Enclosed, please, find revised version of our manuscript entitled “Herichová I., Jendrisková
S., Pidíková P., Kršková L., Olexová L., Morová M., Stebelová K., Štefánik P.: Effect
of 17β-estradiol on the daily pattern of ACE2, ADAM17, TMPRSS2 and estradiol receptor
transcription in the lungs and colon of male rats”.
We are very grateful for the opportunity to improve our manuscript (MS) and greatly
appreciate all comments and suggestions proposed by the editors and reviewers. All
suggestions, including 17β-estradiol (E2) measurement and providing of in vitro evidence,
have been accepted and incorporated into MS.
We believe that results demonstrating how E2 and the circadian system shape the daily
pattern in expression of molecules facilitating SARS-CoV-2 entry into the cell is
very up-today and interesting for wide spectrum of readers.
Our data indicate that E2 and the circadian system interact in regulation of ACE2
and TMPRSS2 mRNA expression while expression of ADAM17 mRNA seems to be mainly under
control of the circadian system and exerts a distinct daily rhythm in expression without
regard to E2 treatment. Capacity of E2 to influence gene expression is now also supported
by in vitro evidence.
We also revealed significant tissue specific effect of E2 administration on expression
of its own receptors. Similarly, E2 administration influenced expression of clock
genes per2 and bmal1.
We believe that the abovementioned results can be useful in Covid-19 management. Moreover,
our data can at least partly elucidate inconsistencies reported earlier on E2 induced
effects on expression of molecules facilitating SARS-CoV-2 entry into the cell as
daily pattern was not taken into consideration previously.
Concerning funding information, we have to acknowledge APVV-16-0209, APVV-20-0241,
VEGA 1/0679/1 and the Operation Program of Integrated Infrastructure for the project,
Advancing University Capacity and Competence in Research, Development and Innovation,
ITMS2014+: 313021X329, co-financed by the European Regional Development Fund.
We appreciate if text provided below is available with the MS (if MS is accepted of
course): “Research was supported by grants APVV-16-0209, APVV-20-0241, VEGA 1/0679/19
and by the Operation Program of Integrated Infrastructure for the project, Advancing
University Capacity and Competence in Research, Development and Innovation, ITMS2014+:
313021X329, co-financed by the European Regional Development Fund.”
We hope that you will find revised version of manuscript suitable for publishing in
your prominent and reputable journal PLOS ONE.
Yours sincerely,
authors
Comments from the editorial board
Discuss relevance of transcription as readout.
We investigated regulation of ACE2, TMPRSS2 and ADAM17 levels at the transcriptional
level as both, the circadian system as well as E2 exert the most of their effect via
regulation of transcription. The circadian feed-back loop influences transcription
via E-box and E2 via ERE regulatory region. Moreover, according published literature,
it is possible to expect that protein translation will follow levels of mRNA. We provide
several references showing positive correlation between mRNA and protein levels of
studied genes:
ACE2
Gao, H., Tanchico, D. T., Yallampalli, U., & Yallampalli, C. (2016). A Low-Protein
Diet Enhances Angiotensin II Production in the Lung of Pregnant Rats but not Nonpregnant
Rats. Journal of pregnancy, 2016, 4293431.
Xue, T., Wei, N., Xin, Z., & Qingyu, X. (2014). Angiotensin-converting enzyme-2 overexpression
attenuates inflammation in rat model of chronic obstructive pulmonary disease. Inhalation
toxicology, 26(1), 14–22.
Khoury, E. E., Knaney, Y., Fokra, A., Kinaneh, S., Azzam, Z., Heyman, S. N., & Abassi,
Z. (2021). Pulmonary, cardiac and renal distribution of ACE2, furin, TMPRSS2 and ADAM17
in rats with heart failure: Potential implication for COVID-19 disease. Journal of
cellular and molecular medicine, 25(8), 3840–3855.
Bellamine, A., Pham, T., Jain, J., Wilson, J., Sahin, K., Dallaire, F., Seidah, N.
G., Durkee, S., Radošević, K., & Cohen, É. A. (2021). L-Carnitine Tartrate Downregulates
the ACE2 Receptor and Limits SARS-CoV-2 Infection. Nutrients, 13(4), 1297.
Martins, F. L., Tavares, C., Malagrino, P. A., Rentz, T., Benetti, A., Rios, T., Pereira,
G., Caramelli, B., Teixeira, S. K., Krieger, J. E., & Girardi, A. (2021). Sex differences
in the lung ACE/ACE2 balance in hypertensive rats. Bioscience reports, 41(12), BSR20211201.
Pedrosa, M. A., Valenzuela, R., Garrido-Gil, P., Labandeira, C. M., Navarro, G., Franco,
R., Labandeira-Garcia, J. L., & Rodriguez-Perez, A. I. (2021). Experimental data using
candesartan and captopril indicate no double-edged sword effect in COVID-19. Clinical
science (London, England : 1979), 135(3), 465–481.
Wang, K., Xu, Y., Yang, W., & Zhang, Y. (2017). Insufficient hypothalamic angiotensin-converting
enzyme 2 is associated with hypertension in SHR rats. Oncotarget, 8(12), 20244–20251.
Zhang, W., Xu, Y. Z., Liu, B., Wu, R., Yang, Y. Y., Xiao, X. Q., & Zhang, X. (2014).
Pioglitazone upregulates angiotensin converting enzyme 2 expression in insulin-sensitive
tissues in rats with high-fat diet-induced nonalcoholic steatohepatitis. TheScientificWorldJournal,
2014, 603409.
ADAM17
Khoury, E. E., Knaney, Y., Fokra, A., Kinaneh, S., Azzam, Z., Heyman, S. N., & Abassi,
Z. (2021). Pulmonary, cardiac and renal distribution of ACE2, furin, TMPRSS2 and ADAM17
in rats with heart failure: Potential implication for COVID-19 disease. Journal of
cellular and molecular medicine, 25(8), 3840–3855.
Zheng, D. Y., Zhao, J., Yang, J. M., Wang, M., & Zhang, X. T. (2016). Enhanced ADAM17
expression is associated with cardiac remodeling in rats with acute myocardial infarction.
Life sciences, 151, 61–69.
TMPRSS2
Khoury, E. E., Knaney, Y., Fokra, A., Kinaneh, S., Azzam, Z., Heyman, S. N., & Abassi,
Z. (2021). Pulmonary, cardiac and renal distribution of ACE2, furin, TMPRSS2 and ADAM17
in rats with heart failure: Potential implication for COVID-19 disease. Journal of
cellular and molecular medicine, 25(8), 3840–3855.
Bellamine, A., Pham, T., Jain, J., Wilson, J., Sahin, K., Dallaire, F., Seidah, N.
G., Durkee, S., Radošević, K., & Cohen, É. A. (2021). L-Carnitine Tartrate Downregulates
the ACE2 Receptor and Limits SARS-CoV-2 Infection. Nutrients, 13(4), 1297.
ESR1
Khaksari, M., Hajializadeh, Z., Mahani, S. E., Soltani, Z., & Asadikaram, G. (2021).
Estrogen receptor agonists induce anti edema effects by altering α and β estrogen
receptor gene expression. Acta neurobiologiae experimentalis, 81(3), 286–294.
Soares, T. S., Fernandes, S. A., Lima, M. L., Stumpp, T., Schoorlemmer, G. H., Lazari,
M. F., & Porto, C. S. (2013). Experimental varicocoele in rats affects mechanisms
that control expression and function of the androgen receptor. Andrology, 1(5), 670–681.
Fernandes, S. A., Gomes, G. R., Siu, E. R., Damas-Souza, D. M., Bruni-Cardoso, A.,
Augusto, T. M., Lazari, M. F., Carvalho, H. F., & Porto, C. S. (2011). The anti-oestrogen
fulvestrant (ICI 182,780) reduces the androgen receptor expression, ERK1/2 phosphorylation
and cell proliferation in the rat ventral prostate. International journal of andrology,
34(5 Pt 1), 486–500.
Harvey, C. N., Chen, J. C., Bagnell, C. A., & Uzumcu, M. (2015). Methoxychlor and
its metabolite HPTE inhibit cAMP production and expression of estrogen receptors α
and β in the rat granulosa cell in vitro. Reproductive toxicology (Elmsford, N.Y.),
51, 72–78.
GPER1 (GPR30)
Hsieh, Y. C., Yu, H. P., Frink, M., Suzuki, T., Choudhry, M. A., Schwacha, M. G.,
& Chaudry, I. H. (2007). G protein-coupled receptor 30-dependent protein kinase A
pathway is critical in nongenomic effects of estrogen in attenuating liver injury
after trauma-hemorrhage. The American journal of pathology, 170(4), 1210–1218.
Lo, E., Lee, J. C., Turner, P. C., & El-Nezami, H. (2021). Low dose of zearalenone
elevated colon cancer cell growth through G protein-coupled estrogenic receptor. Scientific
reports, 11(1), 7403.
Jala, V. R., Radde, B. N., Haribabu, B., & Klinge, C. M. (2012). Enhanced expression
of G-protein coupled estrogen receptor (GPER/GPR30) in lung cancer. BMC cancer, 12,
624.
PER2
Harbour, V. L., Weigl, Y., Robinson, B., & Amir, S. (2014). Phase differences in expression
of circadian clock genes in the central nucleus of the amygdala, dentate gyrus, and
suprachiasmatic nucleus in the rat. PloS one, 9(7), e103309.
Harbour, V. L., Weigl, Y., Robinson, B., & Amir, S. (2013). Comprehensive mapping
of regional expression of the clock protein PERIOD2 in rat forebrain across the 24-h
day. PloS one, 8(10), e76391.
Wang, X., Wang, L., Yu, Q., Xu, Y., Zhang, L., Zhao, X., Cao, X., Li, Y., & Li, L.
(2016). Alterations in the expression of Per1 and Per2 induced by Aβ31-35 in the suprachiasmatic
nucleus, hippocampus, and heart of C57BL/6 mouse. Brain research, 1642, 51–58.
BMAL1
Asher G, Gatfield D, Stratmann M, Reinke H, Dibner C, Kreppel F, Mostoslavsky R, Alt
FW, Schibler U. SIRT1 regulates circadian clock gene expression through PER2 deacetylation.
Cell. 2008 Jul 25;134(2):317-28. doi:
Cha, S., Wang, J., Lee, S.M. et al. Clock-modified mesenchymal stromal cells therapy
rescues molecular circadian oscillation and age-related bone loss via miR142-3p/Bmal1/YAP
signaling axis. Cell Death Discov. 8, 111 (2022).
Liu, S., Zhou, Y., Chen, Y., Liu, Y., Peng, S., Cao, Z., & Xia, H. (2022). Bmal1 promotes
cementoblast differentiation and cementum mineralization via Wnt/β-catenin signaling.
Acta histochemica, 124(3), 151868.
Please provide the rationale of the determination of animal anxiety.
Nearly all molecules facilitating SARS-CoV-2 entry into the cell have been suggested
as therapeutic targets to handle Covid-19 infection. If E2 is also to be used in this
way it is of importance to learn which side effects can be expected. It has been previously
shown that E2 administration can induce anxiety like behaviour in rodents (Borrow
and Handa, 2017), and our results confirmed this earlier observation. This aspect
of possible E2 treatment can be relevant and should not be ignored.
Borrow AP, Handa RJ. Estrogen Receptors Modulation of Anxiety-Like Behavior. Vitam
Horm. 2017; 103: 27–52.
Add transcription and male rats to the title.
The title of MS has been changed accordingly.
Provide the experimental data to confirm the intake amount of E2 in the rats.
Levels of E2 in drinking water and plasma of experimental and control animals were
measured by Elisa. Chapters “Methods” and “Results” were modified accordingly. Supplementary
figure 1 showing water consumption was replaced by new figure showing E2 intake. This
figure is labelled as supplementary figure 2 now as we incorporated scheme of experiment
and sampling, as it was requested by reviewer #2, as a supplementary figure 1.
Provide the experimental data to validate the activation of ESR downstream genes
New in vitro experiment testing dose dependent effect of E2 on ACE2, ADAM17, TMPRSS2,
E2 receptors and clock genes was performed as it was requested and incorporated into
MS. Chapters “Methods” and “Results” were modified accordingly. Results are summarised
in supplementary figures 6 and 7. In vivo and in vitro data are compared in the chapter
“Results”.
Consider shortening introduction and discussion sections.
Chapters “Introduction” and “Discussion” were shortened as it was suggested by reviewers
#2 and #3.
Provide technical and statistical details as requested by the reviewers.
All requested statistical and technical details have been incorporated in the chapter
“Methods”.
• A rebuttal letter that responds to each point raised by the academic editor and
reviewer(s). You should upload this letter as a separate file labelled 'Response to
Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original
version. You should upload this as a separate file labelled 'Revised Manuscript with
Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload
this as a separate file labelled 'Manuscript'.
• We recommend that you deposit your laboratory protocols in protocols.io.
Response to reviewers and manuscript with and without changes were uploaded. We also
deposited protocols for isolation of mRNA and miRNA from cells (isolation from single
wells of 24 well plate) and protocol for E2 dilution in protocols.io and referred
to them in the chapter “Methods”.
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also supported by the Operation Program of Integrated Infrastructure for the project,
Advancing University Capacity and Competence in Research, Development and Innovation,
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https://www.apvv.sk/?lang=en, to IH; APVV-20-0241, The Slovak Research and Development Agency, https://www.apvv.sk/?lang=en, to IH; VEGA 1/0679/19, Scientific Grant Agency of the Ministry of Education, Science,
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Reviewer #1:
Major comments:
The study was aimed at the determination of oscillations in transcription of genes
associated with corona-virus attachment to target cells, and the impact of E2 on this
behavior. However, I regret the authors did not evaluate how these changes in transcriptional
oscillations affected gross protein expression, either by western blotting or immunostaining.
We focused on regulation at the transcriptional level as both, the clock genes as
well as E2 exerts most of their effects via regulation of transcription. The circadian
feed-back loop is exerting this effect via E-box and E2 via ERE regulatory region.
Moreover, according to published literature, it is possible to expect that protein
translation will follow levels of mRNA of genes included in the study. We provide
several references showing positive correlation between mRNA and protein levels of
studied genes:
ACE2
Gao, H., Tanchico, D. T., Yallampalli, U., & Yallampalli, C. (2016). A Low-Protein
Diet Enhances Angiotensin II Production in the Lung of Pregnant Rats but not Nonpregnant
Rats. Journal of pregnancy, 2016, 4293431.
Xue, T., Wei, N., Xin, Z., & Qingyu, X. (2014). Angiotensin-converting enzyme-2 overexpression
attenuates inflammation in rat model of chronic obstructive pulmonary disease. Inhalation
toxicology, 26(1), 14–22.
Khoury, E. E., Knaney, Y., Fokra, A., Kinaneh, S., Azzam, Z., Heyman, S. N., & Abassi,
Z. (2021). Pulmonary, cardiac and renal distribution of ACE2, furin, TMPRSS2 and ADAM17
in rats with heart failure: Potential implication for COVID-19 disease. Journal of
cellular and molecular medicine, 25(8), 3840–3855.
Bellamine, A., Pham, T., Jain, J., Wilson, J., Sahin, K., Dallaire, F., Seidah, N.
G., Durkee, S., Radošević, K., & Cohen, É. A. (2021). L-Carnitine Tartrate Downregulates
the ACE2 Receptor and Limits SARS-CoV-2 Infection. Nutrients, 13(4), 1297.
Martins, F. L., Tavares, C., Malagrino, P. A., Rentz, T., Benetti, A., Rios, T., Pereira,
G., Caramelli, B., Teixeira, S. K., Krieger, J. E., & Girardi, A. (2021). Sex differences
in the lung ACE/ACE2 balance in hypertensive rats. Bioscience reports, 41(12), BSR20211201.
Pedrosa, M. A., Valenzuela, R., Garrido-Gil, P., Labandeira, C. M., Navarro, G., Franco,
R., Labandeira-Garcia, J. L., & Rodriguez-Perez, A. I. (2021). Experimental data using
candesartan and captopril indicate no double-edged sword effect in COVID-19. Clinical
science (London, England : 1979), 135(3), 465–481.
Wang, K., Xu, Y., Yang, W., & Zhang, Y. (2017). Insufficient hypothalamic angiotensin-converting
enzyme 2 is associated with hypertension in SHR rats. Oncotarget, 8(12), 20244–20251.
Zhang, W., Xu, Y. Z., Liu, B., Wu, R., Yang, Y. Y., Xiao, X. Q., & Zhang, X. (2014).
Pioglitazone upregulates angiotensin converting enzyme 2 expression in insulin-sensitive
tissues in rats with high-fat diet-induced nonalcoholic steatohepatitis. TheScientificWorldJournal,
2014, 603409.
ADAM17
Khoury, E. E., Knaney, Y., Fokra, A., Kinaneh, S., Azzam, Z., Heyman, S. N., & Abassi,
Z. (2021). Pulmonary, cardiac and renal distribution of ACE2, furin, TMPRSS2 and ADAM17
in rats with heart failure: Potential implication for COVID-19 disease. Journal of
cellular and molecular medicine, 25(8), 3840–3855.
Zheng, D. Y., Zhao, J., Yang, J. M., Wang, M., & Zhang, X. T. (2016). Enhanced ADAM17
expression is associated with cardiac remodeling in rats with acute myocardial infarction.
Life sciences, 151, 61–69.
TMPRSS2
Khoury, E. E., Knaney, Y., Fokra, A., Kinaneh, S., Azzam, Z., Heyman, S. N., & Abassi,
Z. (2021). Pulmonary, cardiac and renal distribution of ACE2, furin, TMPRSS2 and ADAM17
in rats with heart failure: Potential implication for COVID-19 disease. Journal of
cellular and molecular medicine, 25(8), 3840–3855.
Bellamine, A., Pham, T., Jain, J., Wilson, J., Sahin, K., Dallaire, F., Seidah, N.
G., Durkee, S., Radošević, K., & Cohen, É. A. (2021). L-Carnitine Tartrate Downregulates
the ACE2 Receptor and Limits SARS-CoV-2 Infection. Nutrients, 13(4), 1297.
ESR1
Khaksari, M., Hajializadeh, Z., Mahani, S. E., Soltani, Z., & Asadikaram, G. (2021).
Estrogen receptor agonists induce anti edema effects by altering α and β estrogen
receptor gene expression. Acta neurobiologiae experimentalis, 81(3), 286–294.
Soares, T. S., Fernandes, S. A., Lima, M. L., Stumpp, T., Schoorlemmer, G. H., Lazari,
M. F., & Porto, C. S. (2013). Experimental varicocoele in rats affects mechanisms
that control expression and function of the androgen receptor. Andrology, 1(5), 670–681.
Fernandes, S. A., Gomes, G. R., Siu, E. R., Damas-Souza, D. M., Bruni-Cardoso, A.,
Augusto, T. M., Lazari, M. F., Carvalho, H. F., & Porto, C. S. (2011). The anti-oestrogen
fulvestrant (ICI 182,780) reduces the androgen receptor expression, ERK1/2 phosphorylation
and cell proliferation in the rat ventral prostate. International journal of andrology,
34(5 Pt 1), 486–500.
Harvey, C. N., Chen, J. C., Bagnell, C. A., & Uzumcu, M. (2015). Methoxychlor and
its metabolite HPTE inhibit cAMP production and expression of estrogen receptors α
and β in the rat granulosa cell in vitro. Reproductive toxicology (Elmsford, N.Y.),
51, 72–78.
GPER1 (GPR30)
Hsieh, Y. C., Yu, H. P., Frink, M., Suzuki, T., Choudhry, M. A., Schwacha, M. G.,
& Chaudry, I. H. (2007). G protein-coupled receptor 30-dependent protein kinase A
pathway is critical in nongenomic effects of estrogen in attenuating liver injury
after trauma-hemorrhage. The American journal of pathology, 170(4), 1210–1218.
Lo, E., Lee, J. C., Turner, P. C., & El-Nezami, H. (2021). Low dose of zearalenone
elevated colon cancer cell growth through G protein-coupled estrogenic receptor. Scientific
reports, 11(1), 7403.
Jala, V. R., Radde, B. N., Haribabu, B., & Klinge, C. M. (2012). Enhanced expression
of G-protein coupled estrogen receptor (GPER/GPR30) in lung cancer. BMC cancer, 12,
624.
PER2
Harbour, V. L., Weigl, Y., Robinson, B., & Amir, S. (2014). Phase differences in expression
of circadian clock genes in the central nucleus of the amygdala, dentate gyrus, and
suprachiasmatic nucleus in the rat. PloS one, 9(7), e103309.
Harbour, V. L., Weigl, Y., Robinson, B., & Amir, S. (2013). Comprehensive mapping
of regional expression of the clock protein PERIOD2 in rat forebrain across the 24-h
day. PloS one, 8(10), e76391.
Wang, X., Wang, L., Yu, Q., Xu, Y., Zhang, L., Zhao, X., Cao, X., Li, Y., & Li, L.
(2016). Alterations in the expression of Per1 and Per2 induced by Aβ31-35 in the suprachiasmatic
nucleus, hippocampus, and heart of C57BL/6 mouse. Brain research, 1642, 51–58.
BMAL1
Asher G, Gatfield D, Stratmann M, Reinke H, Dibner C, Kreppel F, Mostoslavsky R, Alt
FW, Schibler U. SIRT1 regulates circadian clock gene expression through PER2 deacetylation.
Cell. 2008 Jul 25;134(2):317-28. doi:
Cha, S., Wang, J., Lee, S.M. et al. Clock-modified mesenchymal stromal cells therapy
rescues molecular circadian oscillation and age-related bone loss via miR142-3p/Bmal1/YAP
signaling axis. Cell Death Discov. 8, 111 (2022).
Liu, S., Zhou, Y., Chen, Y., Liu, Y., Peng, S., Cao, Z., & Xia, H. (2022). Bmal1 promotes
cementoblast differentiation and cementum mineralization via Wnt/β-catenin signaling.
Acta histochemica, 124(3), 151868.
After all, this is likely what matters regarding viral homing to target cells and
to the susceptibility to severe COVID disease.
We did not make any statement about viral homing, however, without mRNA there is no
protein to facilitate homing.
The oscillation in gene expression is not dramatically affected by E2, thus the importance
of these observations is questionable.
We cannot agree that changes induced by E2 are not dramatic. E2 administration induced
significant daily rhythm in ACE2 mRNA and TMPRSS2 mRNA expression in lungs and strongly
modulated expression of its own receptors in both investigated tissues. Originality
of MS underlines also discovery of daily rhythm in ADAM17 which is present in both,
control and experimental group, showing the same acrophase and amplitude that implicates,
that this rhythmicity can be expected also in other tissues. All these changes can
be too subtle to influence severe Covid disease, however, with omicron variant importance
of mild disease rises. Less aggressive variants of SARS-CoV-2 should not be underestimated
as they can, similarly like former more aggressive variants, induce long-time Covid
symptoms (Morioka et al., 2022).
Post COVID-19 condition of the Omicron variant of SARS-CoV-2; Shinichiro Morioka,
Shinya Tsuzuki, Michiyo Suzuki, Mari Terada, Masako Akashi, Yasuyo Osanai, Chika Kuge,
Mio Sanada, Keiko Tanaka, Taketomo Maruki, Kozue Takahashi, Sho Saito, Kayoko Hayakawa,
Katsuji Teruya, Masayuki Hojo, Norio Ohmagari. medRxiv preprint 2022, DOI: https://doi.org/10.1101/2022.05.12.22274990,
https://www.medrxiv.org/content/10.1101/2022.05.12.22274990v1
Accordingly, in the title "expression" might be replaced by "transcription".
- “expression” in the title was replaced by “transcription”
Methods line 187: what was the average daily intake of E2? Could the nocturnal rise
in E2 quoted in Ref 44 reflect the circadian nocturnal active phase of the rat, consuming
more water at nighttime? In this respect, could documented circadian fluctuations
in gene expression might reflect the hour-to hour change in E2 ingestion (likely increasing
during the D period), and not necessarily a true physiologic oscillator phenomenon.
A mini-pump for drug delivery could overcome this potential technical drawback.
As it was reported in earlier studies, in males and females E2 levels in plasma exert
a daily rhythm (Bao et al., 2003; Wijetilleka et al., 2016; Rahman et al., 2019) with
maximum during the active phase of LD cycle (Bao et al., 2003; Wijetilleka et al.,
2016).
In the revised version of MS levels of E2 in drinking water and plasma of experimental
and control animals were measured by Elisa as requested. Supplementary figure 1 showing
water consumption was replaced by new figure showing E2 intake (S2 Fig). This figure
is labelled as supplementary figure 2 now as we incorporated scheme of experiment
and sampling as a supplementary figure 1 (it was requested by reviewer #2).
Bao AM, Liu RY, van Someren EJW, Hofman MA, Cao YX, Zhou JN. Diurnal rhythm of free
estradiol during the menstrual cycle. Eur J Endocrinol. 2003;148: 227–232.
Rahman SA, Grant LK, Gooley JJ, Rajaratnam SMW, Czeisler CA, Lockley SW. Endogenous
Circadian Regulation of Female Reproductive Hormones. J Clin Endocrinol Metab. 2019;104:
6049–6059.
Wijetilleka S, Mon A, Khan M, Joseph F, Robinson A. Circadian Rhythm of Oestradiol:
Impact on the Bone Metabolism of Adult Males. In: J Clin Mol Endocrinol. 2016; p.
7.
Similarly, like in humans, we observed a significant daily rhythm in E2 levels in
plasma of control rats with the maximum during the active phase of LD cycle. Our aim
was not to disturb daily profile in E2 levels in the circulation and to avoid supraphysiological
E2 concentrations. We were successful in reaching this goal as E2 treatment increased
amplitude and mesor of daily pattern in E2 levels in the circulation, however, acrophase
was the same in both groups. Overall E2 levels were approximately 3-4 times higher
in E2 treated rats compared to control and we observed a significant increase in E2
levels in every specific time-point during 24h cycle (please, see S2 Table and S3
Fig).
Mini-pump delivery would very likely diminish naturally occurring daily rhythm in
E2 plasma levels.
Minor comments:
You may consider quoting Maksimowski, PLoS ONE, 2020, 15, e0241534, Borges, Life Sci.
2017, 191, 1–8 and Chanana, Am. J Physiol. Endocrinol. Metab. 2020, 319, E562–E567,
collectively addressing enhanced expression of angiotensin AT1R with testosterone,
but enhanced ACE2, as well as AT2R and MASR with estrogen (both serving as receptors
to Ang 1-7).
You may also quote Abassi, Front physiol 2020, 11, 574753, who further emphasizes
the susceptibility of males to develop AKI as compared with female gender and links
it to estrogen-derived enhanced expression of the ACE2-Ang 1-7-MasR axis in the female
gender
- thank you for the advice, suggested references were incorporated into MS.
Methods line 219: Please provide the rationale of the determination of animal anxiety.
Nearly all molecules facilitating SARS-CoV-2 entry into the cell have been suggested
as therapeutic targets to handle Covid-19 infection. If E2 is also to be used in this
way it is of importance to learn which side effects can be expected. It has been previously
shown that E2 administration can induce anxiety like behaviour in rodents (Borrow
and Handa, 2017), and our results confirmed this earlier observation. This aspect
of possible E2 treatment can be relevant and should not be ignored. This explanation
is now part of the chapter “Discussion” (lines 558 – 559).
Borrow AP, Handa RJ. Estrogen Receptors Modulation of Anxiety-Like Behavior. Vitam
Horm. 2017; 103: 27–52.
Methods lines 231-2. I assume L and D refer to light and dark. Please make it clearer,
perhaps also adding in parenthesis the ZT.
- thank you for the comment, ZT in parenthesis was added as requested
Reviewer #2:
This manuscript presented a study to investigate the effect 17β-estradiol on the expression
pattern of Ace2, Tmprss2, Adam17, the estradiol receptors and the clock gene in the
lung and colon tissues of male rats. In general, this manuscript could be better written.
A number of pitfalls need to be overcome before consideration of publication.
1. Title: Since male rats were used in this study, “male rats” needs to be specified
in the title. Also, “male rats” should also be specified in the conclusions.
- thank you for the comment, using of male rats was specified in the title and in
conclusions.
2. The authors need to explain why male rats were selected for this study.
- male rats were used as males have worse Covid-19 prognosis compared to women (Williamson
et al., 2020, Brandi et al., 2021, Ma et al., 2021) and according to some authors,
it can be related to lower E2 levels (Costeira et al., 2021). Our in vivo experiment
mimics situation in human male probands however using of animals allowed us to measure
whole 24h cycle in tissue gene expression that is not possible in patients or healthy
volunteers. This assumption and supporting references are mentioned in the chapter
“Introduction” (94 – 96 and 144 – 149).
Williamson EJ, Walker AJ, Bhaskaran K, Bacon S, Bates C, Morton CE, et al. Factors
associated with COVID-19-related death using OpenSAFELY. Nature. 2020;584: 430–436.
Brandi ML. Are sex hormones promising candidates to explain sex disparities in the
COVID-19 pandemic? Rev Endocr Metab Disord. 2021.
Ma Q, Hao ZW, Wang YF. The effect of estrogen in coronavirus disease 2019. Am J Physiol
- Lung Cell Mol Physiol. 2021;321.
Costeira R, Lee KA, Murray B, Christiansen C, Castillo-Fernandez J, Lochlainn MN,
et al. Estrogen and COVID-19 symptoms: Associations in women from the COVID Symptom
Study. PLoS One. 2021;16.
3. The introduction section is too long and should be more concise.
- thank you for the suggestion. The chapter “Introduction” was significantly shortened
and we did our best to make it more concise.
4. Since E2 was administrated in the drinking water, the authors should provide the
experimental data to confirm the intake amount of E2 in the rats.
Levels of E2 in drinking water and plasma of experimental and control animals were
measured by Elisa. Chapters “Methods” and “Results” were modified accordingly. Supplementary
figure 1 showing water consumption was replaced by new figure showing E2 intake. This
figure is labelled as supplementary figure 2 now as we incorporated scheme of experiment
and sampling, as a supplementary figure 1 as it was requested bellow (point 8).
5. Refer to Point 4, since the water consumption was decreased upon the start of E2
treatment, would the reduced water consumption affect the intake amount of E2 in the
rats as previously planned?
- as effect of E2 on water intake is known for a long time we expected it and included
it into our initial calculations. Therefore, we were able to administer dose very
exactly.
6. Refer to Point 4, the authors need to provide the experimental data to validate
the activation of ESR downstream genes so as to confirm the successful induction of
the E2 treatment in this system. Without this confirmation, it is unknown whether
the results could reflect the original study design.
New in vitro experiment testing dose dependent effect of E2 on ACE2, ADAM17, TMPRSS2,
E2 receptors and clock genes was performed as it was requested and incorporated into
MS. Chapters “Methods” and “Results” were modified accordingly. Results of in vitro
experiment are summarised in the supplementary figures 6 and 7. In vivo and in vitro
data are compared in the chapter “Results”.
7. Would the reduced water consumption affect the gene expression in the rats?
- as water was provided ad libitum and consumed volume of water in E2 treated rats
was still perfectly in the physiological range (Claasen et al., 1994) this parameter
can hardly influence gene expression measured in this study in the lungs and colon.
Claassen V.: Techniques in the Behavioral and Neural Sciences, 12 - Food and water
intake. Elsevier, Volume 12, 1994, Pages 267-287,
8. The treatment timing and the sampling timing are complicated. The authors should
better provide a schematic diagram to illustrate the treatment and sampling timings.
- thank you for the suggestion. Scheme of experiment and sampling was created and
referred as supplementary figure 1 mentioned in the chapter “Methods”.
9. The daily rhythm was only evaluated on Day 7 (Line 193). The authors should explain
whether the rhythm on the other days would be similar to that on Day 7. The authors
should better provide the experimental data to explain this concern.
- the locomotor activity was monitored during the whole experiment. We provide representative
data from two days, but the pattern was the same during the whole experiment. As animals
were exposed to LD cycle substantial changes in daily pattern of their locomotor activity
were not expected and, accordingly, were not observed. This information was provided
in the chapter “Results” (line 258). Tissue sampling for gene expression determination
requires euthanasia of animals and therefore only one 24h cycle was measured. This
is a typical end-point experiment and this design has been well accepted previously,
please, see references below:
Herichová I, Tesáková B, Kršková L, Olexová L. Food reward induction of rhythmic clock
gene expression in the prefrontal cortex of rats is accompanied by changes in miR-34a-5p
expression. Eur J Neurosci. 2021 Nov;54(10):7476-7492. doi: 10.1111/ejn.15518. Epub
2021 Nov 10. PMID: 34735028.
Herichová, I. and Hasáková, K. and Mravec, B. and Kavická, D. Tissue specific regulation
of egr1 rhythmic expression in the prefrontal cortex in comparison with other rat
brain regions. Activitas Nervosa Superior Rediviva. 2017; 59(2):51-56.
Herichová I, Hasáková K, Lukáčová D, Mravec B, Horváthová Ľ, Kavická D. Prefrontal
cortex and dorsomedial hypothalamus mediate food reward-induced effects via npas2
and egr1 expression in rat. Physiol Res. 2017 Dec 30;66(Suppl 4):S501-S510. doi: 10.33549/physiolres.933799.
PMID: 29355377.
Zeman M, Molcan L, Herichova I, Okuliarova M. Endocrine and cardiovascular rhythms
differentially adapt to chronic phase-delay shifts in rats. Chronobiol Int. 2016;33(9):1148-1160.
doi: 10.1080/07420528.2016.1203332. Epub 2016 Jul 26. PMID: 27459109.
Herichová I, Ambrušová J, Molčan Ľ, Veselá A, Svitok P, Zeman M. Different effects
of phase advance and delay in rotating light-dark regimens on clock and natriuretic
peptide gene expression in the rat heart. Physiol Res. 2014;63(Suppl 4):S573-84. doi:
10.33549/physiolres.932937. PMID: 25669688.
Herichova I, Zsoldosova K, Vesela A, Zeman M. Effect of angiotensin II infusion on
rhythmic clock gene expression and local renin-angiotensin system in the aorta of
Wistar rats. Endocr Regul. 2014 Jul;48(3):144-51. doi: 10.4149/endo_2014_03_144. PMID:
25110213.
Soltésová D, Veselá A, Mravec B, Herichová I. Daily profile of glut1 and glut4 expression
in tissues inside and outside the blood-brain barrier in control and streptozotocin-treated
rats. Physiol Res. 2013;62(Suppl 1):S115-24. doi: 10.33549/physiolres.932596. PMID:
24329691.
Herichová I, Šoltésová D, Szántóová K, Mravec B, Neupauerová D, Veselá A, Zeman M.
Effect of angiotensin II on rhythmic per2 expression in the suprachiasmatic nucleus
and heart and daily rhythm of activity in Wistar rats. Regul Pept. 2013 Sep 10;186:49-56.
doi: 10.1016/j.regpep.2013.06.016. Epub 2013 Jul 12. PMID: 23850797.
Šoltésová D, Monošíková J, Koyšová L, Veselá A, Mravec B, Herichová I. Effect of streptozotocin-induced
diabetes on clock gene expression in tissues inside and outside the blood-brain barrier
in rat. Exp Clin Endocrinol Diabetes. 2013 Aug;121(8):466-74. doi: 10.1055/s-0033-1349123.
Epub 2013 Jul 17. PMID: 23864491.
Szántóová K, Zeman M, Veselá A, Herichová I. Effect of phase delay lighting rotation
schedule on daily expression of per2, bmal1, rev-erbα, pparα, and pdk4 genes in the
heart and liver of Wistar rats. Mol Cell Biochem. 2011 Feb;348(1-2):53-60. doi: 10.1007/s11010-010-0636-x.
Epub 2010 Nov 14. PMID: 21076970.
Benova M, Herichova I, Stebelova K, Paulis L, Krajcirovicova K, Simko F, Zeman M.
Effect of L-NAME-induced hypertension on melatonin receptors and melatonin levels
in the pineal gland and the peripheral organs of rats. Hypertens Res. 2009 Apr;32(4):242-7.
doi: 10.1038/hr.2009.12. Epub 2009 Feb 27. PMID: 19262491.
Monosíková J, Zeman M, Veselá A, Herichová I. Down regulation of angiotensin II receptor
AT1 expression in the pancreas of diabetic rat. Exp Clin Endocrinol Diabetes. 2009
Sep;117(8):438-9. doi: 10.1055/s-0029-1216376. Epub 2009 May 26. PMID: 19472100.
Monosíková J, Herichová I, Mravec B, Kiss A, Zeman M. Effect of upregulated renin-angiotensin
system on per2 and bmal1 gene expression in brain structures involved in blood pressure
control in TGR(mREN-2)27 rats. Brain Res. 2007 Nov 14;1180:29-38. doi: 10.1016/j.brainres.2007.08.061.
Epub 2007 Sep 6. Erratum in: Brain Res. 2009 Jan 28;1251:296-7. PMID: 17915197.
Zeman M, Szántóová K, Stebelová K, Mravec B, Herichová I. Effect of rhythmic melatonin
administration on clock gene expression in the suprachiasmatic nucleus and the heart
of hypertensive TGR(mRen2)27 rats. J Hypertens Suppl. 2009 Aug;27(6):S21-6. doi: 10.1097/01.hjh.0000358833.41181.f6.
PMID: 19633447.
Zeman M, Szántóová K, Herichová I. Ontogeny of circadian oscillations in the heart
and liver in chicken. Comp Biochem Physiol A Mol Integr Physiol. 2009 Sep;154(1):78-83.
doi: 10.1016/j.cbpa.2009.05.005. Epub 2009 May 15. PMID: 19447189.
Zeman M, Petrák J, Stebelová K, Nagy G, Krizanova O, Herichová I, Kvetnanský R. Endocrine
rhythms and expression of selected genes in the brain, stellate ganglia, and adrenals
of hypertensive TGR rats. Ann N Y Acad Sci. 2008 Dec;1148:308-16. doi: 10.1196/annals.1410.069.
PMID: 19120123.
Herichová I, Monosíková J, Zeman M. Ontogeny of melatonin, Per2 and E4bp4 light responsiveness
in the chicken embryonic pineal gland. Comp Biochem Physiol A Mol Integr Physiol.
2008 Jan;149(1):44-50. doi: 10.1016/j.cbpa.2007.10.006. Epub 2007 Oct 11. PMID: 17996471.
Monosíková J, Herichová I, Mravec B, Kiss A, Zeman M. Effect of upregulated renin-angiotensin
system on per2 and bmal1 gene expression in brain structures involved in blood pressure
control in TGR(mREN-2)27 rats. Brain Res. 2007 Nov 14;1180:29-38. doi: 10.1016/j.brainres.2007.08.061.
Epub 2007 Sep 6. Erratum in: Brain Res. 2009 Jan 28;1251:296-7. PMID: 17915197.
Herichová I, Mravec B, Stebelová K, Krizanová O, Jurkovicová D, Kvetnanský R, Zeman
M. Rhythmic clock gene expression in heart, kidney and some brain nuclei involved
in blood pressure control in hypertensive TGR(mREN-2)27 rats. Mol Cell Biochem. 2007
Feb;296(1-2):25-34. doi: 10.1007/s11010-006-9294-4. Epub 2006 Aug 15. PMID: 16909304.
Herichová I, Zeman M, Stebelová K, Ravingerová T. Effect of streptozotocin-induced
diabetes on daily expression of per2 and dbp in the heart and liver and melatonin
rhythm in the pineal gland of Wistar rat. Mol Cell Biochem. 2005 Feb;270(1-2):223-9.
doi: 10.1007/s11010-005-5323-y. PMID: 15792371.
Seres J, Herichová I, Roman O, Bornstein S, Jurcovicová J. Evidence for daily rhythms
of the expression of proopiomelanocortin, interleukin-1-beta and interleukin-6 in
adenopituitaries of male long-evans rats: effect of adjuvant arthritis. Neuroimmunomodulation.
2004;11(5):316-22. doi: 10.1159/000079412. PMID: 15316242.
Roman O, Seres J, Herichova I, Zeman M, Jurcovicova J. Daily profiles of plasma prolactin
(PRL), growth hormone (GH), insulin-like growth factor-1 (IGF-1), luteinizing hormone
(LH), testosterone, and melatonin, and of pituitary PRL mRNA and GH mRNA in male Long
Evans rats in acute phase of adjuvant arthritis. Chronobiol Int. 2003 Sep;20(5):823-36.
doi: 10.1081/cbi-120021085. PMID: 14535356.
10. U6 was used for normalization (Line 215). U6 is better to be the housekeeping
gene for normalization of small RNA. For gene expression, the housekeeping gene should
better be Gapdh or Actb.
We used U6 to normalise gene expression in our previous studies since U6 shows constant
expression during 24h cycle.
Herichova I, Reis R, Hasakova K, Vician M, Zeman M. Sex-dependent regulation of estrogen
receptor beta in human colorectal cancer tissue and its relationship with clock genes
and VEGF-A expression. Physiol Res. 2019 Dec 20;68(Suppl 3):S297-S305.
Hasakova K, Vician M, Reis R, Zeman M, Herichova I. Sex-dependent correlation between
survival and expression of genes related to the circadian oscillator in patients with
colorectal cancer. Chronobiol Int. 2018 Sep;35(10):1423-1434.
Hasakova K, Vician M, Reis R, Zeman M, Herichova I. The expression of clock genes
cry1 and cry2 in human colorectal cancer and tumor adjacent tissues correlates differently
dependent on tumor location. Neoplasma. 2018 Nov 15;65(6):986-992.
U6 is transcribed similarly like mRNA and its length allows use random hexamers in
transcription. Therefore, methodologically, it can provide internal control for mRNA
transcription. However, we appreciate reviewer suggestion and added another housekeeper
– beta actin, which was measured in both tissues and cells and used for normalisation
along with U6. All graphs have been changed accordingly and statistics was calculated
using new data. Using both housekeepers certainly improves quality of results. Information
about using two housekeepers was added into chapter „Methods“.
11. Figure 1D: Statistical significant difference was not presented in the chart,
which was different from the statement “ACE2 mRNA expression in the colon was significantly
increased in rats exposed to E2 treatment (line 270-273).
- thank you for the comment. The difference mentioned in lines 270-273 of former MS
version is related to comparison of all control vs. all E2 treated animals and finding,
that E2 increased overall ACE2 mRNA expression in colon. As we did not find a decent
way how to put this information into the graph (too long text), we changed S2 Table
and provided an overview of statistical analysis in additional column. The title of
S2 Table has been changed accordingly.
12. Figure 3: Statistical significant differences were not marked in the charts.
- thank you for the comment, results of Cosinor analysis were added in all graphs
as requested.
13. The authors need to explain how the daily rhythm of the gene expression could
be related to the SARS-CoV-2 infection and the COVID-19 treatment.
Thank you for the comment. Text bellow was incorporated into chapter “Discussion”
(line 604 – 610):
Daily rhythms in ACE2, ADAM17 and TMPRSS2 mRNA expression can directly influence SARS-CoV-2
entrance into the cell during the 24-h cycle as ACE2 and TMPRSS2 facilitate this process
while ADAM17 decreases levels of membrane-bound ACE2 and in this way inhibits process
of SARS-CoV-2 cell entry. Changes in BMAL1 expression have also been associated with
efficiency of virulence. Our results implicate that E2 has capacity to influence these
processes and therefore its use in Covid-19 treatment should be evaluated with respect
to daily pattern in expression of E2-responsive molecules.
Moreover, there is a paragraph in chapter “Discussion” (lines 528 – 538) describing
in detail effect of changed BMAL1 mRNA expression on virulence and symptoms related
to Covid-19 disease:
“It was shown that bmal1 deletion disrupted glucocorticoid signalling on the CXCL5
promoter and reduced the efficiency of bacterial clearance in lung epithelial club
cells [73]. Several studies reported that bmal1 deletion contributes significantly
to lung damage and inflammation in response to viral infection [34]. Involvement of
BMAL1 in the regulation of neutrophil infiltration and course of influenza infection
has been demonstrated lately in knockout animal and cell models. Deletion of bmal1
from the genome causes, in addition to pronounced changes in the rhythmic transcriptome,
elevated pulmonary neutrophilia and a deregulation of reaction to inflammatory stimuli
[46]. Majority of the in vivo and in vitro experimental evidence implicates an association
of low levels of BMAL1 with enhanced viral virulence [74,75]. On the other hand, it
was reported that silencing or pharmacological inhibition of BMAL1 reduces SARS-CoV-2
cell entry and replication in human lung Calu-3 epithelial cells [3].”
The role of the circadian system in the Covid-19 management is supported by references
73 - 80 in the chapter “Discussion” (lines 546 – 549):
“…the immune system is under the control of the circadian system [73,76], a proper
synchronisation of organ systems could at least contribute to effective timing of
anti-inflammatory drugs. In addition, the role of the circadian system in the treatment
of viral infection and Covid-19 specifically issue from experimental as well as epidemiological
evidence [77–80].”
14. Line 387-388: The references for the published experimental evidences need to
be cited.
- thank you for the comment. We apologise for misunderstanding. A sentence from lines
387-388 (former MS) was just supposed to introduce next 4 paragraphs (lines 418 –
458 recent MS) discussing our results and results of references 30 and 50-58. To avoid
this misunderstanding sentence “Unfortunately, previously published experimental evidence
focused on the effect of E2 on the expression of ACE2, ADAM17 and TMPRSS2 is rather
scarce and not completely consistent.“ was deleted from revised version of MS. All
references were preserved.
15. The authors should also confirm the results in the female rats as the estradiol
hormone therapy was given to the female subjects (Line 138-139).
- sentence from lines 138-139 “Part of the rationale for why increased E2 levels can
exert a beneficial effect after SARS-CoV-2 infection issues from studies employing
probands subjected to estradiol hormone therapy (EHT). It was shown that in women
above 50 years of age, EHT reduced the risk of fatality from Covid-19 by more than
50%.“ referring to the paper of Seeland at al., 2020 was used just to show origins
of idea that E2 administration to Covid-19 patients could be beneficial. However,
in clinical trials focused on possible E2 benfits for Covid-19 patients, both sexes
are being tested (e.g. NCT04865029 and NCT04853069). The major goal of recent MS is
to reveal if E2 administration to males can change their internal milliou in such
a way that males would be able coop with Covid-19 disease better. Our results implicate
that expression of genes coding molecules determing efficientcy of SARS-CoV-2 spread
are influenced by E2 and that single point study cannot reveal complexity of this
regulation.
Reviewer #3:
To Academic Editor of PLOS One
The manuscript # PONE-D-22-06577 entitled "Effect of 17β-estradiol on the daily pattern
of ACE2, ADAM17, TMPRSS2 and estradiol receptor expression in the lungs and colon
of rats" by Herichova I et al.
The authors hypothesized that a better Covid-19 survival rate in females can be attributed
to the presence of higher 17β-estradiol (E2) levels in women than in men. Based on
the fact that cellular entry of SARS-CoV-2 virus is facilitated by the use of ACE2
and the expression of several RAAS components has been shown to exert a rhythmic pattern;
the authors aimed to elucidate possible interference between E2 signaling and the
circadian system in the regulation of expression of ACE2 mRNA and functionally related
molecules. They found that following E2 administration, a rhythmic pattern in molecules
facilitating SARS-CoV-2 entry into the cell, clock genes and E2 receptor. They concluded
that the daily pattern of components of the SARS-CoV-2 entrance pathway and their
responsiveness to E2 should be considered in the timing of pharmacological therapy
for Covid-19.
Analysis of the research:
1. Hypothesis rationale: Comprehensive and is based on their experience and other
studies.
2. Study design: The power of the study to identify > 5% change among the study groups.
The Protocol and the methods are well designed.
3. Results: Very interesting and coincide with the accumulating knowledge of gender
differences in the prognosis of COVID patients.
4. Discussion: the discussion is comprehensive and includes comparisons to the relevant
articles.
Comments:
1. The introduction is very long and difficult to follow the rationale behind this
interesting study. I recommend to summarize the introduction and focus on the importance
of gender differences on COVID severity and prognosis and the effect of E2 on rhythmic
pattern of molecules facilitating SARS-CoV-2 entry into the cell, clock genes and
E2 receptor.
- thank you for the suggestion. Chapter “Introduction” was shortened substantially.
We focused mainly on the role of E2 in generating gender differences in Covid-19 prognosis.
E2 modulated expression of molecules facilitating SARS-CoV-2 entry into the cell,
clock genes and E2 receptors were pointed out in this context.
2. Recently, a manuscript that was published and showed that in human organoids, hrsACE2
can significantly block early stages of SARS-CoV-2 infections (Monteil V, Kwon H,
Prado P, Hagelkrüys A, Wimmer RA, Stahl M, Leopoldi A, Garreta E, Hurtado Del Pozo
C, Prosper F, Romero JP, Wirnsberger G, Zhang H, Slutsky AS, Conder R, Montserrat
N, Mirazimi A, Penninger JM. Inhibition of SARS-CoV-2 Infections in Engineered Human
Tissues Using Clinical-Grade Soluble Human ACE2. Cell. 2020 May 14;181(4):905-913).
- than you for the suggestion, this interesting article was incorporated in revised
version of MS.
3. The author should clarify whether they examined whole lung lysate or alternatively,
they cultures alveolar epithelial cells.
- thank you for the comment. Gene expression in tissues was analysed in samples from
the whole lungs or colon. Updated MS includes also in vitro study using DLD1 cell
line. Chapter “Methods” was modified to avoid any misunderstanding.
4. Table S2 should be clarified. Data on E2 values should be noted instead of mentioning
"ns".
- thank you for the comment. All results of statistical analysis have been provided.
As Reviewer #2 suggested to use another housekeeping gene, numbers slightly differ
from the previous version of MS, however, major results were not influenced by adding
new housekeeper.
5. Figure 4. The tissue variation doesn't seem, in my eyes, to be circadian.
- thank you for the comment. Figure 4 actually does not show a circadian rhythm. Instead
it shows relative expression of E2 receptors. We changed Figure 4 description to avoid
misunderstanding. In case, that reviewer meant graph D from figure 2 we provide graph
shown together with Cosinor fit and details of statistical analysis.
Graph shows averaged ESR1 mRNA expression (empty circles connected with dotted line)
and SEM together with cosinor fit that significantly corresponds with experimental
data
(P = 0.005, S2 Table).
Best-fit values
m 0.531
a 0.09164
c -0.1678
Std. Error
m 0.01718
a 0.02473
c 0.2603
95% CI (asymptotic)
m 0.4954 to 0.5667
a 0.04035 to 0.1429
c -0.7075 to 0.3719
Goodness of Fit
Degrees of Freedom 22
R squared 0.3843
Sum of Squares 0.1618
Sy.x 0.08576
Constraints
m m > 0
a a > 0
c c < 6.28
Number of points
# of X values 30
# Y values analyzed 25
6. Discussion is too long and doesn't concentrate mainly on the study findings; but
also discuss speculations that may weaken the study. I advise the authors to be concise
and concentrate more on the interesting findings of this study.
- thank you for very much for suggestion. Chapter “Discussion” was rewritten. We did
our best to omit speculations that can be avoided and focused on major findings of
the study.
7. On the conclusion section, I would add a sentence regarding future therapy with
E2 on COVID patients.
- thank you for the comment, text below was added in the section “Conclusions” (line
604 – 610).
“Daily rhythms in ACE2, ADAM17 and TMPRSS2 mRNA expression can directly influence
SARS-CoV-2 entrance into the cell during the 24-h cycle as ACE2 and TMPRSS2 facilitates
this process while ADAM17 decreases levels of membrane bound ACE2 and in this way
inhibits process of SARS-CoV-2 cell entry. Changes in BMAL1 expression have also been
associated with efficiency of virulence (also requested by reviewer #2). Our results
implicate that E2 has capacity to influence these processes and therefore its use
in Covid-19 treatment should be evaluated with respect to daily pattern in expression
of E2-responsive molecules.”
8. Please, go over the English typos and make corrections. For example, line 36 add
"the" between "with" and "use". Same wise on line 40, to add "the" between "of" and
"expression". Line 356, to delete "a" that follows "significant". Line 524, to replace
"is" with "it". Line 594, to correct "influence" to "influenced".
- thank you for the comment, we did our best to correct all typos, whole MS was carefully
controlled.
- Attachments
- Attachment
Submitted filename: Response to Reviewers.doc