Comp

Question 1

Name the initiator caspases

Answer 1

Caspase-2, 8, 9, 10

Question 2

Name the effector caspases

Answer 2

Caspase-3, 6, 7

Question 3

What is the function of initiator caspases?

Answer 3

To cleave pro-caspases 3, 6, and 7

Question 4

What is the function of effector caspases?

Answer 4

To cleave cellular substrates and dismantle cells.

Question 5

What are caspases?

Answer 5

Caspases are cysteine proteases that are expressed as inactive precursor enzymes with an N-terminal prodomain followed by a two-subunit effector domain

Question 6

How is genomic DNA cleaved through caspase -3, 7 function?

Answer 6

Cleaved between histones. Caspase-activated DNase is the actual enzyme that cleaves DNA, but it’s inhibitor (iCAD) is cleaved by Caspase 3 and 7.

Question 7

What substrates are cleaved for each apoptotic morphology change?

Answer 7

Lamins, the scaffold proteins of the nuclear envelope, are cleaved by effector caspases, leading to nuclear shrinkage and fragmentation.
Loss of overall cell shape is probably caused by the cleavage of cytoskeleton proteins.
Focal adhesion protein cleavage leads to disassociation with neighbouring cells.
Membrane blebbing occurs through activation of an actin depolymerizing enzyme.
A combined effect of down-regulation of a phospholipid translocase activity and activation of a lipid scramblase, which are observed in apoptotic lymphocytes, may contribute to PS exposure

Question 8

Which caspases molecularly define apoptosis?

Answer 8

The molecular definition of apoptosis can logically be based on the proteolytic activity of certain caspases (caspase-2, -3, -6, -7, -8, -9, and -10) because these enzymes mediate the process of apoptotic cell death.

Question 9

What does autophagic cell death look like?

Answer 9

The morphological characteristics of autophagy include vacuolization, degradation of cytoplasmic contents, and slight chromatin condensation.

Question 10

Why is plasma membrane damage a good indicator of cell death?

Answer 10

The loss of structural integrity of the plasma membrane is a hallmark of necrosis and represents the common final endpoint at which a cell can no longer maintain its discrete identity from the environment

Question 11

What is the biggest difference in the outcome of cell death between pyroptosis and apoptosis?

Answer 11

Pyroptosis features rapid plasma-membrane rupture and release of proinflammatory intracellular contents. This is in marked contrast to the packaging of cellular contents and non-inflammatory phagocytic uptake of membrane-bound apoptotic bodies that characterizes apoptosis

Question 12

What are the main differences between LPS and GMCSF stimulation of moncytes?

Answer 12

Monocytes cultured in IFN-gamma progressively lost their activity by 2 days but monocytes in GM-CSF or IL-3 maintained their high level of anticandidal activity throughout the whole length of culture. Therefore, GM-CSF and IL-3 not only enhanced fresh monocyte anticandidal activity, but maintained monocyte function for a longer period. These results suggest that GM-CSF and IL-3 may act on monocytes via a different pathway than does IFN-gamma.

Question 13

Why is the phagolysosome insufficient in eliminating engulfed yeast C. alb?

Answer 13

C. alb. actively and passively alter the maturation of the phagosome. Mannans on the surface mask glucans that are more easily recognized by macrophages (mutants for O-mannans have more exposed beta-glucans). This leads to delayed Rab7 aquisition, delayed acidification, and severely impairs cathepsin B delivery to phagosomes, while actively inducing the recycling of cathepsin D out of the phagosome. Candida impairs vATPase delivery to the phagosome, but even when it can’t, once Candida senses a drop in phagosomal pH, it also secretes ammonia that alkilizes the phagosome, preventing acidification.
Hyphal formation is stimulated by this, and perforates the phagosome, as well as the macrophage itself eventually.

Question 14

For mouse study, what are the sign of illness to look for?

Answer 14

These include weight loss, ruffled coat due to reduced grooming, increased/decreased movement, abnormal posture (e.g., hunched back), and trembling.

Question 15

For mouse survival and histopath studies, what are the end points and why?

Answer 15

For survival studies, sacrifice mice when they have reached a permissible percentage loss of body weight (normally 20% to 25%) or when the mice look moribund. Record date of death as the following day. For tissue burden and histology studies, sacrifice mice at a pre-determined end point.

Question 16

What are come sites of colonization in the mouse?

Answer 16

Stomach, esophagus, and intestines are the primary sites of colonization.

Question 17

How do your results line up with what has been published about GI colonization with Candida?

Answer 17

GI colonization has been reported to persist up to 20 days after the initial gavage (10^2 to 10^5 CFU/g intestinal tissue), with levels dropping precipitously (by 3 to 4 log units) within the first 24 h.

Question 18

Do you expect persorption to occur in wild-type mice at the dose you used? When do you expect dissemination?

Answer 18

Translocation to extraintestinal organs, such as the livers, kidneys, and spleens, occurs almost uniformly within the first 6 to 72 h of infection but appears to be transient.

Question 19

Is neutropenia alone sufficient for dissemination of Candida from the gut

Answer 19

No. There needs to be both neutrophil depletion and epithelial/mucosal damage.

Question 20

What other pathogens use a Trojan horse?

Answer 20

TB, cryptococcus.

Question 21

What are other animal models used to study Candida?

Answer 21

Zebrafish - are transparent, have an adaptive immune response.
Wax moth and silk worm larva - only innate responses, used for large screens of Candida mutants.
Flies - Toll was discovered here.
C. elegans.
All are farther than mice on an evolutionary time scale.

Question 22

Name 3 mechanisms to cross the BBB

Answer 22

i) direct fungal interactions with brain endothelial cells, leading to endocytosis and subsequent transcytosis of free fungi; (ii) disruption of BBB endothelial cell junctions, allowing paracellular passage of free fungi; and (iii) “Trojan horse” crossing, where fungi traverse the BBB within infected phagocytes, either transcellularly or paracellularly.

Question 23

What are the steps in PCR? How does it amplify DNA?

Answer 23

Denaturation, primer annealing, extension. It doubles the amplicon with every cycle.

Question 24

What is rt-PCR?

Answer 24

A combination of PCR and spectrofluorometery. Measures the amount of DNA in a PCR reaction continuously. during the amplification.

Question 25

What detectors are used in qPCR?

Answer 25

Specific Detection Detective Probe - Taqman (specific to the sequence you are amplifying). DNA single-stranded probe coupled to a fluorophore at the 5’ end and a quencher at the 3’ end. Quencher keeps the 5’ end dark. The probe binds to a specific sequence, the main primers bind, and the DNA polymerase elongates the strand, it cleaves the fluorophore away from the probe as it degrades the probes (thus separating it from the quencher) via its 5’ to 3’ endonuclease activity. Fluorescence from reporter dye is directly proportional to the number of amplicons generated. Allows multiplexing in a single tube it detective probes have different colours.

Non-specific dye like Syber Green. Sensitive to dsDNA, but non-specific. So if the primers aren’t specific, you will get a false positive.

Question 26

What is RT-PCR?

Answer 26

Reverse transcription - PCR.
mRNA is extracted and RT’ed into cDNA.
RT makes mRNA into ssDNA, then a DNA polymerase makes double stranded DNA from this ssDNA = complementaryDNA.= cDNA

Question 27

What makes rt-PCR quantitative?

Answer 27

Reading of the amplification curve. As the amount of DNA is measured after every cycle of PCR. The threshold line is the most important - Ct is the cycle at which the amplification curve reaches the threshold (when the fluorescence of the sample rises above background noise). This value tells you how much DNA was in the sample to begin with - more DNA (more mRNA) means a smaller Ct.

Question 28

What does Oligo(dT) bind

Answer 28

3’ poly-A tail of mRNA transcripts. Can give a 3’ bias to the cDNAs that result (often used in combination with random hexamers)

Question 29

What are random hexamer primers?

Answer 29

Bind everywhere, amplify everything. Give a good representation of the population. (often used in combination with Oligo (dT))

Question 30

Where should you set the threshold of a qPCR amplification plot?

Answer 30

Where the precision is highest, during geometric amplification. Ideally all the curves will be parallel here. Not too low in the noise, and not too high in the plateau phase where data becomes unpredictable.