# =============================================================================
# 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)
#
# =============================================================================

# =============================================================================
# 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"
      name: "OX:001 measurement results"
      reactive_oxygen_species:
        value: "1.0"
        unit:
          id: "UO:0000193"
          name: "fold change"
    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"
      name: "OX:002 measurement results"
      reactive_oxygen_species:
        value: "2.8"
        unit:
          id: "UO:0000193"
          name: "fold change"
    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: "CHEBI:74481"
          name: "PM2.5"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0000274"
            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"
      name: "OX:003 measurement results"
      malondialdehyde_level:
        value: "1.9"
        unit:
          id: "UO:0000193"
          name: "fold change"
    assay_date: "2024-01-16"
    has_exposure_condition:
      - id: "EXPOSURE:pm25-24h"
        name: "PM2.5 10 ug/cm2 for 24 hours"
        exposure_agent:
          id: "CHEBI:74481"
          name: "PM2.5"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0000274"
            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"
      name: "EGFR:001 measurement results"
      egfr_phosphorylation_y1068:
        value: "1.0"
        unit:
          id: "UO:0000193"
          name: "fold change"
    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"
      name: "EGFR:002 measurement results"
      egfr_phosphorylation_y1068:
        value: "3.5"
        unit:
          id: "UO:0000193"
          name: "fold change"
    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"
      name: "EGFR:003 measurement results"
      total_egfr_protein:
        value: "1.05"
        unit:
          id: "UO:0000193"
          name: "fold change"
    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"
      name: "GCM:001 measurement results"
      muc5ac_mrna_expression:
        value: "1.0"
        unit:
          id: "UO:0000193"
          name: "fold change"
    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"
      name: "GCM:002 measurement results"
      muc5ac_mrna_expression:
        value: "4.2"
        unit:
          id: "UO:0000193"
          name: "fold change"
    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"
      name: "GCM:003 measurement results"
      goblet_cell_percentage:
        value: "32"
        unit:
          id: "UO:0000187"
          name: "percent"
    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"
      name: "GCM:004 measurement results"
      mucus_viscosity:
        value: "185"
        unit:
          id: "UO:0000019"
          name: "centipoise"
    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"
      name: "CILIARY:001 measurement results"
      beat_frequency_hz:
        value: "12.5"
        unit:
          id: "UO:0000106"
          name: "hertz"
    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:0000064"
                name: "micrograms per milliliter"
          - id: "soma:supp-aop-002"
            name: "Heparin"
            supplement_type: growth_factor
            concentration:
              value: "4"
              unit:
                id: "UO:0000064"
                name: "micrograms 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:
      id: "CILIARY:002-output"
      name: "CILIARY:002 measurement results"
      beat_frequency_hz:
        value: "9.8"
        unit:
          id: "UO:0000106"
          name: "hertz"
    assay_date: "2024-01-16"
    has_exposure_condition:
      - id: "EXPOSURE:pm25-24h"
        name: "PM2.5 10 ug/cm2 for 24 hours"
        exposure_agent:
          id: "CHEBI:74481"
          name: "PM2.5"
        exposure_concentration:
          value: "10"
          unit:
            id: "UO:0000274"
            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:0000169"
            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"
      name: "CILIARY:003 measurement results"
      beat_frequency_hz:
        value: "7.2"
        unit:
          id: "UO:0000106"
          name: "hertz"
    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"
      name: "CILIARY:004 measurement results"
      percentage_ciliated_cells:
        value: "45"
        unit:
          id: "UO:0000187"
          name: "percent"
    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"
      name: "MCC:001 measurement results"
      transport_rate:
        value: "52"
        unit:
          id: "UO:0000103"
          name: "micrometer per second"
      transport_directionality: normal
    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"
      name: "MCC:002 measurement results"
      transport_rate:
        value: "38"
        unit:
          id: "UO:0000103"
          name: "micrometer per second"
      transport_directionality: normal
    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"
      name: "MCC:003 measurement results"
      transport_rate:
        value: "18"
        unit:
          id: "UO:0000103"
          name: "micrometer per second"
      transport_directionality: variable
    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"
      name: "MCC:004 measurement results"
      percentage_active_transport:
        value: "35"
        unit:
          id: "UO:0000187"
          name: "percent"
    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"
      name: "LF:001 measurement results"
      fev1:
        value: "78"
        unit:
          id: "UO:0000187"
          name: "percent predicted"
    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:0000022"
            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"
      name: "LF:002 measurement results"
      fev1:
        value: "74"
        unit:
          id: "UO:0000187"
          name: "percent predicted"
    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"
      name: "LF:003 measurement results"
      fev1_fvc_ratio:
        value: "0.68"
        unit:
          id: "UO:0000196"
          name: "ratio"
    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"
      name: "LF:004 measurement results"
      decline_rate:
        value: "65"
        unit:
          id: "UO:0000098"
          name: "milliliter per 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"