Human mutations in the SOX18 gene give rise to Hypotrichosis-Lymphedema-Telangiectasia and Renal syndrome (HLTRS) (Irrthum et al., 2003; Moalem et al., 2015). Most disease-causing SOX18 mutations lead to a premature truncation at the C-terminus due to an early stop codon, producing defective protein products that act in a dominant negative fashion. The mutated SOX18 protein can still bind to chromatin but fails to recruit its protein partner(s) to transactivate target genes, and thereby disrupts the endogenous function of SOX18. Further, the dominant-negative mutant prevents other SOX transcription factors to act redundantly and rescue the molecular pathway (Hosking et al., 2009). From birth to adolescence HLTRS patients develop symptoms such as lymphedema and abnormalities to hair and nails for which very limited effective treatment options exist. Patients suffering from HLTRS display a certain level of variability in their phenotypic characteristics with distinct clinical manifestations (see for review Valenzuela et al., 2018) that likely depend on the specific type of SOX18 mutation and how it interferes with SOX18 transcriptional regulation.

A recent report of an HLTRS patient described symptoms so mild that the initial diagnosis failed to identify this syndrome, mostly due to the absence of lymphedema up until late adolescence (Wünnemann et al., 2016). Eventually, the patient was subjected to whole-exome sequencing, which led to the discovery of a premature stop-codon in SOX18, p.Q161*; with such a mutation the patient would be expected to present a severe phenotype. A search for a potential cause of the milder course of the syndrome in this patient revealed that she had been treated with high doses propranolol since a young age due to a significant dilation of the thoracic artery causing a high blood pressure. The milder phenotype observed in this HLTRS patient coincident with the course of the propranolol treatment raised the possibility that this drug has a SOX18-dependent molecular mode of action in addition to its β-blocking activity.

The main indications for propranolol therapy are hypertension, ischemic heart disease, arrhythmia, migraine and essential tremor. Sensitivity to propranolol does not always correlate with the expression levels of the β-adrenergic receptors (Wolter et al., 2014; MacDonald et al., 2006; Chang et al., 2015; Powe et al., 2010). Propranolol use in obstructive hypertrophic cardiomyopathy revealed its beneficial effects in infantile hemangioma (Léauté-Labrèze et al., 2008), raising the possibility that β-adrenergic receptor signaling, and thus by inference, propranolol, modulates VEGF levels (Chen et al., 2013; Ozeki et al., 2013; Lavine et al., 2013). Conclusive proof of such a mode of action is still lacking. Further, this drug is a racemic mixture with R(+) and S(-) enantiomers in equal proportions. The S(-) form displays most of the anti-β-adrenergic activity, whereas the R(+) enantiomer is carried over during drug synthesis. This makes the mode of action of propranolol even more elusive since therapeutic outcomes may result from a combination of effects from the two enantiomers.

In this study, we report an additional case of HLTRS successfully treated with propranolol and describe a R(+) enantiomer-specific mode of action that acts through interference of SOX18 transcriptional activity and thereby functions as an anti-angiogenic agent whereas the S(-) enantiomer does not. This finding paves the way for new therapeutic applications using SOX18 transcription factor as a molecular target in the context of vascular diseases.

Based on the observation that a previous case of HLTRS may have benefited from a long-term exposure to propranolol (Wünnemann et al., 2016), we considered propranolol therapy for another HLTRS patient (Bastaki et al., 2016) in an attempt to mitigate the severity of his symptoms. The patient was diagnosed with HLTRS at 11 months of age, after presenting with alopecia, edema and telangiectasia (Bastaki et al., 2016). Exome sequencing revealed that this patient had a truncation mutation in SOX18 (SOX18 c.492_505dup). In September 2015, at 17 months of age, he developed progressive pericardial effusion over 3 weeks, which required pericardiocentesis. Fluid analysis showed transudate. Two weeks after the procedure, pericardial effusion re-accumulated progressively over 6 weeks, despite strong anti-inflammatory regimens including 3 weeks of oral ibuprofen and 10 days of oral prednisone. Past clinical experience in HLTRS suggested the progression of this pericardial effusion was intractable. The effusion was large enough to cause hemodynamic compromise and to necessitate another pericardiocentesis. After obtaining parental consent, oral propranolol was introduced and increased gradually every 2–3 weeks from 0.8 mg/kg/day in three divided doses to reach a maximum of 4.1 mg/kg/day, as tolerated clinically (pulmonary wheezing). We observed spontaneous and rapid resolution of the pericardial effusion after reaching a dose threshold of 3 mg/kg/day. Bronchial side effects limited the maximal dosing in this patient to 4 mg/kg/day (Figure 1A and B). The positive outcome in this second HLTRS patient harboring SOX18 mutation suggested propranolol might act via an alternative pathway than the well characterized β-adrenergic receptor blockade.

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We next investigated the potential of propranolol to exert β-adrenergic independent effects in cancer cell lines and in human endothelial cells (Figure 1C–D). Survival of primary placentally-derived endothelial colony forming cells (ECFC) was measured in presence of increasing concentrations (5–500 μM) of propranolol and specific β1 (Atenolol) and β2 (ICI 118,551) adrenergic-receptor blockers. Results showed that pharmacological inhibition of specific β-adrenergic receptors did not replicate the marked effect propranolol had in the ECFC survival assay (Figure 1C).

To further explore β-adrenergic independent effects of propranolol we knocked down ADRB2 in a propranolol-sensitive angiosarcoma cell line (ISO-HAS, Figure 1—figure supplement 1A). The cytotoxic activity of propranolol (Figure 1D) and its enhancement of vinblastine cytotoxicity (Figure 1E) were unaltered when ADRB2 was silenced. These results show that the anti-survival and sensitization to chemotherapy activities of propranolol are independent of β-adrenergic receptor expression. Of note, ADRB1 is expressed at low levels in the angiosarcoma cell line (not shown) but is expressed at higher levels in the SHEP neuroblastoma cell line, where its expression can be reduced by siRNA (Figure 1—figure supplement 1B). Similar results were obtained when using a non-endothelial cell type (SHEP neuroblastoma cell line) whereby knock-down of ADRB1 and ADRB2 alone or in combination did not affect the ability of propranolol to block the proliferative response and its ability to enhance response to chemo-therapies (Figure 1—figure supplement 1C and 1D).

The observation that adrenergic receptors are not required for the cellular response to propranolol combined with the clinical benefit of propranolol treatment in the context of HLTRS suggests that this FDA approved drug may act by directly modulating SOX18 protein activity .

Although SOX18 is involved in a variety of patho-physiological processes (Olbromski et al., 2018), HLTRS is a rare and severe vascular disorder. In order to further investigate propranolol in this disease scenario, we used a mouse model of HLTRS, SOX18 Ragged Opossum (RaOp) (Pennisi et al., 2000; Slee, 1957). This mouse mutant is considered a murine counterpart of human HLTRS syndrome since it displays the same range of cardio-vascular and hair follicle defects and exhibits the same type of dominant-negative mutation in the Sox18 gene (Pennisi et al., 2000; Downes et al., 2009; François et al., 2008; Villani et al., 2017). The Ragged mouse is characterized by pathologic corneal neo-vascularisation (CNV, Figure 2—figure supplement 1A-C) (François and Ramchandran, 2012). This phenotypic hallmark offers an indirect biological readout in vivo for aberrant SOX18 function in the control of vessel outgrowth. Propranolol was tested on CNV in the RaOp mouse model to validate its therapeutic potential. Because of early postnatal onset of HLTRS defects (between postnatal day (P)8–22), oral treatment with propranolol (25 mg/kg/day), or vehicle PBS, was initiated at P8 and continued daily through to P28. Both wild type and mutant littermates were subjected to identical treatment schemes in order to safeguard consistent maternal feeding and grooming.

Treatment with propranolol (21 days) did not affect the gross morphological appearance of either wild type or RaOp pups, nor did it significantly affect the weight of the RaOp animals (Figure 1—figure supplement 2A and B, n = 5–10 mice per group). We did not observe any obvious side effects, consistent with the safety profile of propranolol established over the past decades.

Propranolol treatment of RaOp mice, however, led to an almost complete rescue of the CNV phenotype (Figure 2A). The corneas from propranolol-treated mutant animals were devoid of blood vessels (Figure 2A, Endomucin- and ERG-positive cells) in five out of five mice, and one mutant pup only had a mild penetration of blood vessels into the cornea (Figure 2A). These pre-clinical data indicate that propranolol treatment is an effective therapeutic strategy to block aberrant vessel growth in a mouse model of HLTRS and is indicative of on-target engagement on SOX18 protein in vivo.

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These in vivo data prompted us to assess the transcriptional activity of Sox18 in presence of propranolol. Sox18 was overexpressed in COS-7 cells, along with a synthetic construct containing a luciferase reporter gene fused to the Vcam1 promoter fragment, a direct target of Sox18 (Hosking et al., 2004). In this assay, we interfere with wild type Sox18 function using the RaOp dominant-negative protein. We found that RaOp completely abolished the activity of the wild type protein at ratios as low as 1:30 RaOp:Sox18 WT (Figure 2B). We next tested whether propranolol could rescue the dominant-negative inhibitory effect of RaOp reproduced in this cell-based system. Propranolol restored SOX18 wild type functionality in a dose-dependent manner; it significantly increased Vcam1 reporter promoter activity to base line levels at 15 µM (p-value≤0.001) (Figure 2B).

Lastly, to demonstrate the molecular mode of action of propranolol on SOX18 protein partner recruitment, we used an ALPHAScreen assay to measure pairwise protein-protein interaction (PPI). SOX18 homodimer and SOX18/RaOp (Q161*) heterodimer formation was measured in absence or presence of propranolol (Figure 2C). Propranolol showed a mild efficacy at disrupting SOX18 homodimer and was able to interfere more effectively with the assembly of the non functional SOX18/RaOp protein complex. An effector of the NOTCH signaling pathway (RBPJ) was recently shown to act as a SOX18 protein partner (Overman et al., 2017; Fontaine et al., 2017). In addition to disrupting SOX18 homodimer assembly (Figure 2C), we found that propranolol inhibits the association of SOX18 with RBPJ at low micro-molar range (5 μM) (Figure 2—figure supplement 2C). Taken together these in vivo pre-clinical data and in vitro results strongly suggest that one mechanism by which propranolol mediates its anti-angiogenic effects is via direct interference with a SOX18-dependent transcriptional activation.

We have previously identified, developed and validated a small compound, Sm4, that disrupts SOX18 homodimer formation in turn affecting SOX18 ability to recruit RBPJ. Further, we have shown potential therapeutic applications in managing vascular over-growth in pre-clinical model of breast cancer metastasis (Overman et al., 2017). When using Sm4 as a reference, propranolol was equally effective at disrupting SOX18/RBPJ PPI however not as effective at blocking SOX18 homodimer formation (Figure 2—figure supplement 2C). Altogether, these results suggest that the same molecular mode of action of propranolol accounts for both the rescue of dominant-negative SOX18 loss of function by disruption of the non-functional SOX18/RaOp (Q161*) complex, and the disruption of functional SOX18 wild type PPIs, namely SOX18/RBPJ and SOX18/SOX18 dimers. In the context of the Ragged heterozygous mutation dominant negative mutation, the rescue process corresponds to the inhibition by propranolol of an inhibitor (RaOp) which in turn results to the partial restoration of the SOX18 wild type active form.

This novel mode of action of propranolol, independent of its anti-adrenergic effects, prompted us to examine its mechanism of action in infantile hemangiomas, the most common vascular tumor in infancy. Although the molecular mode of action of propranolol in infantile hemangioma therapy is largely unknown, it is the mainstay of treatment for infantile hemangioma. (Léauté-Labrèze et al., 2008; Léauté-Labrèze et al., 2015a; Léauté-Labrèze et al., 2015b) A variety of propranolol-induced cellular effects have been observed in infantile hemangioma-derived stem cells (HemSCs), hemangioma endothelial cells (HemECs) and hemangioma pericytes (Lamy et al., 2010; Lee et al., 2014; Tu et al., 2013; Wong et al., 2012). To learn if SOX18 is involved, we first explored the expression of SOX18 in vivo in hemangioma tissue sections from three patients and in vitro in different cell populations derived from infantile hemangiomas: SOX18 protein was observed in a sup-population of CD31-positive endothelial cells (Figure 3A, arrows) and SOX18 mRNA was expressed in ECFC (positive control) and in HemEC, whereas HemSC showed low expression and hemangioma pericytes were completely devoid of SOX18 transcripts (Figure 3—figure supplement 1).

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Figure 3—source data 1

Supplemental table for Figure 3D (p-values).

https://doi.org/10.7554/eLife.43026.012

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To interrogate whether propranolol inhibits SOX18 in this setting (Figure 3B), we induced patient derived HemSCs to differentiate into HemECs using VEGF-B and low serum media (Khan et al., 2008). CDH5 (a pan-endothelial marker), SOX18, and ADBR2 mRNAs were increased in VEGF-B-treated HemSC (Figure 3C) further showing a process by which differentiation of stem cells results in endothelial cells expressing SOX18. This was extended to other genes such as CD31, NOTCH1, PLXND1 and VEGFR1 (Table 1 and Figure 3—source data 1), each of which are expressed in infantile hemangioma endothelium in vivo (Boscolo et al., 2011; Nakayama et al., 2015; Wu et al., 2010), further confirming the successful differentiation of HemSCs into HemECs (Figure 3D and Figure 3—figure supplement 2A). Using this differentiation assay as a read-out, we next evaluated the capacity of propranolol to inhibit the differentiation of HemSCs isolated from four infantile hemangioma patients. The pharmacologic treatments of HemSC included: propranolol, Sm4 (SOX18 small molecule inhibitor), aspirin (a negative control [Overman et al., 2017; Fontaine et al., 2017]) compared to vehicle control. Of note, SOX18 has been shown to directly up-regulate Notch1 transcription via an intronic enhancer during arterial specification in vertebrates (Chiang et al., 2017), hence transcriptional output of this target gene indirectly reflects SOX18 activity. Propranolol significantly decreased VEGF-B induced expression of all five endothelial markers in HemSC to levels also achieved by Sm4 as compared to controls (see p-values in Figure 3—source data 1).

Propranolol exists as a 1:1 mixture of the S(-) and R(+) enantiomers. The S(-) form displays potent anti-β-adrenergic activity whereas the R(+) enantiomer presents strongly reduced antagonist activity towards β2 adrenergic receptors on peripheral arteries (Stoschitzky et al., 1995). To test whether HemSC were affected by propranolol in a stereoselective manner, we assessed VEGF-B-induced HemSC to HemEC differentiation from two infantile hemangioma in the presence of separated enantiomers.

The R(+) enantiomer recapitulated propranolol and Sm4-induced inhibition on all markers, whereas the S(-) enantiomer showed only weak or no inhibition on hemangioma markers (Figure 3D and Figure 3—source data 1). These results are consistent with a SOX18-dependent, β-adrenergic receptor independent blockade of hemangioma endothelial differentiation, which strongly suggests that blockade of SOX18 activity is the main mode of action of propranolol in this setting. Further, analysis of VEGF-R2 expression and phosphorylation levels in ECFC and HemSC to EC differentiation assay ruled out that the propranolol racemic mixture, the R(+) enantiomer or Sm4 interfere with the VEGF signaling pathway (Figure 3—figure supplement 2B-D).

As a final demonstration of the therapeutic utility of the R(+) enantiomer we performed a corneal neo-vascularisation rescue assay using the Ragged Opossum mutant mouse model. In this in vivo setting both propranolol and the R(+) enantiomer prevented aberrant vessel growth in the corneal tissue of the heterozygous mutant animals. Of note the S(-) enantiomer was also active at disrupting corneal vessel outgrowth, suggesting that conformational changes that occur in the SOX18 mutated protein may attenuate the discrepancy in enantiomeric activities previously observed in the hemangioma endothelial differentiation model (Figure 3—figure supplement 3).

In order to investigate whether propranolol treatment might improve the morphogenesis defects of other vascular beds, we analysed the patterning of the lymphatic vasculature in the ear skin tissue of the adult RaOp animals. As previously reported the RaOp mouse presented with a highly disorganized lymphatic vascular network (François et al., 2008) (Figure 3—figure supplement 4, top graph). In the skin, neither the propranolol racemic mixture nor its enantiomers rescued the lymphatic vascular phenotype caused by the RaOp mutation (Figure 3—figure supplement 4); this is most likely due to the post-natal timing of the treatment: lymphatic vascular defects are acquired in the RaOp mutant during the initial steps of embryonic lymphangiogenesis (early to mid-organogenesis), which precedes the drug treatment window.

In summary, we suggest the R(+) enantiomer of propranolol is a potent selective inhibitor of SOX18 activity and it is sufficient to achieve the effects of propranolol racemic mixture in the context of infantile hemangioma and other SOX18-dependent vascular diseases.

Despite being the front line therapy for infantile hemangioma, approximately 20% of the patient population fails to respond to propranolol treatment and/or experience tumor regrowth when the medication is discontinued too early (Bagazgoitia et al., 2011; Xiao et al., 2013). Our finding opens up new treatment options using enantiopure R(+) propranolol instead of the racemic mixture. This could potentially allow a dose adjustment to achieve optimal benefit for the patients while mitigating adverse events that may result from long-term propranolol treatment, particularly in the refractory population of patients that currently do not respond to the highest dose of propranolol. In addition, the side effects associated with long-term exposure to propranolol could be greatly reduced if β-adrenergic receptors blockage would be minimized.

This discovery is of broad interest because propranolol, comprised of 2 enantiomers, has been used clinically for decades. Clinical trials of the R(+) versus the S(-) form have been performed and deemed safe with full toxicology data available (Stensrud and Sjaastad, 1976). Pharmacokinetic studies suggest that propranolol therapy results in an accumulation of the S(-) enantiomer following drug metabolism, which could be problematic if one is interested in the activity of the R(+) form (Mehvar and Brocks, 2001). Indeed toxicology studies further suggest that propranolol in its racemic form is more toxic than either enantiomer alone (Stoschitzky et al., 1992). This study presents indirect evidence that in a HLTRS patient treated with propranolol, SOX18 wild type function is at least partly restored. The observation that the S(-) enantiomer also has the capability to rescue the corneal neo-vascularisation phenotype in a pre-clinical model of HTLRS suggests that the effects of propranolol as a racemic mixture are likely to be a combination of the blockade of beta-adrenergic receptor activity and SOX18 RaOp mutant protein inhibition. Of importance, in the hemangioma HemSC-EC differentiation assay the R(+) enantiomer was as efficient as propranolol and more efficient than the S (-) enantiomer at blocking this differentiation process. This may suggest that the SOX18 wild type protein blockade displays some enantio-selectivity, whereas conformational changes in the SOX18/RaOp complex caused by the RaOp mutation sensitize the dominant-negative protein complex to both R(+) and S(-) enantiomers.

The studies in human patients with HLTRS and the murine RaOP model suggest that there may be critical time windows in which treatment with R(+) propranolol would be most efficient as an anti-angiogenic compound via a blockade of SOX18 transcriptional activity.

The low number of patients with HLTRS – less than 10 documented survivors have been reported – makes it difficult to establish genotype-phenotype relationships in greater detail. In contrast, the unambiguous phenotypic rescue by propranolol racemic mixture in two human patients and by the R(+) enantiomer in the Ragged Opossum mouse model is strongly supported by our in vitro mechanistic results. Our findings outline that the R(+) enantiomer is a SOX18 small molecule inhibitor with weak anti-beta adrenergic activity whereas the S(-) molecule is both a beta-blocker and a weak SOX18 inhibitor which introduces variability in net efficacy of propranolol treatment.

The strong monogenic effect of human SOX18 mutations and their rescue with propranolol allowed us to identify its mode of action. We suggest the dosing and duration of propranolol therapy may be adjusted by using R(+) propranolol in potential future trials of HLTRS and infantile hemangioma.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Jeroen Overman

   Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Writing—original draft, Project administration, Writing—review and editing

   Contributed equally with

   Frank Fontaine

   Competing interests

   No competing interests declared

2. Frank Fontaine

   Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing—review and editing

   Contributed equally with

   Jeroen Overman

   Competing interests

   No competing interests declared

3. Jill Wylie-Sears

   Vascular Biology Program, Department of Surgery, Boston Children's Hospital, Harvard Medical School, Boston, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Mehdi Moustaqil

   Single Molecule Science, Lowy Cancer Research Centre, The University of New South Wales, Sydney, Australia

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

5. Lan Huang

   Vascular Biology Program, Department of Surgery, Boston Children's Hospital, Harvard Medical School, Boston, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

6. Marie Meurer

   Centre de Recherche en Cancérologie de Marseille (CRCM Marseille Cancer Research Centre), Inserm UMR1068, CNRS UMR7258, Aix-Marseille University UM105, Marseille, France

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

7. Ivy Kim Chiang

   Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

   Contribution

   Formal analysis, Visualization

   Competing interests

   No competing interests declared

8. Emmanuelle Lesieur

   Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

   Contribution

   Formal analysis, Visualization

   Competing interests

   No competing interests declared

9. Jatin Patel

   Translational Research Institute, Diamantina Institute, The University of Queensland, Brisbane, Australia

   Contribution

   Formal analysis, Investigation, Writing—original draft

   Competing interests

   No competing interests declared

10. Johannes Zuegg

    Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

    Contribution

    Resources, Formal analysis, Investigation

    Competing interests

    No competing interests declared

11. Eddy Pasquier

    Centre de Recherche en Cancérologie de Marseille (CRCM Marseille Cancer Research Centre), Inserm UMR1068, CNRS UMR7258, Aix-Marseille University UM105, Marseille, France

    Contribution

    Formal analysis, Investigation, Writing—original draft

    Competing interests

    No competing interests declared

12. Emma Sierecki

    Single Molecule Science, Lowy Cancer Research Centre, The University of New South Wales, Sydney, Australia

    Contribution

    Resources, Formal analysis, Supervision, Methodology

    Competing interests

    No competing interests declared

13. Yann Gambin

    Single Molecule Science, Lowy Cancer Research Centre, The University of New South Wales, Sydney, Australia

    Contribution

    Resources, Supervision, Methodology

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-7378-8976

14. Mohamed Hamdan

    Dubai Healthcare City, Dubai, United Arab Emirates

    Contribution

    Resources, Supervision, Investigation, Methodology, Writing—original draft, Project administration

    Competing interests

    No competing interests declared

15. Kiarash Khosrotehrani

    Translational Research Institute, Diamantina Institute, The University of Queensland, Brisbane, Australia

    Contribution

    Supervision, Funding acquisition, Investigation, Writing—original draft, Project administration

    Competing interests

    No competing interests declared

16. Gregor Andelfinger

    Department of Pediatrics, University of Montreal, Ste-Justine University Hospital Centre, Montréal, Canada

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

    Competing interests

    No competing interests declared

17. Joyce Bischoff

    Vascular Biology Program, Department of Surgery, Boston Children's Hospital, Harvard Medical School, Boston, United States

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    joyce.bischoff@childrens.harvard.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-6367-1974

18. Mathias Francois

    Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    m.francois@imb.uq.edu.au

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9846-6882

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