RNA-based thermal regulation is an important strategy for organisms to cope with temperature changes. Inhabiting the intertidal rocky shore, a key interface of the ocean, atmosphere and terrestrial environments, intertidal species have developed variable thermal adaptation mechanisms; however, adaptions at the RNA level remain largely uninvestigated. To examine the relationship between mRNA structural stability and species distribution, in the present study, the secondary structure of cytosolic malate dehydrogenase (cMDH) mRNA of Echinolittorina malaccana, Echinolittorina radiata and Littorina brevicula was determined using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), and the change in folding free energy of formation (ΔGfold) was calculated. The results showed that ΔGfold increased as the temperature increased. The difference in ΔGfold (ΔΔGfold) between two specific temperatures (25 versus 0°C, 37 versus 0°C and 57 versus 0°C) differed among the three species, and the ΔΔGfold value of E. malaccana was significantly lower than those of E. radiata and L. brevicula. The number of stems of cMDH mRNA of the snails decreased with increasing temperature, and the breakpoint temperature of E. malaccana was the highest among these. The number of loops was also reduced with increasing temperature, while the length of the loop structure increased accordingly. Consequently, these structural changes can potentially affect the translational efficiency of mRNA. These results imply that there were interspecific differences in the thermal stability of RNA secondary structures in intertidal snails, and these differences may be related to snail distribution.

Ambient temperature is an important environmental factor that determines whether organisms can survive and grow in a specific habitat (Fields et al., 2015). All types of macromolecules – proteins, RNAs, lipids and sugars – play important roles in sensing and adaptation to temperature (Becskei and Rahaman, 2022; Somero, 2022; Somero et al., 2017). At the RNA level, structural perturbations in RNA molecules induced by temperature change may have important biological implications in an organism's response to temperature (Becskei and Rahaman, 2022; Halder et al., 2019; Liao et al., 2021; Chursov et al., 2012).

Messenger RNA (mRNA) transmits protein-coding information and is also a key carrier of translational regulatory information. mRNAs are widely involved in the co-translational folding of proteins and gene expression regulation, and have a significant impact on the structure and function of proteins, which in turn affect the temperature adaptation of organisms (Keller et al., 2012; Boël et al., 2016; Dvir et al., 2013). At the same time, mRNAs are also dependent on their own secondary structures to function and can respond to temperature stress at the secondary structure level (Batey, 2006; Righetti et al., 2016). Many studies in bacteria have shown that mRNA secondary structure contributes to the efficient regulation of translation and the binding of ligands, such as proteins, or metabolites, in response to different signals (e.g. temperature, pH, etc.) (Chiaruttini and Guillier, 2020). Therefore, structural changes of RNA are an important response to environmental temperature.

Secondary structural features of mRNAs can help mRNAs tolerate conformational changes in response to environmental stress (Ding et al., 2014). RNA secondary structures are highly autonomous, largely as a result of the strong interactions between base pairs (Becskei and Rahaman, 2022). As temperature increases, the equilibrium of RNA stem-loops shifts from the folded to the unfolded state (Stephenson et al., 2014; Becskei and Rahaman, 2022). As previous studies described, the secondary structures in the untranslated regions (UTRs) unfold under temperature stress, and can affect ribosome binding and translation (Kortmann and Narberhaus, 2012; Su et al., 2018). Within the coding sequences (CDS) of the yeast genome, the RNA secondary structure revealed a higher prevalence of secondary structures, and a noteworthy correlation was observed between the RNA structural characteristics and the efficiency of mRNA translation (Kertesz et al., 2010). Therefore, identifying mRNA secondary structure features is of great significance for understanding the adaptability of organisms to temperature.

Determination of RNA secondary structure is still a large challenge. Many studies have developed algorithms to simulate RNA secondary structure in silico (Oluoch et al., 2018; Liao et al., 2021). For example, based on thermodynamic methods, ‘RNAstructure’ software can calculate the change of folding free energy (ΔGfree) to predict structural stability (Reuter and Mathews, 2010). Although in silico prediction can provide a certain understanding of RNA secondary structure, it still needs to be verified by experiments to better explore the relationship between RNA secondary structure and biological temperature adaptability. An in vitro method for RNA secondary structure determination, named selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), is based on the nucleophilicity of the ribose 2′-position to the adjacent 3′-phosphodiester group (Wilkinson et al., 2006; Chamberlin et al., 2002; Merino et al., 2005). Compared with based-paired or other constrained nucleotides, unconstrained nucleotides have more conformations that enhance the nucleophilicity of the 2′-hydroxyl group (Wilkinson et al., 2006). Therefore, hydroxyl-selective electrophiles (i.e. N-methylisatoic anhydride) form stable 2′-O-adducts more rapidly with flexible RNA nucleotides (Wilkinson et al., 2006, 2005; Merino et al., 2005). For example, a recent study validated the regulatory role of mRNA structural features in Arabidopsis thaliana by SHAPE chemical analysis (Liu et al., 2021). Another study used the SHAPE method to analyze the mRNA secondary structure of the 5′-untranslated region and the N-terminal nsp1 coding sequence of mouse hepatitis virus (Yang et al., 2015). All in all, accurate determination of RNA secondary structure will help to better understand the relationship between RNA structural features and biological temperature adaptability.

The intertidal rocky shore is a key interface of the ocean, atmosphere and terrestrial environments, and species inhabiting the shore usually have high thermal resistance to thermal stress (Dong et al., 2022; Helmuth et al., 2006; Stillman and Somero, 1996). The periwinkle snails Echinolittorina malaccana, Echinolittorina radiata and Littorina brevicula are distributed widely along the coast of the northwestern Pacific (Fig. 1), and have different distribution areas and upper thermal limits (Reid, 2007; Li, 2012; Takada, 2003). In the summer, these species frequently suffer from high temperature over 50°C during low tides (Ng et al., 2017). Compared with E. malaccana and E. radiata in the splash zone, the high intertidal species L. brevicula exhibits relatively low upper thermal tolerance (Dong et al., 2022).

Fig. 1.

The location of the study species. (A) The biogeographical ranges of the three intertidal snails, Echinolittorina malaccana, Echinolittorina radiata and Littorina brevicula, in the northwestern Pacific. (B) The vertical distribution of the three intertidal snails. Echinolittorina malaccana and E. radiata are mainly distributed in the splash zone; L. brevicula is mainly distributed in the high intertidal.

Fig. 1.

The location of the study species. (A) The biogeographical ranges of the three intertidal snails, Echinolittorina malaccana, Echinolittorina radiata and Littorina brevicula, in the northwestern Pacific. (B) The vertical distribution of the three intertidal snails. Echinolittorina malaccana and E. radiata are mainly distributed in the splash zone; L. brevicula is mainly distributed in the high intertidal.

The enzyme cytoplasmic malate dehydrogenase (cMDH; EC 1.1.1.37) plays important roles in many metabolic pathways, and has been shown to exhibit adaptative variation related to temperature in many marine invertebrates (Musrati et al., 1998; Dong and Somero, 2009; Fields et al., 2006; Dong et al., 2018; Somero et al., 2017). In the present study, we aimed to determine the secondary structure of cMDH mRNA in vitro in the three intertidal periwinkles at different temperatures for investigating the thermal adaptation of RNA structures in thermophilic animals.

Sample collection

Three species of gastropods were used in the present study: Echinolittorina malaccana (R. A. Philippi 1847), a southern species, and Echinolittorina radiata (Souleyet 1852), a cosmopolitan species along China's coastline, distributed in the splash zone, and with a high upper lethal temperature of ∼55°C (Chen et al., 2021; Liao et al., 2019); and Littorina brevicula (R. A. Philippi 1844), a northern species, distributed on the high intertidal shore with relatively low upper thermal limit of 50°C (Dong et al., 2017). Specimens of E. malaccana and E. radiata were collected from natural rocky intertidal habitats in Xiamen (24°28′N, 118°05′E), and L. brevicula were collected from Qingdao (36°06′N, 120°31′E), China. All individuals were transported to the laboratory within 24 h and subjected to at least 1 week of indoor acclimation (room temperature, ∼25°C). All individuals were sprayed with fresh seawater three times a week.

Construction of recombinant plasmids containing T7 promoter

To construct recombinant plasmids containing the cMDH gene, we sequenced all of the cMDH orthologs as described previously (Liao et al., 2017). Total RNA was extracted from ∼10 mg of each muscle sample with 1 ml TRIzol reagent (Invitrogen). Freshly isolated RNA was reverse transcribed into cDNA using the PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time; Takara). PCR for amplification of cMDH CDS of was performed as follows: 25 μl Premix Taq (Ex Taq version 2.0, Takara), 2 μl (<500 ng) template cDNA, 0.4 μmol l−1 primers (final concentration) (Table 1), to a final volume of 50 μl with ddH2O. Target DNA fragments were purified with AxyPrep DNA gel recovery kit (Axygen).

Table 1.

Primers used for cMDH PCR and reverse transcription

Primers used for cMDH PCR and reverse transcription
Primers used for cMDH PCR and reverse transcription

For L. brevicula, the target DNA fragment was ligated into pGM-T TA cloning vector (Tiangen). For E. malaccana and E. radiata, the target DNA fragment was first ligated into the pUC19 vector (Takara), and then subcloned into the pET-32a(+) vector containing the T7 promoter. Finally, the recombinant vectors of these species were transformed into E. coli TOP10/DH5α (Takara/Tiangen), and the positive clones were selected and Sanger sequenced (Sangon).

In vitro transcription

To imitate in vivo transcription and synthesize a large quantity of pure RNA molecules, in vitro transcription was performed. The extracted plasmid was single-digested with a suitable restriction endonuclease (SphI: E. malaccana and E. radiata, SpeI: L. brevicula; TransGen). The linear template DNA was purified using the E.Z.N.A.® Cycle Pure Kit (Omega). An in vitro transcription reaction was performed using a T7 mRNA Synthesis Kit (Takara) as follows: 2 μl 10× transcription buffer, 2 μl each of ATP, GTP, CTP and UTP solution, 0.5 μl RNase inhibitor, 2 μl T7 RNA polymerase, 20 ng to 1 μg linear template DNA, to a final volume of 20 μl with RNase-free dH2O. The reaction product was purified using the RNeasy Mini Kit (Qiagen) (Mustoe et al., 2018).

RNA modification and precipitation

For in vitro RNA modification, the following steps were carried out. A total of 9 μl purified RNA was added to each tube. Subsequently, 2 μl of 130 mmol l−1N-methylisatoic anhydride (NMIA; Sigma-Aldrich) was added to the (+)NMIA tube, while the same volume of dimethyl sulfoxide (DMSO) (Sangon) was added to the (−)NMIA tube as a control. The solutions in both tubes were incubated at 37°C for 45 min using a dry bath incubator (Major Science). To precipitate RNA, 500 μl of an ethanol precipitation mixture (9 ml RNase-free water, 40 ml absolute ethanol, 400 μl 5 mol l−1 NaCl and 40 μl 0.5 mol l−1 EDTA solution) was added, with 1.5 μl of glycogen (Invitrogen). The mixture was allowed to settle at −80°C for 30 min, and then centrifuged at 14,000 rpm (4°C) for 30 min. Finally, the RNA precipitate was dissolved in enzyme-free water and stored at −80°C for subsequent analysis.

SHAPE

To gain local nucleotide flexibility at all positions within the RNA, the SHAPE experiment was performed (Wilkinson et al., 2006). Firstly, 10 μl DEPC-H2O, 1 μl modified RNA and 1 μl specific reverse transcription primer were mixed together (Table 1), and the following program was run in a thermal cycler (Biometra TADVANCED): 65°C, 5 min; 35°C, 5 min. Secondly, 6 μl of reverse transcription reaction solution (5×SSIV buffer, 10 mmol l−1 dNTP mix and 100 mmol l−1 DTT with a ratio of 4:1:1, prepared in advance) was added and then incubated at 52°C for 1 min. After incubation, 1 μl reverse transcriptase SuperScript™ IV Reverse Transcriptase (200 U μl−1; Invitrogen), 1 μl ribonuclease inhibitor (Invitrogen) and 1 μl ddATP (10 mmol l−1) were added and heated with the following program: 52°C, 10 min; 80°C, 10 min. Finally, 1 μl E. coil RNase H (Invitrogen) was added to remove RNA, and the reaction was performed at 37°C for 20 min. The final reaction system was sequenced using capillary electrophoresis (Sangon). The SHAPE procedure is summarized in Fig. 2A.

Fig. 2.

SHAPE analysis of cMDH. (A) Flow chart of the 2′-hydroxyl acylation analyzed by primer extension (SHAPE) experiment. The experiment was divided into four steps: (1) extraction of total RNA and acquisition of target gene; (2) construction of recombinant plasmids containing the T7 promoter and in vitro transcription; (3) RNA modification and SHAPE; (4) sequence and data analysis (see Materials and Methods for details). (B) Change in folding free energy (ΔGfold) of cytosolic malate dehydrogenase (cMDH) mRNA in the three intertidal snails under different temperatures. The gray shading represents the 95% confidence interval. (C) The differences in ΔGfold (ΔΔGfold) between different simulation temperatures (25 versus 0°C, 37 versus 0°C, 57 versus 0°C). Different letters indicate significant differences (P<0.001).

Fig. 2.

SHAPE analysis of cMDH. (A) Flow chart of the 2′-hydroxyl acylation analyzed by primer extension (SHAPE) experiment. The experiment was divided into four steps: (1) extraction of total RNA and acquisition of target gene; (2) construction of recombinant plasmids containing the T7 promoter and in vitro transcription; (3) RNA modification and SHAPE; (4) sequence and data analysis (see Materials and Methods for details). (B) Change in folding free energy (ΔGfold) of cytosolic malate dehydrogenase (cMDH) mRNA in the three intertidal snails under different temperatures. The gray shading represents the 95% confidence interval. (C) The differences in ΔGfold (ΔΔGfold) between different simulation temperatures (25 versus 0°C, 37 versus 0°C, 57 versus 0°C). Different letters indicate significant differences (P<0.001).

Construction of cMDH mRNA models

The detected capillary electrophoresis results were analyzed using QuShape software (Karabiber et al., 2013; Leonard et al., 2013). The SHAPE information was used as a parameter to constrain the predicted RNA secondary structure. The fold and efn2 plugins in the RNAstructure software (Mathews et al., 1999; Reuter and Mathews, 2010) were used to calculate ΔGfold and every substructure for each cMDH mRNA model at different temperatures.

Statistical analysis

The trend of ΔGfold with temperature variations was subjected to linear regression analysis for fitting. The difference in ΔGfold (ΔΔGfold) between two specific temperatures (25 versus 0°C, 37 versus 0°C and 57 versus 0°C) among the three species was subjected to statistical analysis using one-way analysis of variance (ANOVA).

To investigate the impact of temperature on the number of stem structures, a line graph was plotted to show the variation of different structures with temperature, and the segmented package in the R software was used to calculate the breakpoint temperature (Muggeo, 2008). The trend of other types of structure changing with temperature was also represent by a line graph. All statistical analysis was performed using R software (http://www.R-project.org/). RNA secondary structures were visualized using ViennaRNA web services (Kerpedjiev et al., 2015).

ΔGfold and ΔΔGfold

Sequences of cMDH mRNA were highly conserved in the three intertidal gastropods (Fig. S1). The SHAPE experiment indicated that the absolute values of folding free energy for all three species decreased with increasing temperature. However, there were significant differences in the slopes of the linear relationship between free energy and temperature among the three species (linear regression, P<0.001, F=43.344): 7.49, 7.34 and 7.71 for E. malaccana, E. radiata and L. brevicula, respectively (Fig. 2B).

ΔΔGfold for different temperatures was also calculated (Fig. 2C). In all temperature ranges (25 versus 0°C, 37 versus 0°C, and 57 versus 0°C), the ΔΔGfold values of cMDH mRNA of E. malaccana were significantly lower than those of E. radiata and L. brevicula (P<0.05).

Changes in RNA secondary structure in the three intertidal gastropods

The number of stem structures remained stable until reaching the breakpoint temperature in all three species (E. malaccana, 28.57±1.13°C; E. radiata, 18.54±0.64°C; L. brevicula, 13.50±1.78°C) (Fig. 3A,C and Table 2), and then decreased as the temperature further decreased (Fig. 3A,C). The number of hairloop structures decreased as temperature increased (Fig. 3B,D) in all three species. The number of interloops and multiloop structures in E. malaccana, E. radiata and L. brevicula remained relatively stable as temperature increased (Figs S2 and S3).

Fig. 3.

Changes in RNA secondary structure. (A,B) The number of stem structures (A) and hairloop structures (B) in the RNA of the three intertidal snails changed with temperature. The gray shading represents the 95% confidence interval. (C,D) Line plots representing the segmented relationship between temperature and the number of stem structures (C) and hairloop structures (D). In C, the estimated breakpoint is indicated by a black circle, representing the point where the regression line experiences a significant change.

Fig. 3.

Changes in RNA secondary structure. (A,B) The number of stem structures (A) and hairloop structures (B) in the RNA of the three intertidal snails changed with temperature. The gray shading represents the 95% confidence interval. (C,D) Line plots representing the segmented relationship between temperature and the number of stem structures (C) and hairloop structures (D). In C, the estimated breakpoint is indicated by a black circle, representing the point where the regression line experiences a significant change.

Table 2.

Breakpoint temperature of cMDH mRNA for the three intertidal snails

Breakpoint temperature of cMDH mRNA for the three intertidal snails
Breakpoint temperature of cMDH mRNA for the three intertidal snails

Transformation from stem to loop

Around the temperature breakpoints, cMDH mRNA tended to form longer single-stranded regions (Fig. 4). As the temperature increased from 29 to 30°C (Fig. 4A,B), the RNA secondary structure of E. malaccana underwent changes starting from the 81st nucleotide. For E. radiata, as the temperature increased from 20 to 21°C (Fig. 4C,D), the RNA secondary structure underwent changes starting from the 339th nucleotide. For L. brevicula, as the temperature increased from 14 to 15°C (Fig. 4E,F), the RNA secondary structure of underwent changes starting from the 174th nucleotide.

Fig. 4.

Changes in cMDH RNA secondary structure with increases in temperature for the three intertidal snails. Combination charts show the cMDH RNA secondary structure of E. malaccana at 29°C (A) versus 30°C (B); E. radiata at 20°C (C) versus 21°C (D); and L. brevicula at 14°C (E) versus 15°C (F). Red arrows indicate the nucleotide where these changes started. The corresponding sequences are shown below.

Fig. 4.

Changes in cMDH RNA secondary structure with increases in temperature for the three intertidal snails. Combination charts show the cMDH RNA secondary structure of E. malaccana at 29°C (A) versus 30°C (B); E. radiata at 20°C (C) versus 21°C (D); and L. brevicula at 14°C (E) versus 15°C (F). Red arrows indicate the nucleotide where these changes started. The corresponding sequences are shown below.

We integrated the SHAPE experiment with in silico simulation to explore the relationship between RNA secondary structure of cMDH and thermal adaptation in three intertidal snails with different geographical distribution. This technology had significant utility in the sense that SHAPE reactivity could serve as a valuable constraint for refining the results of secondary structure prediction algorithms (Wilkinson et al., 2006). In other words, the utilization of SHAPE information has often proved adequate for deducing or robustly constraining possible secondary structure models for many RNAs (Wilkinson et al., 2005, 2006; Merino et al., 2005; Badorrek and Weeks, 2005). Therefore, compared with in silico simulation, this technology can improve the accuracy of RNA secondary structure models.

In the present study, we observed that the absolute values of the change in RNA folding free energy (ΔGfold) decreased with increasing temperature in all three intertidal species. At the same time, we also found significant differences in the slopes of ΔGfold–temperature relationships among the three species. These findings indicate that while all species exhibited decreased stability of RNA secondary structures at higher temperatures, the rate at which this stability changed differed among them (Liao et al., 2021).

Furthermore, we investigated the differences in ΔGfold between two specific temperatures and observed that E. malaccana exhibited the smallest difference, further indicating the highest structural stability and thermodynamic favorability among the three intertidal species (Liao et al., 2021). Besides these differences at the RNA level, a previous study indicated that E. malaccana and E. radiata had higher thermal stability than other intertidal snails, in terms of both physiology and protein (Liao et al., 2019). Greater stability and heat resistance in E. malaccana may allow these snails to survive in a higher temperature environment (∼55°C), potentially explaining their presence in locations with elevated temperatures (Liao et al., 2017; Chen et al., 2021). In contrast, E. radiata and L. brevicula, with different thermal responses, might be better suited to other thermal niches and distribution ranges.

The primary reason for changes in folding free energy was the alteration of RNA secondary structure. The free energy change was typically approximated with a nearest neighbor model in which ΔGfold was the sum of free energy increments for the various nearest neighbor motifs (e.g. stacked base pairs in an RNA helix) that occurred in a structure (Mathews and Turner, 2006; Mathews et al., 2010). RNA secondary structure exists in a dynamic equilibrium and can respond to changes in intracellular environment (e.g. temperature and pH) and interactions with RNA-binding proteins (Harmon, 2017). Temperature is known to have an essential effect on mRNA secondary structure (Irvine, 2020). For instance, the mRNA secondary structure melts with increasing temperature in many prokaryotic genes, thus liberating the ribosome binding site (RBS) (Krajewski and Narberhaus, 2014). Additionally, the integrity of the stem is more essential than that of the loop structure for stabilization (Xia et al., 2002), so organisms respond to an abrupt temperature increase by breaking the hydrogen bonds of the mRNA secondary structure. In the present study, we observed that changes in the stem structure of E. malaccana, E. radiata and L. brevicula followed a similar trend, initially showing a gradual decrease followed by a point of inflection where the rate of decrease accelerated. However, it is important to note that the temperature at which this inflection point occurred differed among the three species, with E. malaccana having a higher inflection temperature compared with E. radiata and L. brevicula. The observed pattern suggests that E. malaccana can maintain higher RNA structural stability under high-temperature conditions, while E. radiata and L. brevicula exhibit faster structural instability at lower temperatures. This may indicate that E. malaccana is adapted to higher-temperature environments and heat stress, while E. radiata and L. brevicula may be better suited for lower-temperature ecological conditions.

In response to high-temperature stress, mRNA secondary structures tend to have more single-strandedness and longer maximal loop lengths, indicating greater structural flexibility for coping with environmental change (Ding et al., 2014; Bevilacqua et al., 2016; Flynn et al., 2016). In the present study, we found that near the temperature breakpoint, the RNA secondary structure of cMDH in the three intertidal snails underwent a relatively pronounced transition, tending to form single-stranded loop structures. Compared with paired bases, the single-stranded structure has greater flexibility and can be folded into a variety of spatial structures. However, movement of ribosomes – cellular structures responsible for converting RNA into proteins – requires ‘reading’ of the RNA sequence and synthesis of proteins (Alberts et al., 2002); when RNA secondary structures are unfolded, ribosomes can more easily access the coding information on the RNA, thereby accelerating the process of protein synthesis. Additionally, longer single-stranded regions may provide more binding sites, making ribosomal movement on RNA more efficient (Krajewski and Narberhaus, 2014). The transformation of secondary structures of cMDH mRNA was an adaptation to the change of ambient temperature (Thomas et al., 2022). Therefore, we infer that when the environmental temperature changes, these intertidal snails can adapt to the higher temperature by altering the types of secondary structure and increasing the flexibility of the mRNA structure.

In conclusion, both changes in folding free energy and changes in RNA secondary structure are crucial for the adaptation of organisms to environmental temperature. These adaptive changes can help organisms respond quickly to maintain their normal physiological functions, and are potentially connected with species' geographical distribution. In future studies, we will continue to explore the relationship between the temperature adaptability of biological RNA structures and the biogeographical distribution of organisms.

The authors thank Shuang-En Yu and Xiao-Lu Zhu from Ocean University of China for their support with data analysis and figures.

Author contributions

Conceptualization: M.-L.L., Y.-W.D.; Methodology: Y.-J.Z.; Software: Y.-J.Z., M.-L.L.; Formal analysis: M.-L.L.; Investigation: Y.-J.Z.; Resources: Y.-J.Z.; Data curation: Y.-J.Z., M.-L.L.; Writing - original draft: Y.-J.Z., M.-L.L.; Writing - review & editing: Y.-J.Z., M.-L.L., Y.-W.D.; Supervision: M.-L.L., Y.-W.D.; Project administration: M.-L.L.; Funding acquisition: M.-L.L., Y.-W.D.

Funding

This study was supported by National Natural Science Foundation of China (grant nos 42106112 and 42025604) and the Fundamental Research Funds for the Central Universities.

Data availability

All relevant data can be found within the article and its supplementary information.

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Competing interests

The authors declare no competing or financial interests.

Supplementary information