TS7: Challenges for the biosciences

Question 1

What is synthetic biology?

Answer 1

Uses nature’s solutions, deciphers these solutions, and re-purposes them for our own advantage.

Question 2

What is a toggle switch in synthetic biology?

Answer 2

A synthetic, bistable gene-regulatory network that allows for gene expression to be turned on or off depending on the conditions applied.

Question 3

Describe the process of a modern SynBio development.

Answer 3

1. Design: AI/CAD tools and extensive sequence databases to find parts.
2. Build: creating libraries rather than single constructs.
3. Test: high precision single-cell analysis in bioreactors/robotics.
4. Learn: support from algorithms with designs that learn on their own with directed evolution.

Question 4

Why aren’t we good at synthetic biology?

Answer 4

• Complexity of cells
• Can’t predict intracellular responses
• Won’t work in all systems e.g., bacteria to human
• Cells have limited resources
• Cells have multiple interacting processes
• Changing behaviours can destabilize circuits

Question 5

How can we overcome the issues facing synthetic biology?

Answer 5

Cell variability –> Feedback control: nature uses feedback control to deal with changing and complex environments without falling apart.

Combine the high-level programming of computers with the best parts of biological components.

Question 6

What is steered evolution in synthetic biology?

Answer 6

Using technology to observe and optimize mutations and selection pressures to achieve the best possible solution, rather than an intermediate solution.

Question 7

What’s the difference between vaccines and anti-malarial drugs?

Answer 7

Vaccines act via the immune system’s immunological memory. Anti-malarial drugs kill the parasite itself.

Question 8

What is the difference between whole-organism and subunit vaccine approaches?

Answer 8

Whole-organism is the traditional approach where the whole organism is inactivated for vaccine use.

Subunit vaccine strategies identify a target from the pathogen and administer is with some signal to activate the immune system.

Question 9

What is a human challenge study?

Answer 9

A clinical trial for a vaccine where healthy volunteers are vaccinated and then infected. The parasites are monitored by qPCR in the hopes that with a vaccine administered, there will be a reduction or elimination of the parasite.

Question 10

What are transmission-blocking vaccines?

Answer 10

Type of vaccine that blocks the ability of diseases to infect others, rather than protecting the individual who receives the vaccine.

They work by stimulating the immune system to produce antibodies that specifically target the disease-causing organism and will enter the blood meal of the vector insect to bind the pathogen within the vector’s gut.

Question 11

Describe the standard membrane feeding assay.

Answer 11

Technique to evaluate the efficacy of transmission-blocking vaccines.

Infected vectors are artificially fed a blood sample containing the TBV. At the end of an incubation period, the vectors are dissected and the number of parasites in the gut is quantified.

Question 12

What are anti-sporozoite vaccines?

Answer 12

Vaccines that target the sporozoite stage of the malaria parasite’s life cycle.

Sporozoites are the form of malaria that are injected into the human bloodstream by the bite of an infected mosquito. Anti-sporozoite vaccines work by stimulating the immune system to produce antibodies that specifically target the sporozoites, preventing them from infecting liver cells and developing into the next stage of the malaria parasite’s life cycle.

Question 13

What’s the difference between the RTS,S and R21 vaccine?

Answer 13

Both target the antigen on the surface of liver-invasive sporozoites, but R21 has more of the antigens present on its surface which gives it a much higher efficacy.

Question 14

What is blood-stage immunity and why is it a new focus for malaria vaccines?

Answer 14

The immune response that targets the malaria parasite during the blood stage of the infection, when the parasite is multiplying within red blood cells.

It’s being used in combination with sporozoite immunity, because every sporozoite that slips through will lead to blood-stage infection.

Question 15

What are anti-merozoite vaccines?

Answer 15

Merozoites are the form of the malaria parasite that are released from infected red blood cells and then invade other red blood cells.

Anti-merozoite vaccines stimulate the production of antibodies to prevent merozoites from invading red blood cells.

Question 16

What are the 3 key challenges in developing blood-stage malaria vaccines?

Answer 16

1. Antigenic polymorphism and redundant invasion pathways - need to identify better antigens for targeting.
2. Need to learn more about the immunology.
3. Need to induce an extremely high antibody concentration to protect, and to maintain this for a useful duration of immunity.

Question 17

Why was the discovery of RH5 so fundamental in malaria vaccine development?

Answer 17

RH5 is the first conserved target within the blood-stage merozoite to be susceptible to vaccine-induced antibodies.

As it’s conserved, it makes it like the Achilles’ heel of malaria.

Question 18

How has SpyTag technology been used in the development of malaria vaccines?

Answer 18

RH5 is a conserved target in blood-stage merozoites, and it was found that antibodies capable of killing the parasite do so most successfully when they block RH5’s ability to bind its receptor.

Antibodies can be created to block this binding site (called the tip), but inducing this via a vaccine was proving difficult.

SpyTag technology was used to decorate a hepatitis B vaccine with the tip protein instead. This saw a 10-fold improvement in antibody potency.

Question 19

How has rapid progress been made with next-generation blood-stage vaccines?

Answer 19

• Identifying essential and conserved target antigens (e.g., RH5)
• Identifying assays that predict protective outcome and thereby enable rational protein vaccine design improvements.
• Optimizing design and delivery of protein/adjuvant vaccines to maximize quantity, quality and longevity of vaccine-induced antibody responses.

Question 20

Define biological ageing.

Answer 20

The progressive loss of bodily function over time that increases the probability of death.

Question 21

What causes Progeroid Werner syndrome?

Answer 21

Mutations in DNA repair, recombination and replication cause cells to undergo premature cell senescence.

This results in patients with gene expression patterns similar to those of an elderly person.

Question 22

What is SIRT6 and how is it being targeted for age-related diseases?

Answer 22

SIRT6 is a protein that has been shown to regulate the ageing process by promoting genomic stability and suppressing cellular senescence.

• Small molecule activators of SIRT6
• Gene therapy to increase SIRT6 expression

Question 23

How are senescent cells a driver of ageing pathology?

Answer 23

• Secrete collagenase, making invasion of blood vessels by cancer much easier
• Fail to repair DNA damage so tissue cannot be replaced

Question 24

What are the therapeutic approaches to tackle cell senescence?

Answer 24

• Avoid telomere shortening
• Replace stem cells
• Reverse modifications leading to senescence to make them behave like ‘young’ cells
• Suppress inflammatory molecules
• Kill senescent cells

Question 25

Why might we not want to kill off all senescent cells?

Answer 25

The % of senescent cells increases dramatically as we age, and we don’t know how much of these cells make up, for example, an 80 year old brain. This could make matters worse rather than better.

Question 26

How do methylation patterns (the methylome) change with age?

Answer 26

One common pattern is that methylation tends to increase with age at CpG dinucleotides in gene promoter regions which can result in decreased gene expression.

Question 27

How is mTOR impacted as cells age? What does this mean for targeting senescent cells?

Answer 27

As cells age, mTOR is upregulated.

Drugging senescent cells with rapamycin makes them look younger and increases proliferative capacity.

Question 28

How do senescent cells affect ability for a person to become infected by a virus?

Answer 28

Senescent cells are more vulnerable to viral infection.

They also secrete inflammatory molecules that contributes to low-grade chronic inflammation. This can create a ‘primed’ immune system that’s more likely to overreact in response to a pathogen, leading to development of a cytokine storm.

Question 29

How are the following drugs being used to treat ageing and cell senescence:
- Senolytics
- Senomodifiers
- Autophagy inducers
- Immune enhancers

Answer 29

Senolytics: eliminate senescent cells from the body.

Senomodifiers: alter the behaviour of cells to reduce the harmful effects on senescence-inducing signals.

Autophagy inducers: stimulate autophagy, which declines with age, in the hopes of removing harmful substances associated with ageing.

Immune enhancers: improve the function of the immune system.

Question 30

How is cerebral blood flow regulation associated with Alzheimer disease?

Answer 30

Cerebral blood flow is regulated by various mechanisms, including neurovascular coupling. This is the mechanism by which neural activity triggers the dilation of blood vessels and increases CBF to supply oxygen and glucose to the active brain regions.

The disruption of NVC in AD is caused by several factors, including the accumulation of amyloid beta plaques and neurofibrillary tangles in the brain, leading to inflammation and oxidative stress.

Question 31

Define electromicrobiology.

Answer 31

The study of microbial electron exchange with external electronic devices.

Question 32

What are microbial fuel cells?

Answer 32

Devices that have microorganisms on the anode, donating electrons, which are then passed to a cathode. This occurs in two half-cells, separated by a semi-permeable membrane for the movement of hydrogen ions.

These devices harvest electricity from organic matter.

Question 33

What is feedstock?

Answer 33

The organic material that’s used as a source of fuel for the microorganisms that generate electricity.

Question 34

What is the main challenge for microbial fuel cells, and how is this overcome?

Answer 34

Getting electrons from the bacteria to the anode.

1. Mediator to shuttle the electrons.
2. Direct contact e.g., Geobacter nanowires.

Question 35

Why are Geobacter so useful for microbial fuel cells?

Answer 35

They have external electron transfer as part of their normal lifestyle, putting electrons out into the environment rather than using final electron acceptors.

Question 36

Describe Geobacter nanowires. What are they made from? How do they conduct electrons? How do they overcome electron transfer in biofilms?

Answer 36

Type IV pilli composed of PilA monomeric subunits.

Proposed to transfer electrons by a metallic-like electron transfer mechanism, thanks to their stacked aromatic residues.

Pilli interact with one another, so these electrons could be transferred across vast distances. This allows for a greater density of bacteria to be compacted around the fuel cell because they can still produce the electrons and export them outside of the biofilm.

Question 37

What are the problems with microbial fuel cells?

Answer 37

• Difficult to scale up.
• Produce negligible amounts of energy compared to what we require.
• Still need a suitable feedstock that has to come from somewhere…
• Waste? (Perhaps not as important as they’re metabolizing the sugar as we humans would)

Question 38

How are specific Geobacter that live with methanogens being used for microbial fuel cells?

Answer 38

These Geobacter can take electrons from the environment and feed them into an ETC on the cathode.

Question 39

What are the pros and cons of anaerobic digestion of waste?

Answer 39

+ produces methane gas that can be burnt to produce electricity
+ produces solid waste that’s high in nitrogen-based compounds so can be used as fertilizer

• Slow process so requires batch processing
• Need to be cutting back on food waste as well
• Contents of food waste differs

Question 40

How are algae factories being used? What’s the main con to these?

Answer 40

In these factories, the microorganisms are genetically modified to produce the desired compound, which can range from biofuels and pharmaceuticals to bioplastics and other materials. They are powered by renewable energy sources, such as sunlight, making them more sustainable than traditional manufacturing processes. Using photosynthesis, algae remove carbon from the atmosphere, converting carbon dioxide into biomass.

• Requires land mass that could be occupied by other uses e.g., making food.

Question 41

How are tropical plants being studied to try to improve efficiency of RuBisCo in photosynthesis?

Answer 41

Some tropical plants fix carbon by fixing it into malate which is then moved into another cell and converted back into pyruvate, releasing carbon dioxide (C4 metabolism). The net effect is that the second cell has a much higher concentration of carbon dioxide than normal that can be used to overcome the oxygenase reaction.