates and representative images are shown.</p

Simultaneous Enhancement of Energy Storage and Hardness Performances in (Na_0.5Bi_0.5)_0.7Sr_0.3TiO₃‑Based Relaxor Ferroelectrics Via Multiscale Regulation

For (Na0.5Bi0.5)0.7Sr0.3TiO3-based (BNST) energy storage materials, a critical bottleneck is the early polarization saturation and low breakdown electric field (Eb), which severely limits further development in the field of advancing pulsed power capacitors. Herein, a strategy, via multiscale regulation, including synergistically manipulation of the domain configuration and microstructure evolution in BNST-based ceramics, is propounded through introducing LiTaO3(LT). The composition-driven fine domain size, as demonstrated by macroscale (size effect and dielectric response) and mesoscale (domains relaxor behavior) analysis, provides robust evidence of delayed polarization saturation and large polarization difference. Theoretical simulations and experimental results confirm that the fine grain size, uniform grain size distribution, and insignificant secondary phase contribute to the enhancements of Eb. Further analyses of the intrinsic electronic structure reveal the intrinsic mechanism for enhancing Eb via first-principles calculations on the basis of density functional theory. Consequently, owing to improved Eb, delayed polarization saturation, and refined grain size, excellent comprehensive performances [high Wrec of 5.52 J/cm3, large η of 85.68%, high hardness H of 7.06 GPa, and broad operating temperature range (20–140 °C)] are realized. We believe that these findings can provide a thorough understanding of the origins of excellent comprehensive performances in BNST-based ceramics as well as some guidance in the exploration of materials with high-performance lead-free capacitors for application in future pulsed power systems

Additional file 2: Figure S1. of Hyperphosphorylation of ribosomal protein S6 predicts unfavorable clinical survival in non-small cell lung cancer

The prognostic value of clinical characteristics in NSCLC patients. (TIFF 594 kb

Discovery of Highly Potent and BMPR2-Selective Kinase Inhibitors Using DNA-Encoded Chemical Library Screening

The discovery of monokinase-selective inhibitors for patients is challenging because the 500+ kinases encoded by the human genome share highly conserved catalytic domains. Until now, no selective inhibitors unique for a single transforming growth factor β (TGFβ) family transmembrane receptor kinase, including bone morphogenetic protein receptor type 2 (BMPR2), have been reported. This dearth of receptor-specific kinase inhibitors hinders therapeutic options for skeletal defects and cancer as a result of an overactivated BMP signaling pathway. By screening 4.17 billion “unbiased” and “kinase-biased” DNA-encoded chemical library molecules, we identified hits CDD-1115 and CDD-1431, respectively, that were low-nanomolar selective kinase inhibitors of BMPR2. Structure–activity relationship studies addressed metabolic lability and high-molecular-weight issues, resulting in potent and BMPR2-selective inhibitor analogs CDD-1281 (IC50 = 1.2 nM) and CDD-1653 (IC50 = 2.8 nM), respectively. Our work demonstrates that DNA-encoded chemistry technology (DEC-Tec) is reliable for identifying novel first-in-class, highly potent, and selective kinase inhibitors