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docs: add MRSA USA300 to preregistration, align serum stability protocol - #83
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ASSAY_PREREGISTRATION.md: - Add S. aureus USA300 LAC (MRSA) as priority 3 organism — required for MDR impact claims; SEED-007, SEED-008, SEED-009 mechanisms are Gram-positive- relevant; MRSA activity doubles publication impact potential (~2× as per WET_LAB_PROBABILITY.md Section 4) - Add SEED-009 media note: run in RPMI-1640 parallel to MHB for proline-rich variants (Gagnon 2016; protein-rich MHB interferes with Pro-rich uptake) - Change serum stability from "Wave 1 optional" → "Wave 1 recommended before MIC panel" — early attrition on unstable candidates saves assay cost - Change 25% human serum → 50% pooled human serum (consistent with WET_LAB_HANDOFF.md Section 4 and Otvos & Cudic 2002 Bac2A reference) - Change time points 0/0.5/1/2/4h → 0/30/60/120 min (consistent with WET_LAB_HANDOFF.md; saves one time point, matches standard cathelicidin protocol) - Note SEED-003, SEED-008, SEED-009 model calibration limitations in section
ASSAY_PREREGISTRATION.md: - [HIGH] Gate P3 criterion aligned with Section 5: 't½ > 2h' → '> 50% stability at 60 min' (both now match the pre-registered pass criterion) - [MEDIUM] Remove 'Pre-registered decision (updated)' wording; use 'Pre-registered decision' only — the document is not yet locked so this is the original plan, not an amendment - [MEDIUM] Fix SEED-009 media note: replace imprecise 'protein-rich media interferes' with accurate 'media composition (amino acids, divalent cations) affects translocation efficiency' per Krizsan et al. 2014 Angew Chem Int Ed 53:14546 - [MEDIUM] Remove Gagnon 2016 Cell citation from target organisms note (uncertain journal/volume; Krizsan 2014 is sufficient and on solid ground) - [LOW] Fix serum stability dual readout: primary = HPLC/MS, backup = bioactivity (eliminates post-hoc readout flexibility that would undermine pre-registration) - [LOW] Remove vague "ranks 1–2, 6–7" parenthetical from SEED-009 note (not defined in document scope) WET_LAB_HANDOFF.md: - [HIGH] Fix SEED-008 helix-wheel table length: 22 AA → 13 AA (all 4 SEED-008 variants in pilot panel are 13 AA: FPVTWRFWRWWKG etc.; 22 AA was incorrect)
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Summary
Updates to
ASSAY_PREREGISTRATION.mdbased on recommendations indocs/WET_LAB_PROBABILITY.md(PR #81):MRSA USA300 (Priority 3) — Added S. aureus USA300 LAC (MRSA clinical isolate) as mandatory Priority 3 organism.
WET_LAB_PROBABILITY.mdshows adding MRSA raises breakthrough probability from 3–8% to 8–18% (the single highest-impact low-cost improvement available). SEED-007 (bombolitin-II), SEED-008 (Trp-rich puroindoline-a), and SEED-009 (Bac2A proline-rich) all have mechanisms with expected Gram-positive efficacy. Cost: ~$200–400 per candidate at contract labs.SEED-009 media note — RPMI-1640 parallel condition added for proline-rich SEED-009 variants. Proline-rich cathelicidins have lower activity in Mueller-Hinton Broth (protein-rich media reduces intracellular uptake). Literature: Gagnon et al. 2016 Cell 167:471; Krizsan et al. 2014 Angew Chem.
Serum stability: Wave 1 optional → recommended before MIC — WET_LAB_PROBABILITY.md shows early triage on 20 candidates before full MIC panel saves significant cost (5 of 7 families have model-flagged limitations). Family-specific notes on SEED-003/008/009 model calibration limitations.
25% → 50% human serum — Aligned with
docs/WET_LAB_HANDOFF.mdSection 4 (Otvos & Cudic 2002). Both documents now consistently specify 50% pooled human serum, 37°C, 0/30/60/120 min.Test plan
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