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Biotechnology > Quiz 2 + Final Study Guide > Flashcards

Quiz 2 + Final Study Guide Flashcards

1
Q

What caused COVID-19?

A

severe acute respiratory syndrome SARS-CoV-2
1. infects ACE-2 expressing nasal epithelial cells in the upper respiratory tract
2. infects ACE-2 expressing alveolar epithelial cells and patients exhibit pneumonitis
3. severe disease involves disruption of epithelial-endothelial barrier and hyperinflammation

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2
Q

Characteristics of SARS-CoV-2

A

enveloped, positive-sense (direct translation by host ribosome), single stranded RNA (+ssRNA) virus

Components
- S1: receptor binding domain
- S2: membrane fusion subunits

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3
Q

What are the general COVID-19 immunopathologies?

A

while most infections are mild, severe reactions include:
1. fatal systemic inflammation
2. tissue damage
3. cytokine storm: IL-6, IL-10
4. acute respiratory distress syndrome

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4
Q

Specific COVID-19 immunopathologies?

A
  1. lymphopenia: decrease WBC, more prone to infection
  2. T-cell activation
  3. lymphocyte dysfunction
  4. increased cytokine production, multiple organ dysfunction from inflammation
  5. increased antibodies
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5
Q

What do the most severe cases of COVID-19 exhibit?

A

extreme increase in inflammatory cytokines
- IL-6, IL-10
- TNF-alpha

initiates inflammation-induced organ dysfunction across multiple organs
- lung/kidney/liver injury, respiratory failure

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6
Q

What is the difference between alpha, delta, and omicron variants?

A

alpha: 10 changes in spike sequence; RBDs stay up; easier to enter

delta: 3 mutations in RBD that improve binding to ACE2 and evade immune response

omicron: 32 mutations in spike protein; increased infectivity and immune escape

Case fatality rate (CFR) was highest in beta

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7
Q

What is the life cycle of SARS-CoV-2?

A
  1. spike binds to host ACE2
  2. virus enters via endocytosis or direct fusion to plasma membrane
  3. viral genome is released and RNA is translated
  4. viral structural proteins produced and virus assembles
  5. exocytosis of virions
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8
Q

Describe fusion of SARS-CoV-2

A
  1. binds to ACE2
  2. Furin cleaves S1/S2 and TMPRSS2 cleaves S2’ of spike protein
  3. Activates S2 domain causes fusion of viral and host membranes
  4. viral capsid disassembles, releasing the viral RNA genome into the host cell’s cytoplasm
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9
Q

Describe endocytosis of SARS-CoV-2

A

absence of TMPRSS2
1. SARS-CoV-2 binds to ACE2
2. additional attachment factors (NRP-1)
3. Cathepsin L cleaves S protein and releases virus from the endosome

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10
Q

What is required for S binding to ACE2?

A

only one monomer needs to be in up/open position
- RBD in S1

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11
Q

Therapies for SARS-CoV-2

A

target several stages of the virus’s lifecycle: entry, replication, immune response
1. drugs targeting spike protein or binding to ACE2
2. antivirals that inhibit SARS-CoV-2 main protease, MPro
3. Vaccines: increase humoral response

differential receptor localization means that targeted therapies don’t work on all cell types

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12
Q

What is AD?

A
  • A neurodegenerative disorder marked by cognitive and behavioral impairment
  • Characterized by the deposition of amyloid-b (Ab) plaques and hyperphosphorylated tau neurofibrillary tangles
  • 90% of cases are sporadic
  • 10% of cases are hereditary (APP, PSEN1/2, APOE4)
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13
Q

What are the two subtypes of AD?

A
  1. Familial: usually early-onset, 5-10% of all cases; 5-10% of these involve mutations in APP, PSEN1/2, APOE4
  2. Sporadic: late-onset
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14
Q

How is AD identified?

A
  1. Psychological evaluations (short-term memory)
  2. Plasma levels of phosphorylated tau protein
  3. Amyloid beta and p-tau in CSF
  4. mutations in PSEN1/2, APOE4
  5. Neuroinflammation
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15
Q

What are the causes of AD?

A
  1. AB cascade hypothesis
  2. Neurofibrillary tau tangles
  3. Neuroinflammation: cytokine release, microglial dysfunction
  4. Genetics that cause AB clearance dysfunction
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16
Q

What is the amyloid-beta cascade hypothesis?

A

increase in AB with reduced clearance leads to oligomerization, inflammatory response (microglial/astrocyte), synaptic/neuronal injury, oxidative injury, hyperphosphorylation of tau, cell death, dementia with plaque and tangles

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17
Q

Where is APP found? How is amyloid precursor protein processed?

A

APP is found mostly on membranes of vesicles

APP is cleaved first by beta-secretase instead of alpha-secretase, leading to subsequent cleavage by gamma-secretase and formation of amyloid beta (AB) peptides

Alpha: good; cleaves transmembrane domain
Beta: bad (encoded by PSEN1/2)
Gamma: bad

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18
Q

Amyloid beta oligomers and plaques

A

damage neurons by neuroinflammation (activation of astrocytes/microglia), oxidative injury, and altered kinase/phosphatase activity (tau)

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19
Q

Neurofibrillary tangles (phosphorylated tau)

A

normal: binds and stabilizes microtubules
AD: MT depolymerization, aggregation, NFTs, neuron death

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20
Q

Neuroinflammation

A

activation of microglia and astrocytes by AB plaques leads to production of pro-inflammatory cytokines –> neuron damage + death

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21
Q

Theoretical Model for how Tau and AB converge?

A
  1. AB is produced by cleavage of APP
  2. AB forms oligomers that disrupt synapses
  3. fibrils of AB aggregate into plaques around neurons and activate microglia and release of inflammatory molecules
  4. misfolded tau aggregates into NFTs inside neurons
  5. NFTs can pass through synapses to other neurons
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22
Q

Genetics / AB clearance

A
  1. elevate AB production: APP, PSEN1, PSEN2 (gamma-secretase)
  2. cholesterol transport to neurons and AB clearance: APOE e4
  3. neuroinflammantion: TREM2, CD33
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23
Q

APOE e4

A

secreted by astrocytes, binds to AB then binds to TREM2 on microglia, phagocytosis + cleavage
- 3 isoforms: e2 is protective bc it binds AB and TREM2 with high affinity
- e4 has low affinity, bad clearance

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24
Q

How is AB cleared?

A
  1. APOE binds to AB
  2. TREM2 on microglia binds APOE/lipid complex with AB
  3. binding promotes phagocytosis and cleavage
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25
Q

CD33

A

inhibitor of microglial phagocytosis

during AD, CD33 levels increase
- AB can bind to CD33

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26
Q

What is the physiological role of AB?

A

protection against oxidation, regulation of cholesterol transport, antibiotic

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27
Q

3 therapeutic strategies for AD

A
  1. target neurotransmitters (acetylcholinesterase inhibitor drugs)
  2. target amyloid pathway
    - tau aggregation
    - secretase inhibitors
    - antibodies targeting AB
  3. target things not in the AB pathway
    - CNS inflammation
    - brain clearance with sonography
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28
Q

What is an AChe inhibitor?

A

Acetylcholinesterase (AChe) inhibitor prevents breakdown of acetylcholine
- treats symptoms, can’t reverse brain damage, but helps with cognition/behavior

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29
Q

What are tau therapies?

A
  1. OGA inhibitors to prevent NFTs
    - OGA removes sugars + OGT adds them; without sugars, Tau gets P and forms tangles
    - promote sugars to prevent P
  2. antibodies targeting tau
    - how to get them across BBB = anti-oligomers in micelles, nanobodies, intrabodies (fragments, ScFv)
  3. targeting tau for destruction
    - TTCM2 micelle enters and binds tau oligomers, binds to TRIM21, ubiquitin ligase, degredation
    - PROTAC: small molecule with protein binding ligand + linker + E3 binding ligand, ubiquitinated, degredation
  4. Tau vaccine: generate antibodies against P-tau
30
Q

Secretase inhibitors?

A

inhibit gamma-secretase to prevent APP into AB peptide
- resulted in massive CNS side effects bc gamma cuts other substrates

31
Q

Antibodies targeting AB?

A

most have failed
- crossing BBB, may induce ARIA-E, and they mostly target beta-sheet when toxic AB is the alpha sheet

32
Q

What is ARIA?

A

amyloid related imaging abnormalities
- ARIA-E: with edema; ARIA-H with hemorrhage
- amyloid in cerebral blood vessel walls

33
Q

Evidence for rapid climate change? Consequences?

A
  1. global temperature rise
  2. shrinking ice sheets
  3. glacial retreat
  4. decreased snow cover
  5. warming oceans
  6. increase in greenhouse gases

sea level rising, extreme weather events, ocean acidification

34
Q

How do we know that CO2 is from fossil fuel?

A

burning fossil fuels enriches atmosphere with C-12 over C-14
- living orgs can absorb C-14
- coal and oil made from dead organisms with C-12

35
Q

What about past CO2 spikes in Earth’s history?

A

Earth’s orbit switches from circular to elliptical every 100k years, tilt varies, and it wobbles

36
Q

What is biofuel? What are the three main modalities?

A

fuel derived from living matter
- carbon in biofuels comes from the atmosphere, captured by plants during the growing season

  1. bioethanol
  2. microalgae
  3. E.coli biofuels
37
Q

What are bioethanols?

A
  • made from corn, sugarcane, cellulose, sorghum
  • low amount of stored energy, corrosive
  • fuels that compete with food cause farmers to expand into forests
  • Cellulosic EtOH are not edible (stalks)

biodiesel: made from fats and oils

38
Q

What are microalgae? Compare the cultivation systems?

A

ex) microorganisms such as C. vulgaris

open advantages: low operating cost, easy to scale, cooling through direct contact with atmosphere, good gas interchange
open disadvantages: high evaporation rate, requires lots of land, high loss of CO2, high harvesting cost, low production performance, contamination

photobioreactors (PBR) = closed ADV: lower contamination risk, high production performance, low harvesting cost, requires low land to produce, low loss of CO2
DISADV: high operating cost, high construction cost, difficult to scale

39
Q

E.coli biofuels?

A

modify fatty acid synthesis for generation of desired product: isobutanol, isopropanol, propanol, bioethanol, biodiesel

ADV: rapid growth, genetic engineering
DISADV: products can be toxic, hard to recover

40
Q

Cyanobacteria biofuels?

A

engineered to secrete hydrocarbons of various lengths
- photosynthetic: placed near areas of high CO2 to make fuel

41
Q

What are the main challenges in agriculture?

A
  1. Food security
  2. Fertilizer (declining efficiency)
  3. Climate Change
  4. Monoculture and disease
  5. Lack of diversification and prohibitive costs for new crops
  6. Geopolitical conflicts
42
Q

What is heterosis (hybrid vigor)?

A

hybrid offspring exhibit phenotypic performance that is superior to that of their parents
- action of multiple genetic loci to complement variations in alleles / gene expression
- molecular basis not well understood

43
Q

What is transgenesis?

A

insertion of foreign gene with promoter by biolistic particle delivery = BPD (gene gun) or agrobacterium into a new organism
- into germ cell chromosome

genes can be expressed at different levels and in different cells by choosing appropriate promoter, terminator, regulatory elements

provide useful alleles not present in the gene pool, accelerate breeding programs, backcrossing
ex) large corncobs (P2) into elite agronomic background (P1)

44
Q

How does transgenesis via agrobacterium work?

A
  1. one binary vector with T-DNA containing a selectable marker (KanamycinR) and gene of interest between LB and RB
  2. non-oncogenic disarmed Ti plasmid containing bacterial virulence genes for T-DNA transferability; permanently in the agrobacterium
    - auxin and cytokinin genes removed so no tumor growth
  3. T-DNA section of Ti plasmid is inserted randomly into a plant chromosome

ex) endotoxin to kill insects, vitamin A in Golden Rice

45
Q

Mutation breeding?

A

use of chemical or physical agents to induce novel mutations in plants
- mutations are made randomly in plant genome (fast, but dirty)
- screening of mutant plants to ID plants with trait of interest
- backcross to WT plants to eliminate the detrimental mutation

46
Q

Advantages and disadvantages of mutation breeding?

A

ADV: first and easiest method, create useful variants not in gene pool, not considered GMOs

DISADV: types of mutations are limited, not precise, large number of mutations, segregate beneficial mutations

47
Q

CRISPR in plant improvement?

A

targeted mutations, specific types of mutations, inactivate gene or alter sequence to modulate protein expression or activity
- NHEJ: random INDELs
- HR: precision breeding (gene targeting)

48
Q

Multiplex CRISPR/Cas9?

A

multiple genes edited at the same time with Cas9 + multiple different gRNAs can target different genes
- used to create large deletions
- promoter bashing, exon skipping, gene skipping

49
Q

Gene targeting?

A

modifying endogenous gene using HR
- create genetic variation at any given locus in a genome
- donor template contains specific mutations and provided exogenously
- using CRISPR/Cas9

50
Q

RNA interference (RNAi)?

A

allows for silencing of mRNA target through complementary siRNAs

  1. dsRNA cleaved by dicer into siRNAs
  2. RISC binds to complementary mRNA and cleaves it
  3. degredation

ex) RNAi to make longer-lasting food, inhibits ripening hormone formation
- genome defense against viruses

51
Q

Advantages and disadvantages of RNAi?

A

Adv: induce phenotypes without altering DNA, silence any gene, no transgene insertion, non-GMO lowers regulatory costs

Disadv: issues with specificity, cost of producing dsRNAs, needs to be constantly expressed/applied unlike DNA modifications
- cannot freely diffuse into cells (large and - charge), unstable in cells (higher dose), oral administration is not possible

52
Q

Apomixis?

A

asexual, clonal reproduction through seeds from maternal side

allows for generating clones of an elite agro background through seeds

53
Q

What is an antibody?

A

secreted glyco-proteins that have very specific binding to small molecular structures (epitopes) on target antigens
- contain complementary determining region (CDR)

produced by B-cells

54
Q

Antibody structure and function?

A

Fab (fragment antigen binding): variable region that binds antigen

Fc (fragment constant): constant region that provides biological function
- neutralize, opsonize (tag), complement fixing (IS), activate effector cells, generate ROS

5 main classes by their heavy chain (Fc region)

55
Q

How do you make a monoclonal?

A

single antibody against single epitope; produced by hybridoma cells (B + myeloma)

  1. inject antigen into HGPRT+ mouse
  2. remove the spleen (B cell rich)
  3. fuse spleen cells with myeloma cells (HGPRT-)
  4. Select HGPRT+ hybridoma cells
  5. ELISA screen for hybridoma producing Ab to antigen
  6. Harvest Ab
56
Q

how to reduce monoclonal immunogenicity?

A

design module that does not evoke immune response
mouse–>chimeric–>humanized–> fully human

57
Q

What are single chain antibodies?

A

scFv: single chain variable fragments
- contains variable regions of both heavy and light chain

can penetrate tissues easier, more efficient to produce

58
Q

How do you make scFv?

A
  1. create a phage display library: phage with scFvs
  2. link VH and VL with peptide linker
  3. expose library to antigen, screen for binding
59
Q

What is an approved monoclonal antibody?

A

Herceptin: humanized monoclonal antibody to HER2
(15-25% of breast cancer)

60
Q

Variation in therapeutic mAb?

A
  1. Fc fusions: proteins fused to the Fc region of antibody to enhance effector function + stability
  2. Bispecific antibodies: binds two different antigens
  3. Nanobodies: single-domain, small size, high stability, tissue penetration
  4. Antibody-drug conjugates (ADCs): linked to cytotoxic drugs (MT destabilizers, DNA damaging agents, protein toxin)
61
Q

What is the difference between linkers in ADCs?

A

cleavable: the entire Ab, linker, and drug are internalized but the drug is released by protease
- drug can leak out of cell; more off-target killings

non-cleavable: entire Ab, linker, and drug is internalized and released after lysosome degredation
- keeps drugs inside cells, but is slow

62
Q

How does naive T cell activation work?

A
  1. Activation: T-cell receptor complex binds to either
    MHC-I with CD8
    MHC-2 with CD4
    on an antigen-presenting cell (APC)
  2. Survival: CD28 binds to B7 on the APC (Co-stimulation)
  3. Differentiation: cytokine release from APC
63
Q

What determines is a T cell is activated or inhibited?

A
  • Naive T cells express CD28 (GAS)
  • after priming in the draining lymph nodes (TCR-MHC +co-stim +/- cytokines), T cells migrate to tissue and begin effector functions
  • CD28 is downregulated after activation and CTLA-4 and PD-1 are upregulated (BRAKES)
64
Q

What is T cell exhaustion?

A

chronic TCR stimulation leads to T cell “exhaustion”
- dysfunctional and can’t clear chronic infections or cancers

65
Q

How do you develop or activate immune cells to target cancer cells?

A
  1. Immune checkpoint inhibition, CPI
  2. ACT: adoptive cell transfer
  3. CAR T: Chimeric antigen receptor T cell
66
Q

Immune checkpoints? CPI?

A

to prevent overstimulation and activation, T cells are down regulated by an inhibitory signaling pathway
- surface receptor proteins CTLA-4 and PD-1

B7 binds to CTLA-4 and PD-L1 binds to PD-1 and suppresses T cells; expressed on APCs and tumor cells

CPI: to combat exhaustion, release brakes on the T cell to reinvigorate them
- activated T cells release IFN-g which induces PD-L1 on target cells, making them more susceptible to CPI

67
Q

ACT: adoptive cell transfer?

A

cancer cells produce neoantigens by oncogenic viruses, mutations, epigenetic modifications

  1. identify neoantigens
  2. induce or expand neoantigen specific T cells and select with activation marker
  3. provide in combination checkpoint blockade
68
Q

CAR T-cells?

A

determine common antigens on specific tissues, use a virus to insert CAR gene into T-cells, and reinserted into patient

has binding domain (ScFv), transmembrane domain, signaling domain (kill)
- release cytotoxic molecules

independent from patient’s MHC

69
Q

What are the “kill” signals?

A

IFNg, TNF, perforins, granzymes

70
Q

ACT vs. CAR T cell therapies?

A

Both require engineering T cells to recognize cancer epitopes

ACT needs neoantigen and TCR that recognizes it

CAR T cells recognize common antigens expressed by cancer type (HER2)

71
Q

Drawbacks of CAR T cells?

A

target specific proteins expressed by cancer cells

limited efficacy against solid tumors

life-threatening toxicities: cytokine release syndrome and neurologic pathologies

limited persistence, expensive

Biotechnology (3 decks)

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