PLTW Medical Interventions EOC Review

UNIT 1: HOW TO FIGHT INFECTION

Activities 1.1-1.5: The Mystery Infection

  • Dilution Problems:
    • A 100 \, \text{ug/mL} antigen solution is diluted. The task is to determine the total tube dilution, final dilution, and antigen concentration for each well.
    • Tube dilutions involve calculating the ratio of sample volume to total volume (sample + diluent).
    • Final dilution is the product of individual tube dilutions in a serial dilution.
    • Antigen concentration is calculated based on the dilution factor applied to the original concentration.
  • Serial Dilution Problem:
    • 50 µL of a sample is added to 100 µL of diluent, resulting in a concentration of 500 \, \text{ng/mL}.
    • A serial dilution is performed by adding 50 µL of sample into 100 µL of diluent for three more tubes.
    • The final dilution of the last tube (tube 4) needs to be determined.
    • Each dilution step involves a 50 µL sample added to 100 µL diluent, making each tube's dilution factor 1/3.
    • After four serial dilutions, the final dilution is (1/3)^4 = 1/81.
  • Serial Dilution in Tubes:
    • A serial dilution is performed with an initial concentration of 500 \, \text{mg/mL} in the first tube.
    • Each tube contains 100 µL of diluent.
    • 50 µL is withdrawn from one tube and added to the next tube.
    • This process continued up to tube 11, then 50µL discarded, leaving 100µL in Tube 12.
    • The task: complete a table which includes tube dilution, final dilution, and antigen concentration for each tube.
    • Tube dilution is calculated as \frac{50 \, \mu L}{150 \, \mu L} = \frac{1}{3}
    • Final dilution is the cumulative product of tube dilutions. For example, final dilution of tube 3 = \frac{1}{3} \cdot \frac{1}{3} \cdot \frac{1}{3} = \frac{1}{27}
    • Antigen concentration is calculated by multiplying the previous tube's concentration by the tube dilution (\frac{1}{3}).
    • Thus, concentration in Tube 2 is 500 \, \text{mg/mL} \cdot \frac{1}{3} = 166.67 \, \text{mg/mL}.
  • ELISA Results Analysis:
    • ELISA results are compared to a standard strip to determine antigen concentration.
    • Patients are ranked based on antigen concentration to determine the sequence of sickness.

ELISA Test

  • Purpose: To detect and quantify a specific substance, such as an antigen or antibody.
  • Antigens: Substances that can trigger an immune response in the body.
  • Antibodies: Proteins produced by the immune system to recognize and bind to specific antigens.

ELISA Assay

  • Components:
    • Well: A small container in which the assay is performed.
    • Enzyme: A protein that catalyzes a biochemical reaction, used to produce a detectable signal.
    • Secondary Antibody: An antibody that binds to the primary antibody and is conjugated to an enzyme.
    • Antigen: The substance being detected in the sample.
    • Primary Antibody: An antibody that specifically binds to the antigen of interest.
    • Substrate: A molecule that is acted upon by the enzyme to produce a detectable signal.
    • Positive Result Color: Usually a color change, indicating the presence of the antigen.
  • Steps:
    1. Add antigen of interest to wells to make a positive control, then fill other wells with patient samples. (Step 2/b)
    2. Make serial dilutions of antigen of interest for comparison with test wells (Step 5/e)
    3. Add primary antibodies to all wells. Let develop, then wash. (Step 6/f)
    4. Add secondary antibodies with attached enzyme to all wells. Let develop, then wash. (Step 1/a)
    5. Add TMB substrate to all wells. (Step 3/c)
    6. Add Stop solution once color change has occurred to all wells. (Step 4/d)
    7. Compare coloration of test samples to determine concentration. (Step 7/g).

ELISA Results and Infection Path

  • ELISA test results from three students (S1, S2, S3) are analyzed for the presence of N. meningitidis antigen.
  • The path of infection is determined based on the relative antigen concentrations in each student's sample.
  • The student with the highest antigen concentration was infected first, followed by the student with the next highest, and so on.

ELISA Standard Curve and Sample Concentration

  • An ELISA test uses a standard curve with serially diluted concentrations (Well 5: 80 mg/mL, Well 4: 40 mg/mL, Well 3: 20 mg/mL, Well 2: 10 mg/mL, Well 1: 5 mg/mL).
  • The concentration of a sample is determined by comparing its signal intensity to the standard curve.
  • Sample A-E contains 40 mg/mL which can be determined by identifying which well has the same concentration.

Activities 1.6-1.9: Antibiotic Treatment

  • Bacterial Cell Parts and Functions:

    • Nucleoid: Contains the bacterial DNA, which is typically a single, circular chromosome.
    • Plasmid: Small, circular DNA molecules that carry extra genes separate from the bacterial chromosome.
    • Ribosome: Synthesizes proteins by translating mRNA into amino acid sequences.
    • Cell Membrane: Encloses the cytoplasm and regulates the transport of substances in and out of the cell.
    • Cell Wall: Provides structural support and protection to the cell.
    • Capsule: A sticky outer layer that enhances the bacterium's ability to cause disease.
    • Flagella: Long, whip-like appendages that enable the bacteria to move.
    • Pilus: Short, hair-like structures on the cell surface involved in attachment and genetic material transfer via conjugation.
    • Endotoxins: Toxic substances released when the bacterial cell is disrupted or destroyed.

  • Gram-Positive vs. Gram-Negative Bacteria:

    • Gram-positive: Bacteria with a thick peptidoglycan layer in their cell wall, which stains purple in a Gram stain.
    • Gram-negative: Bacteria with a thin peptidoglycan layer and an outer membrane containing lipopolysaccharides (LPS), which stains pink in a Gram stain.

  • Antibiotic Mechanisms of Action:

    • Inhibition of Protein Synthesis:
      • Some antibiotics bind to the ribosome, blocking the attachment of tRNA and preventing protein synthesis.
    • Inhibition of Folic Acid Synthesis:
      • Some antibiotics prevent the synthesis of folic acid by competitively inhibiting the incorporation of PABA into folic acid.
    • Inhibition of Cell Wall Synthesis:
      • Some antibiotics inhibit enzymes involved in the final steps of cell wall synthesis, preventing peptidoglycan strands from connecting.
    • Inhibition of DNA Replication:
      • Some antibiotics inhibit topoisomerases, which maintain the supercoiling of chromosomal DNA, thus preventing essential cell processes.

  • Antibiotics and Viruses: Antibiotics cannot be used to treat viruses because viruses have different structures and mechanisms than bacteria; antibiotics target bacterial-specific processes.

  • Antibiotic Resistance:

    • Definition: The ability of a bacterium to withstand the effects of an antibiotic.
    • Growth on Petri Dish: A resistant bacterium will grow on a petri dish exposed to the antibiotic.

  • Antibiotic Susceptibility:

    • Definition: The vulnerability of a bacterium to an antibiotic.
    • Growth on Petri Dish: A susceptible bacterium will not grow on a petri dish exposed to the antibiotic.

  • Zone of Inhibition: A clear area around an antibiotic disk on a petri dish, indicating that the antibiotic is effective against the bacteria.

    • The larger the zone of inhibition, the more effective the antibiotic.
    • Determining Effectiveness:
      • Antibiotic A: Most effective treatment since having the largest zone of inhibition.
      • Antibiotic B: Bacterial strain is resistant to this antibiotic since there is no zone of inhibition around this antibiotic.

  • Mechanisms of Bacterial Resistance: Bacteria can share DNA through various mechanisms to create more antibiotic-resistant cells

    • Conjugation:
      • Direct transfer of genetic material (usually a plasmid) between two bacterial cells via a pilus.
      • This allows for the rapid spread of antibiotic resistance genes within a bacterial population.
    • Transformation:
      • Uptake of free DNA from the environment by a bacterial cell.
      • The DNA may contain antibiotic resistance genes, which are then incorporated into the recipient cell's genome.
    • Transduction:
      • Transfer of genetic material from one bacterium to another by a bacteriophage (virus that infects bacteria).
      • If the bacteriophage carries antibiotic resistance genes, it can introduce these genes into a new host cell.

  • Ways Bacteria Develop Resistance:

    • Target Modification: Bacteria alter the structure of the antibiotic's target site, reducing its affinity for the antibiotic.
    • Enzymatic Inactivation: Bacteria produce enzymes that degrade or modify the antibiotic, rendering it inactive.
    • Reduced Permeability: Bacteria decrease the permeability of their cell membrane, preventing the antibiotic from entering the cell.
    • Efflux Pumps: Bacteria use efflux pumps to actively pump the antibiotic out of the cell, reducing its intracellular concentration.
    • Bypass Pathways: Bacteria develop alternative metabolic pathways that bypass the steps inhibited by the antibiotic.

  • Predicting Bacterial Growth Patterns: The experiment involves two strains of E. coli:

    • E. coli I: contains a plasmid gene coding for ciprofloxacin resistance.
    • E. coli II: contains a chromosomal gene coding for penicillin resistance.
    • Each strain must be streaked on plates with either ciprofloxacin or penicillin, and the growth patterns are predicted after 24 hours at 37°C.

Activities 1.10-1.12: Aftermath: Hearing Loss

  • Sound Waves:

    • Loud sound: high amplitude
    • High-pitched sound: high frequency
    • Quiet sound: low amplitude
    • Low-pitched sound: low frequency

  • Ear Anatomy and Function:

    • Outer Ear:
      • Pinna: Collects and funnels sound waves into the auditory canal. (F,G)
      • Auditory Canal: Funnels and amplifies sound to the eardrum. (G)
      • Tympanic Membrane (Eardrum): Vibrates when struck by sound waves, transmitting vibrations to the middle ear. (H)
    • Middle Ear:
      • Tympanic Cavity: Space filled with the ossicles. (J)
      • Ossicles (Malleus, Incus, Stapes): Small bones that amplify and transmit vibrations from the eardrum to the inner ear. (D)
      • Eustachian Tube: Connects the middle ear to the back of the nasal cavity, equalizing air pressure in the middle ear. (I,J)
      • Oval Window: Membrane that connects the middle ear with the cochlea, allowing vibrations to enter the inner ear. (B)
    • Inner Ear:
      • Cochlea: Converts stimuli from outside to nerve impulses using internal hair cells. (E)
      • Auditory Nerve: Transmits electrical signals from the cochlea to the brain. (C)
      • Semicircular Canals: Contain fluid that helps with perception of balance and position in space. (A)

  • Types of Hearing Loss:

    • Sensorineural Hearing Loss:
      • Definition/Cause: Damage to the inner ear or auditory nerve, often caused by aging, noise exposure, or genetics.
      • Part(s) of Ear Malfunctioning: Inner ear (cochlea) or auditory nerve.
    • Conductive Hearing Loss:
      • Definition/Cause: Blockage or damage to the outer or middle ear, preventing sound from reaching the inner ear.
      • Part(s) of Ear Malfunctioning: Outer or middle ear.

  • Rinne Test:

    • Compares air conduction (AC) and bone conduction (BC) hearing.
    • Normally, AC should be greater than BC.
    • Sensorineural hearing loss: AC > BC in both ears, but both are reduced compared to normal values.
    • Conductive hearing loss: BC > AC in the affected ear.

  • Audiogram Analysis:

    • Type of Hearing Loss: Determined by comparing air and bone conduction thresholds.
      • Conductive: Air conduction thresholds are worse than bone conduction thresholds.
      • Sensorineural: Air and bone conduction thresholds are equally affected.
    • Severity of Hearing Loss: Determined by the degree of threshold elevation.
      • Mild, Moderate, Severe, Profound, etc.
    • Affected Pitches/Frequencies: Identified by noting the frequencies at which thresholds are elevated on the audiogram.
    • Ear Specificity: Each ear is assessed separately, and findings are documented for both the left and right ears.

Lesson 1.13-1.15: Vaccination

  • Vaccine Mechanism: Vaccines introduce weakened or inactive pathogens (antigens) into the body, triggering an immune response and the production of antibodies, providing long-term protection against future infections.

  • Herd Immunity: When a large portion of a population is vaccinated, it creates a buffer of immunity that protects unvaccinated individuals by reducing the likelihood of disease spread.

  • Recombinant DNA: A technology that combines DNA molecules from different sources to create new genetic combinations.

    • Restriction Enzymes: Cut DNA at specific sequences.
    • Ligase: An enzyme that joins DNA fragments together.

  • Restriction Enzyme Cut Sites:

    • Sticky Ends: Restriction enzymes make staggered cuts, producing fragments with overhanging single-stranded ends.
    • Blunt Ends: Restriction enzymes make straight cuts, producing fragments with no overhanging ends.

  • Study Types:

    • Cohort Study: A prospective study that follows a group of individuals over time to determine the incidence of a disease or outcome.
      • Example: Following a group of farmworkers exposed to pesticides over ten years to see who develops asthma.
    • Case-Control (Retrospective) Study: A study that compares a group of individuals with a disease (cases) to a group without the disease (controls) to identify risk factors.
      • Example: Comparing obese and non-obese individuals to see how many servings of vegetables a day they eat.

  • Restriction Enzyme Analysis:

    • Restriction enzymes cut DNA at specific sequences which result in different cut sites.
    • Determining whether to use each enzyme involves assessing:
      • Number of cuts in plasmid DNA.
      • Number of cuts in viral DNA.
      • If enzymes not to be used, you won't use them due to multiple cuts, or irrelevant cut sites.

UNIT 2: HOW TO Screen What Is In Your Genes

Activities 2.1-2.4: Genetic Testing

  • Types of Genetic Diseases:

    • Single Gene - Recessive: Requires two copies of the mutated gene to cause the disease. Carriers have one copy but do not show symptoms. (e.g., Cystic Fibrosis)
    • Single Gene - Dominant: Only one copy of the mutated gene is needed to cause the disease. Affected individuals have at least one affected parent. (e.g., Huntington’s disease)
    • Single Gene - Sex-Linked: Mutation is located on a sex chromosome (usually X). Males are more often affected because they have only one X chromosome. (e.g., Hemophilia)
    • Chromosomal: Caused by abnormalities in chromosome number or structure. (e.g., Down syndrome)

  • Karyotype Analysis:

    • A karyotype is a visual representation of an individual's chromosomes, arranged in a standardized format.
    • Diagnoses can be made if abnormalities are present such as extra chromosomes (Down Syndrome), missing chromosomes (Turner Syndrome), or structural abnormalities.
    • Sex of the patient can also be confirmed based on the presence of XX (female) or XY (male) chromosomes.

  • Genotype vs. Phenotype:

    • Genotype: The genetic makeup of an organism (e.g., the specific alleles of a gene). (e.g., BB, Bb, bb)
    • Phenotype: The observable characteristics or traits of an organism, resulting from the interaction of the genotype with the environment. (e.g., brown eyes, tall height).

  • Pedigree Analysis:

    • Pedigrees show family relationships and inheritance patterns of genetic traits.
    • Determine if the trait is dominant or recessive by analyzing the pattern of inheritance.
    • Dominant traits show affected individuals in every generation, while recessive traits may skip generations.
    • Determine genotypes based on the phenotypes and inheritance patterns of family members.

  • Familial Hypercholesterolemia (FH) Example:

    • Assuming the pedigree tracks Familial Hypercholesterolemia, a dominant trait.
    • Determine the genotype of individual I-1 based on the pedigree information.

  • Chance of Inheriting FH:

    • Calculate the probability of individual II-1 having a child with FH (if they marry someone who does not have FH).

  • PCR Steps:

    1. Denaturation: Heating the DNA to separate the double strands.
    2. Annealing: Cooling the DNA to allow primers to bind to the target sequence.
    3. Extension: DNA polymerase extends the primers, creating new DNA strands.

  • DNA Copies after PCR:

    • After the 4th cycle: 2^4 = 16 copies.
    • After 30 cycles: 2^{30} = 1,073,741,824 copies.

  • Gel Electrophoresis:

    • Labeling:
      1. Positive end.
      2. Negative end.
      3. Wells.
      4. DNA Marker/Ladder.
      5. Smallest DNA fragment.
      6. Largest DNA fragment.

  • Gel Electrophoresis for Genotype Determination:

    • Gel electrophoresis separates DNA fragments based on size. By comparing the banding patterns of individuals with known genotypes, one can determine the genotypes of others.
    • If lane B shows a Homozygous Dominant individual and lane C shows a Homozygous Recessive Individual, then the genotypes for lanes D, E, and F can be determined based on their banding patterns relative to lanes B and C.

  • SNP Analysis:

    • Identifying Single Nucleotide Polymorphisms (SNPs) helps determine genotypes.
    • The restriction site for HaeIII is GG|CC, and CC|GG.
    • An allele can be cut if it contains the HaeIII restriction site.
    • Determine whether the Taster (T) or Nontaster (t) allele contains the HaeIII restriction site, and thus which allele can be cut.

Activities 2.5-2.6: Our Genetic Future

  • Vector Selection for Gene Therapy:

    • Given that the gene for NF1 is approximately 8,400 base pairs long.
    • The best candidate vector for gene therapy should be determined as a vector that can accommodate larger sequences.

UNIT 3: HOW TO Conquer Cancer

Activities 3.1-3.5: Detecting Cancer

  • Cancer Genes and Cell Cycle Regulation:

    • Cell division is a highly regulated process.
    • Some genes that control cell division are tumor suppressor genes and proto-oncogenes.
    • Proto-oncogenes send signals to the cell that tells it when to divide, only activated when cell division needs to occur.
    • Tumor suppressor genes tell cells when NOT to divide and determine when a cell is unhealthy or too old to continue with mitosis.
    • P53 is a tumor suppressor gene that checks a cell’s DNA for mutations and tells cells to do apoptosis if damage is too extensive to continue.
    • For that reason, p53 is called the guardian of the genome.
    • Mutations in proto-oncogenes and tumor suppressor genes which cause cells to lose the ability to control cell growth can lead to cancer.
    • When a proto oncogene is mutated we call it an oncogene.
    • Oncogenes are constantly activated so cells are always getting the signal to divide.
    • This is analogous to when the gas pedal in a car is stuck down. The cell is constantly being told to GO (divide) even though it isn’t safe or necessary.
    • Mutations in tumor suppressor genes make it so that cells can’t stop dividing when they have damage.
    • This is analogous to when the brake pedal in a car is broken. The cell needs to STOP dividing but is unable to prevent the process from happening.
    • These mutations lead to cells that ignore the normal signals and continue to divide without any regulation.
    • If the constantly dividing cells grow so much that they start to spread into surrounding tissues that is what we call invasion.
    • If the cells enter the bloodstream and spreads to other organs or tissues then this is called metastasis.

  • Gene Expression: The process by which the information encoded in a gene is used to synthesize a functional gene product (protein or RNA). The amount of protein produced in the cell indicates gene expression.

  • DNA Microarray: A tool used to measure the expression levels of thousands of genes simultaneously.

  • Microarray Colors and Gene Expression:

    • Upregulated Gene in Cancer Cells: The gene would be more expressed in the cancer cells so you will see red.
    • Downregulated Gene in Cancer Cells: The gene would be less expressed in the cancer cells so you will see green.
    • Genes Not Expressed in Healthy Nor in Cancerous Cells: The gene would show as black.
    • Genes Expressed Equally in Both Healthy and Cancerous Cells: The gene would show as yellow.

  • Gene Expression Predictions in Pancreatic Cancer Cells:

    • Cyclin E: Predicted to be upregulated because it is a proto-oncogene that promotes cell division, and is often overexpressed in cancer.
    • Surfactant B: Predicted to be equally expressed because it assists in breathing and is not involved in cell cycle regulation.
    • p53: Predicted to be downregulated because it is a tumor suppressor gene that induces apoptosis, and is often mutated or inactivated in cancer.
    • INS gene: Predicted to be equally expressed because it produces insulin for glucose control and is not directly involved in cell cycle regulation.

  • Microarray Results Analysis:

    • Gene expression is examined (up, down, or equally regulated) with microarray results from the genes above.

  • Base 2 Logarithm for Gene Expression:

    • Base 2 Logarithm are used to determine gene expression ratios.

  • Base 2 Logarithmic Expression of 0:

    • A base 2 logarithmic expression of 0 indicates that the gene is equally expressed in both normal and cancerous tissues.

  • Base 2 Logarithmic Expression of -3:

    • A base 2 logarithmic expression of -3 indicates that the gene is downregulated and would appear green on the microarray.

  • Pearson Correlation Coefficient for Chemotherapy Effectiveness:

    • The Pearson correlation coefficient is used to determine the relationship between Joe Smith’s gene expression and 4 other lung cancer patients.
    • How Joe would likely respond to each of the chemotherapy drugs that worked for the other 4 patients can be determined by analyzing the correlation coefficient data.

Activities 3.6-3.10: Reducing Cancer Risk

  • Types of Cancer Risk Factors:

    • Behavioral Risk Factor: Lifestyle choices or habits that increase the risk of cancer. (e.g., smoking, poor diet)
    • Biological Risk Factor: Inherent characteristics of an individual that increase the risk of cancer. (e.g., age, genetics)
    • Environmental Risk Factor: External conditions or substances that increase the risk of cancer. (e.g., radiation, pollution).
    • Sporadic Cancer: Cancer that occurs due to random mutations and is not inherited.
    • Familial Cancer: Cancer that occurs more frequently in a family than expected, but without a clear pattern of inheritance.
    • Hereditary Cancer: Cancer caused by inherited genetic mutations, with a clear pattern of inheritance.

  • Marker Analysis:

    • Marker analysis helps determine whether other family members share a similar BRCA mutation.

Activities 3.11-3.17: Treating Cancer

  • Cancer Treatment Methods:

    • Chemotherapy: Uses drugs to kill cancer cells throughout the body.
    • Radiation: Uses high-energy rays to kill cancer cells in a specific area.
    • Surgery: Physically removes cancerous tumors from the body.

UNIT 4: HOW TO Prevail When Organs Fail

Lesson 4.1: Transplant

  • Organ Allocation Scenarios (OPTN Guidelines):

    • Scenario a: 32-year-old former drug addict (4 years on the list) vs. 25-year-old single mother (3 years on the list).
      • The single mother gets the transplant because the drug addict has a history of substance abuse.
    • Scenario b: 9-year-old girl in remission from leukemia (1 year on the list) vs. 35-year-old ophthalmologist (2 years on the list).
      • The child gets the transplant because children get prioritized, in general, over adults.
    • Scenario c: Both 26-year-old are single, and one lives closer to the organ donor.
      • The single teacher goes first because shorter transportation time.

  • HLA Tissue Typing:

    • Matching HLA (Human Leukocyte Antigen) types is essential for organ transplantation to minimize the risk of rejection.
    • The best tissue match is determined by comparing the HLA antigens of the transplant patient with those of potential donors.
    • A higher number of matching HLA antigens indicates a better match and a lower risk of rejection.
    • Donor 7 is the best tissue match due to having the most antigens in common with the transplant patient.

  • Blood Typing:

    • Understanding Agglutination: The chart can be completed by completing the different blood types based on agglutination.

  • Blood Type and Organ Donation:

    • Using blood group compatibility rules when organ donation is needed, blood group compatible can be useful to see potential donors.
    • Patient 1 needs an organ and patients 2-5 are willing to donate to them. Potential donors based on blood type can be determined by cross-matching:
    • If Patient 2 needs an organ, potential donors can also be determined through the cross-matching process.