{"gene":"NDUFS8","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":1997,"finding":"NDUFS8 (TYKY subunit) contains two clusters of four conserved cysteine residues and encodes a 210-amino-acid precursor with a 34-amino-acid N-terminal mitochondrial targeting presequence; the mature protein is 92% identical to the bovine subunit and 72% to the Rhodobacter capsulatus NuoI counterpart, establishing it as an iron-sulfur subunit of Complex I.","method":"cDNA sequencing, deduced protein analysis, chromosomal mapping","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — sequence analysis plus cross-species conservation and structural domain identification, single lab","pmids":["9116042"],"is_preprint":false},{"year":1997,"finding":"Deletion of the NuoI (TYKY ortholog) gene or mutation of a conserved cysteine (C74S) in Rhodobacter capsulatus abolishes Complex I activity, eliminates EPR signals for FeS clusters N1 and N2, and prevents assembly of peripheral subunits, demonstrating that NuoI is required for connecting the peripheral and membraneous domains of Complex I.","method":"Homologous recombination deletion, site-directed mutagenesis (C74S), EPR spectroscopy, immunochemical analysis, trans-complementation","journal":"European journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — deletion and point-mutagenesis with EPR, immunochemistry, and complementation rescue across multiple mutants in a single rigorous study","pmids":["9428698"],"is_preprint":false},{"year":1997,"finding":"The TYKY/NDUFS8 homolog in Neurospora crassa is closely associated with the peripheral arm of Complex I, as shown by co-purification and antiserum-based localization.","method":"Heterologous expression in E. coli, purification, antibody production, biochemical fractionation","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single lab, co-fractionation only, but consistent with broader Complex I literature","pmids":["9452770"],"is_preprint":false},{"year":1998,"finding":"The mature NDUFS8/TYKY protein has a molecular mass of ~22 kDa and a pI of 4.9–5.0, as determined by 2D gel electrophoresis and immunodetection; the gene spans ~6 kb with seven exons, and its promoter contains Sp1 and NRF1 binding site motifs in the first intron, with ubiquitous expression predominant in heart and skeletal muscle.","method":"Genomic sequencing, 2D gel electrophoresis, immunodetection","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein characterization by 2D gel plus genomic structural analysis, single lab","pmids":["9666055"],"is_preprint":false},{"year":2002,"finding":"Transcription of NDUFS8 is driven by a minimal 247-bp basal promoter containing three Sp1 sites and one YY1 site; gel-shift analysis and site-directed mutagenesis showed that the YY1 site and two adjacent Sp1 sites are the primary drivers of promoter activity.","method":"Primer extension, reporter gene assay (HeLa and C2C12 cells), gel-shift (EMSA), site-directed mutagenesis","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal methods (EMSA, reporter assay, mutagenesis) in a single focused promoter study","pmids":["11955626"],"is_preprint":false},{"year":2003,"finding":"The NuoI (TYKY) subunit of Complex I binds two distinct [4Fe-4S] clusters, named N2a and N2b, each coordinated by a separate set of conserved cysteine residues; cysteine mutants C67S and C106S each caused a 50% decrease in the EPR N2 signal, demonstrating that both N2 clusters reside on this subunit.","method":"Site-directed mutagenesis of five cysteine residues, EPR spectroscopy of membrane fractions, NADH:ubiquinone oxidoreductase activity assays","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic cysteine mutagenesis combined with EPR spectroscopy, multiple mutant classes, ortholog study firmly establishing FeS binding","pmids":["12615348"],"is_preprint":false},{"year":2004,"finding":"Compound heterozygous mutations in NDUFS8 in a Leigh syndrome patient cause reduced levels of the NDUFS8 polypeptide and secondary reductions in other nuclear-encoded Complex I subunits, indicating NDUFS8 is essential for the assembly or stability of the Complex I holoenzyme.","method":"Mutation analysis, Western blot of patient-derived tissue","journal":"Neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Western blot from a single patient, single lab, but directly ties protein loss to Complex I assembly defect","pmids":["15159508"],"is_preprint":false},{"year":2021,"finding":"TAT-fused NDUFS8 fusion proteins are transduced into mitochondria in a membrane-potential-independent manner, are correctly processed, rescue Complex I assembly, and partially restore Complex I activity in NDUFS8-deficient cells.","method":"Recombinant TAT-fusion protein production, cell transduction, mitochondrial fractionation, in-gel activity assay, oxygen consumption assay","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional assays (assembly, in-gel activity, OCR) with deficient cell rescue, single lab","pmids":["34204592"],"is_preprint":false},{"year":2022,"finding":"Disease-associated mutations in NDUFS8 (mapped to the nuoI locus in E. coli Complex I) disrupt Complex I assembly; co-immunoprecipitation and time-delayed expression assays confirmed that certain mutations act at subunit interfaces, and compound heterozygote modeling identified the more deleterious mutation in each pair.","method":"Site-directed mutagenesis in E. coli homolog (nuoI), deamino-NADH oxidase activity, co-immunoprecipitation, assembly assay","journal":"Mitochondrion","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bacterial reconstitution with multiple orthogonal assays, but indirect (E. coli surrogate model)","pmids":["36462614"],"is_preprint":false},{"year":2024,"finding":"NDUFS8 knockdown or knockout in endothelial cells reduces Complex I activity, decreases ATP production, depolarizes mitochondria, and increases ROS; these effects impair the Akt-mTOR signaling cascade, which is rescuable by exogenous ATP. Conversely, NDUFS8 overexpression promotes Akt-mTOR activation, cell proliferation, migration, and tube formation. Endothelial-specific NDUFS8 knockdown in vivo inhibits retinal angiogenesis.","method":"shRNA/CRISPR-Cas9 KO, oxygen consumption assay, ATP assay, ROS measurement, mitochondrial membrane potential assay, Akt-mTOR pathway analysis, constitutively-active Akt1 rescue, AAV-mediated in vivo knockdown, retinal angiogenesis assay","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (KO, OE, in vitro rescue, in vivo AAV), consistent mechanistic pathway from Complex I activity to Akt-mTOR to angiogenesis","pmids":["38594244"],"is_preprint":false},{"year":2025,"finding":"HUWE1 is an E3 ubiquitin ligase that ubiquitinates NDUFS8 at lysine 88, regulating its protein stability; NDUFS8 localizes to mitochondria, promotes Complex I activity and ATP production in hepatocellular carcinoma cells, and its loss increases ROS, disrupts redox homeostasis, and induces apoptosis.","method":"Mass spectrometry, co-immunoprecipitation, ubiquitination assay, mitochondrial fractionation, shRNA/KO/OE functional assays, xenograft mouse model","journal":"Translational oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified interactor confirmed by co-IP and ubiquitination assay, single lab, multiple functional readouts","pmids":["40914145"],"is_preprint":false},{"year":2026,"finding":"NRF2 regulates NDUFS8 transcription by binding both ARE and non-ARE motifs in the NDUFS8 promoter; cytoplasmic NRF2 also stabilizes NDUFS8 protein post-translationally. AAV-mediated NDUFS8 restoration in basal forebrain rescues spatial memory deficits in chronic cerebral hypoperfusion rats by enhancing mitochondrial oxidative phosphorylation.","method":"Dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP), computational simulation, stereotaxic AAV injection with gain/loss-of-function, behavioral memory assay, mitochondrial OXPHOS measurement","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and reporter assay for transcriptional mechanism, AAV in vivo rescue for functional relevance, single lab","pmids":["41355955"],"is_preprint":false}],"current_model":"NDUFS8 (TYKY/CI-23k) is a nuclear-encoded core subunit of mitochondrial Complex I that harbors two [4Fe-4S] clusters (N2a and N2b), each coordinated by a distinct set of conserved cysteine residues; it is required for the assembly and stability of both the peripheral and membrane domains of Complex I, and its loss reduces NADH:ubiquinone oxidoreductase activity, lowers ATP production, and elevates ROS. Transcription of NDUFS8 is driven by YY1 and Sp1 at a minimal promoter, and NRF2 additionally activates the gene via ARE/non-ARE elements while also stabilizing the protein post-translationally. NDUFS8 protein stability is further regulated by the E3 ubiquitin ligase HUWE1, which ubiquitinates it at lysine 88. Downstream of Complex I, NDUFS8-dependent ATP production supports Akt-mTOR signaling in endothelial cells to drive angiogenesis, and NDUFS8 activity in the basal forebrain supports cognitive function by maintaining mitochondrial oxidative phosphorylation."},"narrative":{"mechanistic_narrative":"NDUFS8 (TYKY) is a nuclear-encoded core iron-sulfur subunit of mitochondrial Complex I that is essential for assembly of the holoenzyme and for NADH:ubiquinone oxidoreductase activity [PMID:9116042, PMID:15159508]. It carries two distinct [4Fe-4S] clusters, N2a and N2b, each coordinated by a separate set of conserved cysteine residues, and mutation of these cysteines diminishes the N2 EPR signal and abolishes electron transfer activity [PMID:12615348]. The subunit physically connects the peripheral and membrane domains of Complex I; loss of the ortholog or disease-associated mutations at subunit interfaces prevent assembly and cause secondary loss of other nuclear-encoded subunits [PMID:9428698, PMID:15159508, PMID:36462614]. Functionally, NDUFS8-dependent Complex I activity sustains ATP production and mitochondrial integrity, and its loss depolarizes mitochondria and elevates ROS; in endothelial cells this ATP supply drives Akt-mTOR signaling to support proliferation, migration, and angiogenesis [PMID:38594244], and restoration of NDUFS8 in the basal forebrain rescues memory deficits by enhancing oxidative phosphorylation [PMID:41355955]. Transcription is controlled by a minimal promoter in which a YY1 site and adjacent Sp1 sites are the primary drivers [PMID:11955626], with NRF2 additionally activating the gene through ARE and non-ARE motifs and stabilizing the protein post-translationally [PMID:41355955], while the E3 ligase HUWE1 ubiquitinates NDUFS8 at lysine 88 to regulate its stability [PMID:40914145]. Compound heterozygous mutations in NDUFS8 cause Leigh syndrome through Complex I deficiency [PMID:15159508].","teleology":[{"year":1997,"claim":"Establishing NDUFS8's molecular identity was the first step: cloning showed it encodes a mitochondrially-targeted iron-sulfur subunit with two conserved cysteine clusters, predicting a metal-cofactor role in Complex I.","evidence":"cDNA sequencing, deduced protein and cross-species conservation analysis","pmids":["9116042"],"confidence":"Medium","gaps":["Did not demonstrate which clusters the cysteines coordinate","No direct functional or assembly data"]},{"year":1997,"claim":"Whether the subunit is functionally required was answered by ortholog deletion and cysteine mutagenesis, showing it is essential for Complex I activity, FeS cluster signals, and assembly bridging the peripheral and membrane arms.","evidence":"Homologous recombination deletion, C74S mutagenesis, EPR, and trans-complementation in Rhodobacter capsulatus","pmids":["9428698"],"confidence":"High","gaps":["Performed in a bacterial ortholog rather than human protein","Did not resolve the number of FeS clusters carried"]},{"year":1998,"claim":"Gene/protein structural characterization defined the mature ~22 kDa product and identified Sp1 and NRF1 motifs, opening the question of how transcription is controlled.","evidence":"Genomic sequencing, 2D gel electrophoresis, immunodetection","pmids":["9666055"],"confidence":"Medium","gaps":["Promoter motifs not functionally validated","Tissue-specificity correlative only"]},{"year":2002,"claim":"The basal transcriptional control was resolved, identifying a minimal promoter where a YY1 site and adjacent Sp1 sites drive expression.","evidence":"Reporter assays, EMSA, and site-directed mutagenesis in HeLa and C2C12 cells","pmids":["11955626"],"confidence":"High","gaps":["Did not address inducible or stress-responsive regulation","No in vivo promoter validation"]},{"year":2003,"claim":"The cofactor content was settled by systematic cysteine mutagenesis with EPR, demonstrating the subunit carries two distinct [4Fe-4S] clusters, N2a and N2b.","evidence":"Site-directed mutagenesis of five cysteines, EPR spectroscopy, NADH:ubiquinone oxidoreductase assays in ortholog","pmids":["12615348"],"confidence":"High","gaps":["Ortholog system; human cluster assignment inferred","Functional role of each cluster in electron transfer not separated"]},{"year":2004,"claim":"The human disease relevance was established: compound heterozygous mutations cause Leigh syndrome with reduced NDUFS8 and secondary loss of other subunits, confirming its role in holoenzyme assembly/stability.","evidence":"Mutation analysis and Western blot of patient tissue","pmids":["15159508"],"confidence":"Medium","gaps":["Single patient","Assembly defect inferred from steady-state protein levels, not assembly intermediates"]},{"year":2021,"claim":"A delivery-based rescue addressed therapeutic restoration, showing exogenous TAT-NDUFS8 enters mitochondria potential-independently and partially restores Complex I assembly and activity in deficient cells.","evidence":"TAT-fusion protein transduction, mitochondrial fractionation, in-gel activity and oxygen consumption assays","pmids":["34204592"],"confidence":"Medium","gaps":["Only partial activity restoration","Single cell model"]},{"year":2022,"claim":"Structure-function mapping of patient mutations clarified that specific variants act at subunit interfaces to block assembly, and ranked the more deleterious allele in compound heterozygotes.","evidence":"nuoI mutagenesis in E. coli, co-immunoprecipitation, assembly and deamino-NADH oxidase assays","pmids":["36462614"],"confidence":"Medium","gaps":["E. coli surrogate model","Interface assignments not confirmed in human Complex I"]},{"year":2024,"claim":"The downstream physiological consequence was defined: NDUFS8-driven ATP production feeds Akt-mTOR signaling to control endothelial proliferation and angiogenesis in vitro and in vivo.","evidence":"shRNA/CRISPR KO and overexpression, OCR/ATP/ROS assays, constitutively-active Akt rescue, AAV in vivo retinal angiogenesis","pmids":["38594244"],"confidence":"High","gaps":["Mechanistic link is ATP-dependent but molecular coupling to Akt unresolved","Restricted to endothelial context"]},{"year":2025,"claim":"Post-translational regulation was identified, with HUWE1 ubiquitinating NDUFS8 at K88 to control its stability and influence ATP/ROS balance and apoptosis in cancer cells.","evidence":"Mass spectrometry, co-IP, ubiquitination assay, functional KO/OE, xenograft","pmids":["40914145"],"confidence":"Medium","gaps":["Single lab interactor","Consequence of K88 ubiquitination on degradation kinetics not fully mapped"]},{"year":2026,"claim":"Stress-responsive transcriptional and protein-level regulation was added, showing NRF2 both activates the promoter via ARE/non-ARE motifs and stabilizes the protein, with restoration rescuing cognitive deficits.","evidence":"Dual-luciferase, ChIP, AAV gain/loss-of-function in basal forebrain, behavioral and OXPHOS assays","pmids":["41355955"],"confidence":"Medium","gaps":["Mechanism of cytoplasmic NRF2 protein stabilization unresolved","Non-ARE binding mode not structurally defined"]},{"year":null,"claim":"How NDUFS8's two FeS clusters mediate electron transfer within the human Complex I architecture, and how its transcriptional, ubiquitin, and NRF2-mediated regulatory layers are integrated under physiological stress, remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of human NDUFS8 within assembled Complex I in the corpus","Interplay between HUWE1 ubiquitination and NRF2 stabilization not reconciled"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[1,5]},{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[5,1]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,6]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,7,10]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[5,9]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[6,8]}],"complexes":["Mitochondrial respiratory Complex I (NADH:ubiquinone oxidoreductase)"],"partners":["HUWE1","NRF2","YY1","SP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O00217","full_name":"NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial","aliases":["Complex I-23kD","CI-23kD","NADH-ubiquinone oxidoreductase 23 kDa subunit","TYKY subunit"],"length_aa":210,"mass_kda":23.7,"function":"Core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) which catalyzes electron transfer from NADH through the respiratory chain, using ubiquinone as an electron acceptor (PubMed:22499348). Essential for the catalytic activity and assembly of complex I (PubMed:22499348)","subcellular_location":"Mitochondrion inner membrane","url":"https://www.uniprot.org/uniprotkb/O00217/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/NDUFS8","classification":"Common Essential","n_dependent_lines":615,"n_total_lines":1208,"dependency_fraction":0.5091059602649006},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NDUFS8","total_profiled":1310},"omim":[{"mim_id":"619382","title":"LEBER-LIKE HEREDITARY OPTIC NEUROPATHY, AUTOSOMAL RECESSIVE 1; LHONAR1","url":"https://www.omim.org/entry/619382"},{"mim_id":"618222","title":"MITOCHONDRIAL COMPLEX I DEFICIENCY, NUCLEAR TYPE 2; MC1DN2","url":"https://www.omim.org/entry/618222"},{"mim_id":"618202","title":"DNAJ/HSP40 HOMOLOG, SUBFAMILY C, MEMBER 30; DNAJC30","url":"https://www.omim.org/entry/618202"},{"mim_id":"617228","title":"COMBINED OXIDATIVE PHOSPHORYLATION DEFICIENCY 31; COXPD31","url":"https://www.omim.org/entry/617228"},{"mim_id":"603846","title":"NADH-UBIQUINONE OXIDOREDUCTASE Fe-S PROTEIN 3; NDUFS3","url":"https://www.omim.org/entry/603846"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mitochondria","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NDUFS8"},"hgnc":{"alias_symbol":["TYKY","CI-23k"],"prev_symbol":[]},"alphafold":{"accession":"O00217","domains":[{"cath_id":"-","chopping":"47-80","consensus_level":"medium","plddt":94.6403,"start":47,"end":80},{"cath_id":"3.30.70.3270","chopping":"97-206","consensus_level":"high","plddt":95.8188,"start":97,"end":206}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O00217","model_url":"https://alphafold.ebi.ac.uk/files/AF-O00217-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O00217-F1-predicted_aligned_error_v6.png","plddt_mean":88.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NDUFS8","jax_strain_url":"https://www.jax.org/strain/search?query=NDUFS8"},"sequence":{"accession":"O00217","fasta_url":"https://rest.uniprot.org/uniprotkb/O00217.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O00217/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O00217"}},"corpus_meta":[{"pmid":"15159508","id":"PMC_15159508","title":"Late-onset Leigh syndrome in a patient with mitochondrial complex I NDUFS8 mutations.","date":"2004","source":"Neurology","url":"https://pubmed.ncbi.nlm.nih.gov/15159508","citation_count":71,"is_preprint":false},{"pmid":"9116042","id":"PMC_9116042","title":"cDNA sequence and chromosomal localization of the NDUFS8 human gene coding for the 23 kDa subunit of the mitochondrial complex I.","date":"1997","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/9116042","citation_count":34,"is_preprint":false},{"pmid":"9428698","id":"PMC_9428698","title":"The NuoI subunit of the Rhodobacter capsulatus respiratory Complex I (equivalent to the bovine TYKY subunit) is required for proper assembly of the membraneous and peripheral domains of the enzyme.","date":"1997","source":"European journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9428698","citation_count":31,"is_preprint":false},{"pmid":"9666055","id":"PMC_9666055","title":"Genomic structure of the human NDUFS8 gene coding for the iron-sulfur TYKY subunit of the mitochondrial NADH:ubiquinone oxidoreductase.","date":"1998","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/9666055","citation_count":20,"is_preprint":false},{"pmid":"12615348","id":"PMC_12615348","title":"Two EPR-detectable [4Fe-4S] clusters, N2a and N2b, are bound to the NuoI (TYKY) subunit of NADH:ubiquinone oxidoreductase (Complex I) from Rhodobacter capsulatus.","date":"2003","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/12615348","citation_count":20,"is_preprint":false},{"pmid":"38594244","id":"PMC_38594244","title":"The requirement of the mitochondrial protein NDUFS8 for angiogenesis.","date":"2024","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/38594244","citation_count":18,"is_preprint":false},{"pmid":"23430795","id":"PMC_23430795","title":"NDUFS8-related Complex I Deficiency Extends Phenotype from \"PEO Plus\" to Leigh Syndrome.","date":"2012","source":"JIMD reports","url":"https://pubmed.ncbi.nlm.nih.gov/23430795","citation_count":15,"is_preprint":false},{"pmid":"11955626","id":"PMC_11955626","title":"YY1 and Sp1 activate transcription of the human NDUFS8 gene encoding the mitochondrial complex I TYKY subunit.","date":"2002","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/11955626","citation_count":13,"is_preprint":false},{"pmid":"35887413","id":"PMC_35887413","title":"Role of the Gene ndufs8 Located in Respiratory Complex I from Monascus purpureus in the Cell Growth and Secondary Metabolites Biosynthesis.","date":"2022","source":"Journal of fungi (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/35887413","citation_count":9,"is_preprint":false},{"pmid":"9452770","id":"PMC_9452770","title":"Identification of the TYKY homologous subunit of complex I from Neurospora crassa.","date":"1997","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/9452770","citation_count":9,"is_preprint":false},{"pmid":"34204592","id":"PMC_34204592","title":"TAT-Conjugated NDUFS8 Can Be Transduced into Mitochondria in a Membrane-Potential-Independent Manner and Rescue Complex I Deficiency.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/34204592","citation_count":8,"is_preprint":false},{"pmid":"36462614","id":"PMC_36462614","title":"Analysis of compound heterozygous and homozygous mutations found in peripheral subunits of human respiratory Complex I, NDUFS1, NDUFS2, NDUFS8 and NDUFV1, by modeling in the E. coli enzyme.","date":"2022","source":"Mitochondrion","url":"https://pubmed.ncbi.nlm.nih.gov/36462614","citation_count":4,"is_preprint":false},{"pmid":"36135178","id":"PMC_36135178","title":"Higher NADH Dehydrogenase [Ubiquinone] Iron-Sulfur Protein 8 (NDUFS8) Serum Levels Correlate with Better Insulin Sensitivity in Type 1 Diabetes.","date":"2022","source":"Current issues in molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/36135178","citation_count":4,"is_preprint":false},{"pmid":"39707499","id":"PMC_39707499","title":"Human umbilical mesenchymal stem cells ameliorate atrophic gastritis in aging mice by participating in mitochondrial autophagy through Ndufs8 signaling.","date":"2024","source":"Stem cell research & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/39707499","citation_count":3,"is_preprint":false},{"pmid":"36101822","id":"PMC_36101822","title":"Expansion of the clinical and neuroimaging spectrum associated with NDUFS8-related disorder.","date":"2022","source":"JIMD reports","url":"https://pubmed.ncbi.nlm.nih.gov/36101822","citation_count":3,"is_preprint":false},{"pmid":"38428158","id":"PMC_38428158","title":"Mitochondrial complex I subunit NDUFS8.2 modulates responses to stresses associated with reduced water availability.","date":"2024","source":"Plant physiology and biochemistry : PPB","url":"https://pubmed.ncbi.nlm.nih.gov/38428158","citation_count":2,"is_preprint":false},{"pmid":"40239028","id":"PMC_40239028","title":"NDUFS8-Related Leigh Syndrome Mimicking a Leukodystrophy.","date":"2025","source":"Journal of child neurology","url":"https://pubmed.ncbi.nlm.nih.gov/40239028","citation_count":1,"is_preprint":false},{"pmid":"40914145","id":"PMC_40914145","title":"NDUFS8 facilitates hepatocellular carcinoma growth by enhancing mitochondrial function and escaping HUWE1-dependent degradation.","date":"2025","source":"Translational oncology","url":"https://pubmed.ncbi.nlm.nih.gov/40914145","citation_count":0,"is_preprint":false},{"pmid":"41355955","id":"PMC_41355955","title":"Targeting NDUFS8 in basal forebrain ameliorates cognitive decline related to chronic cerebral hypoperfusion.","date":"2026","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/41355955","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10729,"output_tokens":3137,"usd":0.039621,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10582,"output_tokens":3483,"usd":0.069992,"stage2_stop_reason":"end_turn"},"total_usd":0.109613,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"NDUFS8 (TYKY subunit) contains two clusters of four conserved cysteine residues and encodes a 210-amino-acid precursor with a 34-amino-acid N-terminal mitochondrial targeting presequence; the mature protein is 92% identical to the bovine subunit and 72% to the Rhodobacter capsulatus NuoI counterpart, establishing it as an iron-sulfur subunit of Complex I.\",\n      \"method\": \"cDNA sequencing, deduced protein analysis, chromosomal mapping\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — sequence analysis plus cross-species conservation and structural domain identification, single lab\",\n      \"pmids\": [\"9116042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Deletion of the NuoI (TYKY ortholog) gene or mutation of a conserved cysteine (C74S) in Rhodobacter capsulatus abolishes Complex I activity, eliminates EPR signals for FeS clusters N1 and N2, and prevents assembly of peripheral subunits, demonstrating that NuoI is required for connecting the peripheral and membraneous domains of Complex I.\",\n      \"method\": \"Homologous recombination deletion, site-directed mutagenesis (C74S), EPR spectroscopy, immunochemical analysis, trans-complementation\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — deletion and point-mutagenesis with EPR, immunochemistry, and complementation rescue across multiple mutants in a single rigorous study\",\n      \"pmids\": [\"9428698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"The TYKY/NDUFS8 homolog in Neurospora crassa is closely associated with the peripheral arm of Complex I, as shown by co-purification and antiserum-based localization.\",\n      \"method\": \"Heterologous expression in E. coli, purification, antibody production, biochemical fractionation\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single lab, co-fractionation only, but consistent with broader Complex I literature\",\n      \"pmids\": [\"9452770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The mature NDUFS8/TYKY protein has a molecular mass of ~22 kDa and a pI of 4.9–5.0, as determined by 2D gel electrophoresis and immunodetection; the gene spans ~6 kb with seven exons, and its promoter contains Sp1 and NRF1 binding site motifs in the first intron, with ubiquitous expression predominant in heart and skeletal muscle.\",\n      \"method\": \"Genomic sequencing, 2D gel electrophoresis, immunodetection\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein characterization by 2D gel plus genomic structural analysis, single lab\",\n      \"pmids\": [\"9666055\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Transcription of NDUFS8 is driven by a minimal 247-bp basal promoter containing three Sp1 sites and one YY1 site; gel-shift analysis and site-directed mutagenesis showed that the YY1 site and two adjacent Sp1 sites are the primary drivers of promoter activity.\",\n      \"method\": \"Primer extension, reporter gene assay (HeLa and C2C12 cells), gel-shift (EMSA), site-directed mutagenesis\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal methods (EMSA, reporter assay, mutagenesis) in a single focused promoter study\",\n      \"pmids\": [\"11955626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The NuoI (TYKY) subunit of Complex I binds two distinct [4Fe-4S] clusters, named N2a and N2b, each coordinated by a separate set of conserved cysteine residues; cysteine mutants C67S and C106S each caused a 50% decrease in the EPR N2 signal, demonstrating that both N2 clusters reside on this subunit.\",\n      \"method\": \"Site-directed mutagenesis of five cysteine residues, EPR spectroscopy of membrane fractions, NADH:ubiquinone oxidoreductase activity assays\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic cysteine mutagenesis combined with EPR spectroscopy, multiple mutant classes, ortholog study firmly establishing FeS binding\",\n      \"pmids\": [\"12615348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Compound heterozygous mutations in NDUFS8 in a Leigh syndrome patient cause reduced levels of the NDUFS8 polypeptide and secondary reductions in other nuclear-encoded Complex I subunits, indicating NDUFS8 is essential for the assembly or stability of the Complex I holoenzyme.\",\n      \"method\": \"Mutation analysis, Western blot of patient-derived tissue\",\n      \"journal\": \"Neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Western blot from a single patient, single lab, but directly ties protein loss to Complex I assembly defect\",\n      \"pmids\": [\"15159508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TAT-fused NDUFS8 fusion proteins are transduced into mitochondria in a membrane-potential-independent manner, are correctly processed, rescue Complex I assembly, and partially restore Complex I activity in NDUFS8-deficient cells.\",\n      \"method\": \"Recombinant TAT-fusion protein production, cell transduction, mitochondrial fractionation, in-gel activity assay, oxygen consumption assay\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional assays (assembly, in-gel activity, OCR) with deficient cell rescue, single lab\",\n      \"pmids\": [\"34204592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Disease-associated mutations in NDUFS8 (mapped to the nuoI locus in E. coli Complex I) disrupt Complex I assembly; co-immunoprecipitation and time-delayed expression assays confirmed that certain mutations act at subunit interfaces, and compound heterozygote modeling identified the more deleterious mutation in each pair.\",\n      \"method\": \"Site-directed mutagenesis in E. coli homolog (nuoI), deamino-NADH oxidase activity, co-immunoprecipitation, assembly assay\",\n      \"journal\": \"Mitochondrion\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bacterial reconstitution with multiple orthogonal assays, but indirect (E. coli surrogate model)\",\n      \"pmids\": [\"36462614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NDUFS8 knockdown or knockout in endothelial cells reduces Complex I activity, decreases ATP production, depolarizes mitochondria, and increases ROS; these effects impair the Akt-mTOR signaling cascade, which is rescuable by exogenous ATP. Conversely, NDUFS8 overexpression promotes Akt-mTOR activation, cell proliferation, migration, and tube formation. Endothelial-specific NDUFS8 knockdown in vivo inhibits retinal angiogenesis.\",\n      \"method\": \"shRNA/CRISPR-Cas9 KO, oxygen consumption assay, ATP assay, ROS measurement, mitochondrial membrane potential assay, Akt-mTOR pathway analysis, constitutively-active Akt1 rescue, AAV-mediated in vivo knockdown, retinal angiogenesis assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (KO, OE, in vitro rescue, in vivo AAV), consistent mechanistic pathway from Complex I activity to Akt-mTOR to angiogenesis\",\n      \"pmids\": [\"38594244\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HUWE1 is an E3 ubiquitin ligase that ubiquitinates NDUFS8 at lysine 88, regulating its protein stability; NDUFS8 localizes to mitochondria, promotes Complex I activity and ATP production in hepatocellular carcinoma cells, and its loss increases ROS, disrupts redox homeostasis, and induces apoptosis.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, ubiquitination assay, mitochondrial fractionation, shRNA/KO/OE functional assays, xenograft mouse model\",\n      \"journal\": \"Translational oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified interactor confirmed by co-IP and ubiquitination assay, single lab, multiple functional readouts\",\n      \"pmids\": [\"40914145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"NRF2 regulates NDUFS8 transcription by binding both ARE and non-ARE motifs in the NDUFS8 promoter; cytoplasmic NRF2 also stabilizes NDUFS8 protein post-translationally. AAV-mediated NDUFS8 restoration in basal forebrain rescues spatial memory deficits in chronic cerebral hypoperfusion rats by enhancing mitochondrial oxidative phosphorylation.\",\n      \"method\": \"Dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP), computational simulation, stereotaxic AAV injection with gain/loss-of-function, behavioral memory assay, mitochondrial OXPHOS measurement\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and reporter assay for transcriptional mechanism, AAV in vivo rescue for functional relevance, single lab\",\n      \"pmids\": [\"41355955\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NDUFS8 (TYKY/CI-23k) is a nuclear-encoded core subunit of mitochondrial Complex I that harbors two [4Fe-4S] clusters (N2a and N2b), each coordinated by a distinct set of conserved cysteine residues; it is required for the assembly and stability of both the peripheral and membrane domains of Complex I, and its loss reduces NADH:ubiquinone oxidoreductase activity, lowers ATP production, and elevates ROS. Transcription of NDUFS8 is driven by YY1 and Sp1 at a minimal promoter, and NRF2 additionally activates the gene via ARE/non-ARE elements while also stabilizing the protein post-translationally. NDUFS8 protein stability is further regulated by the E3 ubiquitin ligase HUWE1, which ubiquitinates it at lysine 88. Downstream of Complex I, NDUFS8-dependent ATP production supports Akt-mTOR signaling in endothelial cells to drive angiogenesis, and NDUFS8 activity in the basal forebrain supports cognitive function by maintaining mitochondrial oxidative phosphorylation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NDUFS8 (TYKY) is a nuclear-encoded core iron-sulfur subunit of mitochondrial Complex I that is essential for assembly of the holoenzyme and for NADH:ubiquinone oxidoreductase activity [#0, #6]. It carries two distinct [4Fe-4S] clusters, N2a and N2b, each coordinated by a separate set of conserved cysteine residues, and mutation of these cysteines diminishes the N2 EPR signal and abolishes electron transfer activity [#5]. The subunit physically connects the peripheral and membrane domains of Complex I; loss of the ortholog or disease-associated mutations at subunit interfaces prevent assembly and cause secondary loss of other nuclear-encoded subunits [#1, #6, #8]. Functionally, NDUFS8-dependent Complex I activity sustains ATP production and mitochondrial integrity, and its loss depolarizes mitochondria and elevates ROS; in endothelial cells this ATP supply drives Akt-mTOR signaling to support proliferation, migration, and angiogenesis [#9], and restoration of NDUFS8 in the basal forebrain rescues memory deficits by enhancing oxidative phosphorylation [#11]. Transcription is controlled by a minimal promoter in which a YY1 site and adjacent Sp1 sites are the primary drivers [#4], with NRF2 additionally activating the gene through ARE and non-ARE motifs and stabilizing the protein post-translationally [#11], while the E3 ligase HUWE1 ubiquitinates NDUFS8 at lysine 88 to regulate its stability [#10]. Compound heterozygous mutations in NDUFS8 cause Leigh syndrome through Complex I deficiency [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing NDUFS8's molecular identity was the first step: cloning showed it encodes a mitochondrially-targeted iron-sulfur subunit with two conserved cysteine clusters, predicting a metal-cofactor role in Complex I.\",\n      \"evidence\": \"cDNA sequencing, deduced protein and cross-species conservation analysis\",\n      \"pmids\": [\"9116042\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not demonstrate which clusters the cysteines coordinate\", \"No direct functional or assembly data\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Whether the subunit is functionally required was answered by ortholog deletion and cysteine mutagenesis, showing it is essential for Complex I activity, FeS cluster signals, and assembly bridging the peripheral and membrane arms.\",\n      \"evidence\": \"Homologous recombination deletion, C74S mutagenesis, EPR, and trans-complementation in Rhodobacter capsulatus\",\n      \"pmids\": [\"9428698\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Performed in a bacterial ortholog rather than human protein\", \"Did not resolve the number of FeS clusters carried\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Gene/protein structural characterization defined the mature ~22 kDa product and identified Sp1 and NRF1 motifs, opening the question of how transcription is controlled.\",\n      \"evidence\": \"Genomic sequencing, 2D gel electrophoresis, immunodetection\",\n      \"pmids\": [\"9666055\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Promoter motifs not functionally validated\", \"Tissue-specificity correlative only\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"The basal transcriptional control was resolved, identifying a minimal promoter where a YY1 site and adjacent Sp1 sites drive expression.\",\n      \"evidence\": \"Reporter assays, EMSA, and site-directed mutagenesis in HeLa and C2C12 cells\",\n      \"pmids\": [\"11955626\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address inducible or stress-responsive regulation\", \"No in vivo promoter validation\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The cofactor content was settled by systematic cysteine mutagenesis with EPR, demonstrating the subunit carries two distinct [4Fe-4S] clusters, N2a and N2b.\",\n      \"evidence\": \"Site-directed mutagenesis of five cysteines, EPR spectroscopy, NADH:ubiquinone oxidoreductase assays in ortholog\",\n      \"pmids\": [\"12615348\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ortholog system; human cluster assignment inferred\", \"Functional role of each cluster in electron transfer not separated\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"The human disease relevance was established: compound heterozygous mutations cause Leigh syndrome with reduced NDUFS8 and secondary loss of other subunits, confirming its role in holoenzyme assembly/stability.\",\n      \"evidence\": \"Mutation analysis and Western blot of patient tissue\",\n      \"pmids\": [\"15159508\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single patient\", \"Assembly defect inferred from steady-state protein levels, not assembly intermediates\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"A delivery-based rescue addressed therapeutic restoration, showing exogenous TAT-NDUFS8 enters mitochondria potential-independently and partially restores Complex I assembly and activity in deficient cells.\",\n      \"evidence\": \"TAT-fusion protein transduction, mitochondrial fractionation, in-gel activity and oxygen consumption assays\",\n      \"pmids\": [\"34204592\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Only partial activity restoration\", \"Single cell model\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Structure-function mapping of patient mutations clarified that specific variants act at subunit interfaces to block assembly, and ranked the more deleterious allele in compound heterozygotes.\",\n      \"evidence\": \"nuoI mutagenesis in E. coli, co-immunoprecipitation, assembly and deamino-NADH oxidase assays\",\n      \"pmids\": [\"36462614\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E. coli surrogate model\", \"Interface assignments not confirmed in human Complex I\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The downstream physiological consequence was defined: NDUFS8-driven ATP production feeds Akt-mTOR signaling to control endothelial proliferation and angiogenesis in vitro and in vivo.\",\n      \"evidence\": \"shRNA/CRISPR KO and overexpression, OCR/ATP/ROS assays, constitutively-active Akt rescue, AAV in vivo retinal angiogenesis\",\n      \"pmids\": [\"38594244\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic link is ATP-dependent but molecular coupling to Akt unresolved\", \"Restricted to endothelial context\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Post-translational regulation was identified, with HUWE1 ubiquitinating NDUFS8 at K88 to control its stability and influence ATP/ROS balance and apoptosis in cancer cells.\",\n      \"evidence\": \"Mass spectrometry, co-IP, ubiquitination assay, functional KO/OE, xenograft\",\n      \"pmids\": [\"40914145\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab interactor\", \"Consequence of K88 ubiquitination on degradation kinetics not fully mapped\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Stress-responsive transcriptional and protein-level regulation was added, showing NRF2 both activates the promoter via ARE/non-ARE motifs and stabilizes the protein, with restoration rescuing cognitive deficits.\",\n      \"evidence\": \"Dual-luciferase, ChIP, AAV gain/loss-of-function in basal forebrain, behavioral and OXPHOS assays\",\n      \"pmids\": [\"41355955\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of cytoplasmic NRF2 protein stabilization unresolved\", \"Non-ARE binding mode not structurally defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NDUFS8's two FeS clusters mediate electron transfer within the human Complex I architecture, and how its transcriptional, ubiquitin, and NRF2-mediated regulatory layers are integrated under physiological stress, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of human NDUFS8 within assembled Complex I in the corpus\", \"Interplay between HUWE1 ubiquitination and NRF2 stabilization not reconciled\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [5, 1]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 7, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [5, 9]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [6, 8]}\n    ],\n    \"complexes\": [\"Mitochondrial respiratory Complex I (NADH:ubiquinone oxidoreductase)\"],\n    \"partners\": [\"HUWE1\", \"NRF2\", \"YY1\", \"SP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}