# =============================================================================
# Comprehensive AOP Example: PM2.5 Exposure → Impaired Lung Function
# =============================================================================
#
# This example demonstrates an Adverse Outcome Pathway (AOP) for particulate
# matter (PM2.5) exposure leading to impaired lung function. The pathway
# includes:
#
#   MIE (Molecular Initiating Event): Oxidative stress
#   KE1 (Key Event 1): EGFR activation/phosphorylation
#   KE2 (Key Event 2): Goblet cell hyperplasia and mucin hypersecretion
#   KE3 (Key Event 3): Impaired mucociliary clearance
#   AO  (Adverse Outcome): Reduced lung function
#
# Study Design:
#   - In vitro: Primary human bronchial epithelial (HBE) cells at ALI
#   - In vivo: Clinical cohort with spirometry
#   - Exposure: PM2.5 (10 ug/cm2) vs. clean air control
#   - Timepoints: 4h (early), 24h (intermediate), 7d (late)
#
# How the results are recorded:
#   Every output record names the experimental group it belongs to and the
#   exposure condition it was measured under, so a value is never separated
#   from the condition that produced it. Control and exposed groups are
#   written as separate assay records here because each was run and reported
#   separately; the change between them lives in response_comparisons at the
#   end of this file, never inside the assay data.
#
# =============================================================================

# =============================================================================
# PROTOCOLS - Detailed procedures with equipment
# =============================================================================

protocols:
  - id: "PROTOCOL:007"
    name: "DCFDA fluorescence protocol"
    equipment_required:
      - "Fluorescence plate reader"
      - "DCFDA reagent kit"

  - id: "PROTOCOL:008"
    name: "Western blot protocol"
    equipment_required:
      - "SDS-PAGE apparatus"
      - "Chemiluminescence imager"

  - id: "PROTOCOL:009"
    name: "qRT-PCR protocol"
    equipment_required:
      - "QuantStudio real-time PCR system"

  - id: "PROTOCOL:010"
    name: "High-speed microscopy protocol"
    equipment_required:
      - "High-speed camera (>200 fps)"
      - "Inverted microscope with 20x objective"

  - id: "PROTOCOL:011"
    name: "Microsphere tracking protocol"
    equipment_required:
      - "Fluorescence microscope"
      - "1-um fluorescent microspheres"

  - id: "PROTOCOL:012"
    name: "Spirometry protocol"
    equipment_required:
      - "Spirometer (ATS/ERS compliant)"

# =============================================================================
# OXIDATIVE STRESS ASSAYS (MIE)
# =============================================================================

oxidative_stress_assays:
  # Control - 4h
  - id: "OX:001"
    name: "ROS level - Control 4h"
    description: "Intracellular ROS in control HBE cells at 4 hours"
    has_specified_output:
      - id: "OX:001-output-control"
        name: "Clean air control ROS measurement"
        experimental_group: control
        measured_under: "EXPOSURE:aop-clean-air-4h"
        reactive_oxygen_species:
          value: "1.0"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 6
    has_exposure_condition:
      - id: "EXPOSURE:aop-clean-air-4h"
        name: "Clean air control, 4 hours"
        exposure_duration:
          value: "4"
          unit:
            id: "UO:0000032"
            name: "hour"
    ros_probe_type: "DCFDA"
    assay_date: "2024-01-15"
    informs_on_key_event:
      id: "KE:mie-oxidative-stress"
      name: "Increased oxidative stress"
      biological_action: increased
      level_of_biological_organization: molecular
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      anatomical_origin:
        id: "UBERON:0002031"
        name: "bronchus"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: MolecularAssayProtocol
        id: "PROTOCOL:001"
        name: "DCFDA ROS detection in ALI cultures"
        detection_method: "fluorescence plate reader"
        normalization_method: "cell count normalization"

  # PM2.5 exposed - 4h
  - id: "OX:002"
    name: "ROS level - PM2.5 4h"
    description: "Intracellular ROS in PM2.5-exposed HBE cells at 4 hours"
    has_specified_output:
      - id: "OX:002-output-pm25"
        name: "PM2.5 4h ROS measurement"
        experimental_group: treated
        measured_under: "EXPOSURE:pm25-4h"
        reactive_oxygen_species:
          value: "2.8"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 6
    ros_probe_type: "DCFDA"
    assay_date: "2024-01-15"
    informs_on_key_event:
      id: "KE:mie-oxidative-stress"
      name: "Increased oxidative stress"
      biological_action: increased
      level_of_biological_organization: molecular
    has_exposure_condition:
      - id: "EXPOSURE:pm25-4h"
        name: "PM2.5 10 ug/cm2 for 4 hours"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0010071"
            name: "microgram per square centimeter"
        exposure_duration:
          value: "4"
          unit:
            id: "UO:0000032"
            name: "hour"
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: MolecularAssayProtocol
        id: "PROTOCOL:001"
        name: "DCFDA ROS detection in ALI cultures"
        detection_method: "fluorescence plate reader"
        normalization_method: "cell count normalization"

  # Lipid peroxidation marker - PM2.5 24h
  - id: "OX:003"
    name: "MDA level - PM2.5 24h"
    description: "Malondialdehyde (lipid peroxidation marker) after PM2.5 exposure"
    has_specified_output:
      - id: "OX:003-output-pm25"
        name: "PM2.5 24h MDA measurement"
        experimental_group: treated
        measured_under: "EXPOSURE:pm25-24h"
        malondialdehyde_level:
          value: "1.9"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 6
    assay_date: "2024-01-16"
    has_exposure_condition:
      - id: "EXPOSURE:pm25-24h"
        name: "PM2.5 10 ug/cm2 for 24 hours"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0010071"
            name: "microgram per square centimeter"
        exposure_duration:
          value: "24"
          unit:
            id: "UO:0000032"
            name: "hour"
        timing_post_exposure:
          value: "0"
          unit:
            id: "UO:0000032"
            name: "hour"
    informs_on_key_event:
      id: "KE:mie-oxidative-stress"
      name: "Increased oxidative stress"
      biological_action: increased
      level_of_biological_organization: molecular
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: MolecularAssayProtocol
        id: "PROTOCOL:001"
        name: "DCFDA ROS detection in ALI cultures"
        detection_method: "fluorescence plate reader"

# =============================================================================
# EGFR SIGNALING ASSAYS (KE1)
# =============================================================================

egfr_signaling_assays:
  # Control - 4h
  - id: "EGFR:001"
    name: "pEGFR Y1068 - Control 4h"
    description: "EGFR phosphorylation at Y1068 in control cells at 4 hours"
    has_specified_output:
      - id: "EGFR:001-output-control"
        name: "Clean air control pEGFR measurement"
        experimental_group: control
        measured_under: "EXPOSURE:aop-clean-air-4h"
        egfr_phosphorylation_y1068:
          value: "1.0"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 6
    has_exposure_condition:
      - id: "EXPOSURE:aop-clean-air-4h"
        name: "Clean air control, 4 hours"
        exposure_duration:
          value: "4"
          unit:
            id: "UO:0000032"
            name: "hour"
    normalization_reference: "beta-actin"
    phosphorylation_site: "Y1068"
    assay_date: "2024-01-15"
    informs_on_key_event:
      id: "KE:ke1-egfr-activation"
      name: "EGFR activation"
      biological_action: activated
      level_of_biological_organization: molecular
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: MolecularAssayProtocol
        id: "PROTOCOL:002"
        name: "Western blot for phospho-EGFR"
        detection_method: "chemiluminescence"
        antibodies_used:
          - "anti-pEGFR Y1068 (Cell Signaling #3777)"
          - "anti-total EGFR (Cell Signaling #4267)"
        normalization_method: "total protein normalization"

  # PM2.5 exposed - 4h
  - id: "EGFR:002"
    name: "pEGFR Y1068 - PM2.5 4h"
    description: "EGFR phosphorylation at Y1068 after PM2.5 exposure at 4 hours"
    has_specified_output:
      - id: "EGFR:002-output-pm25"
        name: "PM2.5 4h pEGFR measurement"
        experimental_group: treated
        measured_under: "EXPOSURE:pm25-4h"
        egfr_phosphorylation_y1068:
          value: "3.5"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 6
    has_exposure_condition:
      - id: "EXPOSURE:pm25-4h"
        name: "PM2.5 10 ug/cm2 for 4 hours"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0010071"
            name: "microgram per square centimeter"
        exposure_duration:
          value: "4"
          unit:
            id: "UO:0000032"
            name: "hour"
    normalization_reference: "beta-actin"
    phosphorylation_site: "Y1068"
    assay_date: "2024-01-15"
    informs_on_key_event:
      id: "KE:ke1-egfr-activation"
      name: "EGFR activation"
      biological_action: activated
      level_of_biological_organization: molecular
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: MolecularAssayProtocol
        id: "PROTOCOL:002"
        name: "Western blot for phospho-EGFR"
        detection_method: "chemiluminescence"
        antibodies_used:
          - "anti-pEGFR Y1068 (Cell Signaling #3777)"
        normalization_method: "total protein normalization"

  # Total EGFR protein (normalization control)
  - id: "EGFR:003"
    name: "Total EGFR - PM2.5 4h"
    description: "Total EGFR protein expression (loading control)"
    has_specified_output:
      - id: "EGFR:003-output-pm25"
        name: "PM2.5 4h total EGFR measurement"
        experimental_group: treated
        measured_under: "EXPOSURE:pm25-4h"
        total_egfr_protein:
          value: "1.05"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 6
    has_exposure_condition:
      - id: "EXPOSURE:pm25-4h"
        name: "PM2.5 10 ug/cm2 for 4 hours"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0010071"
            name: "microgram per square centimeter"
        exposure_duration:
          value: "4"
          unit:
            id: "UO:0000032"
            name: "hour"
    assay_date: "2024-01-15"
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: MolecularAssayProtocol
        id: "PROTOCOL:002"
        name: "Western blot for phospho-EGFR"
        detection_method: "chemiluminescence"

# =============================================================================
# GOBLET CELL ASSAYS (KE2)
# =============================================================================

goblet_cell_assays:
  # MUC5AC mRNA - Control 24h
  - id: "GCM:001"
    name: "MUC5AC mRNA - Control 24h"
    description: "MUC5AC mRNA expression in control cells at 24 hours"
    has_specified_output:
      - id: "GCM:001-output-control"
        name: "Clean air control MUC5AC mRNA measurement"
        experimental_group: control
        measured_under: "EXPOSURE:aop-clean-air-24h"
        muc5ac_mrna_expression:
          value: "1.0"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 6
    has_exposure_condition:
      - id: "EXPOSURE:aop-clean-air-24h"
        name: "Clean air control, 24 hours"
        exposure_duration:
          value: "24"
          unit:
            id: "UO:0000032"
            name: "hour"
    assay_date: "2024-01-16"
    informs_on_key_event:
      id: "KE:ke2-goblet-hyperplasia"
      name: "Goblet cell hyperplasia"
      biological_action: increased
      level_of_biological_organization: cellular
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: StainingProtocol
        id: "PROTOCOL:stain-aop-001"
        name: "AB-PAS staining for goblet cells"
        staining_type: "AB-PAS"
        fixation_method: "4% paraformaldehyde"

  # MUC5AC mRNA - PM2.5 24h
  - id: "GCM:002"
    name: "MUC5AC mRNA - PM2.5 24h"
    description: "MUC5AC mRNA expression after PM2.5 exposure at 24 hours"
    has_specified_output:
      - id: "GCM:002-output-pm25"
        name: "PM2.5 24h MUC5AC mRNA measurement"
        experimental_group: treated
        measured_under: "EXPOSURE:pm25-24h"
        muc5ac_mrna_expression:
          value: "4.2"
          unit:
            id: "STATO:0000169"
            name: "fold change"
          central_tendency: mean
          sample_size: 6
    has_exposure_condition:
      - id: "EXPOSURE:pm25-24h"
        name: "PM2.5 10 ug/cm2 for 24 hours"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0010071"
            name: "microgram per square centimeter"
        exposure_duration:
          value: "24"
          unit:
            id: "UO:0000032"
            name: "hour"
    assay_date: "2024-01-16"
    informs_on_key_event:
      id: "KE:ke2-goblet-hyperplasia"
      name: "Goblet cell hyperplasia"
      biological_action: increased
      level_of_biological_organization: cellular
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: StainingProtocol
        id: "PROTOCOL:stain-aop-001"
        name: "AB-PAS staining for goblet cells"
        staining_type: "AB-PAS"

  # Goblet cell percentage - PM2.5 7d
  - id: "GCM:003"
    name: "Goblet cell percentage - PM2.5 7d"
    description: "Percentage of goblet cells after 7-day PM2.5 exposure"
    has_specified_output:
      - id: "GCM:003-output-pm25"
        name: "PM2.5 7d goblet cell percentage measurement"
        experimental_group: treated
        measured_under: "EXPOSURE:pm25-7d"
        goblet_cell_percentage:
          value: "32"
          unit:
            id: "UO:0000187"
            name: "percent"
          central_tendency: mean
          sample_size: 6
    has_exposure_condition:
      - id: "EXPOSURE:pm25-7d"
        name: "PM2.5 10 ug/cm2 daily for 7 days"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0010071"
            name: "microgram per square centimeter"
        exposure_duration:
          value: "7"
          unit:
            id: "UO:0000033"
            name: "day"
    assay_date: "2024-01-22"
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: StainingProtocol
        id: "PROTOCOL:stain-aop-001"
        name: "AB-PAS staining for goblet cells"
        staining_type: "AB-PAS"

  # Mucus viscosity - PM2.5 7d
  - id: "GCM:004"
    name: "Mucus viscosity - PM2.5 7d"
    description: "Mucus viscosity after 7-day PM2.5 exposure showing hypersecretion phenotype"
    has_specified_output:
      - id: "GCM:004-output-pm25"
        name: "PM2.5 7d mucus viscosity measurement"
        experimental_group: treated
        measured_under: "EXPOSURE:pm25-7d"
        mucus_viscosity:
          value: "185"
          unit:
            id: "UCUM:cP"
            name: "centipoise"
          central_tendency: mean
          sample_size: 6
    has_exposure_condition:
      - id: "EXPOSURE:pm25-7d"
        name: "PM2.5 10 ug/cm2 daily for 7 days"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0010071"
            name: "microgram per square centimeter"
        exposure_duration:
          value: "7"
          unit:
            id: "UO:0000033"
            name: "day"
    assay_date: "2024-01-22"
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: StainingProtocol
        id: "PROTOCOL:stain-aop-001"
        name: "AB-PAS staining for goblet cells"
        staining_type: "AB-PAS"

# =============================================================================
# CILIARY FUNCTION ASSAYS (KE3 - part 1)
# =============================================================================

ciliary_function_assays:
  # CBF - Control baseline
  - id: "CILIARY:001"
    name: "CBF - Control baseline"
    description: "Ciliary beat frequency in control HBE cells at baseline"
    has_specified_output:
      - id: "CILIARY:001-output-control"
        name: "Clean air control CBF measurement"
        experimental_group: control
        measured_under: "EXPOSURE:aop-clean-air-baseline"
        beat_frequency_hz:
          value: "12.5"
          unit:
            id: "UO:0000106"
            name: "hertz"
          central_tendency: mean
          sample_size: 6
    has_exposure_condition:
      - id: "EXPOSURE:aop-clean-air-baseline"
        name: "Clean air control, baseline"
    assay_date: "2024-01-15"
    informs_on_key_event:
      id: "KE:ke3-impaired-ciliary"
      name: "Decreased ciliary function"
      biological_action: decreased
      level_of_biological_organization: cellular
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
      primary_cell:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      anatomical_origin:
        id: "UBERON:0002031"
        name: "bronchus"
      cell_culture_growth_mode: air_liquid_interface
      days_at_differentiation: 21
      donor_info: "Healthy non-smoker, male, age 42, no respiratory disease"
      replicates_per_donor: 3
      substrate_type: transwell_insert
      passage_number: 2
      seeding_density:
        value: "200000"
        unit:
          id: "UO:0000210"
          name: "cells per square centimeter"
      culture_conditions:
        id: "soma:conditions-aop-001"
        name: "Standard ALI culture conditions"
        days_at_air_liquid_interface: 21
        substrate_type: transwell_insert
        cell_culture_growth_mode: air_liquid_interface
        replicates_per_donor: 3
      culture_media:
        id: "soma:media-aop-001"
        name: "PneumaCult-ALI medium"
        base_medium: "PneumaCult-ALI"
        manufacturer: "STEMCELL Technologies"
        supplements:
          - id: "soma:supp-aop-001"
            name: "Hydrocortisone"
            supplement_type: hormone
            concentration:
              value: "0.48"
              unit:
                id: "UO:0000274"
                name: "microgram per milliliter"
          - id: "soma:supp-aop-002"
            name: "Heparin"
            supplement_type: growth_factor
            concentration:
              value: "4"
              unit:
                id: "UO:0000274"
                name: "microgram per milliliter"
    follows_protocols:
      - protocol_type: ImagingProtocol
        id: "PROTOCOL:004"
        name: "CBF measurement by high-speed microscopy"
        imaging_frame_rate:
          value: "200"
          unit:
            id: "UO:0000105"
            name: "hertz"
        imaging_duration:
          value: "5"
          unit:
            id: "UO:0000010"
            name: "second"
        temperature_control:
          value: "37"
          unit:
            id: "UO:0000027"
            name: "degree Celsius"
        evaporation_prevention: "perfluorocarbon overlay"

  # CBF - PM2.5 + ozone co-exposure 24h (demonstrates multivalued exposure)
  - id: "CILIARY:002"
    name: "CBF - PM2.5 + ozone 24h"
    description: "Ciliary beat frequency after PM2.5 and ozone co-exposure at 24 hours"
    has_specified_output:
      # Co-exposure: the assay lists both agents as exposure conditions, but
      # measured_under is single-valued, so it names the primary agent. The
      # full co-exposure is what has_exposure_condition below describes.
      - id: "CILIARY:002-output-pm25-ozone"
        name: "PM2.5 + ozone co-exposure CBF measurement"
        experimental_group: treated
        measured_under: "EXPOSURE:pm25-24h"
        beat_frequency_hz:
          value: "9.8"
          unit:
            id: "UO:0000106"
            name: "hertz"
          central_tendency: mean
          sample_size: 6
    assay_date: "2024-01-16"
    has_exposure_condition:
      - id: "EXPOSURE:pm25-24h"
        name: "PM2.5 10 ug/cm2 for 24 hours"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0010071"
            name: "microgram per square centimeter"
        exposure_duration:
          value: "24"
          unit:
            id: "UO:0000032"
            name: "hour"
      - id: "EXPOSURE:ozone-100ppb-24h"
        name: "Ozone 100 ppb for 24 hours"
        exposure_agent:
          id: "CHEBI:25812"
          name: "ozone"
        exposure_concentration:
          value: "100"
          unit:
            id: "UO:0000170"
            name: "parts per billion"
        exposure_duration:
          value: "24"
          unit:
            id: "UO:0000032"
            name: "hour"
    informs_on_key_event:
      id: "KE:ke3-impaired-ciliary"
      name: "Decreased ciliary function"
      biological_action: decreased
      level_of_biological_organization: cellular
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
      substrate_type: transwell_insert
    follows_protocols:
      - protocol_type: ImagingProtocol
        id: "PROTOCOL:004"
        name: "CBF measurement by high-speed microscopy"
        imaging_frame_rate:
          value: "200"
          unit:
            id: "UO:0000105"
            name: "hertz"

  # CBF - PM2.5 7d
  - id: "CILIARY:003"
    name: "CBF - PM2.5 7d"
    description: "Ciliary beat frequency after 7-day PM2.5 exposure"
    has_specified_output:
      - id: "CILIARY:003-output-pm25"
        name: "PM2.5 7d CBF measurement"
        experimental_group: treated
        measured_under: "EXPOSURE:pm25-7d"
        beat_frequency_hz:
          value: "7.2"
          unit:
            id: "UO:0000106"
            name: "hertz"
          central_tendency: mean
          sample_size: 6
    has_exposure_condition:
      - id: "EXPOSURE:pm25-7d"
        name: "PM2.5 10 ug/cm2 daily for 7 days"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0010071"
            name: "microgram per square centimeter"
        exposure_duration:
          value: "7"
          unit:
            id: "UO:0000033"
            name: "day"
    assay_date: "2024-01-22"
    informs_on_key_event:
      id: "KE:ke3-impaired-ciliary"
      name: "Decreased ciliary function"
      biological_action: decreased
      level_of_biological_organization: cellular
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
      substrate_type: transwell_insert
    follows_protocols:
      - protocol_type: ImagingProtocol
        id: "PROTOCOL:004"
        name: "CBF measurement by high-speed microscopy"
        imaging_frame_rate:
          value: "200"
          unit:
            id: "UO:0000105"
            name: "hertz"

  # Ciliated cell percentage - PM2.5 7d
  - id: "CILIARY:004"
    name: "Ciliated cell percentage - PM2.5 7d"
    description: "Percentage of ciliated cells after 7-day PM2.5 exposure"
    has_specified_output:
      - id: "CILIARY:004-output-pm25"
        name: "PM2.5 7d ciliated cell percentage measurement"
        experimental_group: treated
        measured_under: "EXPOSURE:pm25-7d"
        percentage_ciliated_cells:
          value: "45"
          unit:
            id: "UO:0000187"
            name: "percent"
          central_tendency: mean
          sample_size: 6
    has_exposure_condition:
      - id: "EXPOSURE:pm25-7d"
        name: "PM2.5 10 ug/cm2 daily for 7 days"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0010071"
            name: "microgram per square centimeter"
        exposure_duration:
          value: "7"
          unit:
            id: "UO:0000033"
            name: "day"
    assay_date: "2024-01-22"
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
      substrate_type: transwell_insert
    follows_protocols:
      - protocol_type: ImagingProtocol
        id: "PROTOCOL:004"
        name: "CBF measurement by high-speed microscopy"
        imaging_frame_rate:
          value: "200"
          unit:
            id: "UO:0000105"
            name: "hertz"

# =============================================================================
# MUCOCILIARY CLEARANCE ASSAYS (KE3 - part 2)
# =============================================================================

mcc_assays:
  # MCT rate - Control baseline
  - id: "MCC:001"
    name: "MCT rate - Control baseline"
    description: "Mucociliary transport rate in control HBE cells at baseline"
    has_specified_output:
      - id: "MCC:001-output-control"
        name: "Clean air control transport rate measurement"
        experimental_group: control
        measured_under: "EXPOSURE:aop-clean-air-baseline"
        transport_rate:
          value: "52"
          unit:
            id: "UCUM:um/s"
            name: "micrometer per second"
          central_tendency: mean
          sample_size: 6
        transport_directionality: normal
    has_exposure_condition:
      - id: "EXPOSURE:aop-clean-air-baseline"
        name: "Clean air control, baseline"
    assay_date: "2024-01-15"
    informs_on_key_event:
      id: "KE:ke3-impaired-mcc"
      name: "Impaired mucociliary clearance"
      biological_action: impaired
      level_of_biological_organization: tissue
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: ImagingProtocol
        id: "PROTOCOL:005"
        name: "MCT rate by microsphere tracking"
        fluorescent_tracer: "1-um fluorescent microspheres"
        particle_tracking_method: "automated particle tracking"
        particle_size:
          value: "1"
          unit:
            id: "UO:0000017"
            name: "micrometer"

  # MCT rate - PM2.5 24h
  - id: "MCC:002"
    name: "MCT rate - PM2.5 24h"
    description: "Mucociliary transport rate after PM2.5 exposure at 24 hours"
    has_specified_output:
      - id: "MCC:002-output-pm25"
        name: "PM2.5 24h transport rate measurement"
        experimental_group: treated
        measured_under: "EXPOSURE:pm25-24h"
        transport_rate:
          value: "38"
          unit:
            id: "UCUM:um/s"
            name: "micrometer per second"
          central_tendency: mean
          sample_size: 6
        transport_directionality: normal
    has_exposure_condition:
      - id: "EXPOSURE:pm25-24h"
        name: "PM2.5 10 ug/cm2 for 24 hours"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0010071"
            name: "microgram per square centimeter"
        exposure_duration:
          value: "24"
          unit:
            id: "UO:0000032"
            name: "hour"
    assay_date: "2024-01-16"
    informs_on_key_event:
      id: "KE:ke3-impaired-mcc"
      name: "Impaired mucociliary clearance"
      biological_action: impaired
      level_of_biological_organization: tissue
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: ImagingProtocol
        id: "PROTOCOL:005"
        name: "MCT rate by microsphere tracking"
        fluorescent_tracer: "1-um fluorescent microspheres"
        particle_tracking_method: "automated particle tracking"

  # MCT rate - PM2.5 7d (severely impaired)
  - id: "MCC:003"
    name: "MCT rate - PM2.5 7d"
    description: "Mucociliary transport rate after 7-day PM2.5 exposure"
    has_specified_output:
      - id: "MCC:003-output-pm25"
        name: "PM2.5 7d transport rate measurement"
        experimental_group: treated
        measured_under: "EXPOSURE:pm25-7d"
        transport_rate:
          value: "18"
          unit:
            id: "UCUM:um/s"
            name: "micrometer per second"
          central_tendency: mean
          sample_size: 6
        transport_directionality: variable
    has_exposure_condition:
      - id: "EXPOSURE:pm25-7d"
        name: "PM2.5 10 ug/cm2 daily for 7 days"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0010071"
            name: "microgram per square centimeter"
        exposure_duration:
          value: "7"
          unit:
            id: "UO:0000033"
            name: "day"
    assay_date: "2024-01-22"
    informs_on_key_event:
      id: "KE:ke3-impaired-mcc"
      name: "Impaired mucociliary clearance"
      biological_action: impaired
      level_of_biological_organization: tissue
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: ImagingProtocol
        id: "PROTOCOL:005"
        name: "MCT rate by microsphere tracking"
        fluorescent_tracer: "1-um fluorescent microspheres"

  # Percentage active transport - PM2.5 7d
  - id: "MCC:004"
    name: "Active transport percentage - PM2.5 7d"
    description: "Percentage of surface with active mucociliary transport after 7-day PM2.5 exposure"
    has_specified_output:
      - id: "MCC:004-output-pm25"
        name: "PM2.5 7d active transport percentage measurement"
        experimental_group: treated
        measured_under: "EXPOSURE:pm25-7d"
        percentage_active_transport:
          value: "35"
          unit:
            id: "UO:0000187"
            name: "percent"
          central_tendency: mean
          sample_size: 6
    has_exposure_condition:
      - id: "EXPOSURE:pm25-7d"
        name: "PM2.5 10 ug/cm2 daily for 7 days"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0010071"
            name: "microgram per square centimeter"
        exposure_duration:
          value: "7"
          unit:
            id: "UO:0000033"
            name: "day"
    assay_date: "2024-01-22"
    study_subject:
      subject_type: CellularSystem
      id: "soma:culture-aop-001"
      name: "Primary HBE ALI culture - Donor 1"
      cell_type:
        id: "CL:0002328"
        name: "bronchial epithelial cell"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
    follows_protocols:
      - protocol_type: ImagingProtocol
        id: "PROTOCOL:005"
        name: "MCT rate by microsphere tracking"
        fluorescent_tracer: "1-um fluorescent microspheres"

# =============================================================================
# LUNG FUNCTION ASSAYS (Adverse Outcome - Clinical, InVivoSubject)
# =============================================================================

lung_function_assays:
  # FEV1 - Subject 1 (PM2.5 exposed community)
  - id: "LF:001"
    name: "FEV1 - Subject 1 baseline"
    description: "FEV1 in subject from high PM2.5 exposure area at baseline"
    has_specified_output:
      - id: "LF:001-output-baseline"
        name: "Enrollment spirometry"
        description: "FEV1 is expressed as percent of predicted."
        experimental_group: baseline
        measured_under: "EXPOSURE:aop-ambient-pm25-baseline"
        fev1:
          value: "78"
          unit:
            id: "UO:0000187"
            name: "percent"
          central_tendency: single_value
          sample_size: 1
    has_exposure_condition:
      - id: "EXPOSURE:aop-ambient-pm25-baseline"
        name: "Residential ambient PM2.5, 15 years at enrollment"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_duration:
          value: "15"
          unit:
            id: "UO:0000036"
            name: "year"
    reference_dataset: "GLI-2012"
    assay_date: "2024-02-01"
    informs_on_key_event:
      id: "KE:ao-decreased-lung-function"
      name: "Decreased lung function"
      biological_action: decreased
      level_of_biological_organization: organ
    study_subject:
      subject_type: InVivoSubject
      id: "SUBJECT:aop-001"
      name: "Subject 1"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
      age:
        value: "52"
        unit:
          id: "UO:0000036"
          name: "year"
      sex: "male"
      sample_type: exhaled_breath_condensate
      collection_site: "pulmonary function laboratory"
      subject_characteristics: "Non-smoker, high PM2.5 exposure area resident, 15 years"
    follows_protocols:
      - protocol_type: SpirometryProtocol
        id: "PROTOCOL:006"
        name: "Pre- and post-bronchodilator spirometry"
        spirometry_standard: "ATS/ERS 2019"
        bronchodilator_agent: "albuterol"
        bronchodilator_dose:
          value: "400"
          unit:
            id: "UO:0000023"
            name: "microgram"

  # FEV1 - Subject 1 follow-up
  - id: "LF:002"
    name: "FEV1 - Subject 1 1-year follow-up"
    description: "FEV1 decline after 1 year continued PM2.5 exposure"
    has_specified_output:
      - id: "LF:002-output-followup"
        name: "One-year follow-up spirometry"
        description: "FEV1 is expressed as percent of predicted."
        experimental_group: treated
        measured_under: "EXPOSURE:aop-ambient-pm25-followup"
        fev1:
          value: "74"
          unit:
            id: "UO:0000187"
            name: "percent"
          central_tendency: single_value
          sample_size: 1
    has_exposure_condition:
      - id: "EXPOSURE:aop-ambient-pm25-followup"
        name: "Residential ambient PM2.5, 16 years at 1-year follow-up"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_duration:
          value: "16"
          unit:
            id: "UO:0000036"
            name: "year"
    reference_dataset: "GLI-2012"
    assay_date: "2025-02-01"
    study_subject:
      subject_type: InVivoSubject
      id: "SUBJECT:aop-001"
      name: "Subject 1"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
      age:
        value: "53"
        unit:
          id: "UO:0000036"
          name: "year"
      sex: "male"
      subject_characteristics: "Non-smoker, high PM2.5 exposure area resident, 16 years"
    follows_protocols:
      - protocol_type: SpirometryProtocol
        id: "PROTOCOL:006"
        name: "Pre- and post-bronchodilator spirometry"
        spirometry_standard: "ATS/ERS 2019"

  # FEV1/FVC ratio - Subject 1
  - id: "LF:003"
    name: "FEV1/FVC ratio - Subject 1"
    description: "FEV1/FVC ratio indicating obstructive pattern"
    has_specified_output:
      - id: "LF:003-output-followup"
        name: "One-year follow-up FEV1/FVC ratio"
        experimental_group: treated
        measured_under: "EXPOSURE:aop-ambient-pm25-followup"
        fev1_fvc_ratio:
          value: "0.68"
          unit:
            id: "UO:0000190"
            name: "ratio unit"
          central_tendency: single_value
          sample_size: 1
    has_exposure_condition:
      - id: "EXPOSURE:aop-ambient-pm25-followup"
        name: "Residential ambient PM2.5, 16 years at 1-year follow-up"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_duration:
          value: "16"
          unit:
            id: "UO:0000036"
            name: "year"
    assay_date: "2025-02-01"
    study_subject:
      subject_type: InVivoSubject
      id: "SUBJECT:aop-001"
      name: "Subject 1"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
      age:
        value: "53"
        unit:
          id: "UO:0000036"
          name: "year"
      sex: "male"
    follows_protocols:
      - protocol_type: SpirometryProtocol
        id: "PROTOCOL:006"
        name: "Pre- and post-bronchodilator spirometry"
        spirometry_standard: "ATS/ERS 2019"

  # FEV1 decline rate - Subject 1
  - id: "LF:004"
    name: "FEV1 decline rate - Subject 1"
    description: "Rate of FEV1 decline over 1 year (accelerated compared to normal aging)"
    has_specified_output:
      - id: "LF:004-output-followup"
        name: "One-year FEV1 decline rate"
        experimental_group: treated
        measured_under: "EXPOSURE:aop-ambient-pm25-followup"
        decline_rate:
          value: "65"
          unit:
            id: "UCUM:mL/a"
            name: "milliliter per year"
          central_tendency: single_value
          sample_size: 1
    has_exposure_condition:
      - id: "EXPOSURE:aop-ambient-pm25-followup"
        name: "Residential ambient PM2.5, 16 years at 1-year follow-up"
        exposure_agent:
          id: "ENVO:01000415"
          name: "fine respirable suspended particulate matter"
        exposure_duration:
          value: "16"
          unit:
            id: "UO:0000036"
            name: "year"
    assay_date: "2025-02-01"
    study_subject:
      subject_type: InVivoSubject
      id: "SUBJECT:aop-001"
      name: "Subject 1"
      model_species:
        id: "NCBITaxon:9606"
        name: "Homo sapiens"
      age:
        value: "53"
        unit:
          id: "UO:0000036"
          name: "year"
      sex: "male"
      subject_characteristics: "Accelerated decline >2x normal (25-30 mL/year)"
    follows_protocols:
      - protocol_type: SpirometryProtocol
        id: "PROTOCOL:006"
        name: "Pre- and post-bronchodilator spirometry"
        spirometry_standard: "ATS/ERS 2019"

# =============================================================================
# RESPONSE COMPARISONS (Analysis Layer)
# =============================================================================
#
# Every step of this pathway compares a clean-air control against a PM2.5
# exposure. The control and exposed groups are written as separate assay
# records here because each was run and reported separately, so each
# comparison references one output record from each assay - the change itself
# is never stored inside the assay data.

response_comparisons:
  # MIE: oxidative stress
  - id: "COMP:aop-ros-4h"
    name: "Change in ROS, PM2.5 vs clean air (4h)"
    description: "Cross-assay comparison between the control assay OX:001 and the exposed assay OX:002."
    derived_from_assay: "OX:002"
    compared_measurement: "reactive_oxygen_species"
    control_output: "OX:001-output-control"
    treated_output: "OX:002-output-pm25"
    change_type: fold_change
    change_direction: increased
    change_value:
      value: "2.8"
      unit:
        id: "STATO:0000169"
        name: "fold change"
    p_value: 0.001
    statistical_test: "two-sample t-test"
    derivation: reported_in_paper

  # KE1: EGFR activation
  - id: "COMP:aop-pegfr-4h"
    name: "Change in EGFR Y1068 phosphorylation, PM2.5 vs clean air (4h)"
    description: "Cross-assay comparison between the control assay EGFR:001 and the exposed assay EGFR:002."
    derived_from_assay: "EGFR:002"
    compared_measurement: "egfr_phosphorylation_y1068"
    control_output: "EGFR:001-output-control"
    treated_output: "EGFR:002-output-pm25"
    change_type: fold_change
    change_direction: increased
    change_value:
      value: "3.5"
      unit:
        id: "STATO:0000169"
        name: "fold change"
    p_value: 0.002
    statistical_test: "two-sample t-test"
    derivation: reported_in_paper

  # KE2: goblet cell hyperplasia
  - id: "COMP:aop-muc5ac-24h"
    name: "Change in MUC5AC mRNA, PM2.5 vs clean air (24h)"
    description: "Cross-assay comparison between the control assay GCM:001 and the exposed assay GCM:002."
    derived_from_assay: "GCM:002"
    compared_measurement: "muc5ac_mrna_expression"
    control_output: "GCM:001-output-control"
    treated_output: "GCM:002-output-pm25"
    change_type: fold_change
    change_direction: increased
    change_value:
      value: "4.2"
      unit:
        id: "STATO:0000169"
        name: "fold change"
    p_value: 0.001
    statistical_test: "two-sample t-test"
    derivation: reported_in_paper

  # KE3 (part 1): ciliary function
  - id: "COMP:aop-cbf-7d"
    name: "Change in ciliary beat frequency, PM2.5 7d vs clean air baseline"
    description: "Cross-assay comparison between the control assay CILIARY:001 and the exposed assay CILIARY:003."
    derived_from_assay: "CILIARY:003"
    compared_measurement: "beat_frequency_hz"
    control_output: "CILIARY:001-output-control"
    treated_output: "CILIARY:003-output-pm25"
    change_type: absolute_difference
    change_direction: decreased
    change_value:
      value: "-5.3"
      unit:
        id: "UO:0000106"
        name: "hertz"
    p_value: 0.001
    statistical_test: "two-sample t-test"
    derivation: computed

  # KE3 (part 2): mucociliary clearance
  - id: "COMP:aop-mct-7d"
    name: "Change in mucociliary transport rate, PM2.5 7d vs clean air baseline"
    description: "Cross-assay comparison between the control assay MCC:001 and the exposed assay MCC:003."
    derived_from_assay: "MCC:003"
    compared_measurement: "transport_rate"
    control_output: "MCC:001-output-control"
    treated_output: "MCC:003-output-pm25"
    change_type: percent_change
    change_direction: decreased
    change_value:
      value: "-65.4"
      unit:
        id: "UO:0000187"
        name: "percent"
    p_value: 0.001
    statistical_test: "two-sample t-test"
    derivation: computed

  # AO: lung function decline
  - id: "COMP:aop-fev1-1yr"
    name: "Change in FEV1 over 1 year of continued ambient PM2.5 exposure"
    description: "Within-subject comparison: LF:001 is the enrollment measurement and LF:002 the 1-year follow-up for the same subject. The change is expressed in percent-of-predicted points (FEV1 percent predicted at follow-up minus at enrollment)."
    derived_from_assay: "LF:002"
    compared_measurement: "fev1"
    control_output: "LF:001-output-baseline"
    treated_output: "LF:002-output-followup"
    change_type: absolute_difference
    change_direction: decreased
    change_value:
      value: "-4"
      unit:
        id: "UO:0000187"
        name: "percent"
    derivation: computed

# =============================================================================
# KEY EVENT RELATIONSHIPS - the topology of the pathway described above
# =============================================================================

key_event_relationships:
  - id: "KER:aop-oxidative-stress-to-egfr"
    name: "Increased oxidative stress leads to EGFR activation"
    upstream_event:
      id: "KE:mie-oxidative-stress"
      name: "Increased oxidative stress"
      biological_action: increased
      level_of_biological_organization: molecular
    downstream_event:
      id: "KE:ke1-egfr-activation"
      name: "EGFR activation"
      biological_action: activated
      level_of_biological_organization: molecular
    relationship_type: "directly leads to"
    evidence_support: strong

  - id: "KER:aop-egfr-to-goblet"
    name: "EGFR activation leads to goblet cell hyperplasia"
    upstream_event:
      id: "KE:ke1-egfr-activation"
      name: "EGFR activation"
      biological_action: activated
      level_of_biological_organization: molecular
    downstream_event:
      id: "KE:ke2-goblet-hyperplasia"
      name: "Goblet cell hyperplasia"
      biological_action: increased
      level_of_biological_organization: cellular
    relationship_type: "directly leads to"
    evidence_support: strong

  - id: "KER:aop-goblet-to-ciliary"
    name: "Goblet cell hyperplasia leads to decreased ciliary function"
    upstream_event:
      id: "KE:ke2-goblet-hyperplasia"
      name: "Goblet cell hyperplasia"
      biological_action: increased
      level_of_biological_organization: cellular
    downstream_event:
      id: "KE:ke3-impaired-ciliary"
      name: "Decreased ciliary function"
      biological_action: decreased
      level_of_biological_organization: cellular
    relationship_type: "indirectly leads to"
    evidence_support: moderate

  - id: "KER:aop-ciliary-to-mcc"
    name: "Decreased ciliary function leads to impaired mucociliary clearance"
    upstream_event:
      id: "KE:ke3-impaired-ciliary"
      name: "Decreased ciliary function"
      biological_action: decreased
      level_of_biological_organization: cellular
    downstream_event:
      id: "KE:ke3-impaired-mcc"
      name: "Impaired mucociliary clearance"
      biological_action: impaired
      level_of_biological_organization: tissue
    relationship_type: "directly leads to"
    evidence_support: strong

  - id: "KER:aop-mcc-to-lung-function"
    name: "Impaired mucociliary clearance leads to decreased lung function"
    upstream_event:
      id: "KE:ke3-impaired-mcc"
      name: "Impaired mucociliary clearance"
      biological_action: impaired
      level_of_biological_organization: tissue
    downstream_event:
      id: "KE:ao-decreased-lung-function"
      name: "Decreased lung function"
      biological_action: decreased
      level_of_biological_organization: organ
    relationship_type: "directly leads to"
    evidence_support: moderate