Cell Signalling: Journal Clubs
BBS2042
Extensive journal club notes with explanations and drawings.
1
,Table of Contents
Journal Club 1: Assessing the receptor-mediated activity of PAHs using AhR-, ERα-
and PPARγ- CALUX bioassays (pages 3–8 + drawing).
Journal Club 2: Nuclear receptors in liver fibrosis (pages 9–21).
Journal Club 3: Histamine stimulates hydrogen peroxide production by bronchial
epithelial cells via histamine H1 receptor and dual oxidase (pages 22–31 + drawing).
Journal Club 4: miR-199a impairs autophagy and induces cardiac hypertrophy
through mTOR activation (pages 32–37).
Journal Club 5: Clustered, Regularly Interspaced Short Palindromic Repeats
(CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a
Novel Role of Neurofibromin in mTOR Signaling (pages 38–44 + drawing).
Journal Club 6: The dynamic control of signal transduction networks in cancer cells
(pages 45–54).
2
, Journal Club No.1:
Assessing the receptor-mediated activity of PAHs using AhR-, ERα- and PPARγ- CALUX
bioassays
01. What is the affiliation of authors?
02. Are there any conflict of interest?
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
03. Are references appropriate?
The references seem to be appropriate, but I believe that placing numbers for each of the
references, would have been easier for the readers to direct themselves to the citation that
they are looking for.
04. What kind of paper? (research/review article) What is the impact factor of the
journal?
a. This is a research article, because the paper has its own experimental set-up, where
they assess the endocrine activity of polycyclic aromatic hydrocarbons (PAHs) using
three different CALUX bioassays.
b. IFJ-a metric used to assess the citation rate of articles published in a particular
journal (Food and Chemical Toxicology) over a specific time-usually 2 years.
For Food and Chemical Toxicology, the impact factor is typically between 4 and 6, which is
considered strong for a toxicology and food safety journal. A "good" impact factor depends
on the field of research. Generally:
➢ Impact Factor (IF) < 2 → Low impact
➢ IF 2–4 → Moderate impact
➢ IF 4–10 → High impact
➢ IF > 10 → Very high impact (top journals in a field)
05. What was the rationale for performing the research?
The aim of this study was to determine the agonistic and antagonistic activity of 9
environmentally relevant PAHs, present either on the U.S. EPA priority list, the WFD priority
list or part of the 6 Borneff PAHs, and readily available, on three nuclear receptors using
three different CALUX bioassays: The AhR-CALUX bioassay, the ERα-CALUX bioassay and
the PPARγ CALUX bioassay, three major targets of environmental contaminants.
3
, 06. Do the authors explicitly state a hypothesis? If so, what is it? If not, what is the
implied research question?
The paper does not explicitly state a hypothesis/ proper hypothesis. However, the implied
aim of this study is:
Do polycyclic aromatic hydrocarbons (PAHs) exhibit agonistic or antagonistic endocrine
activity in AhR, ERα, and PPARγ receptor pathways, as assessed through CALUX
bioassays?
07. What methodology and study design did the researchers use to address the
hypothesis or research question?
● PAHs tested: Benzo(a)pyrene, phenanthrene, naphthalene, fluorene, pyrene,
anthracene, chrysene, benzo(g,h,i)perylene, fluoranthene.
● Control compounds: Reference agonists and antagonists for each receptor were
used to compare results.
The researchers used three reporter gene bioassays to test the interactions of PAHs with
nuclear receptors:
1. AhR-CALUX → Measured AhR (Aryl Hydrocarbon Receptor) activation.
2. ERα-CALUX → Measured estrogen receptor (ERα) activation.
3. PPARγ-CALUX → Measured peroxisome proliferator-activated receptor
gamma (PPARγ) activation.
Methodology:
1. Cells were transfected with receptor-specific luciferase reporter genes.
2. Cells were exposed to different concentrations of PAHs for specific time periods.
3. Luciferase activity was measured to determine receptor activation or inhibition.
4. Agonistic activity: Measured as an increase in luciferase activity.
5. Antagonistic activity: Measured by the ability of PAHs to suppress receptor
activation induced by a known agonist.
Statistical analysis:
Dose-response curves were generated to determine EC50 (effective concentration for 50%
response) and IC50 (inhibitory concentration for 50% response).
Fold induction (FI) was calculated to compare receptor activation relative to controls.
Relative Potency (REP) was determined by comparing EC50 values of PAHs with reference
compounds.
07. Define transfected cells and their significance in this study.
a. PPARy CALUX=> uses U–2OS cells (human osteoblast) that are stably transfected
with human PPARy2 and a luciferase reporter construct under the control of a
receptor specific response element.
b. AhR-CALUX=> uses a recombinant mouse hepatoma (Hepa1c1c7) cell line
(H1L7.5c1) that contains a stably transfected AhR-responsive luciferase reporter
gene (pGudLuc7.5). The H1L7.5c1 cells respond to AhR agonists with the induction
of luciferase in a time-, chemical-, concentration- and AhR-dependent manner.
4
, c. The ERα-CALUX is a reporter gene mammalian bioassay that is based on the
recombinant human breast cancer cell line VM7Luc4E2, which expresses ERα
endog enously and lacks any functional ERβ. VM7Luc4E2 cells contain a stably
transfected estrogen-responsive luciferase reporter gene plasmid which responds to
estrogenic chemicals with the induction of luciferase reporter gene expression.
d. Explain the mechanism of the gene reporter assay used in this study
(luciferase reporter gene).
1. Cells were transfected with receptor-specific luciferase reporter genes.
2. Cells were exposed to different concentrations of PAHs for specific time periods.
3. Luciferase activity was measured to determine receptor activation or inhibition.
4. Agonistic activity: Measured as an increase in luciferase activity.
5. Antagonistic activity: Measured by the ability of PAHs to suppress receptor
activation induced by a known agonist.
e. What do the terms "agonist" and "antagonist" signify in the context of this
research?
Agonist is when the interaction between the ligand and receptor induced activation, whereas
antagonist is when it causes gene suppression.
08. Are the data presented effectively through tables and figures? Yes, they are.
a. Provide an interpretation of Figures 2, 3, and 4.
Fig. 2: Shows concentration-dependent induction of PPARγ-responsive reporter gene
activity for Anthracene, Fluoranthene, Pyrene, and Fluorene compared to the reference
agonist (Rosiglitazone).
1. Pyrene and fluorene showed a weak agonistic activity for PPARγ.
2. Anthracene and fluoranthene showed a weak agonistic trend (weak partial
agonists).
3. B (a)P showed a weak antagonistic activity.
Fig. 3: Depicts concentration-dependent AhR activation for B(a)P, Chrysene, Fluoranthene,
Pyrene, and Anthracene.
1. Chrysene was the only PAH that showed relatively strong agonist activity (except for
B(a)P which was used as a standard).
2. Pyrene, anthracene and fluoranthene showed weak AhR agonist activity.
Figure 4: Displays the estrogenic activity of PAHs using the ERα-CALUX bioassay by
concentration-induction of ERα-responsive reporter gene activity.
1. Fluoranthene had agonistic activity whilst B(a)P exhibited both agonistic and
antagonistic activity (lowering E2 activity by 30%).
2. Phenanthrene and anthracene had weak ERα agonist activities.
b. Describe the contents and significance of Table 2.
Table 2: Summarizes EC50 values and fold induction (FI) for PAHs in PPARγ-
CALUX, AhR-CALUX, and ERα-CALUX assays.
5
BBS2042
Extensive journal club notes with explanations and drawings.
1
,Table of Contents
Journal Club 1: Assessing the receptor-mediated activity of PAHs using AhR-, ERα-
and PPARγ- CALUX bioassays (pages 3–8 + drawing).
Journal Club 2: Nuclear receptors in liver fibrosis (pages 9–21).
Journal Club 3: Histamine stimulates hydrogen peroxide production by bronchial
epithelial cells via histamine H1 receptor and dual oxidase (pages 22–31 + drawing).
Journal Club 4: miR-199a impairs autophagy and induces cardiac hypertrophy
through mTOR activation (pages 32–37).
Journal Club 5: Clustered, Regularly Interspaced Short Palindromic Repeats
(CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a
Novel Role of Neurofibromin in mTOR Signaling (pages 38–44 + drawing).
Journal Club 6: The dynamic control of signal transduction networks in cancer cells
(pages 45–54).
2
, Journal Club No.1:
Assessing the receptor-mediated activity of PAHs using AhR-, ERα- and PPARγ- CALUX
bioassays
01. What is the affiliation of authors?
02. Are there any conflict of interest?
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
03. Are references appropriate?
The references seem to be appropriate, but I believe that placing numbers for each of the
references, would have been easier for the readers to direct themselves to the citation that
they are looking for.
04. What kind of paper? (research/review article) What is the impact factor of the
journal?
a. This is a research article, because the paper has its own experimental set-up, where
they assess the endocrine activity of polycyclic aromatic hydrocarbons (PAHs) using
three different CALUX bioassays.
b. IFJ-a metric used to assess the citation rate of articles published in a particular
journal (Food and Chemical Toxicology) over a specific time-usually 2 years.
For Food and Chemical Toxicology, the impact factor is typically between 4 and 6, which is
considered strong for a toxicology and food safety journal. A "good" impact factor depends
on the field of research. Generally:
➢ Impact Factor (IF) < 2 → Low impact
➢ IF 2–4 → Moderate impact
➢ IF 4–10 → High impact
➢ IF > 10 → Very high impact (top journals in a field)
05. What was the rationale for performing the research?
The aim of this study was to determine the agonistic and antagonistic activity of 9
environmentally relevant PAHs, present either on the U.S. EPA priority list, the WFD priority
list or part of the 6 Borneff PAHs, and readily available, on three nuclear receptors using
three different CALUX bioassays: The AhR-CALUX bioassay, the ERα-CALUX bioassay and
the PPARγ CALUX bioassay, three major targets of environmental contaminants.
3
, 06. Do the authors explicitly state a hypothesis? If so, what is it? If not, what is the
implied research question?
The paper does not explicitly state a hypothesis/ proper hypothesis. However, the implied
aim of this study is:
Do polycyclic aromatic hydrocarbons (PAHs) exhibit agonistic or antagonistic endocrine
activity in AhR, ERα, and PPARγ receptor pathways, as assessed through CALUX
bioassays?
07. What methodology and study design did the researchers use to address the
hypothesis or research question?
● PAHs tested: Benzo(a)pyrene, phenanthrene, naphthalene, fluorene, pyrene,
anthracene, chrysene, benzo(g,h,i)perylene, fluoranthene.
● Control compounds: Reference agonists and antagonists for each receptor were
used to compare results.
The researchers used three reporter gene bioassays to test the interactions of PAHs with
nuclear receptors:
1. AhR-CALUX → Measured AhR (Aryl Hydrocarbon Receptor) activation.
2. ERα-CALUX → Measured estrogen receptor (ERα) activation.
3. PPARγ-CALUX → Measured peroxisome proliferator-activated receptor
gamma (PPARγ) activation.
Methodology:
1. Cells were transfected with receptor-specific luciferase reporter genes.
2. Cells were exposed to different concentrations of PAHs for specific time periods.
3. Luciferase activity was measured to determine receptor activation or inhibition.
4. Agonistic activity: Measured as an increase in luciferase activity.
5. Antagonistic activity: Measured by the ability of PAHs to suppress receptor
activation induced by a known agonist.
Statistical analysis:
Dose-response curves were generated to determine EC50 (effective concentration for 50%
response) and IC50 (inhibitory concentration for 50% response).
Fold induction (FI) was calculated to compare receptor activation relative to controls.
Relative Potency (REP) was determined by comparing EC50 values of PAHs with reference
compounds.
07. Define transfected cells and their significance in this study.
a. PPARy CALUX=> uses U–2OS cells (human osteoblast) that are stably transfected
with human PPARy2 and a luciferase reporter construct under the control of a
receptor specific response element.
b. AhR-CALUX=> uses a recombinant mouse hepatoma (Hepa1c1c7) cell line
(H1L7.5c1) that contains a stably transfected AhR-responsive luciferase reporter
gene (pGudLuc7.5). The H1L7.5c1 cells respond to AhR agonists with the induction
of luciferase in a time-, chemical-, concentration- and AhR-dependent manner.
4
, c. The ERα-CALUX is a reporter gene mammalian bioassay that is based on the
recombinant human breast cancer cell line VM7Luc4E2, which expresses ERα
endog enously and lacks any functional ERβ. VM7Luc4E2 cells contain a stably
transfected estrogen-responsive luciferase reporter gene plasmid which responds to
estrogenic chemicals with the induction of luciferase reporter gene expression.
d. Explain the mechanism of the gene reporter assay used in this study
(luciferase reporter gene).
1. Cells were transfected with receptor-specific luciferase reporter genes.
2. Cells were exposed to different concentrations of PAHs for specific time periods.
3. Luciferase activity was measured to determine receptor activation or inhibition.
4. Agonistic activity: Measured as an increase in luciferase activity.
5. Antagonistic activity: Measured by the ability of PAHs to suppress receptor
activation induced by a known agonist.
e. What do the terms "agonist" and "antagonist" signify in the context of this
research?
Agonist is when the interaction between the ligand and receptor induced activation, whereas
antagonist is when it causes gene suppression.
08. Are the data presented effectively through tables and figures? Yes, they are.
a. Provide an interpretation of Figures 2, 3, and 4.
Fig. 2: Shows concentration-dependent induction of PPARγ-responsive reporter gene
activity for Anthracene, Fluoranthene, Pyrene, and Fluorene compared to the reference
agonist (Rosiglitazone).
1. Pyrene and fluorene showed a weak agonistic activity for PPARγ.
2. Anthracene and fluoranthene showed a weak agonistic trend (weak partial
agonists).
3. B (a)P showed a weak antagonistic activity.
Fig. 3: Depicts concentration-dependent AhR activation for B(a)P, Chrysene, Fluoranthene,
Pyrene, and Anthracene.
1. Chrysene was the only PAH that showed relatively strong agonist activity (except for
B(a)P which was used as a standard).
2. Pyrene, anthracene and fluoranthene showed weak AhR agonist activity.
Figure 4: Displays the estrogenic activity of PAHs using the ERα-CALUX bioassay by
concentration-induction of ERα-responsive reporter gene activity.
1. Fluoranthene had agonistic activity whilst B(a)P exhibited both agonistic and
antagonistic activity (lowering E2 activity by 30%).
2. Phenanthrene and anthracene had weak ERα agonist activities.
b. Describe the contents and significance of Table 2.
Table 2: Summarizes EC50 values and fold induction (FI) for PAHs in PPARγ-
CALUX, AhR-CALUX, and ERα-CALUX assays.
5
