Abstract 84: nAG (a Salamander-Derived Protein) as an Inhibitor of TGF-β Signaling and Fibrotic Responses
Bibliographic record
Abstract
PURPOSE: Salamanders have the amazing ability to regenerate their limbs within 30 days when amputated. The key protein responsible for this regeneration is the nAG protein, which stands for newt anterior gradient. In previous studies, a nAG gene was designed that was suitable for human use that demonstrated local injection of recombinant nAG protein reduces hypertrophic scarring in a rabbit ear model. Fibrotic disorders of the skin such as scleroderma, hypertrophic scarring and keloids are characterized by excessive TGF-β, leading to an increase in deposition of collagen and other extracellular matrix (ECM) components, resulting in functional impairment which is often debilitating and treatment options remain limited. The aim of this study is to assess the effect nAG has on the fibrotic process of human scleroderma fibroblasts. METHODS: Fibroblasts from lesional scleroderma patient skin were treated with nAG protein in doses of 100pM,1nM and 10nM for 24 hrs and were then left untreated or treated with 20 pM of TGF-β. The inhibition of TGF-β-mediated pro-fibrotic responses was determined by measuring the Extracellular Matrix (collagen III, Fibronectin), connective tissue growth factor (CTGF) and myofibroblast alpha smooth muscle actin (α-SMA) protein production by Western blot as well as immunofluorescence and validated at the mRNA level by Quantitative PCR. Activation of the TGF-β pathway was determined by measuring the TGF-β receptor 1 (ALK5) and phosphorylated Smad2/3 levels by using Western blot and immunofluorescence. Luciferase assay was used to measure nAG protein’s capacity to inhibit the thee TGF-β isomers. Cell migration was assessed using a scratch assay and finally colocalization of nAG protein with TGF- β receptor 1 was evaluated using confocal microscopy. RESULTS: Both the Western Blot and the immunofluorescence results revealed that the application of the nAG protein to human scleroderma fibroblasts in the presence of TGF-β successfully inhibited the fibrotic response shown by a decrease in the fibrotic factors such as collagen III, alpha smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) and fibronectin. In addition, immunofluorescence and western blot after 1 hour of treatment with nAG revealed a significant decrease in Phosphorylated Smad2/3, and a decrease in the TGF-β receptor 1 (ALK5) inhibiting the TGF- β pathway. Luciferase assay revealed nAG’s inhibition of the canonical TGF-β pathway to be most specific with TGF-β1 isomer reducing activity by 83%. Cell migration was significantly inhibited with nAG protein treatment and confocal microscopy revealed the colocalization of nAG protein with ALK5 receptors. CONCLUSION: Fibrosis in scleroderma fibroblasts was effectively inhibited when treated with nAG protein, demonstrated by the decrease in ECM using Western Blot and immunofluorescence. Although much about the mechanism of the nAG protein is still unknown, the decrease in pSmad2/3 and ALK5 receptors most potently with TGF-β1 after treatment, inhibition of cell migration and binding of nAG protein with ALK5 receptors suggest that nAG blocks the canonical TGF-β pathway. This research is anticipated to lead to the development of an injectable antifibrotic agent for the treatment of fibrotic disorders such as scleroderma, hypertrophic scars and keloids. A. Al-Qattan: None. L. Lessard: None. A. Philip: None.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.002 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".