Generation Of Cell-Penetrating Heme Oxygenase Proteins To Improve The Resistance Of Steatotic Livers To Reperfusion Injury Following Transplantation
Bibliographic record
Abstract
Liver transplantation is the only life-saving treatment for patients with end-stage liver\ndisease; however, organ availability is insufficient to meet demands. Steatotic livers are\nextended criteria donor (ECD) organs that could be used for transplantation if not for an\nincreased susceptibility ischemia reperfusion injury (IRI). Heme oxygenase-1 is a gene,\nthat when upregulated has be shown to reduce IRI in animal models of transplantation.\nIncreasing HO-1 activity in steatotic livers by delivery of a functional cell-penetrating\nHO-1 protein (through the use of cell-penetrating peptides) may provide protection\nagainst IRI, making these organs useful for transplantation. The purpose of this thesis\nwas the generation and testing of a cell-penetrating HO-1 protein. HO-1 and EGFP gene\nsequences were cloned into the pET-28B(+) vector in frame with a CPP or TAT\nsequence. Resulting plasmids were cloned into E. coli, and protein expression was\ninduced using IPTG. Proteins were purified using Ni-NTA affinity chromatography\nunder denaturing and non-denaturing conditions. Non-denatured proteins were tested for\nHO-1 activity and the ability of both denatured and non-denatured proteins to transduce\ncells in vitro was tested by fluorescence microscopy. The cell-penetrating ability of nondenatured\nproteins was further tested in J774, HepG2 and HUVEC cells using\nimmunofluorescence. Five HO-1 and two EGFP cell-penetrating proteins were generated\nexpressed and purified successfully. Purified non-denatured HO-1 retains its enzymatic\nactivity. Non-denatured CPP-EGFP and CPP-HO1 penetrated cells more effectively than\ntheir denatured counterparts. CPP-EGFP and CPP-HO1 proteins are able to penetrate\nmultiple cell types in vitro. Successful generation and testing of a cell-penetrating HO-1\nprotein, for use in an animal model of steatotic liver transplantation. This protein\ndemonstrates promise for use as a potential therapeutic agent in the field of liver\ntransplantation.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| 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 source (direct Gemma or distilled Codex), 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".