CoChat
Sign Up
Cited by 1,198Olfactory and Sensory Function StudiesSARS-CoV-2 and COVID-19 ResearchCOVID-19 Clinical Research Studies

Non-neuronal expression of SARS-CoV-2 entry genes in the olfactory system suggests mechanisms underlying COVID-19-associated anosmia

Open access
Brann, David H., Tsukahara, Tatsuya, Weinreb, Caleb, Lipovsek, Marcela, Van den Berge, Koen, Gong, Boying, Chance, Rebecca, Macaulay, Iain C. +17 more·2020·Science Advances
DOI 10.1126/sciadv.abc5801PMC PMC10715684

Published version of a preprint

Open this paper in CoChat — free accountOpen PDF

Abstract

Altered olfactory function is a common symptom of COVID-19, but its etiology is unknown. A key question is whether SARS-CoV-2 (CoV-2) – the causal agent in COVID-19 – affects olfaction directly, by infecting olfactory sensory neurons or their targets in the olfactory bulb, or indirectly, through perturbation of supporting cells. Here we identify cell types in the olfactory epithelium and olfactory bulb that express SARS-CoV-2 cell entry molecules. Bulk sequencing demonstrated that mouse, non-human primate and human olfactory mucosa expresses two key genes involved in CoV-2 entry, ACE2 and TMPRSS2. However, single cell sequencing revealed that ACE2 is expressed in support cells, stem cells, and perivascular cells, rather than in neurons. Immunostaining confirmed these results and revealed pervasive expression of ACE2 protein in dorsally-located olfactory epithelial sustentacular cells and olfactory bulb pericytes in the mouse. These findings suggest that CoV-2 infection of non-neuronal cell types leads to anosmia and related disturbances in odor perception in COVID-19 patients.

AI snapshot

Written from the paper’s full text.

TL;DR

Single-cell and bulk RNA-seq plus immunostaining show that SARS-CoV-2 entry genes ACE2 and TMPRSS2 are expressed in non-neuronal cells of the olfactory epithelium and bulb—support cells, stem cells, Bowman’s gland cells, and pericytes—but not in olfactory sensory neurons or bulb neurons. The authors propose that infection of these non-neuronal cell types, rather than direct neuronal infection, may underlie COVID-19-associated anosmia.

Key findings

  • In human nasal scSeq (33,358 cells), ACE2 was detected in 295 cells and TMPRSS2 in 4,953 cells; among 865 immature and mature OSNs, none expressed ACE2 and only 2 (0.23%) expressed TMPRSS2. ACE2 was detected in sustentacular cells (2.9%), olfactory HBCs (0.8%), respiratory HBCs (1.7%), respiratory ciliated cells (3.6%), and respiratory secretory cells (3.9%).
  • In mouse whole olfactory mucosa Drop-seq (29,585 cells), Ace2 was absent from all 17,666 mature OSNs and all 4,674 immature OSNs, but was found in Bowman’s gland cells, olfactory HBCs, and dorsal sustentacular cells; an independent 10x dataset found Ace2 in 2 of 28,769 mature OSNs.
  • In mouse olfactory bulb scSeq (nearly 50,000 single cells integrated), Ace2 was absent from OB neurons and detected only in vascular cells, predominantly pericytes; Tmprss2 was not expressed in the OB.
  • After methimazole injury, activated HBCs expressed Ace2 at higher levels than resting HBCs; in a Smart-seq2 lineage dataset, Ace2 was detected in >0.7% of GBCs, nearly 2% of activated HBCs, and nearly 3% of sustentacular cells, but not in OSNs. Immunostaining confirmed ACE2 protein in dorsal OE sustentacular microvilli, Bowman’s gland cells, and OB pericytes, but not in OSNs or OB neurons.

Methods

  • The study analyzed published and newly generated bulk RNA-seq and single-cell RNA-seq datasets from mouse, macaque, marmoset, and human olfactory mucosa, human nasal biopsy scSeq (Durante et al., GSE139522), mouse whole olfactory mucosa Drop-seq, mouse 10x scSeq, mouse HBC lineage-tracing scSeq, mouse olfactory bulb Drop-seq integrated with a published OB dataset, and Smart-seq2 sequencing of manually sorted OB dopaminergic neurons.
  • Expression of CoV-2 entry genes was quantified per cell type using normalized UMI counts, clustering, UMAP visualization, marker-gene annotation, and percentage of cells with at least one transcript; co-expression of ACE2 and TMPRSS2 was tested with Fisher’s exact test (odds ratio 7.088, p=3.74E-57).
  • Immunostaining and RNAscope were used in mouse and human tissue to validate transcript findings at the protein level, including ACE2 co-staining with cell-type markers in olfactory epithelium, olfactory bulb, and methimazole-injured tissue.

Limitations

  • The paper states that other molecules such as BSG may enable CoV-2 entry independently of ACE2, that ACE2 expression below detection could still permit infection, that syncytia between infected and ACE2-negative cells could damage adjacent neurons, and that inflammation can induce ACE2, so the survey may under-represent ACE2-expressing cell types.
  • The paper states that definitive identification of mechanisms underlying COVID-19-mediated anosmia will require additional research, and that inconsistencies remain about ACE2 expression in human neurons/glia and CoV-2 presence in the brain across models.
  • Evident from the text: the study is largely expression-based and does not directly demonstrate SARS-CoV-2 infection of the identified cells; key protein and injury data are from mouse, with human data limited to biopsy samples and scSeq that the authors note substantially underestimates the fraction of cells expressing Ace2 transcript.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1126/sciadv.abc5801

Cites

134 of 134 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 104 cited sources checked · 0 supported · 0 flagged

  • Findingcitation

    As in humans, Ace2 and other CoV-2 entry-related genes are expressed in the mouse olfactory epithelium.

    “These data demonstrate that, as in humans, Ace2 and other CoV-2 entry-related genes are expressed in the mouse olfactory epithelium.”
    Quote in the paperSubstantial claim
  • Findingown data

    Immunostaining of human olfactory epithelium biopsy tissue revealed an absence of ACE2 protein in olfactory sensory neurons.

    “We confirmed the expression of ACE2 proteins via immunostaining of human olfactory epithelium biopsy tissue, which revealed expression in sustentacular and basal cells, and an absence of ACE2 protein in OSNs (Figs. 2E and S2).”
    Quote in the paperSubstantial claim
  • Findingcitation

    After alignment, human olfactory epithelium sustentacular cells were found to express ACE2 and TMPRSS2 at levels similar to those observed in the remainder of the non-nasal respiratory tract.

    “after alignment, human OE sustentacular cells were found to express ACE2 and TMPRSS2 at levels similar to those observed in the remainder of the non-nasal respiratory tract (44) (Fig. 3C).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [44] W. Sungnak, N. Huang, C. Bécavin, M. Berg, H. C. A. L. B. Network, SARS-CoV-2 entry genes are most highly expressed in nasal goblet and ciliated cells within human airways. arXiv preprint arXiv:2003.06122, (2020).

      Not checked in the free run.

  • Findingown data

    A two-step alignment procedure identified a correspondence between submucosal gland goblet cells in the respiratory epithelium and Bowman's gland cells in the olfactory epithelium (96% mapping probability).

    “This approach revealed a correspondences between submucosal gland goblet cells in the RE and Bowman’s gland cells in the OE (96% mapping probability, see Methods)”
    Quote in the paperSubstantial claim
  • Findingcitation

    In human olfactory epithelium scSeq, ACE2 and TMPRSS2 were detected in sustentacular cells and horizontal basal cells.

    “Neither ACE2 nor TMPRSS2 were detected in mature OSNs, whereas these genes were detected in both sustentacular cells and HBCs (Fig. 2A-D and S1B-E).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [38] M. A. Durante, S. Kurtenbach, Z. B. Sargi, J. W. Harbour, R. Choi, S. Kurtenbach, G. M. Goss, H. Matsunami, B. J. Goldstein, Single-cell analysis of olfactory neurogenesis and differentiation in adult humans. Nat. Neurosci. 23, 323– 326 (2020). 10.1038/s41593-020-0587-9 32066986 PMC7065961 source

      Not checked in the free run.

  • May combine two claims. 36
  • Classified as probably not a substantial claim. 46
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingcitation

    In mouse whole olfactory mucosa, genes used for cell entry by other coronaviruses (except St3gal4) were expressed and de-enriched in purified olfactory sensory neurons.

    “Genes used for cell entry by other CoVs (except St3gal4) were also expressed in WOM, and de-enriched in purified OSNs.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [46] L. R. Saraiva, X. Ibarra-Soria, M. Khan, M. Omura, A. Scialdone, P. Mombaerts, J. C. Marioni, D. W. Logan, Hierarchical deconstruction of mouse olfactory sensory neurons: From whole mucosa to single-cell RNA-seq. Sci. Rep. 5, 18178 (2015). 10.1038/srep18178 26670777 PMC4680959

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    In purified mouse olfactory sensory neuron samples, expression of Ace2, Tmprss2, and cathepsins (with the exception of Ctsb) was much lower, and Ace2 expression was nearly absent.

    “However, expression of these genes (with the exception of Ctsb) was much lower and Ace2 expression was nearly absent in purified OSN samples (Figs. 4A and S4A, see Legend for counts).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [46] L. R. Saraiva, X. Ibarra-Soria, M. Khan, M. Omura, A. Scialdone, P. Mombaerts, J. C. Marioni, D. W. Logan, Hierarchical deconstruction of mouse olfactory sensory neurons: From whole mucosa to single-cell RNA-seq. Sci. Rep. 5, 18178 (2015). 10.1038/srep18178 26670777 PMC4680959

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Key CoV-2 entry-related genes are present in specific cell types in the human olfactory epithelium, but at lower levels of expression than in respiratory epithelium isolated from human nasal mucosa.

    “These results demonstrate the presence of key CoV-2 entry-related genes in specific cell types in the OE, but at lower levels of expression than in RE isolated from the human nasal mucosa.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    In human olfactory epithelium, sustentacular cells exhibited the highest frequency of ACE2 expression in the OE (2.9% of cells), although this frequency was slightly lower than that in respiratory ciliated and secretory cells (3.6% and 3.9%, respectively).

    “Sustentacular cells exhibited the highest frequency of ACE2 expression in the OE (2.9% of cells) although this frequency was slightly lower than that observed in respiratory ciliated and secretory cells (3.6% and 3.9%, respectively).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [38] M. A. Durante, S. Kurtenbach, Z. B. Sargi, J. W. Harbour, R. Choi, S. Kurtenbach, G. M. Goss, H. Matsunami, B. J. Goldstein, Single-cell analysis of olfactory neurogenesis and differentiation in adult humans. Nat. Neurosci. 23, 323– 326 (2020). 10.1038/s41593-020-0587-9 32066986 PMC7065961

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    In mouse whole olfactory mucosa, Ace2 and Tmprss2 were expressed, as were cathepsins Ctsb and Ctsl.

    “The CoV-2 receptor Ace2 and the protease Tmprss2 were expressed in WOM, as were the cathepsins Ctsb and Ctsl (Figs. 4A and S4A) (46).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [46] L. R. Saraiva, X. Ibarra-Soria, M. Khan, M. Omura, A. Scialdone, P. Mombaerts, J. C. Marioni, D. W. Logan, Hierarchical deconstruction of mouse olfactory sensory neurons: From whole mucosa to single-cell RNA-seq. Sci. Rep. 5, 18178 (2015). 10.1038/srep18178 26670777 PMC4680959

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    In human olfactory epithelium, all HBC subtypes expressed ACE2, but the frequency of ACE2 expression was lower in olfactory HBCs (0.8% of cells) than in respiratory HBCs (1.7% of cells).

    “While all HBC subtypes expressed ACE2, the frequency of expression of ACE2 was lower in olfactory HBCs (0.8% of cells) compared to respiratory HBCs (1.7% of cells) (Fig. 2D).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] M. A. Durante, S. Kurtenbach, Z. B. Sargi, J. W. Harbour, R. Choi, S. Kurtenbach, G. M. Goss, H. Matsunami, B. J. Goldstein, Single-cell analysis of olfactory neurogenesis and differentiation in adult humans. Nat. Neurosci. 23, 323– 326 (2020). 10.1038/s41593-020-0587-9 32066986 PMC7065961

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    In mouse whole olfactory mucosa scSeq (Drop-seq), Ace2 and Tmprss2 were expressed in a fraction of sustentacular and Bowman's gland cells, and a very small fraction of stem cells, but not in olfactory sensory neurons (zero of 17,666 identified mature OSNs).

    “Ace2 and Tmprss2 were expressed in a fraction of sustentacular and Bowman’s gland cells, and a very small fraction of stem cells, but not in OSNs (zero of 17,666 identified mature OSNs, Figs. 4C and S4C-D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    An independent mouse scSeq dataset (10x Chromium) confirmed that olfactory sensory neurons did not express Ace2 (2 of 28,769 mature OSNs were positive for Ace2), while expression was observed in a fraction of Bowman's gland cells and HBCs.

    “An independent mouse scSeq data set (obtained using the 10x Chromium platform, see Methods) confirmed that olfactory sensory neurons did not express Ace2 (2 of 28,769 mature OSNs were positive for Ace2), while expression was observed in a fraction of Bowman’s gland cells and HBCs (Figure S5, see Methods).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Staining of mouse whole olfactory mucosa showed ACE2 protein in Bowman's gland cells but not in olfactory sensory neurons, and in subsets of respiratory epithelium cells.

    “Staining was also observed in Bowman’s gland cells but not in OSNs, and in subsets of RE cells (Fig. 5F-G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In mouse olfactory bulb, although other potential CoV proteases were expressed, Tmprss2 was not expressed.

    “Although other potential CoV proteases were expressed in the OB, Tmprss2 was not expressed.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In mouse olfactory bulb scSeq, Ace2 expression was absent from olfactory bulb neurons and was observed only in vascular cells, predominantly pericytes.

    “This analysis revealed that Ace2 expression was absent from OB neurons and instead was observed only in vascular cells, predominantly in pericytes, which are involved in blood pressure regulation, maintenance of the blood-brain barrier, and inflammatory responses (51) (Figs. 7A-D and S7-8).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Immunostaining in the mouse olfactory bulb revealed that blood vessels expressed high levels of ACE2 protein, particularly in pericytes; nearly all pericytes exhibited some degree of staining with ACE2 antibodies.

    “Immunostaining in the OB revealed that blood vessels expressed high levels of ACE2 protein, particularly in pericytes; indeed nearly all pericytes exhibited some degree of staining with ACE2 antibodies.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Staining of mouse whole olfactory mucosa with anti-ACE2 antibodies confirmed that ACE2 protein is expressed in sustentacular cells and is specifically localized to the sustentacular cell microvilli.

    “Staining of the mouse WOM with anti-ACE2 antibodies confirmed that ACE2 protein is expressed in sustentacular cells and is specifically localized to the sustentacular cell microvilli (Fig. 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ACE2-positive sustentacular cells were identified exclusively within the dorsal subregion of the mouse olfactory epithelium; within that region many (and possibly all) sustentacular cells expressed ACE2.

    “ACE2-positive sustentacular cells were identified exclusively within the dorsal subregion of the OE; critically, within that region many (and possibly all) sustentacular cells expressed ACE2 (Fig. 5B-E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    In a re-analyzed mouse HBC lineage tracing dataset, Ace2 was detected in more than 0.7% of GBCs, nearly 2% of activated HBCs, and nearly 3% of sustentacular cells, but was not detected in OSNs.

    “In this dataset, Ace2 was detected in more than 0.7% of GBCs, nearly 2% of activated HBCs and nearly 3% of sustentacular cells but was not detected in OSNs (Figures S6B).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [36] R. B. Fletcher, D. Das, L. Gadye, K. N. Street, A. Baudhuin, A. Wagner, M. B. Cole, Q. Flores, Y. G. Choi, N. Yosef, E. Purdom, S. Dudoit, D. Risso, J. Ngai, Deconstructing Olfactory Stem Cell Trajectories at Single-Cell Resolution. Cell Stem Cell 20, 817– 830.e8 (2017). 10.1016/j.stem.2017.04.003 28506465 PMC5484588

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Mouse vascular pericytes in the olfactory bulb express ACE2, while we did not detect ACE2 in olfactory bulb neurons.

    “Similarly, mouse vascular pericytes in the OB express ACE2, while we did not detect ACE2 in OB neurons.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Nearly all vascular pericytes expressed ACE2 protein, although only a fraction of olfactory bulb pericytes were positive for Ace2 transcripts by scSeq.

    “Similarly, nearly all vascular pericytes also expressed ACE2 protein, although only a fraction of OB pericytes were positive for Ace2 transcripts when assessed using scSeq.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In mouse olfactory epithelium after injury, Sult1c1-positive sustentacular cells have higher levels of Ace2 (p=1.87E-03, Mann-Whitney test) and Ace2-positive sustentacular cells have higher levels of Sult1c1 (p=8.06E-07, Mann-Whitney test).

    “Sult1c1-positive sustentacular cells have higher levels of Ace2 (p=1.87E-03, Mann-Whitney test) and Ace2-positive sustentacular cells have higher levels of Sult1c1 (p=8.06E-07, Mann-Whitney test).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    In human scSeq data, ACE2 transcripts were detected in only a subset of olfactory epithelium cells, and this low level of observed expression matches or exceeds that observed in respiratory cell types that are infected by CoV-2 in COVID-19 patients.

    “Although scSeq revealed ACE2 transcripts in only a subset of OE cells, this low level of observed expression matches or exceeds that observed in respiratory cells types that are infected by CoV-2 in COVID-19 patients (39) (Fig. 3).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [39] W. Sungnak, N. Huang, C. Bécavin, M. Berg, R. Queen, M. Litvinukova, C. Talavera-López, H. Maatz, D. Reichart, F. Sampaziotis, K. B. Worlock, M. Yoshida, J. L. Barnes; HCA Lung Biological Network, SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nat. Med. 26, 681– 687 (2020). 10.1038/s41591-020-0868-6 32327758 PMC8637938 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    At the cell type level, Ace2 mRNA expression accurately predicted presence of ACE2 protein, and Ace2 transcript-negative cell types, including olfactory sensory neurons, did not express ACE2 protein.

    “Although Ace2 transcripts were more rarely detected than protein, there was a clear concordance at the cell type level: expression of Ace2 mRNA in a particular cell type accurately predicted the presence of ACE2 protein, while Ace2 transcript-negative cell types (including OSNs) did not express ACE2 protein.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    295 cells expressed ACE2 and 4953 cells expressed TMPRSS2.

    “295 cells expressed ACE2 and 4953 cells expressed TMPRSS2.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The filtered human scSeq dataset contained 33358 cells.

    “The filtered human scSeq dataset contained 33358 cells.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    A small cluster of excitatory neurons (cluster 13) in the whole olfactory bulb dataset contained neurons from the anterior olfactory nucleus.

    “As previously reported, a small cluster of excitatory neurons (cluster 13) contained neurons from the anterior olfactory nucleus.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    In the manually sorted olfactory bulb dopaminergic neuron dataset, all observed effects were independent of the naris occlusion/sham treatment groups.

    “all observed effects here were independent of these treatment groups.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    ACE2 was reliably detected in at least 2% and TMPRSS2 was expressed in close to 50% of multiple respiratory epithelial subtypes.

    “In contrast, ACE2 was reliably detected in at least 2% and TMPRSS2 was expressed in close to 50% of multiple respiratory epithelial subtypes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Of the 865 identified olfactory sensory neurons, none expressed ACE2 and only 2 (0.23%) expressed TMPRSS2.

    “Of the 865 identified OSNs, including both immature and mature cells, none of the cells express ACE2 and only 2 (0.23%) expressed TMPRSS2.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Interpretationreasoning

    Because CoV-2 can infect cells in the lower respiratory tract, the finding that human olfactory epithelium sustentacular cells express ACE2 at levels similar to the non-nasal respiratory tract is consistent with the possibility that these cells express ACE2 at a level permissive for direct infection.

    “As CoV-2 can infect cells in the lower respiratory tract (40, 45), these results are consistent with the possibility that specific cell types in the human olfactory epithelium express ACE2 at a level that is permissive for direct infection.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [40] Y. J. Hou, K. Okuda, C. E. Edwards, D. R. Martinez, T. Asakura, K. H. Dinnon 3rd, T. Kato, R. E. Lee, B. L. Yount, T. M. Mascenik, G. Chen, K. N. Olivier, A. Ghio, L. V. Tse, S. R. Leist, L. E. Gralinski, A. Schäfer, H. Dang, R. Gilmore, S. Nakano, L. Sun, M. L. Fulcher, A. Livraghi-Butrico, N. I. Nicely, M. Cameron, C. Cameron, D. J. Kelvin, A. de Silva, D. M. Margolis, A. Markmann, L. Bartelt, R. Zumwalt, F. J. Martinez, S. P. Salvatore, A. Borczuk, P. R. Tata, V. Sontake, A. Kimple, I. Jaspers, W. K. O’Neal, S. H. Randell, R. C. Boucher, R. S. Baric, SARS-CoV-2 Reverse Genetics Reveals a Va source

      Not checked in the free run.

    • cites [45] N. Zhu, D. Zhang, W. Wang, X. Li, B. Yang, J. Song, X. Zhao, B. Huang, W. Shi, R. Lu, P. Niu, F. Zhan, X. Ma, D. Wang, W. Xu, G. Wu, G. F. Gao, W. Tan; China Novel Coronavirus Investigating and Research Team, A Novel Coronavirus from Patients with Pneumonia in China, 2019. N. Engl. J. Med. 382, 727– 733 (2020). 10.1056/NEJMoa2001017 31978945 PMC7092803 source

      Not checked in the free run.

    • Rests on an assumption: Cells expressing ACE2 and TMPRSS2 are susceptible to SARS-CoV-2 infection, or such expression is sufficient for viral entry.

  • Classified as probably not a substantial claim.

    Premisecitation

    SARS-CoV-2 infects cells through interactions between its spike (S) protein and the ACE2 protein on target cells.

    “CoV-2 — like SARS-CoV — infects cells through interactions between its spike (S) protein and the ACE2 protein on target cells.”
    Quote in the paperSubstantial claim0 of 7 sources checked
    • cites [4] P. Zhou, X.-L. Yang, X.-G. Wang, B. Hu, L. Zhang, W. Zhang, H.-R. Si, Y. Zhu, B. Li, C.-L. Huang, H.-D. Chen, J. Chen, Y. Luo, H. Guo, R.-D. Jiang, M.-Q. Liu, Y. Chen, X.-R. Shen, X. Wang, X.-S. Zheng, K. Zhao, Q.-J. Chen, F. Deng, L.-L. Liu, B. Yan, F.-X. Zhan, Y.-Y. Wang, G.-F. Xiao, Z.-L. Shi, A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270– 273 (2020). 10.1038/s41586-020-2012-7 32015507 PMC7095418 source

      Not checked in the free run.

    • cites [5] M. Ceccarelli, M. Berretta, E. Venanzi Rullo, G. Nunnari, B. Cacopardo, Differences and similarities between Severe Acute Respiratory Syndrome (SARS)-CoronaVirus (CoV) and SARS-CoV-2. Would a rose by another name smell as sweet? Eur. Rev. Med. Pharmacol. Sci. 24, 2781– 2783 (2020). 32196628

      Not checked in the free run.

    • cites [6] A. Zumla, J. F. W. Chan, E. I. Azhar, D. S. C. Hui, K.-Y. Yuen, Coronaviruses - drug discovery and therapeutic options. Nat. Rev. Drug Discov. 15, 327– 347 (2016). 10.1038/nrd.2015.37 26868298 PMC7097181

      Not checked in the free run.

    • cites [22] M. Hoffmann, H. Kleine-Weber, S. Schroeder, N. Krüger, T. Herrler, S. Erichsen, T. S. Schiergens, G. Herrler, N.-H. Wu, A. Nitsche, M. A. Müller, C. Drosten, S. Pöhlmann, SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 271– 280.e8 (2020). 10.1016/j.cell.2020.02.052 32142651 PMC7102627 source

      Not checked in the free run.

    • cites [23] W. Li, M. J. Moore, N. Vasilieva, J. Sui, S. K. Wong, M. A. Berne, M. Somasundaran, J. L. Sullivan, K. Luzuriaga, T. C. Greenough, H. Choe, M. Farzan, Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450– 454 (2003). 10.1038/nature02145 14647384 PMC7095016 source

      Not checked in the free run.

    • cites [24] W. Li, T. C. Greenough, M. J. Moore, N. Vasilieva, M. Somasundaran, J. L. Sullivan, M. Farzan, H. Choe, Efficient replication of severe acute respiratory syndrome coronavirus in mouse cells is limited by murine angiotensin-converting enzyme 2. J. Virol. 78, 11429– 11433 (2004). 10.1128/JVI.78.20.11429-11433.2004 15452268 PMC521845

      Not checked in the free run.

    • cites [25] K. Kuba, Y. Imai, S. Rao, H. Gao, F. Guo, B. Guan, Y. Huan, P. Yang, Y. Zhang, W. Deng, L. Bao, B. Zhang, G. Liu, Z. Wang, M. Chappell, Y. Liu, D. Zheng, A. Leibbrandt, T. Wada, A. S. Slutsky, D. Liu, C. Qin, C. Jiang, J. M. Penninger, A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat. Med. 11, 875– 879 (2005). 10.1038/nm1267 16007097 PMC7095783 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The interaction between SARS-CoV-2 S protein and ACE2 requires cleavage of the S protein, likely by TMPRSS2, although other proteases such as Cathepsin B and L may also be involved.

    “This interaction requires cleavage of the S protein, likely by the cell surface protease TMPRSS2, although other proteases (such as Cathepsin B and L, CTSB/CTSL) may also be involved (4-6, 22-25).”
    Quote in the paperNot a substantial claim0 of 7 sources checked
    • cites [4] P. Zhou, X.-L. Yang, X.-G. Wang, B. Hu, L. Zhang, W. Zhang, H.-R. Si, Y. Zhu, B. Li, C.-L. Huang, H.-D. Chen, J. Chen, Y. Luo, H. Guo, R.-D. Jiang, M.-Q. Liu, Y. Chen, X.-R. Shen, X. Wang, X.-S. Zheng, K. Zhao, Q.-J. Chen, F. Deng, L.-L. Liu, B. Yan, F.-X. Zhan, Y.-Y. Wang, G.-F. Xiao, Z.-L. Shi, A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270– 273 (2020). 10.1038/s41586-020-2012-7 32015507 PMC7095418 source

      Not checked in the free run.

    • cites [5] M. Ceccarelli, M. Berretta, E. Venanzi Rullo, G. Nunnari, B. Cacopardo, Differences and similarities between Severe Acute Respiratory Syndrome (SARS)-CoronaVirus (CoV) and SARS-CoV-2. Would a rose by another name smell as sweet? Eur. Rev. Med. Pharmacol. Sci. 24, 2781– 2783 (2020). 32196628

      Not checked in the free run.

    • cites [6] A. Zumla, J. F. W. Chan, E. I. Azhar, D. S. C. Hui, K.-Y. Yuen, Coronaviruses - drug discovery and therapeutic options. Nat. Rev. Drug Discov. 15, 327– 347 (2016). 10.1038/nrd.2015.37 26868298 PMC7097181

      Not checked in the free run.

    • cites [22] M. Hoffmann, H. Kleine-Weber, S. Schroeder, N. Krüger, T. Herrler, S. Erichsen, T. S. Schiergens, G. Herrler, N.-H. Wu, A. Nitsche, M. A. Müller, C. Drosten, S. Pöhlmann, SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 271– 280.e8 (2020). 10.1016/j.cell.2020.02.052 32142651 PMC7102627 source

      Not checked in the free run.

    • cites [23] W. Li, M. J. Moore, N. Vasilieva, J. Sui, S. K. Wong, M. A. Berne, M. Somasundaran, J. L. Sullivan, K. Luzuriaga, T. C. Greenough, H. Choe, M. Farzan, Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450– 454 (2003). 10.1038/nature02145 14647384 PMC7095016 source

      Not checked in the free run.

    • cites [24] W. Li, T. C. Greenough, M. J. Moore, N. Vasilieva, M. Somasundaran, J. L. Sullivan, M. Farzan, H. Choe, Efficient replication of severe acute respiratory syndrome coronavirus in mouse cells is limited by murine angiotensin-converting enzyme 2. J. Virol. 78, 11429– 11433 (2004). 10.1128/JVI.78.20.11429-11433.2004 15452268 PMC521845

      Not checked in the free run.

    • cites [25] K. Kuba, Y. Imai, S. Rao, H. Gao, F. Guo, B. Guan, Y. Huan, P. Yang, Y. Zhang, W. Deng, L. Bao, B. Zhang, G. Liu, Z. Wang, M. Chappell, Y. Liu, D. Zheng, A. Leibbrandt, T. Wada, A. S. Slutsky, D. Liu, C. Qin, C. Jiang, J. M. Penninger, A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat. Med. 11, 875– 879 (2005). 10.1038/nm1267 16007097 PMC7095783 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Basal progenitor populations with transcriptional profiles similar to rodent globose and horizontal basal cells are present in adult human olfactory epithelium, suggesting closely related homeostatic and injury-response mechanisms.

    “basal progenitor populations with similar transcriptional profiles are present in adult human olfactory epithelium, suggesting closely related homeostatic and injury-response mechanisms (37, 38).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [37] J. E. Schwob, W. Jang, E. H. Holbrook, B. Lin, D. B. Herrick, J. N. Peterson, J. Hewitt Coleman, Stem and progenitor cells of the mammalian olfactory epithelium: Taking poietic license. J. Comp. Neurol. 525, 1034– 1054 (2017). 10.1002/cne.24105 27560601 PMC5805156

      Not checked in the free run.

    • cites [38] M. A. Durante, S. Kurtenbach, Z. B. Sargi, J. W. Harbour, R. Choi, S. Kurtenbach, G. M. Goss, H. Matsunami, B. J. Goldstein, Single-cell analysis of olfactory neurogenesis and differentiation in adult humans. Nat. Neurosci. 23, 323– 326 (2020). 10.1038/s41593-020-0587-9 32066986 PMC7065961

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    The nasal respiratory epithelium has higher expression of CoV-2 entry genes than the respiratory epithelium that lines the trachea or lungs.

    “It was recently reported that the nasal RE has higher expression of CoV-2 entry genes than the RE that lines the trachea or lungs (44)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [44] W. Sungnak, N. Huang, C. Bécavin, M. Berg, H. C. A. L. B. Network, SARS-CoV-2 entry genes are most highly expressed in nasal goblet and ciliated cells within human airways. arXiv preprint arXiv:2003.06122, (2020).

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Cells from the human upper airway, including nasal respiratory epithelium goblet and ciliated cells, express high levels of ACE2 and TMPRSS2, suggesting these respiratory epithelium cell types may serve as a viral reservoir during CoV-2 infection.

    “It has recently been demonstrated through single cell RNA sequencing analysis (referred to herein as scSeq) that cells from the human upper airway — including nasal RE goblet and ciliated cells —express high levels of ACE2 and TMPRSS2, suggesting that these RE cell types may serve as a viral reservoir during CoV-2 infection (39, 40).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [39] W. Sungnak, N. Huang, C. Bécavin, M. Berg, R. Queen, M. Litvinukova, C. Talavera-López, H. Maatz, D. Reichart, F. Sampaziotis, K. B. Worlock, M. Yoshida, J. L. Barnes; HCA Lung Biological Network, SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nat. Med. 26, 681– 687 (2020). 10.1038/s41591-020-0868-6 32327758 PMC8637938 source

      Not checked in the free run.

    • cites [40] Y. J. Hou, K. Okuda, C. E. Edwards, D. R. Martinez, T. Asakura, K. H. Dinnon 3rd, T. Kato, R. E. Lee, B. L. Yount, T. M. Mascenik, G. Chen, K. N. Olivier, A. Ghio, L. V. Tse, S. R. Leist, L. E. Gralinski, A. Schäfer, H. Dang, R. Gilmore, S. Nakano, L. Sun, M. L. Fulcher, A. Livraghi-Butrico, N. I. Nicely, M. Cameron, C. Cameron, D. J. Kelvin, A. de Silva, D. M. Margolis, A. Markmann, L. Bartelt, R. Zumwalt, F. J. Martinez, S. P. Salvatore, A. Borczuk, P. R. Tata, V. Sontake, A. Kimple, I. Jaspers, W. K. O’Neal, S. H. Randell, R. C. Boucher, R. S. Baric, SARS-CoV-2 Reverse Genetics Reveals a Va source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Recent findings in respiratory epithelium suggest scSeq substantially underestimates the fraction of a given cell type that expresses the Ace2 transcript, but new Ace2-expressing cell types are not discovered with more sensitive forms of analysis.

    “These observations are consistent with recent findings in the respiratory epithelium suggesting that scSeq substantially underestimates the fraction of a given cell type that expresses the Ace2 transcript, but that “new” Ace2-expressing cell types are not discovered with more sensitive forms of analysis (40).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Y. J. Hou, K. Okuda, C. E. Edwards, D. R. Martinez, T. Asakura, K. H. Dinnon 3rd, T. Kato, R. E. Lee, B. L. Yount, T. M. Mascenik, G. Chen, K. N. Olivier, A. Ghio, L. V. Tse, S. R. Leist, L. E. Gralinski, A. Schäfer, H. Dang, R. Gilmore, S. Nakano, L. Sun, M. L. Fulcher, A. Livraghi-Butrico, N. I. Nicely, M. Cameron, C. Cameron, D. J. Kelvin, A. de Silva, D. M. Margolis, A. Markmann, L. Bartelt, R. Zumwalt, F. J. Martinez, S. P. Salvatore, A. Borczuk, P. R. Tata, V. Sontake, A. Kimple, I. Jaspers, W. K. O’Neal, S. H. Randell, R. C. Boucher, R. S. Baric, SARS-CoV-2 Reverse Genetics Reveals a Va source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Many viruses, including coronaviruses, propagate from nasal epithelium past cribriform plate to infect olfactory bulb; this central infection has been suggested to mediate olfactory deficits even without lasting olfactory epithelium damage.

    “Many viruses, including coronaviruses, have been shown to propagate from the nasal epithelium past the cribriform plate to infect the OB; this form of central infection has been suggested to mediate olfactory deficits, even in the absence of lasting OE damage (60-65).”
    Quote in the paperSubstantial claim0 of 6 sources checked
    • cites [60] K. Bohmwald, N. M. S. Gálvez, M. Ríos, A. M. Kalergis, Neurologic Alterations Due to Respiratory Virus Infections. Front. Cell. Neurosci. 12, 386 (2018). 10.3389/fncel.2018.00386 30416428 PMC6212673 source

      Not checked in the free run.

    • cites [61] S. L. Youngentob, J. E. Schwob, S. Saha, G. Manglapus, B. Jubelt, Functional consequences following infection of the olfactory system by intranasal infusion of the olfactory bulb line variant (OBLV) of mouse hepatitis strain JHM. Chem. Senses 26, 953– 963 (2001). 10.1093/chemse/26.8.953 11595672 PMC7110209

      Not checked in the free run.

    • cites [62] R. L. Doty, Systemic diseases and disorders. Handb. Clin. Neurol. 164, 361– 387 (2019). 10.1016/B978-0-444-63855-7.00021-6 31604558

      Not checked in the free run.

    • cites [63] E. M. Barnett, S. Perlman, The olfactory nerve and not the trigeminal nerve is the major site of CNS entry for mouse hepatitis virus, strain JHM. Virology 194, 185– 191 (1993). 10.1006/viro.1993.1248 8386871

      Not checked in the free run.

    • cites [64] J. E. Schwob, S. Saha, S. L. Youngentob, B. Jubelt, Intranasal inoculation with the olfactory bulb line variant of mouse hepatitis virus causes extensive destruction of the olfactory bulb and accelerated turnover of neurons in the olfactory epithelium of mice. Chem. Senses 26, 937– 952 (2001). 10.1093/chemse/26.8.937 11595671 PMC7110028

      Not checked in the free run.

    • cites [65] J. Netland, D. K. Meyerholz, S. Moore, M. Cassell, S. Perlman, Severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ACE2. J. Virol. 82, 7264– 7275 (2008). 10.1128/JVI.00737-08 18495771 PMC2493326 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Two recent studies in mouse models expressing human ACE2 found CoV-2 in brain after intranasal inoculation, although neither specifically queried olfactory bulb.

    “Additionally, two recent studies in mouse models expressing human ACE2 have found CoV-2 in the brain after intranasal inoculation (67, 68), although neither specifically queried the OB; this work stands in contrast to results in a non-human primate model of COVID-19, in which nasal infection did not lead to the presence of identifiable CoV-2 antigens in the brain (83).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [67] R.-D. Jiang, M.-Q. Liu, Y. Chen, C. Shan, Y.-W. Zhou, X.-R. Shen, Q. Li, L. Zhang, Y. Zhu, H.-R. Si, Q. Wang, J. Min, X. Wang, W. Zhang, B. Li, H.-J. Zhang, R. S. Baric, P. Zhou, X.-L. Yang, Z.-L. Shi, Pathogenesis of SARS-CoV-2 in transgenic mice expressing human angiotensin-converting enzyme 2. Cell 182, 50– 58.e8 (2020). 10.1016/j.cell.2020.05.027 32516571 PMC7241398

      Not checked in the free run.

    • cites [68] S.-H. Sun, Q. Chen, H.-J. Gu, G. Yang, Y.-X. Wang, X.-Y. Huang, S.-S. Liu, N.-N. Zhang, X.-F. Li, R. Xiong, Y. Guo, Y.-Q. Deng, W.-J. Huang, Q. Liu, Q.-M. Liu, Y.-L. Shen, Y. Zhou, X. Yang, T.-Y. Zhao, C.-F. Fan, Y.-S. Zhou, C.-F. Qin, Y.-C. Wang, A mouse model of SARS-CoV-2 infection and pathogenesis. Cell Host Microbe 28, 124– 133.e4 (2020). 10.1016/j.chom.2020.05.020 32485164 PMC7250783

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationreasoning

    Vascular pericytes in cranial nerve I also express ACE2.

    “Although CN I was not directly queried in our datasets, it is reasonable to infer that vascular pericytes in CN I also express ACE2, which suggests a possible route of entry for CoV-2 from the nose into the brain.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Infection of subsets of sustentacular cells by SDAV coronavirus in rats leads to disruption of global architecture of olfactory epithelium, suggesting focal coronavirus infection may be sufficient to cause diffuse epithelial damage.

    “For example, infection of subsets of sustentacular cells by the SDAV coronavirus in rats ultimately leads to disruption of the global architecture of the OE, suggesting that focal coronavirus infection may be sufficient to cause diffuse epithelial damage (56).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [56] C. G. Bihun, D. H. Percy, Morphologic changes in the nasal cavity associated with sialodacryoadenitis virus infection in the Wistar rat. Vet. Pathol. 32, 1– 10 (1995). 10.1177/030098589503200101 7725592

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Given the similarities in ACE2 and TMPRSS2 expression patterns between mouse and human, mouse findings suggest that olfactory bulb neurons are likely not a primary site of infection, but that vascular pericytes may be sensitive to CoV-2.

    “Given the extensive similarities detailed above in expression patterns for ACE2 and TMPRSS2 in the mouse and human, these findings (from mouse experiments) suggest that OB neurons are likely not a primary site of infection, but that vascular pericytes may be sensitive to CoV-2”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Cells expressing ACE2 and TMPRSS2 are susceptible to SARS-CoV-2 infection, or such expression is sufficient for viral entry.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Low level expression of ACE2 can support CoV-2 cell entry.

    “In addition, it has recently been reported that low level expression of ACE2 can support CoV-2 cell entry (86); it is possible, therefore, that ACE2 expression beneath the level of detection in our assays may yet enable CoV-2 infection of apparently ACE2 negative cell types.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [86] M. M. Lamers, SARS-CoV-2 productively infects human gut enterocytes. Science, eabc1669 (2020). PMC7199907 32358202 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    SARS-CoV has been shown to infect olfactory bulb in a transgenic mouse model ectopically expressing human ACE2.

    “Although SARS-CoV has been shown to infect the OB in a transgenic mouse model that ectopically expresses human ACE2 (65), it is unclear to what extent similar results will be observed for CoV-2 in these and in recently-developed mouse models expressing human ACE2 that better recapitulate native expression patterns (67-69).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [65] J. Netland, D. K. Meyerholz, S. Moore, M. Cassell, S. Perlman, Severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ACE2. J. Virol. 82, 7264– 7275 (2008). 10.1128/JVI.00737-08 18495771 PMC2493326 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Activated stem cells recruited during injury express ACE2 and do so at higher levels than resting stem cells.

    “These results demonstrate that activated stem cells recruited during injury express ACE2 and do so at higher levels than those in resting stem cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Two anosmic COVID-19 patients had fMRI-identified hyperintensity in both olfactory bulbs that reverted to normal after resolution of anosmia, consistent with central involvement in at least some cases.

    “Two anosmic COVID-19 patients have presented with fMRI-identified hyperintensity in both OBs that reverted to normal after resolution of the anosmia (58, 59), consistent with central involvement in at least some cases.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [58] L. S. Politi, E. Salsano, M. Grimaldi, Magnetic Resonance Imaging Alteration of the Brain in a Patient With Coronavirus Disease 2019 (COVID-19) and Anosmia. JAMA Neurology, (2020). 32469400 source

      Not checked in the free run.

    • cites [59] T. Laurendon, T. Radulesco, J. Mugnier, M. Gérault, C. Chagnaud, A.-A. El Ahmadi, A. Varoquaux, Bilateral transient olfactory bulbs edema during COVID-19-related anosmia. Neurology 10.1212/WNL.0000000000009850 (2020). 10.1212/WNL.0000000000009850 32444492

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    In the WOM dataset, 281 cells containing mixtures of marker genes with no clear gene expression signature were removed.

    “We removed 281 cells containing mixtures of marker genes with no clear gene expression signature.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Mouse Drop-seq cells with less than 500 UMIs or more than 15,000 UMIs, or higher than 5% mitochondrial genes were removed.

    “Cells with less than 500 UMIs or more than 15,000 UMIs, or higher than 5% mitochondrial genes were removed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    In the manually sorted olfactory bulb dopaminergic neuron dataset, the scTransform function in Seurat was used to remove technical batch effects.

    “The scTransform function in Seurat was used to remove technical batch effects.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Four clusters were removed for quality control, including two with low total counts and two with high mitochondrial counts.

    “Four clusters were removed for quality control, including two with low total counts (likely background) and two with high mitochondrial counts (likely stressed or dying cells).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The whole olfactory bulb clusters corresponded to previous cell class and subtype annotations.

    “the clusters corresponded previous cell class and subtype annotations (50).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [50] A. Zeisel, H. Hochgerner, P. Lönnerberg, A. Johnsson, F. Memic, J. van der Zwan, M. Häring, E. Braun, L. E. Borm, G. La Manno, S. Codeluppi, A. Furlan, K. Lee, N. Skene, K. D. Harris, J. Hjerling-Leffler, E. Arenas, P. Ernfors, U. Marklund, S. Linnarsson, Molecular Architecture of the Mouse Nervous System. Cell 174, 999– 1014.e22 (2018). 10.1016/j.cell.2018.06.021 30096314 PMC6086934 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    In the manually sorted olfactory bulb dopaminergic neuron dataset, positive and negative controls were collected for each sorting session.

    “Positive (more than 10 cells) and negative (sample collection procedure without picking a cell) controls were collected for each sorting session.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    ACE2 expression beneath the level of detection in our assays may enable CoV-2 infection of apparently ACE2 negative cell types.

    “In addition, it has recently been reported that low level expression of ACE2 can support CoV-2 cell entry (86); it is possible, therefore, that ACE2 expression beneath the level of detection in our assays may yet enable CoV-2 infection of apparently ACE2 negative cell types.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    In the adult whole olfactory bulb Drop-seq dataset, cells with low numbers of genes (500), low numbers of UMIs (700) or high numbers of UMIs (>10000) were removed (6% of cells).

    “Cells with low numbers of genes (500), low numbers of UMIs (700) or high numbers of UMIs (>10000) were removed (6% of cells).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Only cell-type correspondences with a high Z-score in both mapping directions (Z-score > 25) were used for downstream analysis.

    “Only cell-type correspondences with a high Z-score in both mapping directions (Z-score > 25) were used for downstream analysis.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The population of olfactory bulb cells labeled in DAT-tdTomato mice includes a minor non-dopaminergic calretinin-positive subgroup.

    “The population of olfactory bulb cells labeled in DAT-tdTomato mice is known to include a minor non-dopaminergic calretinin-positive subgroup (113)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [113] J. Ninkovic, L. Pinto, S. Petricca, A. Lepier, J. Sun, M. A. Rieger, T. Schroeder, A. Cvekl, J. Favor, M. Götz, The transcription factor Pax6 regulates survival of dopaminergic olfactory bulb neurons via crystallin αA. Neuron 68, 682– 694 (2010). 10.1016/j.neuron.2010.09.030 21092858 PMC4388427

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Our survey of ACE2 expression might under-represent cell types that express ACE2 under conditions of CoV-2 infection.

    “Finally, it has recently been reported that inflammation can induce expression of ACE2 in human cells (87, 88). It is therefore possible that our survey of ACE2 expression, and other recent reports demonstrating expression of ACE2 in OE support and stem cells but not neurons (81, 89, 90), might under-represent the cell types that express ACE2 under conditions of CoV-2 infection.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Cluster stability for the integrated whole olfactory bulb dataset was analyzed with Clustree on a range of resolution values (0.4 to 1.4), with 0.6 yielding the most stable set of clusters.

    “Cluster stability was analyzed with Clustree on a range of resolution values (0.4 to 1.4), with 0.6 yielding the most stable set of clusters (107).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [107] L. Zappia, A. Oshlack, Clustering trees: A visualization for evaluating clusterings at multiple resolutions. Gigascience 7, (2018). 10.1093/gigascience/giy083 30010766 PMC6057528 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    In the HBC lineage tracing dataset, 219 cells whose experimentally sampled timepoint did not match their position along the HBC differentiation trajectory were removed.

    “Inspection of the data in a three-dimensional UMAP embedding identified two groups of cells whose experimentally sampled timepoint did not match their position along the HBC differentiation trajectory, and these additional 219 cells were also removed from subsequent analyses.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Inflammation can induce expression of ACE2 in human cells.

    “Finally, it has recently been reported that inflammation can induce expression of ACE2 in human cells (87, 88). It is therefore possible that our survey of ACE2 expression, and other recent reports demonstrating expression of ACE2 in OE support and stem cells but not neurons (81, 89, 90), might under-represent the cell types that express ACE2 under conditions of CoV-2 infection.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [87] C. G. K. Ziegler, S. J. Allon, S. K. Nyquist, I. M. Mbano, V. N. Miao, C. N. Tzouanas, Y. Cao, A. S. Yousif, J. Bals, B. M. Hauser, J. Feldman, C. Muus, M. H. Wadsworth 2nd, S. W. Kazer, T. K. Hughes, B. Doran, G. J. Gatter, M. Vukovic, F. Taliaferro, B. E. Mead, Z. Guo, J. P. Wang, D. Gras, M. Plaisant, M. Ansari, I. Angelidis, H. Adler, J. M. S. Sucre, C. J. Taylor, B. Lin, A. Waghray, V. Mitsialis, D. F. Dwyer, K. M. Buchheit, J. A. Boyce, N. A. Barrett, T. M. Laidlaw, S. L. Carroll, L. Colonna, V. Tkachev, C. W. Peterson, A. Yu, H. B. Zheng, H. P. Gideon, C. G. Winchell, P. L. Lin, C. D. B source

      Not checked in the free run.

    • cites [88] S. P. Sajuthi et al., Type 2 and interferon inflammation strongly regulate SARS-CoV-2 related gene expression in the airway epithelium. bioRxiv, 034454 10 April 2020. PMC7550582 33046696

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    The integration of juvenile and adult whole olfactory bulb scRNAseq datasets yielded stable clusters with cells from both datasets, although some cell types were observed with different frequencies.

    “although some cell types were observed with different frequencies, the integration procedure yielded stable clusters with cells from both datasets.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Doublet cell detection was performed per sample using DoubletFinder and Scrublet for both datasets.

    “For both datasets, doublet cell detection was performed per sample using DoubletFinder (100) and Scrublet (101).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [100] C. S. McGinnis, L. M. Murrow, Z. J. Gartner, DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors. Cell Syst. 8, 329– 337.e4 (2019). 10.1016/j.cels.2019.03.003 30954475 PMC6853612 source

      Not checked in the free run.

    • cites [101] S. L. Wolock, R. Lopez, A. M. Klein, Scrublet: Computational Identification of Cell Doublets in Single-Cell Transcriptomic Data. Cell Syst. 8, 281– 291.e9 (2019). 10.1016/j.cels.2018.11.005 30954476 PMC6625319 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    In the manually sorted olfactory bulb dopaminergic neuron dataset, cell-level quality control and filtering was performed in scater.

    “Cell level quality control and cell filtering was performed in scater (112).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [112] D. J. McCarthy, K. R. Campbell, A. T. Lun, Q. F. Wills, Scater: Pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R. Bioinformatics 33, 1179– 1186 (2017). 10.1093/bioinformatics/btw777 28088763 PMC5408845 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    PCA-batch-correction was performed using Patient 4 as a reference.

    “PCA-batch-correction was performed using Patient 4 as a reference, as previously described (93).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [93] C. Weinreb, A. Rodriguez-Fraticelli, F. D. Camargo, A. M. Klein, Lineage tracing on transcriptional landscapes links state to fate during differentiation. Science 367, eaaw3381 (2020). 10.1126/science.aaw3381 31974159 PMC7608074 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    In the manually sorted olfactory bulb dopaminergic neuron dataset, calretinin-expressing cells were excluded from all analyses.

    “so calretinin-expressing cells were excluded from all analyses.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The distribution of cells from each whole olfactory bulb dataset across clusters was homogenous.

    “the distribution of cells from each dataset across clusters was homogenous (Figure S7)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    In the HBC lineage tracing dataset, 271 putative doublets were removed.

    “Doublets were identified using DoubletFinder and 271 putative doublets were removed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Each of the 16 clusters identified contained cells from all 8 replicates in roughly equal proportions.

    “Each of the 16 clusters identified contained cells from all 8 replicates in roughly equal proportions.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    In the adult whole olfactory bulb Drop-seq dataset, potential doublets were identified via Scrublet and removed (3.5% of cells).

    “Potential doublets were identified via Scrublet and removed (3.5% of cells).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Smell loss in COVID-19 can be sudden and precede development of other symptoms.

    “Any reasonable pathophysiological mechanism for COVID-19-associated anosmia must account for the high penetrance of smell disorders relative to endemic viruses (12, 91, 92), the apparent suddenness of smell loss that can precede the development of other symptoms (11, 13), and the transient nature of dysfunction in many patients (11, 17, 18); definitive identification of the disease mechanisms”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [11] J. R. Lechien, C. M. Chiesa-Estomba, D. R. De Siati, M. Horoi, S. D. Le Bon, A. Rodriguez, D. Dequanter, S. Blecic, F. El Afia, L. Distinguin, Y. Chekkoury-Idrissi, S. Hans, I. L. Delgado, C. Calvo-Henriquez, P. Lavigne, C. Falanga, M. R. Barillari, G. Cammaroto, M. Khalife, P. Leich, C. Souchay, C. Rossi, F. Journe, J. Hsieh, M. Edjlali, R. Carlier, L. Ris, A. Lovato, C. De Filippis, F. Coppee, N. Fakhry, T. Ayad, S. Saussez, Olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (COVID-19): A multicenter European study. Eur. Arch. source

      Not checked in the free run.

    • cites [13] G. Spinato, C. Fabbris, J. Polesel, D. Cazzador, D. Borsetto, C. Hopkins, P. Boscolo-Rizzo, Alterations in Smell or Taste in Mildly Symptomatic Outpatients With SARS-CoV-2 Infection. JAMA 323, 2089 (2020). 10.1001/jama.2020.6771 32320008 PMC7177631

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Tuj1 antibody was validated as previously described.

    “The Tuj1 antibody was validated, as previously described (38).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [38] M. A. Durante, S. Kurtenbach, Z. B. Sargi, J. W. Harbour, R. Choi, S. Kurtenbach, G. M. Goss, H. Matsunami, B. J. Goldstein, Single-cell analysis of olfactory neurogenesis and differentiation in adult humans. Nat. Neurosci. 23, 323– 326 (2020). 10.1038/s41593-020-0587-9 32066986 PMC7065961

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Putative doublets were identified using Scrublet and removed (7% of cells).

    “Putative doublets were also identified using Scrublet and removed (7% of cells).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    In the manually sorted olfactory bulb dopaminergic neuron dataset, less than 2.5 hours elapsed between mouse sacrifice and collection of the last cell in any session.

    “Overall, less than 2.5 hours elapsed between mouse sacrifice and collection of the last cell in any session.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Human olfactory mucosa biopsies were obtained via IRB-approved protocol at Duke University School of Medicine.

    “Human olfactory mucosa biopsies were obtained via IRB-approved protocol at Duke University School of Medicine, from nasal septum or superior turbinate during endoscopic sinus surgery.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    In the analysis of scSeq datasets from various brain regions and sensory systems, inspection of all datasets for batch effects was performed using the scater package.

    “Inspection of all datasets for batch effects was performed using the scater package (version 1.10.1) (112).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [112] D. J. McCarthy, K. R. Campbell, A. T. Lun, Q. F. Wills, Scater: Pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R. Bioinformatics 33, 1179– 1186 (2017). 10.1093/bioinformatics/btw777 28088763 PMC5408845 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Cells with co-expression of male and female marker genes were removed as potential doublets from the WOM dataset.

    “Cells with co-expression of male (Ddx3y, Eif2s3y, Kdm5d, and Uty) and female marker genes (Xist) were removed as potential doublets from the WOM dataset.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Potential doublets were removed using Scrublet.

    “Potential doublets were removed using Scrublet.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Mapping Z-scores were highly robust upon varying the number of votes-cast per cell (R>0.98 correlation upon changing vote numbers to 1 or 50 as opposed to 5).

    “The mapping Z-scores were also highly robust upon varying the number of votes-cast per cell (R>0.98 correlation of mapping Z-scores upon changing the vote numbers to 1 or 50 as opposed to 5).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Mapping Z-scores were similar between the two possible mapping directions (R=0.87 Pearson correlation).

    “The resulting Z-scores were similar between the two possible mapping directions (Durante -> Deprez vs. Deprez -> Durante; R=0.87 Pearson correlation of mapping Z-scores).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    In the manually sorted olfactory bulb dopaminergic neuron dataset, cells with <1000 genes, <100,000 reads, >75% reads mapping to ERCC spike-ins, >10% reads mapping to mitochondrial genes or low library complexity were discarded (14% samples).

    “Cells with <1000 genes, <100,000 reads, >75% reads mapping to ERCC spike-ins, >10% reads mapping to mitochondrial genes or low library complexity were discarded (14% samples).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    10x Genomics mtx files were filtered to remove any cells with fewer than 500 total counts.

    “10x Genomics mtx files were filtered to remove any cells with fewer than 500 total counts.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Clustering the two whole olfactory bulb datasets separately yielded similar results.

    “Clustering the two datasets separately yielded similar results.”
    Quote in the paperNot a substantial claim
78 of 78

  • The paper relies on this without support.

    Cells expressing ACE2 and TMPRSS2 are susceptible to SARS-CoV-2 infection, or such expression is sufficient for viral entry.

    “These findings suggest that CoV-2 infection of non-neuronal cell types leads to anosmia and related disturbances in odor perception in COVID-19 patients.”

  • The paper relies on this without support.

    Infection of non-neuronal cells in the olfactory epithelium or olfactory bulb causes anosmia and related disturbances in odor perception in COVID-19 patients.

    “Thus primary infection of non-neuronal cell types — rather than sensory or bulb neurons — may be responsible for anosmia and related disturbances in odor perception in COVID-19 patients.”

  • The paper relies on this without support.

    Cells expressing CoV-2 entry genes are susceptible to CoV-2 infection.

    “The identification of non-neuronal cell types in the OE and OB susceptible to CoV-2 infection suggests four possible, non-mutually-exclusive mechanisms for the acute loss of smell reported in COVID-19 patients.”

  • The authors state this as an assumption.

    All mice used were of normal immune status.

    “All mice were assumed to be of normal immune status.”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Human olfactory sensory neurons do not express ACE2 and TMPRSS2 but express CD147 that could allow SARS-CoV-2 neuroinvasion.

    “Although current literature data reports that human olfactory sensory neurons do not express these proteins, they express CD147 that could allow SARS-CoV-2 neuroinvasion (Fig. 1) [33, 34].”

    From Hyperactivation of P2X7 receptors as a culprit of COVID-19 neuropathology (2021) · cites this paper as [34]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Human OSN TMPRSS2 detection is reported inconsistently

    Minor

    The figure caption says TMPRSS2 was detected in neither immature nor mature OSNs, whereas the methods report detection in two cells among those populations.

1 of 1

Cites

Flagged first, then by citations

Cited by

Sorted by citations

Related

Authors
Brann, D. H., Tsukahara, T., Weinreb, C., Lipovsek, M., Van den Berge, K., Gong, B., Chance, R., Macaulay, I. C., Chou, H.-J., Fletcher, R. B., Das, D., Street, K., de Bezieux, H. R., Choi, Y.-G., Risso, D., Dudoit, S., Purdom, E., Mill, J., Hachem, R. A., Matsunami, H., Logan, D. W., Goldstein, B. J., Grubb, M. S., Ngai, J., & Datta, S. R.
Affiliations
  • Harvard University (US)
  • King's College London (GB)
  • Department of Statistics, University of California, Berkeley, CA 94720
  • University of California, Berkeley (US)
  • Norwich Research Park (GB)
  • Surrozen, Inc. (United States) (US)
  • Genentech (US)
  • Division of Biostatistics, School of Public Health, University of California, Berkeley, CA 94720
  • University of Padua (IT)
  • University of Exeter (GB)
  • Duke University (US)
  • Waltham Petcare Science Institute, Leicestershire LE14 4RT, UK.
  • National Institutes of Health (US)
Corresponding author
  • Brann, David H.
  • Tsukahara, Tatsuya
  • Weinreb, Caleb
  • Datta, Sandeep Robert
ORCID
Venue
Science Advances
Publisher
American Association for the Advancement of Science (AAAS)
Published
2020-07-31
Volume
6
Issue
31
ISSN
2375-2548 (ISSN-L)
DOI
10.1126/sciadv.abc5801
PMC
PMC10715684
OpenAlex
W3044382958
Semantic Scholar
d1e4a11b05a2afeca86ff027d707ab24a63f6c96
Cited by
1,198
References
128
Citations by year
2026: 43 · 2025: 52 · 2024: 97 · 2023: 163 · 2022: 267 · 2021: 345 · 2020: 229 · 2018: 2
FWCI
88.36
Type
journal article
Language
en
Open access
Licence
Locations
  • Science Advances (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • Ghent University Academic Bibliography (Ghent University) (submittedVersion, cc-by)
  • Padua Research Archive (University of Padova) (submittedVersion, cc-by-nc)
  • eScholarship (California Digital Library) (submittedVersion)
  • Research Portal (King's College London) (submittedVersion, cc-by)
  • Royal Devon and Exeter Research Repository (Royal Devon and Exeter) (submittedVersion, cc-by)
  • UEA Digital Repository (University of East Anglia) (submittedVersion)
  • INFM-OAR (INFN Catania) (submittedVersion, cc-by)
  • Zenodo (CERN European Organization for Nuclear Research) (cc-by)
Funders
  • National Institutes of Health (RO11DC016222, U19NS112953, RO1DC007235, RO1DC016859)
  • Leverhulme Trust (RPG-2016-095)
  • European Research Council (725729)
Topics
  • Life Sciences › Neuroscience › Sensory Systems › Olfactory and Sensory Function Studies
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
  • Health Sciences › Medicine › Infectious Diseases › COVID-19 Clinical Research Studies
Keywords
SARS-CoV-2, COVID-19 pandemic, anosmia, olfactory epithelium, olfactory bulb, ACE2, TMPRSS2, viral entry, non-neuronal cells, sustentacular cells, pericytes, olfactory stem cells, olfactory dysfunction
MeSH terms
Betacoronavirus, Angiotensin-Converting Enzyme 2, COVID-19, SARS-CoV-2, Animals, Olfaction Disorders, Callithrix, Humans, Peptidyl-Dipeptidase A, Macaca, Olfactory Mucosa, Pneumonia, Viral, Serine Endopeptidases, Smell, Olfactory Receptor Neurons, Coronavirus Infections, Mice, Virus Internalization, Pandemics

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review