Module 11: Respiratory System

This lesson of the MCAT Respiratory System aligns with the AAMC’s official MCAT content outline, specifically under Foundational Concept 4, Content Category 4A. It covers essential respiratory system topics including gas exchange, ventilation mechanisms, oxygen transport, and acid–base regulation. These principles are frequently tested on the Chemical and Physical Foundations of Biological Systems (C/P) section, especially when evaluating homeostasis, pH buffering, or metabolic demands in the body. You can view the official AAMC MCAT topic guide here.

Overview and Function of the MCAT Respiratory System

The respiratory system is responsible for gas exchange between the environment and the body’s tissues, supporting life-sustaining cellular respiration. In addition to transporting oxygen and removing carbon dioxide, it contributes to several key physiological roles:

• Oxygen Supply: Delivers O₂ to tissues for aerobic metabolism in mitochondria, necessary for ATP production.
• Carbon Dioxide Removal: Expels CO₂, a byproduct of cellular respiration that can acidify the blood if not eliminated.
• pH Regulation: Through control of CO₂ levels, the lungs help regulate blood pH in conjunction with the kidneys.
• Vocalization: Movement of air through the vocal cords (within the larynx) enables speech.
• Olfaction: Nasal passages contain olfactory receptors for the sense of smell.

Structural Divisions:

• Upper Respiratory Tract: Includes the nasal cavity, pharynx, and larynx. These structures condition incoming air by filtering, humidifying, and warming it.
• Lower Respiratory Tract: Comprises the trachea, bronchi, bronchioles, and alveoli within the lungs, where air conduction and gas exchange occur.

Functional Zones:

• Conducting Zone: Extends from the nasal cavity to the terminal bronchioles. Its primary function is to move, filter, and humidify air. No gas exchange occurs here.
• Respiratory Zone: Includes respiratory bronchioles, alveolar ducts, and alveoli. This is where gas exchange with blood occurs.

Example MCAT Application: You may be asked to identify where in this pathway filtration vs. gas exchange occurs, or which muscles are responsible for active inspiration.

MCAT Tip: Know the order of airflow from nasal cavity to alveoli and the structural differences as airways branch.

Pathway of Airflow: External Air to Alveoli (MCAT Respiratory Anatomy)

The primary function of the respiratory system is to enable the exchange of gases (O₂ in, CO₂ out) between the atmosphere and the bloodstream. This begins with the inhalation of air and ends at the alveolar sacs, where oxygen diffuses into pulmonary capillaries. Below is a step-by-step breakdown of this process, with structure, function, and clinical/MCAT insights at each stage.

1. Nasal Cavity (or Oral Cavity)

• Air Entry Point: In most resting conditions, air enters through the nares (nostrils) into the nasal cavity.
• Functions:
  • Filtering: Hairs (vibrissae) trap large particles.
  • Warming: Air is warmed to body temperature.
  • Humidifying: Mucous membranes add moisture to prevent alveolar dryness.
  • Defense: Mucus and cilia trap and move pathogens and particles out.
• MCAT Insight: Be able to describe the role of ciliated epithelium and mucus in innate immunity and air conditioning.

2. Pharynx

• Shared Passageway for both food and air.
  • Divided into: nasopharynx, oropharynx, and laryngopharynx.
• The epiglottis (a flap of elastic cartilage) ensures food is routed into the esophagus, not the airway.

3. Larynx (“Voice Box”)

• Located just below the pharynx.
• Contains the glottis, the opening to the trachea.
• Vocal cords reside here.
• MCAT Tip: Know the epiglottis covers the glottis during swallowing to prevent aspiration.

4. Trachea (“Windpipe”)

• A rigid tube supported by C-shaped cartilaginous rings.
• Lined with ciliated pseudostratified columnar epithelium and goblet cells.
• Mucus traps particles; cilia propel them upward in the mucociliary escalator.
• MCAT Tip: The mucociliary escalator is a classic topic in questions about defense and airway maintenance.

5. Bronchi (Primary, Secondary, Tertiary)

• Primary bronchi (left and right) branch off the trachea at the carina.
• Each bronchus enters a lung and branches into:
  • Secondary (lobar) bronchi → serve lobes of lungs (3 on right, 2 on left).
  • Tertiary (segmental) bronchi → serve bronchopulmonary segments.
• Structure: Still have cartilage and ciliated epithelium.

6. Bronchioles

• Smaller branches of the bronchi that lack cartilage.
• Smooth muscle surrounds the bronchioles and regulates airflow resistance.
  • Sympathetic stimulation → bronchodilation.
  • Parasympathetic stimulation → bronchoconstriction.
• Clinical Insight: Bronchioles are the key site of pathology in asthma (narrowing due to inflammation, mucus, and smooth muscle contraction).

7. Terminal Bronchioles → Respiratory Bronchioles

• Terminal bronchioles are the final part of the conducting zone.
• Respiratory bronchioles begin the respiratory zone, where gas exchange can occur.
• Transition: From pure conduction to gas exchange surfaces.

8. Alveolar Ducts and Alveolar Sacs

• Alveolar ducts end in clusters of alveoli, called alveolar sacs.
• Alveoli:
  • Thin-walled structures (~300 million in human lungs).
  • Lined by Type I pneumocytes (flat cells for gas diffusion).
  • Type II pneumocytes secrete surfactant (reduces surface tension).
  • Surrounded by pulmonary capillaries for gas exchange.

MCAT Strategy Tip: Conducting vs. Respiratory Zones

Conducting Zone: No gas exchange
→ Nasal cavity → pharynx → larynx → trachea → bronchi → terminal bronchioles

Respiratory Zone: Gas exchange possible
→ Respiratory bronchioles → alveolar ducts → alveoli

Be sure you can label these zones and explain which structures belong to each. Questions often hinge on this division.

MCAT Tip: Be able to distinguish clearly between the conducting and respiratory zones and trace airflow: Nasal cavity → Pharynx → Larynx → Trachea → Bronchi → Bronchioles → Alveoli.

MCAT Gas Exchange Mechanics – Partial Pressures and Diffusion

Gas exchange occurs by simple diffusion across the thin barrier separating the alveoli and pulmonary capillaries. The driving force behind this diffusion is the difference in partial pressures of gases, specifically oxygen (O₂) and carbon dioxide (CO₂).

1. Partial Pressure and Dalton’s Law

Dalton’s Law of Partial Pressures states:

The total pressure of a gas mixture is equal to the sum of the partial pressures of each individual gas in the mixture.

$$
P_{\text{total}} = P_{\ce{O_2}} + P_{\ce{CO_2}} + P_{\ce{N_2}} + P_{\ce{H_2O}} + \dots
$$

• Partial pressure (Pₓ) of a gas = Fraction of that gas × Total pressure
• At sea level, atmospheric pressure ≈ 760 mmHg
• Oxygen is ~21% of air → PO2=0.21×760≈160 mmHg
• Nitrogen is ~78%, but is inert for respiratory physiology
• MCAT Tip: Always use partial pressure gradients to explain diffusion, NOT concentration alone.

2. Alveolar Gas Exchange

In the alveoli, gas exchange occurs between the air and pulmonary capillary blood:

Gas	Alveolar Partial Pressure	Capillary Partial Pressure	Net Movement
O₂	~100 mmHg	~40 mmHg	Into blood
CO₂	~40 mmHg	~45 mmHg	Into alveoli

• Oxygen diffuses into the capillary because its partial pressure is higher in the alveolus.
• Carbon dioxide diffuses out of the capillary because its partial pressure is higher in the blood.
• Both gases diffuse down their respective gradients until equilibrium is reached.

MCAT Insight: Remember – diffusion is always from high to low partial pressure, even if the CO₂ concentration in blood is higher than O₂ (CO₂ is more soluble, so partial pressure is what matters).

3. Fick’s Law of Diffusion

Gas exchange rate is governed by Fick’s Law:

$$
\text{Rate of diffusion} \propto \frac{A \cdot \Delta P}{T}
$$

Where:

• A = Surface area of alveoli (huge)
• ΔP = Partial pressure gradient
• T = Thickness of the membrane

4. CO₂ Diffusion is Faster than O₂

Although CO₂ has a smaller pressure gradient, it diffuses more rapidly because it is ~20× more soluble in plasma than O₂.

• MCAT Pitfall: Don’t assume larger gradient = faster exchange without considering solubility.

5. Oxygen Transport in Blood

Only ~1.5% of O₂ is dissolved in plasma. The vast majority is bound to hemoglobin (Hb) in red blood cells.

Hb + O₂ ⇌ HbO₂ (Oxyhemoglobin)

• Each hemoglobin molecule binds up to 4 O₂ molecules.
• This binding is cooperative → once one O₂ binds, the affinity for the next increases (sigmoidal curve).

6. Carbon Dioxide Transport

CO₂ is transported in three main forms:

Form	% of CO₂	Notes
Dissolved in plasma	~10%	Direct diffusion
Bound to hemoglobin	~20%	Forms carbaminohemoglobin
As bicarbonate (HCO₃⁻)	~70%	Via carbonic anhydrase

Key Reaction (in RBCs):

$$
\ce{CO2 + H2O <=> H2CO3 <=> H+ + HCO3^-}
$$

• Catalyzed by carbonic anhydrase
• H⁺ binds to hemoglobin → buffers pH
• HCO₃⁻ exchanged for Cl⁻ (chloride shift) into plasma

Key MCAT Takeaways: Gas Exchange

• Gases move by partial pressure gradients, not total concentration.
• O₂ diffuses into blood; CO₂ diffuses out, driven by pressure differences.
• Hemoglobin is essential for transporting O₂.
• CO₂ is primarily carried as bicarbonate in blood.
• Fick’s Law explains how area, thickness, and gradient affect exchange rate.
• Solubility matters — CO₂ diffuses faster than O₂ despite a smaller gradient.

Mechanics of Breathing (Ventilation) in the MCAT Respiratory System

Breathing, or pulmonary ventilation, refers to the process of moving air into (inspiration) and out of (expiration) the lungs. This is driven by changes in thoracic volume and intrapulmonary pressure, according to Boyle’s Law.

1. Boyle’s Law: Pressure–Volume Relationship

Boyle’s Law states:

At constant temperature, the pressure of a gas is inversely proportional to its volume:

$$
P \propto \frac{1}{V} \quad \text{or} \quad P_1 V_1 = P_2 V_2
$$

• As lung volume increases, pressure decreases → air flows in.
• As lung volume decreases, pressure increases → air flows out.
• Air moves down a pressure gradient, from high to low pressure.

MCAT Insight: This is a classic application of physics to biology. You don’t need to calculate pressure changes — just know increased volume = decreased pressure, and vice versa.

2. Inspiration (Inhalation)

Inspiration is an active process requiring muscle contraction.

Sequence of Events:

1. Diaphragm contracts → flattens downward
2. External intercostal muscles contract → rib cage expands outward and upward
3. Thoracic cavity volume increases
4. Intrapulmonary pressure decreases (below atmospheric)
5. Air rushes in due to pressure gradient

Feature	During Inspiration
Diaphragm	Contracts (flattens)
Thoracic volume	Increases
Intrapulmonary pressure	Drops below atmospheric
Airflow direction	Into lungs

Note: At the end of inspiration, intrapulmonary pressure equilibrates with atmospheric pressure (~760 mmHg), and airflow temporarily stops.

3. Expiration (Exhalation)

Expiration is usually a passive process at rest.

Sequence of Events:

1. Diaphragm and intercostals relax
2. Thoracic volume decreases
3. Intrapulmonary pressure rises (above atmospheric)
4. Air is pushed out of the lungs

Feature	During Expiration
Diaphragm	Relaxes (domes upward)
Thoracic volume	Decreases
Intrapulmonary pressure	Rises above atmospheric
Airflow direction	Out of lungs

Forced Expiration (e.g., during exercise or asthma attack):

• Becomes an active process
• Involves internal intercostals and abdominal muscles to compress thorax

4. Pleural Cavity and Intrapleural Pressure

The pleural cavity is the thin fluid-filled space between:

• Visceral pleura: adheres to lung surface
• Parietal pleura: lines thoracic wall

Intrapleural pressure is always negative relative to atmospheric pressure, helping keep lungs inflated:

• At rest: ~–4 mmHg
• During inspiration: becomes more negative (~–6 mmHg)

MCAT Tip: This negative pressure prevents lung collapse and is essential to normal ventilation.

5. Lung Compliance and Elastic Recoil

• Lung compliance = ease with which lungs expand.
• High compliance = easy to inflate (e.g., in emphysema)
• Low compliance = stiff lungs (e.g., in fibrosis, pulmonary edema)

Elastic recoil is the natural tendency of lungs to collapse inward due to elastic fibers and surface tension in alveoli.

6. Surfactant and Surface Tension

Alveolar surface tension, created by water molecules, tends to collapse alveoli.

Pulmonary surfactant:

• Secreted by Type II alveolar cells
• Reduces surface tension
• Increases alveolar stability and compliance
• Prevents atelectasis (alveolar collapse), especially during expiration

Key MCAT Respiratory System Takeaways: Mechanics of Breathing

• Boyle’s Law explains air movement: ↑ Volume → ↓ Pressure → Inhalation
• Inspiration = active (diaphragm + external intercostals)
• Expiration = passive (at rest), active if forced
• Intrapleural pressure stays negative → keeps lungs inflated
• Surfactant prevents alveolar collapse by reducing surface tension

Control of Breathing: Neural and Chemical Regulation in the MCAT Respiratory System

Breathing is mostly an involuntary, automatic process controlled by the central nervous system. However, it can be voluntarily overridden for speaking, singing, or holding your breath.

The regulation of breathing involves both:

• Neural control centers in the brainstem
• Chemical feedback mechanisms that monitor CO₂, O₂, and blood pH

1. Respiratory Centers in the Brainstem

Breathing rhythm is generated in the medulla oblongata and pons of the brainstem.

Key Brainstem Centers:

Region	Function
Medulla oblongata	Sets the basic rhythm of breathing; contains the dorsal respiratory group (DRG) and ventral respiratory group (VRG)
Pons	Fine-tunes rhythm; apneustic center stimulates inspiration, pneumotaxic center limits it
Cerebral cortex	Allows voluntary control of breathing (e.g., talking, breath-holding)
Hypothalamus/limbic system	Can influence breathing during emotional responses

MCAT Insight: If the medulla is damaged → breathing stops. This is central to understanding respiratory failure in neurological trauma.

2. Negative Feedback Loop: CO₂ and pH Control

The body uses a negative feedback loop to maintain homeostatic levels of blood gases and pH:

1. CO₂ rises (e.g., during exercise or hypoventilation)
2. ↑ CO₂ → ↑ H⁺ → decreased pH
3. Central chemoreceptors detect pH change
4. Ventilation increases
5. CO₂ expelled → pH returns to normal

Example: During intense exercise, CO₂ rises → increased respiratory rate → enhanced CO₂ exhalation → pH stabilized

Acid–Base Balance and Respiratory Compensation in the MCAT Respiratory System

The body must maintain blood pH within a narrow range (7.35–7.45). Even slight deviations can impair enzyme function, alter ion gradients, and disrupt physiological processes.

One of the most important systems regulating pH is the respiratory system, through its control of CO₂ levels.

1. The Bicarbonate Buffer System

At the core of acid–base regulation is the bicarbonate buffer equilibrium:

$$
\ce{CO2 + H2O <=> H2CO3 <=> H+ + HCO3^-}
$$

• Carbonic acid (H₂CO₃) is weak and unstable.
• It dissociates into hydrogen ions (H⁺) and bicarbonate ions (HCO₃⁻).
• This system resists changes in pH by reversibly absorbing or releasing H⁺.

MCAT Insight: This equation explains why CO₂ is considered an acid in the body, because it can generate H⁺ through this pathway.

2. Respiratory Compensation: Fast Regulation of pH

Because the lungs control the level of CO₂, they can alter blood pH rapidly.

Condition	CO₂ Level	pH Effect	Respiratory Compensation
Hypoventilation (↓ breathing)	↑ CO₂	↓ pH → acidosis	↑ ventilation to remove CO₂
Hyperventilation (↑ breathing)	↓ CO₂	↑ pH → alkalosis	↓ ventilation to retain CO₂

• Respiratory acidosis: Common in COPD, sedative overdose, or airway obstruction.
• Respiratory alkalosis: Seen in panic attacks, pain, or high-altitude breathing.

Key Principle: The lungs regulate pH by controlling the partial pressure of CO₂ (PaCO₂).

3. Renal Compensation: Slow but Powerful

When respiratory compensation is insufficient, the kidneys provide backup by:

• Reabsorbing HCO₃⁻
• Excreting or retaining H⁺

Condition	Renal Response
Acidosis	Reabsorb HCO₃⁻, excrete H⁺
Alkalosis	Excrete HCO₃⁻, retain H⁺

MCAT Respiratory System Strategy Tip: If a question mentions a chronic acid–base disturbance, ask yourself whether it’s respiratory or metabolic, and which system (lungs or kidneys) is compensating.

4. Distinguishing Respiratory vs. Metabolic Disturbances

Type	Primary Problem	Compensation
Respiratory acidosis	↑ CO₂	↑ HCO₃⁻ (renal)
Respiratory alkalosis	↓ CO₂	↓ HCO₃⁻ (renal)
Metabolic acidosis	↓ HCO₃⁻	↓ CO₂ (via hyperventilation)
Metabolic alkalosis	↑ HCO₃⁻	↑ CO₂ (via hypoventilation)

5. Henderson-Hasselbalch Equation (MCAT Version)

Used to relate pH, pKa, and the ratio of bicarbonate to CO₂:

$$
\text{pH} = \text{p}K_a + \log\left( \frac{[\ce{HCO3^-}]}{0.03 \times P_{\ce{CO2}}} \right)
$$

Normal values:

• [HCO₃⁻] ≈ 24 mEq/L
• PaCO₂ ≈ 40 mmHg
• pKa ≈ 6.1

MCAT Relevance: You won’t need to calculate exact pH, but you must conceptually understand that:

• ↑ HCO₃⁻ or ↓ CO₂ → ↑ pH (alkalosis)
• ↓ HCO₃⁻ or ↑ CO₂ → ↓ pH (acidosis)

Key MCAT Respiratory System Takeaways: Acid–Base and Breathing

• The bicarbonate buffer system is central to blood pH regulation.
• CO₂ acts like an acid, and breathing controls CO₂ levels.
• Hypoventilation causes acidosis; hyperventilation causes alkalosis.
• The lungs compensate for metabolic disturbances, and the kidneys compensate for respiratory ones.
• Henderson–Hasselbalch gives a quantitative relationship, but focus on directional logic.

The Bicarbonate Buffer System and Its Role in the MCAT Respiratory System

As mentioned earlier in this module, at the core of acid–base regulation is the bicarbonate buffer equilibrium:

$$
\ce{CO2 + H2O <=> H2CO3 <=> H+ + HCO3^-}
$$

Role of Carbonic Anhydrase

• This enzyme is found in high concentration inside red blood cells (RBCs) and renal tubular cells.
• It rapidly catalyzes the conversion of CO₂ and H₂O to carbonic acid (H₂CO₃), which then dissociates into H⁺ and HCO₃⁻.
• This reaction helps:
  • Buffer blood pH
  • Transport CO₂ efficiently as HCO₃⁻ in the plasma

The Chloride Shift

• As HCO₃⁻ builds up in the RBC, it is exchanged for Cl⁻ ions across the membrane to maintain electrical neutrality. This is known as the chloride shift.
• It helps maintain the buffering system’s capacity without disturbing cell charge balance.

The Role of Hemoglobin in Buffering

• Hemoglobin binds the free H⁺ produced by carbonic acid dissociation, preventing sharp drops in pH.
• In tissues: CO₂ → H⁺ produced → deoxyhemoglobin (HHb) buffers it.
• In lungs: O₂ binds Hb → H⁺ released → recombines with HCO₃⁻ to regenerate CO₂ for exhalation.

The Oxygen–Hemoglobin Dissociation Curve

The oxygen–hemoglobin (O₂-Hb) dissociation curve describes the relationship between blood partial pressure of oxygen (PaO₂) and the percent saturation of hemoglobin (SaO₂).

Sigmoidal Shape: Cooperativity

• Hb exhibits positive cooperativity: when one O₂ binds, it increases the affinity for the next.
• This leads to a sigmoid-shaped (S-shaped) curve, steep in the middle and flat at the extremes.

Right Shift vs. Left Shift

Factor	Right Shift	Left Shift
Temperature	↑ (fever, exercise)	↓ (hypothermia)
pH (Bohr effect)	↓ pH (acidic)	↑ pH (alkaline)
CO₂	↑ CO₂ (hypercapnia)	↓ CO₂ (hypocapnia)
2,3-BPG (2,3-bisphosphoglycerate)	↑ (chronic hypoxia)	↓ (stored blood)
Hb Affinity	↓ O₂ affinity	↑ O₂ affinity
Mnemonic	“CADET face Right” → CO₂, Acid, DPG, Exercise, Temperature	Opposite conditions

Right Shift = Releasing O₂

• Promotes oxygen unloading at the tissues
• Occurs during exercise, acidosis, fever, or high altitude
• Helps match oxygen delivery to metabolic demand

Left Shift = Holding O₂

• Increased affinity = oxygen is held tighter
• Occurs in alkalosis, low CO₂, or fetal hemoglobin (HbF)
• Makes it harder to release O₂ at the tissue level

MCAT Strategy Tip: Right shift = more unloading (think exercise); Left shift = more binding (think fetal hemoglobin or alkalosis)

Clinical and Experimental Implications on the MCAT Respiratory System

• Fetal Hemoglobin (HbF): Has a left-shifted curve → higher O₂ affinity helps draw O₂ across the placenta from maternal HbA.
• Stored Banked Blood: May have reduced 2,3-BPG → left shift → poor oxygen unloading in recipients.
• Carbon Monoxide (CO) Poisoning:
  • Binds Hb with >200× the affinity of O₂
  • Left-shifts the curve and reduces O₂ delivery
  • Can lead to tissue hypoxia despite normal PaO₂

Summary Table: O₂–Hemoglobin Curve Shifts

Condition	Curve Shift	Effect on O₂ Affinity	O₂ Delivery to Tissues
↑ CO₂ (hypercapnia)	Right	↓	↑
↑ H⁺ (low pH)	Right	↓	↑
↑ Temperature	Right	↓	↑
↑ 2,3-BPG	Right	↓	↑
↓ CO₂ (hypocapnia)	Left	↑	↓
↓ H⁺ (alkalosis)	Left	↑	↓
HbF or CO poisoning	Left	↑	↓

Lung Volumes and Capacities on the MCAT Respiratory System

Lung volumes refer to individual measurements of air movement in and out of the lungs. Capacities are combinations of two or more volumes. These measurements are critical for assessing lung function.

1. Basic Lung Volumes

Volume	Definition	Approximate Value
Tidal Volume (TV)	Air moved in or out during normal breathing	~500 mL
Inspiratory Reserve Volume (IRV)	Extra air inhaled after a normal breath	~3000 mL
Expiratory Reserve Volume (ERV)	Extra air exhaled after a normal breath	~1200 mL
Residual Volume (RV)	Air remaining in lungs after maximal exhalation	~1200 mL

MCAT Tip: Residual volume cannot be measured by spirometry, because it represents air that cannot be exhaled.

2. Lung Capacities (Combinations of Volumes)

Capacity	Formula	Description	Approx. Value
Inspiratory Capacity (IC)	TV + IRV	Max air inhaled after a normal exhale	~3500 mL
Functional Residual Capacity (FRC)	ERV + RV	Air remaining after normal exhale	~2400 mL
Vital Capacity (VC)	IRV + TV + ERV	Max air exhaled after full inhalation	~4700 mL
Total Lung Capacity (TLC)	VC + RV or IRV + TV + ERV + RV	Total volume of lungs	~5900–6000 mL

MCAT Tips & Strategy

• Gas law relationships (like Boyle’s and Henry’s laws) often appear in passage-based reasoning about changes in altitude, ventilation, or scuba diving.
• Expect conceptual questions on the effect of CO₂ buildup or shifts in the oxygen-Hb curve under different physiological states (e.g., exercise, hyperventilation).
• High-yield mechanism: Be able to walk through CO₂ transport and pH buffering, including the chloride shift and role of carbonic anhydrase.
• Know your volume definitions cold: TV, VC, FRC, and TLC, and what spirometry can and cannot measure.

Common Pitfalls & Mistakes

• Confusing the direction of hemoglobin saturation shifts (Right shift = ↓ affinity = more unloading).
• Forgetting that CO₂ is the main driver of ventilation, not O₂.
• Misapplying Boyle’s Law, remember it’s about pressure–volume changes in a sealed system like the thoracic cavity.
• Mixing up obstructive vs. restrictive lung disease spirometry patterns, obstructive has low FEV₁/FVC; restrictive has low TLC.
• Thinking all lung volumes can be measured by spirometry, residual volume (RV) and any capacity involving it cannot.