sj-docx-1-nvs-10.1177_08997640241233726 – Supplemental material for Achieving Organizationality in Large-Scale Crises: A Comparative Case Study on the Communicative Constitution of Spontaneous Volunteer Collectives

Supplemental material, sj-docx-1-nvs-10.1177_08997640241233726 for Achieving Organizationality in Large-Scale Crises: A Comparative Case Study on the Communicative Constitution of Spontaneous Volunteer Collectives by Christine Carius, Jan Graw and Carsten Schultz in Nonprofit and Voluntary Sector Quarterly</p

Target-Activated Prodrugs (TAPs) for the Autoregulated Inhibition of MMP12

We describe a prodrug concept in which the target enzyme MMP12 produces its own inhibitor in a two-step activation procedure. By using an MMP12-specific peptide sequence and a known sulfonamide drug integrated in the backbone, the active inhibitor is released upon enzyme cleavage. In in vitro experiments, we present proof of concept that the activation proceeds with useful kinetics. The approach is highly selective over the closely related MMP8. If applied in vivo in the future, these prodrugs might release the active entity in a highly specific manner only at such sites where enzyme activity resides

A Bifunctional Noncanonical Amino Acid: Synthesis, Expression, and Residue-Specific Proteome-wide Incorporation

Mapping of weak and hence transient interactions between low-abundance interacting molecules is still a major challenge in systems biology and protein biochemistry. Therefore, additional system-wide acting tools are needed to determine protein interactomics. Most important are reagents that can be applied at any kind of protein interface and the possibility to enrich cross-linked fragments with high efficiency. In this study, we report the synthesis of a novel noncanonical amino acid that features a diazirine group for ultraviolet cross-linking as well as an alkyne group for labeling by click chemistry. This bifunctional amino acid, called PrDiAzK, may be inserted into almost any protein interface with minimal structural perturbation using genetic code expansion. We demonstrate that PrDiAzK can be site-selectively incorporated into proteins in both bacterial and mammalian cell cultures, and we show that PrDiAzK allows protein labeling as well as cross-linking. In addition, we tested PrDiAzK for proteome-wide incorporation via stochastic orthogonal recoding of translation, implying potential applications in system-wide mapping of protein–protein interactions in the future

CFTR Regulates Early Pathogenesis of Chronic Obstructive Lung Disease in βENaC-Overexpressing Mice

<div><h3>Background</h3><p>Factors determining the onset and severity of chronic obstructive pulmonary disease remain poorly understood. Previous studies demonstrated that airway surface dehydration in βENaC-overexpressing (βENaC-Tg) mice on a mixed genetic background caused either neonatal mortality or chronic obstructive lung disease suggesting that the onset of lung disease was modulated by the genetic background.</p> <h3>Methods</h3><p>To test this hypothesis, we backcrossed βENaC-Tg mice onto two inbred strains (C57BL/6 and BALB/c) and studied effects of the genetic background on neonatal mortality, airway ion transport and airway morphology. Further, we crossed βENaC-Tg mice with CFTR-deficient mice to validate the role of CFTR in early lung disease.</p> <h3>Results</h3><p>We demonstrate that the C57BL/6 background conferred increased CFTR-mediated Cl⁻ secretion, which was associated with decreased mucus plugging and mortality in neonatal βENaC-Tg C57BL/6 compared to βENaC-Tg BALB/c mice. Conversely, genetic deletion of CFTR increased early mucus obstruction and mortality in βENaC-Tg mice.</p> <h3>Conclusions</h3><p>We conclude that a decrease or absence of CFTR function in airway epithelia aggravates the severity of early airway mucus obstruction and related mortality in βENaC-Tg mice. These results suggest that genetic or environmental factors that reduce CFTR activity may contribute to the onset and severity of chronic obstructive pulmonary disease and that CFTR may serve as a novel therapeutic target.</p> </div

Survival of βENaC-Tg mice is modified by genetic background.

<p>Survival curves of βENaC-Tg mice and wild-type (WT) littermates on mixed (C3H/He x C57BL/6), C57BL/6 and BALB/c backgrounds (n = 36–59 mice per group). *<i>P</i><0.05 and ^†<i>P</i><0.01 (Kaplan-Meier survival analysis).</p

Genetic background modifies early airway mucus obstruction and epithelial necrosis in neonatal βENaC-Tg mice.

<p>A) Longitudinal sections of tracheae from neonatal (3-day-old) wild-type (WT) and βENaC-Tg mice on the C57BL/6 and BALB/c background. Sections were stained with Alcian blue–periodic acid Schiff (AB-PAS) to determine the presence of mucus and goblet cells. Scale bars  = 200 µm. B–D) Summary of mucus content, as determined from measuring volume density of AB-PAS-positive material in the tracheal lumen (B), goblet cell numbers (C) and Transcript levels of Muc5b (D) (<i>n</i> = 4–13 mice per group). E) Airway histology from neonatal (3-day-old) WT and βENaC-Tg mice on the C57BL/6 and BALB/c background. Sections were stained with hematoxylin and eosin (H&E) and evaluated for degenerative airway epithelial cells (arrows). Scale bars  = 40 µm (upper panels) and 20 µm (lower panels). F) Summary of airway epithelial necrosis as determined from the number of degenerative epithelial cells per mm of the basement membrane (<i>n</i> = 9–11 mice per group). *<i>P</i><0.001 compared with WT mice on same strain background, ^†<i>P</i><0.05 compared with mice of same genotype on C57BL/6 background.</p

Genetic background modulates airway ion transport in wild-type and βENaC-Tg mice.

<p>A) Representative original recordings of the effects of amiloride and cAMP-dependent activation (IBMX/forskolin) on transepithelial voltage (V_te) and transepithelial resistance (R_te) across freshly excised tracheal tissues from neonatal (3-day-old) wild-type (WT) and βENaC-Tg mice on the C57BL/6 and BALB/c background. R_te was determined from V_te deflections obtained by pulsed current injection. (B–F) Summary of basal equivalent short-circuit current (I_sc) (B), amiloride-sensitive I_sc (C), amiloride-insensitive I_sc (D), cAMP-induced I_sc (E) and UTP-induced I_sc (F) in freshly excised tracheal tissues from neonatal WT and βENaC-Tg mice on the C57BL/6 and BALB/c background. Data are presented as mean ± SEM (<i>n</i> = 8–20 mice per group). *<i>P</i><0.05 and **<i>P</i><0.001 compared with WT mice on same strain background, ^†<i>P</i><0.05 and ^††<i>P</i><0.01 compared with mice of same genotype on C57BL/6 background.</p

Genetic background modulates CFTR-mediated Cl⁻ secretion in airways of wild-type and βENaC-Tg mice.

<p>A–C) Effects of genetic background on transepithelial Cl- secretion were determined by adding bumetanide or CFTR_inh-172 to amiloride-pretreated tracheal tissues from neonatal wild-type (WT) and βENaC-Tg mice on the C57BL/6 and BALB/c background. A,B) Summary of bumetanide-sensitive I_sc (A) and CFTR_inh-172-sensitive I_sc (B) in the presence of amiloride (<i>n</i> = 12–23 mice per group). C) Summary of CFTR_inh-172-sensitive I_sc in the presence of amiloride and cAMP-dependent activation (IBMX/forskolin) (<i>n</i> = 5–11 mice per group). Data are presented as mean ± SEM. ^†<i>P</i><0.05 and ^††<i>P</i><0.01 compared with mice of same genotype on C57BL/6 background.</p

PI(3,4,5)P3 analog stimulates detyrosination of microtubules.

<p><b>[A]</b> NIH/3T3 cells were siRNA depleted for GAPDH (control) or PTEN and serum depleted overnight. Cell lysates were analyzed by western blotting against PTEN, phospho-FAK (Y397), total FAK, phospho-PDK1, phospho-Akt (Ser473), total Akt, phospho-GSK3β, total GSK3β and β-Actin. Representative images of 3 independent experiments are shown. <b>[B, C]</b> NIH/3T3 cells were siRNA depleted against GAPDH or PTEN, treated with FAK inhibitors (PF-562271[1 μM], PF-573228 [10 μM]) as indicated and seeded on fibronectin-coated coverslips for 2 h. <b>[B]</b> Representative images of cells immunostained against, detyrosinated tubulin (red), total tubulin (green) and DNA (blue). <b>[C]</b> Percentage of detyrosinated microtubules positive cells. Error bar = StdDev, N = 4 (total of >150 cells each). <b>[D, E]</b> NIH/3T3 cells were serum depleted for 2 days and treated with LPA [10 μM] (positive control) or PIP4-AM as indicated. <b>[D]</b> Representative image of cells immunostained against detyrosinated tubulin (deTyr, red), total tubulin (green) and DNA (blue). <b>[E]</b> Percentage of deTyr positive cells. Error bar = StdDev, N = 3 (total of >150 cells each). Scale bars, 10 μm, ** p<0.01, *** p<0.001.</p

PTEN levels regulate detyrosination of microtubules.

<p>[A] NIH/3T3 cells were siRNA depleted of GAPDH (control) or PTEN, serum-starved and treated with 10 μM LPA (positive control) as indicated. Representative images of cells immunostained for detyrosinated tubulin (deTyr, red), total tubulin (green) and DNA (blue). [B] Percentage of deTyr positive cells depleted of GAPDH or PTEN. Error bar = StdDev, N = 5 (total of >150 cells each). [C] Cell lysates of siRNA-depleted cell were analyzed by western blotting against PTEN, GAPDH and HSP70 as indicated. Error bar = StdDev, N = 3. [D] Percentage of deTyr positive cells treated with PTEN inhibitors (VO-OHpic [100 nM, 1 μM], RV00I (RV) [1μM]) as indicated. Error bar = StdDev, N = 4 (total of >150 cells each). [E] NIH/3T3 cells were transfected with mCherry or mCherry-PTEN-CAAX, seeded on fibronectin-coated coverslips and immunostained against deTyr (red) and mCherry (green). [F] Percentage of deTyr positive cells, transfected with mCherry or mCherry-PTEN-CAAX, seeded on fibronectin-coated coverslips. Error bar = StdDev, N = 4 (total of > 50 cells each). [G] NIH/3T3 cells were siRNA depleted for GAPDH or PTEN and extracted (5 min or 60 min) prior fixation as indicated. Representative images of cells immunostained against detyrosinated tubulin (red), total tubulin (green) and DNA (blue). N = 3 (total of >100 cells each). Scale bars, 10 μm, * p<0.05, *** p<0.001.</p