ARTICLE DISCUSSION

More detailed look at the article

THE INTRODUCTION
BACKGROUND INFORMATION
➤ Information was provided in the introduction
to provide background related to the study
➤ In the form of a short literature review -
citing previous studies from the authors’ lab
and also other labs
➤ This helps put the study in context of what is
already known
RESEARCH QUESTIONS?
➤ Important to then state the research questions
or aims of the study
➤ What was/were the key ideas the authors
wanted to test?
➤ Was there something new they wanted to
study?
THE KEY APPROACHES
COMBINATION OF VARIOUS TECHNIQUES USED
➤ Flow of experiments important and this is dependent on the
techniques used
➤ Combination of
➤ Genetic techniques
➤ Microbiological
➤ Biochemical
➤ Microscopic
➤ …
➤ Step-wise progression from a set of ndings to the next
➤ Building answers to the research question
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 Take me to a
The GENETIC approach illustrated brie y: football match,
please.
Say you are a Martian who has just arrived on Earth.
You watch a football match and became intrigued as to the role of this chap in
black attire running around the eld blowing at his whistle now and then. He does
not seem to be kicking the ball but then stops the game by blowing his whistle.
You wondered what is the role of the chap.
You have some inkling based on what you see as stopping the game when one
player trips another one.
Say you hypothesised that the chap might have a role in regulating the game.
So how would you test this hypothesis?
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 Let me take away
The genetic approach illustrated: the chap with the
whistle!
So how would you test this hypothesis?
Remove the chap in question from the game and see what happens.
i.e. game descends into chaos etc.
Then you put him back.
Order is restored in the game (hopefully)!

So you tested and con rmed your hypothesis by “deleting” the referee from the game and seeing the effects of the
deletion - the de ciencies.
From this, you deduce that normally in the “wild-type” situation, the referee maintains the rules of fair play.
Likewise for the study of a gene function - what process goes wrong when it is not present, so its normal function is to
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 Arh, they need a
referee to maintain
order in the game!

So you tested and con rmed your hypothesis by “deleting” the referee from the game and seeing
the effects of the deletion - the de ciencies.
From this, you deduce that normally in the “wild-type” situation, the referee maintains the rules
of fair play.
Likewise for the study of a gene function - we look at what process goes wrong when the gene is
not present.
Then we deduce its normal function is to maintain that particular process.
Of course this works well if the gene is solely responsible for that process.
- In instances when deleting one gene results in no effects, what can you conclude?
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CULTURE TECHNIQUES
➤ Yeast culture
➤ Pathogen of interest
➤ Microbiological methods
➤ Liquid medium
➤ Solid medium
➤ Cell culture
➤ Host cells - phagocytes
➤ Mammalian cell culture
➤ Co-culture
➤ Host-pathogen interactions
These have been highlighted in the videos
ASSAY METHODS
➤ pH measurements
➤ Ammonia measurements
➤ Survival assay
➤ Microphage cytotoxicity
➤ Lysotracker red
➤ Morphological examinations

These have been highlighted in the videos

THE M&M SECTION
PROTOCOLS
➤ The M&M section provides the protocols to execute the
various techniques
➤ As mentioned brie y, the protocols for speci c technique is
not the same as the experimental design needed to answer a
particular question …
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RESULTS
Fig 1A - signi cance of this rst experiment

Was this experiment needed at all?

The wild-type strain was used in two

different media.

Why bother with this experiment?

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N-acetylglucosamine acts as a control — proves that GlucNAC, similar to amino acids, allows for the neutralisation of yeast pH
Fig 1A - signi cance of this rst experiment

Was this experiment needed at all?

The wild-type strain was used in two different

media.

Why bother with this experiment?

(A) The y-axis is pH of the culture media against the

x-axis that is time in hours.

- Why was measuring the pH in the media part of the

study?
GlucoNAC neutralises pH at a faster rate, but
based on the endpoint, amino acids are slightly
- consider the media as the external environment of
more effective at neutralising the yeast cells and how this relates to the interior of
the phagolysosome

- What can you say about the end-points of the pH in

the two different media?
- What about the pro les of the pH over time?
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FIGURE 1
➤ (B) The y-axis is NH3 ppm against the
x-axis that is time in hours.
➤ What did the data imply about the
relationship between culture media and
pH - eg compare (A) and (B)?
➤ Was the relationship the same or di erent
when di erent medium was used?
➤ Is this important for the study? Recall the
research question raised by the authors

➤ Based on the data in gure 1 taken

together, what statement can you make?
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- Release of NH3 correlates to the change in pH observed in different media
- The increase in pH in GlucNAC correlates with a smaller amount of ammonia
FIGURE 2 (A) AND (B)
➤ How did the data in gure 1 lead the authors to the experiment in gure 2?
➤ They wanted to understand the mechanism behind the NH3 production
➤ Whether NH3 production when grown on GlcNAc is using the same
pathway components as the NH3 production when catabolising amino
acids
Two possibilities on ammonia production in amino acids and GlcNAc, which is why there are FOUR strains (including wild-
type) that are tested in figure 2

Amino acids GlcNAc Amino acids GlcNAc

Gene A Gene X
Gene A
Gene B Gene Y
Gene B
Ammonia production Ammonia production
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 FIGURE 2 (A) AND (B)
➤ Using the genetic approach to test if
the pathways converge
➤ Why were these speci c
mutants included here?
➤ This required understanding
of the background
➤ Normally for scientists in the
eld, they would understand
➤ For others, we can read the
background…
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 Some background
Stp2p, a transcription factor
❖

that activates amino acid

amino acid uptake
permeases - these are needed
to uptake amino acids by C
albicans
Stp2p
❖ Stp2p, a transcription factor
that activates ATO1 and
ATO5 gene expression
amino acid C albicans

metabolism
❖ Ato1p, Ato5p - these likely
function to transport NH3
out of C albicans
❖ note NH3 is produced
upon amino acid
metabolism NH3
SOME BACKGROUND…
➤ No need details
➤ But be able to understand the
various gene products and how
they are functioning in the
GlcNAc pathway -> relevance for
the study
➤ The catabolism of GlcNAc
produces ammonia
➤ hxk1Δ nag1Δdac1Δ (referred to Figure 1. N-acetlyglucosamine (GlcNAc) catabolism in Candida
albicans. Ngt1, GlcNAc-speci c transporter; Hxk1, GlcNAc
as the “h-d mutant”) lacks the kinase; Dac1, GlcNAc-6-phosphate deacetylase; Nag1,
Glucosamine-6-phosphate deaminase; Ngs1, N-acetyltransferase;
three enzymes responsible for Rep1, transcription factor involved in transcription of GlcNAc-
inducible genes. GlcNAc is transported into the cytoplasm by the Ngt1
the conversion of GlcNAc to transporter and is catabolized to fructose-6-phosphate by Hxk1, Dac1,
and Nag1. Ngs1 can bind to GlcNAc and may function as a putative
fructose-6-phosphate, an sensor. Together with the transcription factor, Rep1, Ngs1 induces the
expression of GlcNAc-catabolic genes in the presence of GlcNAc.
intermediate of glycolysis,

https://www.mdpi.com/2309-608X/6/3/129
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FIGURE 2 (A) AND (B)
➤ Using the genetic approach to test
if the pathways converge
➤ What can you conclude from
the gures with respect to the
gene functions in
contributing to pH change in
➤ GlcNAc medium?
➤ Amino acids medium?
- SC5314 and stp2 are NOT implicated in pH neutralisation for GlcNAc
- h-d and ngt components ARE implicated in pH neutralisation for GlcNAc
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FIGURE 2
➤ (C) and (D)
➤ Same strains used but now for
measuring ammonia

➤ What can you say from the data

shown in Figure 2 taken together?
i.e. Can you see any correlation across the figures (A) to (D)?
 FIGURE 3
➤ (A) and (B)
➤ What were the authors measuring?
➤ What is the point made here that is di erent from that
in Figure 2?
➤ Hint: compare the mutants used in Figure 2 with
Figure 3
➤ The genes are mentioned in the previous slide on
STP2

➤ Does this gure add on to the data from the previous

gures?
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FIGURE 4
➤ This shows experiments where there was co-culture
of the pathogens with the macrophages.
➤ Why is this a relevant experiment?
➤ (A) How did the authors measure C albicans survival
here?
➤ What do the data imply about the di erent C albicans
strains when they are phagocytosed by macrophages?
➤ Recall that the study involved experiments
examining the e ects of the phagolysosomes on
the pathogen and also vice versa
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FIGURE 4
➤ (B) How did the authors measure macrophage
killing?
➤ What do the data imply about the killing of the
macrophages by the di erent C albicans strains?
➤ Do you see any correlation between the
data in (A) and (B)?
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 FIGURE 5
➤ (A) Why was it important to
study the morphological Example of yeast Example of hyphae

changes in the yeast?

 Hyphae formation
❖ Generally, hyphal switch can occur under different
conditions
❖ Correlated with pathogenecity of the yeast
❖ Physical structure of the hyphae plays a role in
invasion or perhaps escape from phagocytes
❖ Note also the hyphae formation occurs in
neutral pH
 FIGURE 5
➤ (A)What did hyphae length
correspond to? Example of yeast Example of hyphae

➤ Recall the mutants were

defective in NH3 production
➤ How does this relate to
phagosomes or the original
research question?
FIGURE 5
➤ What techniques are used in 5(B) and 5(C)?
➤ What do the red regions represent? What about the green stainings?
➤ What are the authors trying to show in 5(C)?
FIGURE 5

➤ 5(B) is the quantitation of the data

➤ Do you know what kind of a plot is this?
➤ How do you interpret the data?
➤ What were the authors trying to determine
here?
Example data from another article

Box-plot
 LR assay measurements
macrophage macrophage

yeast

?
FIGURE 5
➤ What are the authors trying to show in 5(C)?
A FEW THINGS TO THINK
ABOUT…
So the article is published, it must be perfect
❖ When we read a research article, are we expecting that
the authors to report a complete, exhaustive study on a
particular topic?
❖ Are the data presented perfect?
❖ So is there anything for readers to critique?
Evaluating different aspects of an article
❖ The premise of the study
❖ Overall design of the study
❖ Methods
Different aspects of the study that you
❖ The quality of the data could comment on, with justifications

❖ Interpretation of data
❖ Conclusions
❖ …?
 Evaluating Design
❖ Overall approach to the study
❖ e.g. Understanding how some pathogens
might escape the innate system due to
functions of speci c genes
❖ what was the main approach?
Is this a reasonable
❖ using cell-line from the innate system design for a short article
that we are reading?
❖ using a fungal pathogen and deletion
strains of it If so, why?
❖ assaying different reciprocal effects of If not, what are other
macrophage-pathogen interactions aspects?

❖ animal infection model

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 Evaluating Methods
❖ Validity and reliability of techniques used to measure
variables of interest
❖ Do we know what the terms mean?
❖ Validity - the tool used is measuring what is intended
to be measured

❖ Reliability - the measurements made using a tool are

reproducible by each experimenter and between
experimenters
 Validity and reliability
❖ The question of validity and reliability.
❖ Is lysotracker a valid indicator of phagosome pH?
❖ i.e. does it measure what it should measure
(pH)?
❖ Here, the accumulation of lysotracker is yeast
correlated with pH, the lower the pH, the
higher the accumulation.
❖ That is the nature of the lysotracker dye.
❖ So there is correlation and one could use it as a
measure of pH. It might not be the only way or
the best way but it seems acceptable in this 1 2
instance (live cells, co-localisation with live
pathogen).
 Validity and reliability
❖ But is lysotracker a reliable method to determine
phagosome pH?
❖ The intensity measurement is done using a
software by drawing a line through the area of
interest. The software will compute the intensity
under the line and give some mean value. yeast

❖ But the experimenter has to decide where to

draw a line through the lysotracker stained
region.
❖ While lysotracker itself might be a reliable
indicator of pH, the execution of the process
might not be. 1 2
❖ So will we always get a reliable reading
between two investigators?
 Validity and reliability
❖ Say, if you (line 2) and your classmate (line 1)
drew the line differently?
❖ Of course one can take 3 measurements and take
mean but there could still be some differences
between experimenters - this means reliability
could be an issue between experimenters as there yeast
is subjectivity involved.
❖ So is this a reliable method to determine the level
of LR staining?

1 2
 Validity and reliability
❖ Look at the range of values
❖ the range could mean a few things
❖ one of which is could be the
drawing of a line through the cells.
 Validity and reliability
❖ The question of validity and reliability.
❖ What about the measurement of LDH as an indicator of macrophage lysis?
❖ Is this valid? Is it reliable?
❖ LDH - this is released when cells lysed. Intact cells will not release LDH.
❖ This has previously been established.
❖ So does LDH release provide us a way to distinguish between lysed and intact cells?
❖ Can the same or different experimenter get readings from similar experiments within a
good range of variations?
❖ What would be an alternative? Look at the cells and count lysed vs intact using the microscope?
That would be direct but not reliable - might be dif cult to judge correctly just with simple light
microscope if cells lysed or not.
❖ Hence the use of something that could be quanti ed to be more objective.
❖ Of course no one method is perfect and researchers are always trying to come up with better
research methods.
❖ One could also use different methods eg count lysed cells and make LDH measurements
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Specificity and Sensitivity
❖ What about the issue of speci city and
sensitivity?
❖ pH electrode - speci c for pH but pH change
could be due to different reasons; sensitivity
could be good, depending on the electrode etc

❖ NH3 ppm - speci c as this is due to the

chemical reaction in the Nessler’s reagent;
sensitivity - needs to measure the amount of
NH3 released - if the reading is zero, does it
mean that there is absolutely no NH3
released?
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 Evaluating Data quality
❖ Quantitative data
❖ The range of data points or error bars
can be large. This could be due to the
limitations of the techniques (see above).
❖ Hence it is always good to have different
types of techniques to measure the same
parameter, if possible.
❖ authors used: pH of media, release of
NH3 and LR - all to try and state that
the mutants are defective in
neutralising pH of external
environment, to make a point.
Evaluating Data quality - biological data
❖ Note that the data are not always
yes or no, eg the % of inhibition in
the article was not always 0 or 100.
❖ This is sometimes the limitations of
methods, experimental design,
redundancy etc.
❖ But generally the nature of
biological experiments is such that it
is not surprising that there are no
absolutes.
 Qualitative data?
❖ Descriptive data
❖ Here there is no
quantitation
❖ The assumption is that
representative cells have
been shown as opposed to
showing only “favourite”
cells.
❖ but the data here is
supported by quantisation
and other experiments in
the article.
 Evaluating Interpretation
❖ This requires some examination of the assertions made by the
authors about the data.
❖ i.e. examine the data, read the authors’ assertions, evaluate if they
made correct interpretations.
❖ e.g. in a few instances, the authors are making correlations across
different experiments. Generally the idea is:
GlcNAC -> catabolism to produce and release NH3 -> leads to
neutralisation of environment -> allowing hyphae switch -> killing of
macrophages -> survival of C albicans

the idea is based on the combination of experiments

 Evaluating Interpretation
❖ Generally the idea is:
GlcNAC -> catabolism to produce and release NH3 -> leads to neutralisation of
environment -> allowing hyphae switch -> killing of macrophages -> survival of C
albicans
the idea is based on the combination of experiments

one could test other ideas to strengthen the authors’ assertions:

- e.g. are there mutants that cannot do dimorphic switch but are able to release NH3
that the authors could use to test if
- it is the neutralisation that is killing the macrophages?
- it is the hyphae formation is that kills macrophage?
- etc etc
 Conclusions
❖ The article started with the authors proposing to test a
hypothesis or answer questions about the GluNAc.
❖ Based on the data you examined, do you think the
authors made the correct conclusions about their
answers to the questions?
❖ Did the data from the experiments answer the
hypothesis?
❖ Go back to the introduction - what is/are the
hypothesis or question the authors wanted to test?
Other take home messages about research data
❖ Last but not least, I think that reading through the
articles you can think of further questions.
❖ So doing research and getting data do not mean having
all the gaps answered in one article.
❖ Also, doing research on one area can open up more
questions.
HOMEWORK
➤ Watch techniques video and complete the technique quiz
➤ Also, watch video explaining research article
➤ Plus review Videos ‘simulating’ experiments in the article
➤ Please through the article
➤ You can answer the question prompts as annotations or
post your own questions or thoughts on the article when
we meet on Friday.
THIS FRIDAY
➤ We will meet at the main LSM2233 MS Teams
➤ Then we will go into the Channels as groups
➤ Then we come back and have a summing up

➤ I will put up a separate document on the tutorial organisation