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3: Dan's Paper 3 > Extra notes > Flashcards

Extra notes Flashcards

1
Q

What are the 3 main things to do when stating the central aims of a paper?

A
  1. See what the title suggests the aim of the paper is.
  2. Give some background - what has been shown before, and what this paper will aim to show.
  3. State the initial hypothesis of the experiment.
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2
Q

What happens when scales vary from one figure to the next with similar conditions?

A

It impairs their ability to be compared and may affect reproducibility.

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3
Q

If a figure axis is logarithmic, should it go down to 0?

A

No.

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4
Q

If there is attrition in a figure, should it be explained?

A

Yes.

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5
Q

What is a general rule of saying things in a scientific paper?

A

Need evidence for it - either another paper if background/comparison, or show it directly.

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6
Q

What should be included in critical discussion?

A
  • Are they showing a big change or is it a small change?
  • Have they included an isotype control?
  • Is there a control from unstained mice which could discredit findings?
  • Did they study all parameters that they could have?
  • Have they used complementary approaches (e.g. gene knockout and antibody depletion)? This is useful when each process is flawed.
  • Did they perform a power analysis to determine the number of animals to use?
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7
Q

Give some points about how findings in a paper may be translated into the clinic.

A
  • What trials would drug need to go through?
  • Does the process you are targeting play an essential role somewhere else in the body?
  • Is the receptor structurally similar in humans and animals? Can you test the drug in an animal model or do you have to use a homologue?
  • Do you need to test it in vitro for binding to the receptor?
  • Does the drug or target need to penetrate the cell membrane?
  • Where in the body do you want the drug or vaccine to act? From there test different routes of administration.
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8
Q

How should an abstract be written?

A
  • Start with the premise, just a couple of sentences saying important background for field. What is not known and therefore why have they set out their hypothesis?
  • Not much detail on the methodology. Just saying which method they have used.
  • Split the methods and results, as examiners are looking for set sections.
  • For the results, just state the major findings. Not every single result, and maybe include some numerical results.
  • For the conclusion, just a couple of sentences. State the results of what they set out to test. Followed by another sentence placing work in the field, and whether this may lead to new treatment and the start of a clinical trial?
  • It is important not to include any limitations or critiques. Do not have a long introduction to the field, this is not the purpose of the abstract.
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9
Q

Give some information about writing a lay summary.

A
  • It can be useful to add in a big statistic if you can?
  • Write a lay summary in the 1st person.
  • Give some background to the research, and what is the impact of the paper?
  • State what the animal model was, or the specific pathogen being used.
  • Finish with the potential applications of the work.
  • Can you use an analogy or helpful term to describe the process?
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10
Q

Can the strengths/weaknesses of the paper use the main result?

A

Yes.

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11
Q

What are cell cultures?

A

Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favourable artificial environment.

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12
Q

What are primary cell cultures?

A

Primary culture refers to the stage of the culture after the cells are isolated from the host tissue.

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13
Q

What are the 2 types of cell culture systems and when are they used?

A
  1. Monolayers on an artificial substrate that allows anchorage (adherent culture, most cells).
  2. Free-floating in culture medium (suspension culture; HSCs).
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14
Q

What are the main 2 advantages of primary cultures?

A

Cells in primary culture closely resemble the parental tissue and are easier to study than whole mount tissue.

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15
Q

What are the 2 main disadvantages of primary cell cultures?

A
  1. Finite life spans due to exhaustion of substrate and nutrients.
  2. Higher risk of contamination than cell lines.
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16
Q

What are the transcription factors that are required for iPSC generation?

A

OCT4, SOX2, KLF4 or MYC, and NANOG or LIN28.

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17
Q

What are the main advantages of iPSCs?

A

Induced pluripotent stem cells are not derived from embryonic stem cells, reducing ethical concerns.

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18
Q

What are the main disadvantages of iPSCs?

A
  • The use of retroviruses to generate iPSCs is associated with cancer.
  • iPSCs often do not fully differentiate, necessitating quality assessments.
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19
Q

What are the main advantages of cell lines?

A

Cell lines offer an easy, inexpensive and stable platform that lasts a long time.

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20
Q

What are the main disadvantages of cell lines?

A
  • Cells might have mutations during sub-culturing.
  • Lack key morphological or functional features, limiting translation to humans.
  • Risk of contamination over time.
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21
Q

What is an advantage of cell fixation?

A

Fixing and permeabilizing cells locks them in place, allowing larger molecules to access the interior.

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22
Q

What is a disadvantage of cell fixation?

A

Fixed and permeabilized cells are dead, losing the ability to observe dynamic biological processes.

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23
Q

What is Phos-Tag? What is it used for?

A

The technique binds the phosphate group of proteins for detection using a biotinylated complex with streptavidin-conjugated HRP.

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24
Q

What are the main advantages of phos-tag?

A

Avoids radioactivity and doesn’t require large antibodies.

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25
Q

What are the main disadvantages of phos-tag?

A

Similar to standard Western blot.

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26
Q

How does immunohistochemistry work?

A

It uses primary antibodies to target the antigen, with a labelled secondary antibody targeting the primary antibody.

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27
Q

What are the main disadvantages of immunohistochemistry?

A

Not good for quantification, laborious, and non-specific binding can occur.

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28
Q

What should be the controls for immunohistochemistry?

A

Positive - tissues known to express the antigen.

Negative - tissues known to not express the antigen.

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29
Q

Name 3 main uses for mass spectrometry.

A

High throughput proteome-wide analysis of proteins, protein-protein interactions, and identification of PTMs.

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30
Q

How do radioimmunoassays work?

A

A known amount of Ag is labelled with a radioactive element and mixed with a known amount of Ab directed against that antigen.

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31
Q

What are the 2 main types of loxP construct? What are they used for?

A

Floxed stop codon - gene expression once Cre is added.

Floxed gene - gene knocked out once Cre is added.

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32
Q

Can genes be switched by Cre/Lox?

A

Yes - one can be excised and replaced with another.

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33
Q

What is the Tet system? Give some background.

A

The Tet system is a conditional gene expression system where transcription is turned on or off in the presence of tetracycline or doxycycline.

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34
Q

What is an advantage and disadvantage of tetracycline promoter systems?

A

It affords very tight control over expression compared to other systems. However, it is not as rapid as other gene expression systems.

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35
Q

What can knock-in mice be used for?

A

Human disease recapitulation, compound efficacy assessment, promoter activity study, and gene replacement.

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36
Q

What is a disadvantage of knock-in mice?

A

Mouse physiology is not identical to that of humans.

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37
Q

What are dominant negative constructs?

A

These are mutated versions of a target protein that inhibit the target’s function.

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38
Q

What is the main disadvantage of dominant negative constructs?

A

They might have their own off-target effects.

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39
Q

How do siRNA knockdowns work?

A

Small double-stranded interfering RNAs (siRNAs) are introduced into the cytoplasm and processed by the RISC complex.

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40
Q

What is the main advantage of siRNA knockdown?

A

It more closely mimics normal physiology compared to knockout.

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41
Q

What are the main disadvantages of siRNA knockdowns?

A

Transient effect and cells may not be fully transfectable.

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42
Q

What is the control for siRNA knockdown studies?

A

This is often in the form of the administration of a scrambled siRNA sequence.

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43
Q

How do optogenetic techniques work?

A

Cells genetically engineered to express a light-sensitive opsin are illuminated with light of the correct frequency.

44
Q

What are the advantages of optogenetic techniques?

A

Temporal resolution on the scale of milliseconds and precise linking of neuron activation to behaviours.

45
Q

What is the main disadvantage of optogenetic techniques? How can this be mitigated?

A

Certain level of invasiveness, mitigated by using light.

46
Q

What happens to opsin-bound retinal at the correct frequency?

A

Opsin-bound retinal undergoes a conformational change, leading to channel opening or pump activation, resulting in cell depolarization or hyperpolarization, and neural activation or silencing.

47
Q

What are the advantages of optogenetic techniques?

A

Temporal resolution on the scale of milliseconds; can be turned on and off; precisely linking a neuron’s activation to behaviors.

48
Q

What is the main disadvantage of optogenetic techniques? How can this be mitigated?

A

Certain level of invasiveness, mitigated by using light permeable organisms such as zebrafish.

49
Q

What is transfection?

A

The artificial introduction of material such as DNA, RNA, or proteins into cells using viral or non-viral methods, resulting in transient or sustained expression of a protein of interest/activity of a molecule within a cell.

50
Q

What are the 2 main viral methods of transfection? How do these work?

A

AAV - DNA exists as episomes, no genome incorporation; expression of the gene is thought to last for a few years.

Lentivirus enables genome incorporation in a stochastic
manner, leading to sustained expression as long as the cell lives.

51
Q

What are the advantages of transfections?

A

Infects a variety of cells; doesn’t affect cellular function with minimal immunogenicity; induces rapid expression compatible with multiple transcriptional promoters.

52
Q

What are the disadvantages of transfections?

A

Non-specific insertion of transgene can occur; if the gene is incorporated under a weak promoter, it may not be expressed; the gene won’t be subject to the same regulation as if it were expressed at the natural locus; risk of insertional mutagenesis.

53
Q

What are the main non-viral methods of transfection?

A

Electroporation, microinjection, and lipofection.

54
Q

What are the controls in transfection studies?

A

Always need to use a non-transfected control and may need to use a positive control to show transfection success.

55
Q

Give some background about GWAS studies.

A

A genome-wide association study (GWAS) is a research approach used to identify genomic variants that are statistically associated with a risk for a disease or a particular trait.

56
Q

What are some disadvantages of GWAS studies?

A

Critics argue that GWAS will eventually implicate the entire genome in disease predisposition; most association signals reflect variants and genes with no direct biological relevance to disease.

57
Q

What are some advantages of GWAS studies?

A

Identify novel biological mechanisms; diverse array of clinical applications; sensitive to low-frequency variants; GWAS data can be easily shared.

58
Q

What are next generation sequencing techniques based on?

A

Next-generation sequencing approaches are derived from the initial sequencing approaches by Sanger in the 1970s using ddNTPs.

59
Q

What are the 2 major second generation sequencing methods?

A

Sequencing by hybridization and sequencing by synthesis (SBS).

60
Q

Give some information about SBS methods.

A

SBS methods are a further development of Sanger sequencing, without the dideoxy terminators.

Individual DNA molecules are distributed to millions of separate wells or tethered to specific locations on a solid substrate.

61
Q

What are the advantages of next generation sequencing?

A

Higher sensitivity to detect low-frequency variants; faster turnaround time for high sample volumes; comprehensive genomic coverage.

62
Q

What are the disadvantages of next generation sequencing?

A

Limited ability to accurately identify variants occurring in regions with high sequence identity to other regions of the genome.

63
Q

What is fluorescence in situ hybridisation (FISH)? How does it work?

A

FISH is a cytogenetic technique that uses fluorescent DNA probes to target specific chromosomal locations within the nucleus, resulting in coloured signals detectable using a fluorescent microscope.

64
Q

What is an advantage of FISH?

A

FISH has become increasingly important in clinical diagnosis due to its simplicity and reliability in evaluating key biomarkers in various tumours.

65
Q

What is a disadvantage of FISH?

A

FISH probes are less sensitive for detecting unbalanced translocations, and adjusting cut-offs for various FISH probes can be technically challenging.

66
Q

How do microarrays work?

A

Microarray involves a microscope slide printed with spots containing known DNA sequences. It detects expression of thousands of genes simultaneously.

67
Q

What can microarrays achieve?

A

Information generated can be used to create gene expression profiles, showing changes in expression of genes in response to a condition.

68
Q

What is the depth of sequencing in RNA seq?

A

The depth is defined as the total number of reads obtained from a high-throughput sequencing run, indicating how many times each reference base is sequenced.

69
Q

What is read length? What do longer reads mean?

A

Read length is determined by the average length of the reads sequenced. Longer reads provide more certainty that a gene is marked correctly.

70
Q

What is the main disadvantage of qPCR?

A

It relies on the use of reference genes such as GAPDH, which have problems, such as inconsistent expression.

71
Q

How does dye-based qPCR work?

A

cDNA template is denatured, dye binds to dsDNA, fluorescent signals are detected, and fluorescence increases with DNA synthesis.

72
Q

What is a non-luciferase reporter? How does it work?

A

The E. coli LacZ operon produces a blue product when cleaved by the Beta-galactosidase enzyme.

73
Q

What is the difference between transmission electron microscopy and scanning electron microscopy?

A

TEM - electrons pass through the sample to image thin sections; SEM - electrons excite the sample, causing electron emission to assess 3D structure.

74
Q

What are the main disadvantages of electron microscopes?

A

Cannot image live tissue; cannot image colour; costly; prone to artefacts.

75
Q

What is the basic premise of confocal microscopy?

A

The key is using spatial filtering techniques to eliminate out-of-focus light in specimens whose thickness exceeds the immediate plane of focus.

76
Q

What are the advantages of confocal microscopy?

A

Allows for rapid, non-invasive, and high resolution imagery; can produce 3D images if a Z stack is taken.

77
Q

What are the advantages of light microscopy?

A

Extremely versatile; can image a wide variety of specimens with minimal preparation; provides information on physical, chemical, and biological attributes.

78
Q

What is the main disadvantage of light microscopy?

A

The principal limitation is its resolving power; using an objective of NA 1.4, the resolution limit is approximately 0.2 μm.

79
Q

How do confocal microscopes work?

A

Light is emitted by the laser system through a pinhole aperture, reflected onto the sample, and focused at the detector pinhole aperture.

80
Q

How does fluorescence recovery after photobleaching (FRAP) work?

A

An area of fluorescently tagged molecules is rapidly photobleached, and the exchange of bleached molecules with unbleached molecules is followed over time.

81
Q

How does fluorescence loss in photobleaching (FLIP) work?

A

In FLIP experiments, a region in the specimen is bleached repetitively, preventing recovery of fluorescence in that region.

82
Q

What is the main advantage of FRAP?

A

FRAP is a simple and fast tool to study molecular diffusion coefficients.

83
Q

What are the disadvantages of FRAP?

A

Challenges include tracking a certain ROI due to cell movement and 3D nature of samples leading to more complex bleaching patterns.

84
Q

How does cryo-electron microscopy work?

A

Cryo-EM cools samples to cryogenic temperatures to preserve native structure, allowing visualization in 3D at various resolutions.

85
Q

What are the advantages of cryo-EM?

A

Eliminates the need for crystals; resolves concerns around purity and heterogeneity; allows investigation of protein function inside the cell.

86
Q

What are the disadvantages of cryo-EM?

A

Low signal-to-noise ratio or poor image contrast is a main challenge, difficult to avoid due to sample sensitivity to radiation.

87
Q

What are the advantages of FRET?

A

FRET is relatively cheap to implement, and measurements at very small distances can be obtained rapidly.

88
Q

What are the disadvantages of FRET?

A

Only works if fluorophores are in the correct orientation; size of the probes can become a problem.

89
Q

How does patch clamp work?

A

Manual electrophysiology with glass microelectrodes that press against the cell membrane, creating a giga-ohm seal resistance.

90
Q

How does voltage clamp work?

A

Uses a feedback circuit to set the membrane potential to a desired value by injecting current opposite to the current flowing through ion channels.

91
Q

What are the advantages of clamp techniques?

A

Allow for understanding of various ionic currents underlying the action potential in detail; provide better electrical access to the inside of a cell.

92
Q

What are the disadvantages of clamp methods?

A

Intracellular contents may be dialyzed by a large volume of the pipette solution after 10 minutes of recording.

93
Q

What is the main disadvantage of patch clamp?

A

The experimental procedures involved in patch-clamping are complicated and time-consuming.

94
Q

What are the advantages of patch clamp?

A

One of the main advantages of patch-clamp is the quality of data it produces, and the multiple different configurations that can be used to generate different forms of data.

95
Q

What are inside-out and outside-out patch clamps?

A

Inside-out = looking at the intracellular surface of ion channels

Outside-out = looking at the extracellular surface of ion channels.

96
Q

What is the disadvantage of lineage tracing?

A

If the reporter or Cre interferes with cellular physiological processes this may cause trans-differentiation.

97
Q

What is the advantage of lineage tracing?

A

The usefulness of this technique is that the reporter is expressed no matter what cell type the cell becomes. This is useful for spotting trans-differentiation reactions.

98
Q

How do MRIs work?

A

MRIs employ powerful magnets which produce a strong magnetic field that forces protons in the body to align with that field.

When a radiofrequency current is then pulsed through the patient, the protons are stimulated, and spin out of equilibrium, straining against the pull of the magnetic field.

When the radiofrequency field is turned off, the MRI sensors are able to detect the energy released as the protons realign with the magnetic field.

The time it takes for the protons to realign with the magnetic field, as well as the amount of energy released, changes depending on the environment and the chemical nature of the molecules.

99
Q

Give a bit of detail about T1 and T2.

A

The timing of radiofrequency pulse sequences used to make T1 images results in images which highlight fat tissue within the body.

The timing of radiofrequency pulse sequences used to make T2 images results in images which highlight fat and water within the body.

100
Q

What are the main advantages of MRIs?

A

MRI is a non-invasive procedure and does not involve radiation, and offers a high level of resolution.

101
Q

What are the main disadvantages of MRIs?

A

MRI machines are very expensive, and require participants to remain very still. This is particularly difficult when studying mice models and children.

102
Q

What are the disadvantages of PET imaging?

A

PET involves radioactivity, and there are only a limited number of tracers available to study different processes.

103
Q

What are the advantages of PET imaging?

A

The procedure is non-invasive and can be performed easily to study metabolic function.

104
Q

What are the disadvantages of clamp methods?

A

The disadvantage is that intracellular contents may be dialyzed by a large volume of the pipette solution after 10 minutes of recording.

105
Q

What is the main disadvantage of patch clamp?

A

The experimental procedures involved in patch-clamping are complicated and time consuming.

3: Dan's Paper 3 (10 decks)

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