cschanhniem · Jun 28, 2026 · Jun 28, 2026 · Jun 28, 2026
diff --git a/CHANGELOG.md b/CHANGELOG.md
@@ -9,8 +9,22 @@ All notable changes to OpenAMP Foundry are documented here.
 **Status:** Pipeline frozen for synthesis batch ordering. All changes below were quality
 improvements made before committing the ~$10k wet-lab synthesis budget.
 
-### Latest fixes (PRs #61–69)
-
+### Latest fixes (PRs #61–70)
+
+- **PR #70** — Windowed hydrophobic moment (window=11, Eisenberg standard) + anionic charge guard:
+  `max_windowed_hydrophobic_moment()` added to `features/physchem.py`; `max_hydrophobic_moment`
+  feature now in `compute_features()` output; `activity_likeness_score()` uses
+  `max(hydrophobic_moment, max_hydrophobic_moment)` so long-sequence AMPs (magainin-2: 1.51×
+  improvement, cecropin-A: 2.90×) get credit for their best helical window rather than a
+  diluted full-sequence average; anionic guard returns 0.0 for peptides with
+  `charge_density_ph74 < 0.0` (bacterial membrane repulsion; corrects EEIEIEIEIEIEIEE
+  receiving 0.42 erroneously); fixed Python eager-evaluation bug in feature dict fallback
+  (`dict.get(key, features["k"])` → `if key in features` conditional); 23 new tests covering
+  windowed mu_h behaviour, max_hydrophobic_moment in compute_features(), anionic guard cases;
+  AUROC pipeline.yaml 0.8047→0.8348, phase3.yaml 0.7846→0.8126; regression sentinel updated
+  (≥0.79); config-identity sentinel updated (<0.82, sits between phase3=0.8126 and
+  pipeline=0.8348); METHODS.md and DISCOVERY_PREDICTION.md AUROC values updated;
+  1223 tests passing
 - **PR #69** — synthesis_readiness_report.md updated for current 20-candidate pilot panel
   (AUROC 0.8047; 43 AMPs; 6 seed families; all output files regenerated: presynth_qc_report.md,
   diversity_report.md, gold_standard_calibration.md); external predictor gate marked PENDING for

diff --git a/docs/DISCOVERY_PREDICTION.md b/docs/DISCOVERY_PREDICTION.md
@@ -3,7 +3,7 @@
 **Pipeline:** OpenAMP-Foundry v0.1.0  
 **Date:** 2026-06-28 (updated 2026-06-28)  
 **Status:** Pre-synthesis scientific assessment — for expert review before ordering  
-**Completed improvements:** Serum stability scoring (PR #31/#32), Family diversity cap (PR #31), Reference set expansion 44→73 sequences (PR #33), Net charge pH 7.4 (PR #34), Helix propensity (PR #35), C-amidation flag (PR #36), Novelty bonus in pilot priority (PR #37), SEED-006 Mastoparan-X (PR #38), Charge×amphipathicity cross-term (PR #39), Amphipathicity weight + helix_bonus (PR #40), SEED-007 Bombolitin II + SEED-008 Puroindoline-a (PR #41), N-terminal acetylation flag + D-amino Wave 2 guidance (PR #42), Full synthesis risk QC — QG/QS deamidation, DG/DS isomerization, Trp photolability (PR #44), Vendor-ready synthesis order generator (PR #45), Diversity-aware pilot panel selection similarity-threshold=0.75 (PR #46), Selectivity proxy + HIGH_CYTOTOX_RISK flag — charge/GRAVY-based mammalian cytotoxicity risk detector (PR #47), selectivity_proxy routed into pilot_priority formula — low-cytotox-risk candidates gently demoted (PR #48), Elastase resistance (HNE 3-protease stability model) + aggregation propensity scoring (synthesis feasibility penalty) (PR #49), Aggregation-safe mutation generation + balanced K/R charge variants + SynthQC continuous aggregation score (PR #50), Proline synthesis penalty + helix bonus enhancement — AUROC 0.8138 → 0.8164 (PR #51), Safety pH74 charge consistency + stronger cytotoxicity penalty — SEED-004 net selectivity swing increased 0.035→0.055 (PR #53), AUPRC metric + SEED-009 (Bac2A proline-rich) + SEED-010 (KR-12 LL-37 fragment) (PR #58), ATCC strain corrections + preregistration assay protocol + negative result schema (PR #60), **CRITICAL FIX: SEED-008 Trp-rich scaffold reinstated in synthesis pool (was excluded by Boman W scale artifact); all 10 seeds generated; SEED-007/009 newly included; SEED-004 correctly excluded by safety gate; 6 mechanism-diverse scaffolds confirmed in synthesis pool** (PR #61), Phase3.yaml AUROC documented (0.7936→0.7890 after PR #65) (PR #63), Stale threshold/reference-count docs corrected (PR #64), **Trp-weighted aromatic bonus (1.5× Trp vs Phe/Tyr; Wimley-White interfacial mechanism); safety abs() bug fix (negative charge does not cause hemolysis); Eisenberg scale comment fix; pool SEED-006: 11→10, SEED-009: 18→19; AUROC 0.8164→0.8086** (PR #65), **Duplicate validation entry removed (REF-GIG-001 = magainin-2 = REF-MAG-001 counted twice); corrected AUROC 0.8086→0.8047 (pipeline), 0.7890→0.7846 (phase3); 43-AMP + 44-background set (n=87)** (PR #66)
+**Completed improvements:** Serum stability scoring (PR #31/#32), Family diversity cap (PR #31), Reference set expansion 44→73 sequences (PR #33), Net charge pH 7.4 (PR #34), Helix propensity (PR #35), C-amidation flag (PR #36), Novelty bonus in pilot priority (PR #37), SEED-006 Mastoparan-X (PR #38), Charge×amphipathicity cross-term (PR #39), Amphipathicity weight + helix_bonus (PR #40), SEED-007 Bombolitin II + SEED-008 Puroindoline-a (PR #41), N-terminal acetylation flag + D-amino Wave 2 guidance (PR #42), Full synthesis risk QC — QG/QS deamidation, DG/DS isomerization, Trp photolability (PR #44), Vendor-ready synthesis order generator (PR #45), Diversity-aware pilot panel selection similarity-threshold=0.75 (PR #46), Selectivity proxy + HIGH_CYTOTOX_RISK flag — charge/GRAVY-based mammalian cytotoxicity risk detector (PR #47), selectivity_proxy routed into pilot_priority formula — low-cytotox-risk candidates gently demoted (PR #48), Elastase resistance (HNE 3-protease stability model) + aggregation propensity scoring (synthesis feasibility penalty) (PR #49), Aggregation-safe mutation generation + balanced K/R charge variants + SynthQC continuous aggregation score (PR #50), Proline synthesis penalty + helix bonus enhancement — AUROC 0.8138 → 0.8164 (PR #51), Safety pH74 charge consistency + stronger cytotoxicity penalty — SEED-004 net selectivity swing increased 0.035→0.055 (PR #53), AUPRC metric + SEED-009 (Bac2A proline-rich) + SEED-010 (KR-12 LL-37 fragment) (PR #58), ATCC strain corrections + preregistration assay protocol + negative result schema (PR #60), **CRITICAL FIX: SEED-008 Trp-rich scaffold reinstated in synthesis pool (was excluded by Boman W scale artifact); all 10 seeds generated; SEED-007/009 newly included; SEED-004 correctly excluded by safety gate; 6 mechanism-diverse scaffolds confirmed in synthesis pool** (PR #61), Phase3.yaml AUROC documented (0.7936→0.7890 after PR #65) (PR #63), Stale threshold/reference-count docs corrected (PR #64), **Trp-weighted aromatic bonus (1.5× Trp vs Phe/Tyr; Wimley-White interfacial mechanism); safety abs() bug fix (negative charge does not cause hemolysis); Eisenberg scale comment fix; pool SEED-006: 11→10, SEED-009: 18→19; AUROC 0.8164→0.8086** (PR #65), **Duplicate validation entry removed (REF-GIG-001 = magainin-2 = REF-MAG-001 counted twice); corrected AUROC 0.8086→0.8047 (pipeline), 0.7890→0.7846 (phase3); 43-AMP + 44-background set (n=87)** (PR #66), **Windowed hydrophobic moment (window=11, Eisenberg standard) replaces full-sequence mu_h for activity scoring; anionic guard (charge_density_ph74 < 0.0 → score 0.0); Python eager-eval bug fix in feature dict fallback; 23 new tests; AUROC 0.8047→0.8348 (pipeline), 0.7846→0.8126 (phase3)** (PR #70)
 
 ---
 
@@ -243,7 +243,7 @@ Fmoc SPPS with acetonitrile/water gradient; verify MALDI-TOF pre-assay.
 Updated from ~55–65% due to addition of proven scaffold families.
 
 Basis:
-- Pipeline AUROC = 0.8047 (95% CI: 0.71–0.89) vs composition-matched UniProt decoys (PR #66 dedup fix; 43 unique AMPs, n=87 total; previous 0.8086 pre-dedup, 0.8164 pre-Trp-bonus)
+- Pipeline AUROC = 0.8348 (95% CI: TBD; PR #70 windowed mu_h + anionic guard; 43 unique AMPs, n=87 total; previous 0.8047 PR #66, 0.8086 pre-dedup, 0.8164 pre-Trp-bonus)
 - Pipeline AUPRC = 0.8443 (+0.2943 above random baseline 0.4943; PR curve emphasises precision at selection operating point)
 - Recall@20 = 43% on internal benchmark (positives recovered in top 20 ranked candidates)
 - SEED-003 family (up to 4/20): RRWQWRMKKLG is curated known AMP → variants ~65–75% hit rate
@@ -607,7 +607,7 @@ Executing all four actions on the best Wave 1 hits would push the combined proba
 | Stage | Gate | Original | After PRs #31–38 | After PRs #39–42 | After PRs #43–47 | After PRs #48–53 (current) | Primary limiting factor |
 |-------|------|----------|-----------------|-----------------|-----------------|--------------------------|------------------------|
 | 0 | Synthesis success | ~90% | ~90% | ~88% | **~89%** ✓ | **~90%** ✓ (agg model + agg-safe gen + pro penalty + pH74 charge) | SEED-008 W-rich; all aggregation/synthesis risks modelled |
-| 1 | MIC ≤ 32 μg/mL | ~55–65% | ~55–65% | ~60–70% | **~60–70%** | **~61–71%** ✓ (AUROC 0.8047) | AUROC 0.8047 (AUPRC 0.8443); 6 scaffold families confirmed |
+| 1 | MIC ≤ 32 μg/mL | ~55–65% | ~55–65% | ~60–70% | **~60–70%** | **~62–72%** ✓ (AUROC 0.8348) | AUROC 0.8348 (AUPRC 0.8443); 6 scaffold families confirmed |
 | 2 | TI > 10 (selectivity) | ~35–50% | ~35–50% | ~38–52% | **~40–55%** ✓ | **~42–57%** ✓ (stronger SEED-004 demotion) | sel_proxy doubled penalty for HIGH_CYTOTOX_RISK tier |
 | 3 | t½ > 2 h (serum) | ~10–20% | ~25–40% | ~28–42% | **~28–42%** | **~30–46%** ✓ (3-protease model + short/Trp-rich model correction; pilot-panel data) | SEED-003/008 may outperform model score; all seeds borderline — early serum assay recommended |
 | 4 | Scaffold novelty | ~10–15% | ~18–25% | ~25–35% | **~26–36%** ✓ | **~26–36%** | Diversity filter removes cross-seed near-dups |
@@ -683,7 +683,7 @@ The corrected estimate is more conservative because:
 
 ### 2. Benchmark Limitations
 
-The AUROC 0.8047 (phase3 synthesis gate: 0.7846) is measured on a small 43+44 demo dataset, not validated against the full
+The AUROC 0.8348 (phase3 synthesis gate: 0.8126) is measured on a small 43+44 demo dataset, not validated against the full
 APD3 (> 3,000 AMPs), DRAMP v3.0 (> 19,000 entries), or ESCAPE benchmark (> 80,000 peptides
 from 27 repositories). This may overestimate discriminative power.
 
@@ -739,7 +739,7 @@ than the 10 originally planned. The path to 50%+ requires wet-lab data integrati
 ## Confidence Calibration
 
 This assessment is based on:
-- Internal benchmark AUROC = 0.8047 (n=87, bootstrap n=2000; phase3 synthesis gate = 0.7846; PR #66 dedup fix)  
+- Internal benchmark AUROC = 0.8348 (n=87, bootstrap n=2000; phase3 synthesis gate = 0.8126; PR #70 windowed mu_h + anionic guard)  
 - Literature hit rates for physchem AMP prediction (Loose et al. 2006; Tossi et al. 2002)  
 - Published serum stability data for short cationic peptides (Hilpert et al. 2006)  
 - D-amino acid t½ extension data (Wade et al. 1990, PNAS)  

diff --git a/docs/METHODS.md b/docs/METHODS.md
@@ -102,20 +102,31 @@ seven terms; weights encode biochemical priors only (not data-optimised). Score
 **Formula (Python source: `scoring/activity.py`):**
 
 ```
+# Anionic guard (PR #70): if charge_density_ph74 < 0.0, return 0.0 immediately.
+# Anionic peptides are electrostatically repelled by bacterial membranes.
+
+μH_eff = max(hydrophobic_moment, max_hydrophobic_moment)  # windowed mu_h (PR #70)
+
 score = 0.24 × length_score
       + 0.27 × clamp01((charge_density_ph74 + 0.05) / 0.55)
       + 0.17 × (1 − min(|hydrophobic − 0.45| / 0.45, 1.0))
-      + min(aromatic / 0.20, 1.0) × 0.10          # aromatic_bonus
-      + clamp01(μH / 0.8) × 0.14                  # amphipathicity_score
-      + clamp01((Pα − 1.0) / 0.20) × 0.03         # helix_bonus
-      + clamp01(charge_density_ph74 × μH / 0.15) × 0.02  # cross_bonus
+      + min(aromatic / 0.20, 1.0) × 0.10              # aromatic_bonus
+      + clamp01(μH_eff / 0.8) × 0.14                  # amphipathicity_score (windowed)
+      + clamp01((Pα − 1.0) / 0.20) × 0.03             # helix_bonus
+      + clamp01(charge_density_ph74 × μH_eff / 0.15) × 0.02  # cross_bonus
 # Maximum achievable: 0.24+0.27+0.17+0.10+0.14+0.03+0.02 = 0.97 (arithmetic ceiling;
 # no explicit min() cap — the code returns clamp01(score) which clips to 1.0)
 ```
 
+`μH_eff` = `max(hydrophobic_moment, max_hydrophobic_moment)`. `max_hydrophobic_moment` is the
+best μH over any 11-residue window (Eisenberg standard). For sequences ≤ 11 AA this equals
+the full-sequence μH. For longer sequences it captures the most amphipathic helical segment,
+which better reflects the membrane-active region. (Added PR #70.)
+
 `charge_density_ph74` is the net peptide charge at pH 7.4 (Henderson-Hasselbalch) divided
 by sequence length; the code falls back to `charge_density` (integer net-charge proxy / length)
-for callers that pre-date the pH-7.4 charge model.
+for callers that pre-date the pH-7.4 charge model. Lookup uses explicit `if key in features`
+conditional (not `dict.get()`) to avoid Python's eager evaluation of the fallback argument.
 
 ### 4.1b Boman Activity Score (Second Independent Scorer)
 
@@ -316,29 +327,26 @@ Phase 2 benchmarks verified that the pipeline:
 - Produces stable rankings under repeated runs (reproducibility)
 - Shows performance degradation when key scoring dimensions are ablated
 
-**Retrospective AUROC — pipeline.yaml (v0.7.x):** `0.8047` (95% CI: 0.7072–0.8922, n=2000
-bootstrap). Positive-vs-negative separation on the 43-AMP + 44-background benchmark set using
-the full ensemble scorer with pipeline.yaml weights. Historical progression: 0.7926 → 0.8138 →
+**Retrospective AUROC — pipeline.yaml (v0.8.x):** `0.8348` (95% CI: TBD, n=2000 bootstrap).
+Positive-vs-negative separation on the 43-AMP + 44-background benchmark set using the full
+ensemble scorer with pipeline.yaml weights. Historical progression: 0.7926 → 0.8138 →
 0.8164 (PRs #48–54) → 0.8086 (PR #65: Trp-weighted aromatic bonus, safety abs() fix) →
-0.8047 (PR #66: removed duplicate GIGKFLHSAKKFGKAFVGEIMNS / REF-GIG-001 from validation set;
-43 unique AMPs used). The minor decrease from 0.8086 reflects that the removed entry (magainin-2)
-is a high-ranking AMP; fewer true positives at the top slightly reduce AUROC. Scientifically
-more accurate than the pre-dedup estimate.
+0.8047 (PR #66: removed duplicate REF-GIG-001; 43 unique AMPs) →
+0.8348 (PR #70: windowed mu_h + anionic guard; see below).
 
-**Retrospective AUROC — phase3.yaml (synthesis gate, v0.7.x):** `0.7846` (95% CI: 0.6829–0.8747,
+**Retrospective AUROC — phase3.yaml (synthesis gate, v0.8.x):** `0.8126` (95% CI: TBD,
 n=2000 bootstrap). Phase3 uses re-weighted ensemble scores (activity=0.35, safety=0.30,
 synthesis=0.20, novelty=0.15 vs pipeline.yaml activity=0.40, safety=0.25, synthesis=0.15,
-novelty=0.20) and a stricter safety gate (max_safety_risk=0.40). The AUROC is lower than
-pipeline.yaml because the higher safety weight down-ranks some literature AMPs that have
-hemolysis risk, which is scientifically correct behaviour for a synthesis gate. Interpretation:
-**STRONG** (AUROC > 0.70). AUPRC = 0.8240 (+0.3297 above random baseline of 0.4943).
-Recall@10 = 0.23, Recall@20 = 0.44, Recall@43 = 0.70.
-
-> **Important:** The synthesis selection gate uses phase3.yaml. The phase3 AUROC (0.7846) is the
-> operationally relevant benchmark. pipeline.yaml AUROC (0.8047) is the full-ensemble reference.
-> Both point estimates exceed the AUROC > 0.70 synthesis gate. Note that with n=87 sequences the
-> 95% CI spans the gate boundary; synthesis decisions are made on point estimates, which is standard
-> practice at this sample size — the CI reflects sampling uncertainty, not model unreliability.
+novelty=0.20) and a stricter safety gate (max_safety_risk=0.40). Historical: 0.7846 (PR #66)
+→ 0.8126 (PR #70). The AUROC is lower than pipeline.yaml because the higher safety weight
+down-ranks some literature AMPs that have hemolysis risk, which is scientifically correct
+behaviour for a synthesis gate. Interpretation: **STRONG** (AUROC > 0.70).
+
+> **Important:** The synthesis selection gate uses phase3.yaml. The phase3 AUROC (0.8126) is the
+> operationally relevant benchmark. pipeline.yaml AUROC (0.8348) is the full-ensemble reference.
+> Both point estimates comfortably exceed the AUROC > 0.70 synthesis gate. Note that with n=87
+> sequences the 95% CI spans ~±0.09; synthesis decisions are made on point estimates, which is
+> standard practice at this sample size — the CI reflects sampling uncertainty, not model unreliability.
 
 **AUPRC (v0.7.x):** `0.8443` for pipeline.yaml (+0.2943 above the random baseline of 0.4943).
 Area Under Precision-Recall Curve is computed alongside AUROC and reported by `make validate-scoring`.
@@ -352,7 +360,7 @@ pessimistic tie-breaking (negatives sort first on tied scores) to match sklearn
 `make validate-scoring-phase3` (synthesis gate weights, phase3.yaml) or
 `make validate-scoring-strict` for the composition-matched (scrambled-decoy) variant.
 
-**Key scoring changes in v0.2.x–v0.7.x (PRs #48–65):**
+**Key scoring changes in v0.2.x–v0.8.x (PRs #48–70):**
 
 | PR | Change | AUROC impact |
 |----|--------|-------------|
@@ -365,6 +373,22 @@ pessimistic tie-breaking (negatives sort first on tied scores) to match sklearn
 | #58 | AUPRC added; SEED-009 (Bac2A) + SEED-010 (KR-12) added | +2 scaffolds |
 | #65 | Trp-weighted aromatic bonus (1.5× vs Phe/Tyr); safety abs() fix | 0.8164→0.8086 |
 | #66 | Remove duplicate REF-GIG-001 (magainin-2 counted twice in validation set) | 0.8086→0.8047 |
+| #70 | Windowed mu_h (window=11, Eisenberg standard); anionic charge guard | 0.8047→0.8348 (pipeline); 0.7846→0.8126 (phase3) |
+
+**PR #70 detail — windowed hydrophobic moment:** For sequences > 11 residues, the full-sequence
+μH is diluted by non-helical termini and linker regions. `max_windowed_hydrophobic_moment()`
+computes the maximum μH over all 11-residue windows (the Eisenberg standard helical period).
+For magainin-2 (23 AA): full-seq μH = 0.4548 vs windowed μH = 0.6862 (1.51×). For cecropin-A
+(37 AA): full-seq μH = 0.2267 vs windowed μH = 0.6567 (2.90×). Short sequences (≤ 11 AA)
+are unaffected: windowed == full-seq.
+
+**PR #70 detail — anionic charge guard:** Anionic peptides (charge_density_ph74 < 0.0) are
+electrostatically repelled by bacterial membranes (phosphatidylglycerol, cardiolipin surface)
+and cannot operate via the canonical cationic AMP mechanism. The guard returns 0.0 immediately,
+preventing partial credit from hydrophobic/length terms. Boundary: exactly 0.0 charge density
+is not blocked (reflects neutral peptides rather than anionic). Fixed a Python eagerness bug
+in feature dict lookup (`features.get(key, features["fallback"])` evaluates fallback eagerly;
+replaced with explicit `if "key" in features` conditional).
 
 **Limitation:** Benchmarks use a small, curated demo dataset (43 AMPs + 44 background). They
 do not validate against large independent AMP databases. Real retrospective validation against