{"artifact":{"id":"experiment_proposal-fa36c80e-5e55-4e39-9406-462337fe58b6","artifact_type":"experiment_proposal","entity_ids":"[\"h-var-08a4d5c07a\"]","title":"Experiment Proposal: h-var-08a4d5c07a","quality_score":0.75,"created_by":"forge_experiment_proposal_generator","provenance_chain":"[]","content_hash":"c265c44c1415b8eb0c8cbcf0319a9fe4ddc9d84d2ff4e78ecb6109d6c010ce62","metadata":{"citations":["29263430","30679038","33875891","33741860","31748742","31043694","27519954","30610225"],"generated_at":"2026-04-28T20:02:35.464403+00:00","intervention":"Next-generation probiotic consortium (Akkermansia muciniphila 1×10^9 CFU + Faecalibacterium prausnitzii 1×10^9 CFU + Bifidobacterium longum 1×10^9 CFU, oral gavage, 5 days/week for 12 weeks) plus MCC950 (50 mg/kg i.p., 3 days/week for 12 weeks); combination and monotherapy arms included","key_controls":["WT C57BL/6J mice receiving identical probiotic+MCC950 regimen to establish baseline variability","NLRP3 knockout mice crossed to APP/PS1 (APP/PS1/Nlrp3−/−) receiving probiotics to confirm inflammasome-dependence of treatment effect","Germ-free APP/PS1 mice with probiotics vs conventional APP/PS1 mice to quantify gut-axis contribution","Heat-killed probiotic consortium in APP/PS1 mice to control for microbial antigen effects independent of colonization","Vehicle-only (saline/DMSO) APP/PS1 mice as positive pathology controls","MCC950-only arm to dissociate direct NLRP3 inhibition from microbiome remodeling effects"],"model_system":"APP/PS1dE9 transgenic mice on C57BL/6J background; both sexes; 3 months (pre-plaque, n=24/group) and 6 months (post-plaque, n=24/group) cohorts; total N=192 animals across 8 groups","hypothesis_id":"h-var-08a4d5c07a","timeline_weeks":14,"_schema_version":1,"null_hypothesis":"Gut microbiome remodeling via next-generation probiotic consortium does not reduce systemic NLRP3 inflammasome priming or attenuate neurodegeneration-associated neuroinflammation compared to vehicle-treated amyloid-deposition mice when administered at symptomatically relevant timepoints","primary_readout":"Cortical and hippocampal IL-1β concentration (pg/mg tissue) measured by ELISA in CD11b+ MACS-isolated microglia; NLRP3 activation assessed by ASC speck count per CD68+ microglial cell via confocal microscopy","estimated_cost_usd":185000.0,"secondary_readouts":["Serum LPS concentration via LAL assay and soluble CD14 (eBioscience) reflecting bacterial translocation","Fecal SCFA profile (acetate, propionate, butyrate) by GC-MS at baseline, week 6, and week 12","Fecal microbiome 16S rRNA sequencing: alpha-diversity (Shannon), beta-diversity (Bray-Curtis), Firmicutes/Bacteroidetes ratio","Amyloid plaque burden: 6E10+ area fraction (%) in cortex and hippocampus by stereology","Soluble Aβ40/Aβ42 in brain homogenates by ELISA (Mesoscale)","Tau phosphorylation at Ser202/Thr205 (AT8), Thr231 (AT180) by ELISA","Plasma NfL and GFAP by Simoa as neurodegeneration/astrogliosis markers","Cognitive assessment: Barnes maze (spatial reference memory), novel object recognition (episodic memory), contextual fear conditioning (hippocampal-dependent) at week 10","qRT-PCR for Nlrp3, Casp1, Il1b, Pycard, Trem2, Cx3cr1 in sorted CD11b+ microglia","Synaptic density: synaptophysin+ puncta in hippocampus by immunohistochemistry"],"expected_effect_size":"Cohen's d = 0.85 for IL-1β reduction in combination arm vs APP/PS1 vehicle based on MCC950 literature (PMID 29263430); power = 0.80, alpha = 0.05, two-tailed; effect on amyloid burden anticipated d = 0.65","statistical_approach":"Mixed-effects ANOVA with time as repeated measure and treatment as between-subject factor; post-hoc Tukey HSD; two-way ANOVA for interaction effects of timing × intervention; Benjamini-Hochberg FDR correction (q<0.05) for secondary readouts; effect sizes reported as Cohen's d with 95% CI","sample_size_rationale":"Power analysis (G*Power 3.1): n=24 per group provides 80% power to detect d=0.80 (alpha=0.05, two-tailed); assumes 15% attrition; sample size anchored to IL-1β outcome variability (SD=0.35) from published MCC950 studies in APP/PS1 mice (PMID 29263430)","falsification_criterion":"Hypothesis falsified if combination therapy fails to reduce microglial IL-1β by ≥40% (pre-registered threshold) and fails to improve Barnes maze performance by ≥20% compared to vehicle APP/PS1 controls (both p<0.05 with BH correction); microbiome-only arm must distinguish gut-axis effect from direct CNS action"},"created_at":"2026-04-28T13:02:35.476173-07:00","updated_at":"2026-04-28T13:02:35.476173-07:00","version_number":3,"parent_version_id":null,"version_tag":null,"changelog":null,"is_latest":1,"lifecycle_state":"active","superseded_by":null,"deprecated_at":null,"deprecated_reason":null,"dependencies":null,"market_price":0.5,"origin_type":"internal","origin_url":null,"lifecycle_changed_at":null,"citation_count":0,"embed_count":0,"derivation_count":0,"support_count":0,"contradiction_count":0,"total_usage":0.0,"usage_score":0.5,"usage_computed_at":null,"quality_status":null,"contributors":[],"answers_question_ids":null,"deprecated_reason_detail":null,"deprecated_reason_code":null,"commit_sha":null,"commit_submodule":null,"last_mutated_at":"2026-05-16T14:51:34.657673-07:00","disputed_at":null,"gap_id":null,"mission_id":null,"intrinsic_priority":null,"effective_priority":null,"artifact_id":"00abf285-9109-4bd8-ba71-42988a6ca02b","artifact_dir":null,"primary_filename":null,"accessory_filenames":null,"folder_layout_version":1,"migrated_to_folder_at":null,"hypothesis_id":null,"authorship":{"kind":"human","contributors":[{"role":"author","actor_ref":"forge_experiment_proposal_generator"}]},"epistemic_tier":"T3_provisional","created_by_agent_id":null},"outgoing_links":[{"target_artifact_id":"h-var-08a4d5c07a","link_type":"derives_from","strength":1.0,"evidence":"{\"source\": \"forge_experiment_proposal_generator\", \"method\": \"generate_experiment_proposal\"}"}],"incoming_links":[],"current_artifact_id":"experiment_proposal-fa36c80e-5e55-4e39-9406-462337fe58b6","is_canonical":true,"supersede_chain":["experiment_proposal-fa36c80e-5e55-4e39-9406-462337fe58b6"]}