RNA modification is the mark and strategy for host-microbe interactions

Abstract

Pathogenic microbes need to adapt to host environments during infection. With advances in sequencing technologies, RNA modifications have been shown to play a pivotal role in pathogen adaptability on a global scale. In this review, we highlight recent advances in RNA modifications that are utilized as the strategy for pathogens to survive in the host. Specifically, we summarize the studies of RNA modifications in the pathogenic bacteria, fungi, viruses, parasites, and host. We also discuss the anti-infection potential of targeting RNA modifications.

Introduction

Pathogens involve a series of processes by which they establish infections and cause diseases in hosts. These processes include migration to the sites of infection, adhesion to host cells, invasion of the host, evasion from host defenses, and assimilation of essential nutrients [1]. During the infection, pathogenic microbes can persist if they adequately adapt to the dynamic host environment and competitive stresses. Host-derived stressors, such as oxidative stress, hypoxia, and immune response, compel pathogenic microbes to adopt a series of strategies [2]. RNA modifications, which are chemical alterations to RNA nucleotides, profoundly impact RNA synthesis, transport, function, and metabolism [3]. Growing evidence indicates that RNA modification reprogramming serves as a microbial survival strategy during host-pathogen interactions, enabling researchers to identify RNA modification-related targets for vaccine and drug development against infectious diseases [4]. In this review, we discuss recent advances in RNA modifications across different pathogens, including bacteria, fungi, viruses, and parasites. Our review enhances understanding of the molecular mechanisms of host-microbe interactions with the involvement of using RNA modifications on various RNA types (mRNA, tRNA, etc.).

RNA modifications for bacteria

Bacterial infections are among of the leading causes of mortality worldwide and represent a pressing public health concern [2]. We will discuss the regulatory roles of RNA modifications for bacterial adaptability, pathogenicity, and antibiotic resistance in some common bacteria.

Adaptability refers to the ability of bacteria to survive within host environments. The host system often recruits immune cells to resist the invading pathogenic bacteria. Hypoxia is a commonly environment due to the inflammatory response by the host immune system. Pathogenic bacteria often encounter hypoxia during infection of the host [4]. Chionh et al. found that exposure to hypoxia can change the tRNA pool and affect tRNA modification status in mycobacteria. The reprogramming of tRNA wobble mo⁵U modification regulated hypoxia-induced dormancy in mycobacteria [5]. Moreover, hypoxia induces oxidative stress by ROS overproduction. Pathogenic bacteria have evolved defense systems to neutralized ROS [6]. Lee et al. utilized menadione to increase the production of superoxide radical and reported that tRNA and rRNA m²A modifications in Enterococcus faecalis were related to the regulation of oxidative stress response. Genetic knockout of RNA-modifying enzyme RlmN in Enterococcus faecalis led to an increase in levels of superoxide dismutase [7]. This study suggests that RNA modification reprogramming can promote the adaptation of Enterococcus faecalis to oxidative stress. When the environmental temperature rises, bacteria trigger the heat shock response, including synthesizing the heat shock proteins and repairing DNA damage [8]. Riquelme-Barrios et al. used nanopore direct RNA sequencing to analyze mRNA, tRNA, and rRNA modifications in Escherichia coli. They observed increased levels of m⁵C, m⁶A, and N⁶,N⁶-dimethyladenosine in 16 S rRNA of Escherichia coli were increased in response to heat shock conditions [8]. These findings suggest that RNA modification reprogramming facilitates Escherichia coli adaptation to heat stress. Bacteria have evolved distinct strategies in response to different host environments, suggesting significant functional differences among various RNA modifications. Developing different interference strategies based on the distinct characteristics of bacteria, such as tolerance to hypoxia, heat, and acid, will be a meaningful bactericidal approach in the future.

Another aspect of bacterial adaptability is overcoming the host nutritional immunity. Host cells restrict bacterial replication by denying bacteria access to nutrients or synthesizing toxic metabolites. Bacteria need to compete with the host and the host microbiome for nutrients to establish a survival advantage [2]. Iron, as an essential nutrient for bacterial growth, plays an important role in determining pathogen survival within host environments. Ten et al. identified selective RNA modification in iron-chelating genes exbD and feoB of Acinetobacter baumannii during infection [9]. Their study revealed that mRNA modifications (m⁵C, m⁶A, and Ψ) in a virulent community-acquired Acinetobacter baumannii strain modulate, iron uptake, and bacterial survival [9]. These findings demonstrate that RNA modification reprogramming enhances nutrient assimilation in Acinetobacter baumannii during infection. An example of the host synthesizing toxic metabolites is 4-thiouracil (4-TU). 4-TU, a pyrimidine analog, is toxic to bacteria. Pyrimidine nucleotides are essential cellular components that play critical roles in pathogenic bacteria infection and survival [10]. Recently, Munneke et al. showed that Clostridioides difficile utilized the thiouracil desulfurase to prevent 4-TU incorporation into tRNA to induce s⁴U modification [10]. Their study reveals a novel detoxification strategy in which Clostridioides difficile converts 4-TU into usable pyrimidines, facilitating gut persistence. In the host gut, expression of the detoxification enzyme provides a competitive growth advantage for Clostridioides difficile. This study is an example showing how Clostridioides difficile has evolved the metabolism to benefit from antibacterial substances. Collectively, these studies indicate that RNA modification reprogramming is a crucial strategy for bacterial adaptability.

Pathogenicity refers to the virulence and invasive capacity of bacteria, it has been reported that RNA modifications are also related to bacterial virulence [1]. Krueger et al. integrated systems-level transcriptomic, proteomic data, ribosome profiling, and virulence datasets from over 400 clinical isolates. The results showed that in the opportunistic pathogen Pseudomonas aeruginosa, GidA-dependent cmnm⁵U modification modulated expression of genes encoding virulence regulators, leading to protein translation toward pathogenic physiological states [11]. This study demonstrates that Pseudomonas aeruginosa enhances its pathogenicity through RNA modification reprogramming, which serves as a regulatory mechanism for virulence expression. Queuosine (Q) modification on tRNA is crucial for protein translation in eukaryotes, and Q de novo biosynthesis only occurs in bacteria [12]. Díaz-Rullo et al. identified Q-related genes across 21 bacteria species, demonstrating that tRNA Q modification precisely regulates the translation of these genes to modulate virulence and biofilm formation in diverse bacterial phyla, particularly in human pathogens [13]. These findings indicate that RNA modification reprogramming can be a crucial strategy for bacterial pathogenicity, which provides a new direction for the development of virulence inhibitors.

Antibiotic resistance is one major cause of antibiotic treatment failure and relapse of bacterial infections. In addition to gene mutation, biofilm formation and efflux pumps are two important mechanisms by which bacteria can develop resistance to antibiotics [14]. In the above, we have mentioned that tRNA Q modification has a broad effect on the biofilm formation of various pathogenic bacteria [13]. Although the structures of biofilm are different between Gram-negative and Gram-positive bacteria, the role of tRNA Q modification in these processes is consistent. As possible mechanism for biofilm formation in all bacteria, tRNA Q modification may hold significant importance for the development of treatments to prevent bacterial infections. Efflux pump is also an important mechanism for bacteria in the host, another study elucidated the role of tRNA modification in the efflux pumps of bacteria [15]. Masuda et al. found that knocking out tRNA m¹G modification enzyme TrmD in Gram-negative bacteria (Escherichia coli and Salmonella) could increase the permeability of the cell membrane and reduce the activity of drug efflux pumps in the cell membrane [15]. These studies suggest that alterations in RNA modifications can enhance the antibiotic resistance of pathogenic bacteria. Therefore, targeting modification enzymes may be a promising strategy for eradicating resistant bacteria.

RNA modifications for viruses

Viruses are non-cellular organisms containing only one type of nucleic acid (DNA or RNA); viruses must parasitize living host cells and proliferate by replication. With the development of viral RNA epitranscriptomics, it has been documented that RNA modifications play an important role in viral replication, innate sensing pathways, and modulation of innate immune responses [16]. Here, we will discuss four extensively studied RNA modifications (m⁶A, m⁵C, ac⁴C, and 2’OMe) as the regulatory strategies for viruses.

As the most extensively studied RNA modification, m⁶A appears is ubiquitous in the mRNAs of viruses that replicate in the nucleus [17]. Kennedy et al. used PAR-CLIP to map binding sites for human YTHDF proteins on HIV-1 genome in infected cells, the results indicated that the recruitment of host proteins could promote viral m⁶A editing [18]. Further evidence demonstrated that mRNA m⁶A modification of HIV-1 played a key role in promoting HIV-1 replication. Recently, Baek et al. employed direct RNA sequencing methods to map an HIV-1 modification landscape. Their results revealed multiple functional m⁶A modification sites across diverse HIV-1 RNA transcripts, suggesting that these modifications may confer additional RNA stability and replication resilience, thereby supporting viral persistence [19]. In addition to promoting HIV replication, m⁶A is involved in regulating HIV-1 full-length RNA packaging [20]. Specifically, HIV-1 Gag and RNA demethylase FTO contribute to full-length RNA demethylation that promoted the incorporation of the HIV-1 full-length RNA into viral particles. These studies suggest that the complex and dynamic regulation of m⁶A on HIV-1 contributes to viral replication and packaging. Endogenous retroviruses are abundant and heterogenous groups of integrated retroviral sequences that affect genome regulation and cell physiology throughout RNA life cycle [21]. Chelmicki et al. employed an unbiased genome-scale CRISPR knockout screen, and they identified m⁶A RNA methylation as a way to restrict retroviruses, their findings indicated that RNA methylation provides a protective effect in maintaining cellular integrity by clearing reactive ERV-derived RNA species [22]. The latest study also indicates that mRNA m⁶A modification promotes the polymerase activity and replication of influenza A virus [23]. These studies suggest that viral m⁶A modification could be used to ensure efficient virus replication, while demethylation facilitates the packaging of the virus, suggesting the precise regulatory role of RNA modifications in the pathogenic processes of viruses.

In addition to mRNA m⁶A modification, mRNA m⁵C modification also plays a significant role in viral pathogenicity. Courtney et al. used UPLC-MS/MS to quantify 11 m⁵C residues in HIV-1 and mapped the m⁵C modification profile by PA-m⁵C-seq [24]. Their further evidence demonstrated that the inactivation of the m⁵C modification enzyme inhibited HIV-1 replication. Recent study has also shown that mRNA m⁵C modification can be used as a critical mediator of the epsilon elements function in hepatitis B virus for virion production and reverse transcription [25]. They also found that a small molecule (drug disruptor) of m⁵C methylation can significantly inhibit hepatitis B virus replication. These studies suggest that viral m⁵C modifications play a regulatory role in viral replication.

Another typical modification reported on viral RNAs is ac⁴C, which is a crucial RNA modification widely present in eukaryotic RNA. Tsai et al. used PA-ac⁴C-seq and PAR-CLIP-seq to map the ac⁴C modification profile in HIV-1 transcripts, and they identified 11 ac⁴C modification sites [26]. Remodelin, the inhibitor of ac⁴C modification enzyme NAT10, could inhibit HIV-1 replication at levels that have no effect on host cell viability, indicating the potential of RNA modification in viral disease treatment [27]. Enterovirus 71, as one of the causative agents of hand-foot-and-mouth disease, is another non-enveloped virus. Hao et al. discovered that mRNA ac4C modification enhanced enterovirus 71 replication by boosting RNA translation, RNA stability and RNA binding to RNA-dependent RNA polymerase. Notably, they found that ac⁴C-deficient mutant enterovirus 71 showed reduced pathogenicity in vivo [27]. Taken together, these studies suggest that ac⁴C modification plays a crucial role in the viral RNA stability and replication.

2’OMe is a common RNA modification found in rRNA, tRNA, snRNA and both the Cap- and body of mRNA. During viral infections, 2’OMe modification serves as a key mechanism by which viruses evade from the innate immune response of the host. Roland et al. demonstrated that human and mouse coronavirus mutants lacking 2’OMe modification upregulated the expression of type I interferon and were sensitive to type I interferon treatment [28]. Mathieu et al. observed a similar phenomenon in HIV-1. They discovered that TRBP recruited FTSJ3 to viral RNA and led to 2’OMe modification, represented an unexpected mechanism by which HIV-1 evaded innate immune recognition [29]. Yu et al. demonstrated that the 2’-O-methyltransferase activity of the SARS-CoV depended on the nsp16/nsp10 protein complex, a feature unique to coronaviruses [30]. This study provides a basis for the development of antiviral drugs that target coronavirus protein interfaces. These studies demonstrate that 2’OMe modifications of viral RNAs exploited by certain viruses as self-signal enabling evasion of interferon-mediated antiviral responses.

In addition to m⁶A, m⁵C, ac⁴C, and 2’OMe, other typical RNA modifications have been studied in viruses [17]. Altering the expression of RNA modification enzymes can affect the replication and packaging of the virus. Therefore, investigating the modulation of RNA modification enzymes represents a significant avenue for developing antiviral strategies.

RNA modifications for fungi

Fungal infections are on the rise worldwide and represent a substantial threat to human health [31]. Despite their ubiquitous nature, studies on RNA modifications in human pathogenic fungi are relatively scarce compared to other pathogens. We will discuss the RNA modification strategies of three common pathogenic fungi (Candida albicans, Aspergillus fumigatus, and Aspergillus flavus).

Candida albicans is one of the major pathogenic fungi of severe infections, and the virulence of Candida albicans is closely linked to RNA modifications [31]. Böttcher et al. found that the expression of tRNA-modifying enzymes was different between highly pathogenic Candida albicans and lowly pathogenic Candida dubliniensis [31]. Their further study found that tRNA t⁶A modification enzyme Hma1 influenced the translation efficiency of many hyphae- and pathogenicity-associated genes, thereby affecting morphogenesis, invasion, and virulence of Candida albicans. Another study on Candida albicans revealed that the accumulation of tRNA mcm⁵s²U modification was conducive to enhancing the virulence of Candida albicans. In support of these findings, strains lacking tRNA mcm⁵s²U modification failed to infect mice and to damage human cells [32]. These findings underscore the potential of tRNA-modifying enzymes as targets for novel antifungal therapeutics against Candida albicans.

Aspergillus fumigatus is an opportunistic pathogen and poses a major threat to immunocompromised individuals in clinical environments. Aspergillus fumigatus has evolved a diverse range of sophisticated strategies to adapt to environmental constraints and to survive within and colonize the host [33]. Bruch et al. found that deletion of Aspergillus fumigatus tRNA-modifying isopentenyl transferase ortholog Mod5 led to altered stress response and unexpected resistance against the antifungal drug 5-fluorocytosine, suggesting that tRNA i⁶A hypomodification can facilitate 5-fluorocytosine resistance via cross-pathway control system activation in Aspergillus fumigatus [34]. This study expands the limited perspective on fungal drug resistance that focuses solely on genetic mutations. Zhang et al. found that mcm⁵s²U modification at tRNA wobble base U₃₄ mediated the hyphal growth, conidiation, exopolysaccharide galactosaminogalactan production, adhesion, and virulence of Aspergillus fumigatus [33]. This study reflects that minor alterations in microbial nucleotide modifications can have extensive impacts, leading to aberrant protein translation and virulence states, which suggests that the global strategy targeting translational regulation will be significantly superior to the link from a single gene to a single phenotype.

Aflatoxin, a potent mycotoxin produced by Aspergillus flavus, has been associated with severe human heath consequences, including developmental delays and hepatocellular carcinoma [35]. In a pivotal study, Liang et al. found that cycloleucine significantly suppresses growth, conidiation, mycelial biomass accumulation, and aflatoxin biosynthesis in Aspergillus flavus by altering mRNA m⁶A patterns [35]. This study has shown that mRNA m⁶A modification is significantly involved in multiple aspects of the pathogenicity of Aspergillus flavus. Yang et al. examined the RNA modifications of Aspergillus flavus under different temperature conditions, and they found that 12 RNA modifications were significantly changed under the treatment [36]. Their further study showed that mRNA m⁶A modification promoted aflatoxin biosynthesis, fungal conidiation, sclerotia formation, and colonization within the host by using meRIP-Seq. These studies demonstrate illustrate how a single RNA modification can coordinately regulate multiple biological processes in Aspergillus flavus, highlighting the broad systemic influence of translational regulation. In other words, targeting RNA modification enzymes will have a ripple effect that impacts the entire system.

RNA modifications for parasites

Parasitic infections and their global burden on human health are often underestimated [37]. Due to the interaction between multicellular parasites and their hosts are highly complex, current research on RNA modifications in parasites is primarily focused on unicellular parasites. We will discuss the current research progress on RNA modifications in four common medical protozoa (Trypanosoma brucei, Plasmodium falciparum, Entamoeba histolytica, and Leishmania).

Trypanosoma brucei is the causative agent of sleeping sickness, cycling between the hosts requires adaptation to environmental and temperature changes [38]. Rajan et al. found that differential Ψ modifications contributed to the function of noncoding RNAs involved in rRNA processing, rRNA modification, protein synthesis, and protein translocation during cycling of Trypanosoma brucei between two hosts [38]. This study highlights the important role of Ψ modifications in different developmental stages of Trypanosoma brucei. In eukaryotes, 5’ mRNA capping is one of the essential co-transcriptional modifications to produce mature mRNA [39]. Anna et al. found that N6, N6-2’-O trimethyladenosine modification in cap 4 enhanced guanylyltransferase activity of TbCe1, which is a cytoplasmic recapping enzyme in Trypanosoma brucei [40]. This mechanism facilitates the regeneration of translatable mRNA in Trypanosoma brucei. In addition to adapting to the host environment, parasite requires the evasion of host immune system [41]. Viegas et al. found that mRNA m⁶A was present in the poly(A) tail of Trypanosoma brucei RNA, and the modification was enriched in the mRNA of variant surface glycoproteins. This unique strategy can help Trypanosoma brucei escape the immune system [41]. These studies highlight the crucial role of RNA modifications in the survival and pathogenesis of Trypanosoma brucei.

Plasmodium falciparum, the causative agent of human malaria, modulates stress tolerance and drug resistance through the regulation of RNA-modifying enzymes [42]. Hammam et al. demonstrated that loss of m⁵C methyltransferase Pf-DNMT2 induced sexual commitment in Plasmodium falciparum [42]. This study also identified a specific tRNA cytosine methylation site that maintained the homeostasis of Plasmodium falciparum against various stresses. Liu et al. mapped the transcriptome profile of m⁵C modifications and described the stage-specific dynamics in two species of Plasmodium, and they found that the m⁵C modification enzyme NSUN2 promoted mRNA stability and gametocytes development of Plasmodium [43]. These studies suggest that m⁵C modification reprogramming can serve as an adaptive strategy for Plasmodium within host cells. Recently, Small-Saunders et al. showed that Plasmodium falciparum used tRNA mcm⁵s²U modification reprogramming for codon bias translation to survive against dihydroartemisinin treatment [44]. This study found that tRNA mcm⁵s²U reprogramming led to decreased carbohydrate metabolism, translation and growth. These characteristics were the same as quiescent, artemisinin-resistant Plasmodium falciparum. Overall, RNA modification reprogramming is an important strategy for development, adaptation, and antibiotic resistance of Plasmodium.

Entamoeba histolytica, the caustive agent of human amebiasis, represents a significant global health burden [45]. Nagaraja et al. found that queuine, as a tRNA Q precursor, can impair Entamoeba histolytica virulence by downregulating the expression of genes associated with virulence, further analysis showed that queuine can be efficiently incorporated into Entamoeba histolytica tRNAs as Q modification to protect the parasite against oxidative stress [45]. This study further demonstrated that queuine compromises Entamoeba histolytica survival in the large intestine of mice. Mammals cannot synthesize queuine and have to salvage it from gut microbes [12], suggesting that the microbiota may resist infection by regulating pathogen tRNA modifications. In the future, selecting probiotics with high queuine production may represent a new therapeutic approach for the treatment of amoebiasis.

Leishmania causes leishmaniases, which is a neglected tropical disease causing 0.7 to 1.2 million infections worldwide [46]. Recently, Rajan et al. obtained the landscape of Ψs on rRNA from Leishmania in promastigote, and they found that Ψ modification was differentially regulated in two life stages of all three Leishmania species [47]. Besides, they also proved that Leishmania rRNA Ψ modification was shown to affect ribosome structure and mRNA translation when lacking a single pseudouridine modification [47]. This study elucidates the exquisite regulatory role of Ψ modification from the perspective of molecular structure.

RNA modifications for host-microbe interactions

Host-microbe interactions can be divided into two aspects. One is the effects of host on microbe, and the other aspect addresses the effects of microbe on the host. Our recent study found that gut microbiota can dynamically reprogram host gene expression via multiple post-transcriptional regulatory factors [48]. When the microbiota undergoes alterations, such as antibiotics-induced dysbiosis, we found that mRNA m⁶A epitranscriptome in host multiple tissues were directly affected by alterations in metabolic pathways [49]. In addition to the influence of gut microbiota, viral infections can also affect the host’s epitranscriptome. For example, Srinivas et al. showed that herpes simplex virus ICP27 disrupted mRNA m⁶A pathway in host through redistribution of nuclear methyltransferase components into the cytoplasm [50]. This study demonstrated that HSV-1 disrupted gene expression by modulating host mRNA m⁶A modification to promote viral replication. Recently, Zheng et al. found that during Siniperca chuatsi rhabdovirus infection, YTHDC2 could promote the STING protein degradation and repress STING mediated antiviral responses, thereby promote viral replication via m⁶A modification [51]. These results indicate that the host seemed to passively undergo changes in RNA modifications following pathogenic microbial infection, and these changes are beneficial to the pathogen but detrimental to the host. However, other studies have demonstrated that this is not always the case. Liu et al. found that the host cell suppressed the activity of m⁶A demethylase ALKBH5 to reduce the expression of the α-ketoglutarate dehydrogenase gene and the production of itaconate, thereby inhibit viral replication [52]. Further study revealed that ALKBH5 facilitated neutrophil accumulation and migration to the site of infection in antibacterial defense [53]. These results indicate that the host can also actively mobilizes defensive pathways against pathogen invasion through dynamic m⁶A modification reprogramming. Overall, host RNA modification reprogramming plays a significant role in host-microbe interactions. With the increased manipulability of host RNA modifications, targeting RNA modifications will be an effective strategy for preventing pathogen infections.

Outlook

Pathogens in the environment are predominantly small organisms with limited energy and nutrition. In contrast, their hosts are giant, multicellular organisms equipped with various defense mechanisms. During long-term interaction and competition with hosts, pathogens have evolved various ingenious reprogramming mechanisms, enabling them to survive and proliferate. RNA epitranscriptome has been an important layer to interpret the adaptive strategy of pathogenic microbes. Accumulating evidence suggests that RNA modification reprogramming is a key strategy for various pathogenic microbes to survive in the host. As a new layer of gene expression regulation, the role of RNA modifications during host-microbe interactions can be extended to various microbes, including bacteria, viruses, fungi, parasites, etc. During host-microbe interactions, RNA modifications are critically involved in key microbial process including infectivity, replication, host immune evasion, and metabolism, etc (Fig. 1). It is known that mRNA was considered as unfeasible for clinical use because of its instability, but reprogramming the epitranscriptome has emerged as a promising strategy for infectious disease treatment in clinical practice. For example, Katalin et al. found that incorporating 1-methyl-pseudouridine modification could enhance the stability of RNA vaccines [54]. Based on this mechanism, COVID-19 RNA vaccine BNT162b1 demonstrates a favorable neutralizing effect against the virus [55]. Recently, Marcin et al. described that the modifications of 5’-cap and 3’-end were extremely important for the regulation of translation and mRNA durability [56]. It has become evident that chemical modifications play a pivotal role in stabilizing the RNA molecules, which is expected to establish mRNA as an ideal tool for gene therapy. Nonetheless, our understanding of RNA modifications and corresponding modifying enzymes is still limited. The remarkable dimensions and diversity of the viral and fungal epitranscriptome far exceed the handful of modifications that have been reported. Besides, the function of the RNA epitranscriptome in multicellular parasites remains to be investigated. Although it is a challenging endeavor, we believe that scientists will be capable of detecting, quantifying, and even editing RNA modifications with the development of new high-throughput technologies. We also believe that it will be helpful to target RNA modifications as an effective strategy against microbial infections in the future.

Fig. 1.

RNA modification can be utilized as a strategy for pathogens to survive in the host

Authors’ contributions

Xiaoyun Wang designed the idea for the article. Ye Tian performed the literature search and drafted the manuscript with the help from Xiaoyun Wang.

Funding

This work was supported by Guangzhou Science and Technology Project (2024A04J6265), and Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme for X.W.

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors agreed with the final manuscript and gave explicit consent to submit. All authors obtained consent from the responsible authorities at the institute where the work has been carried out.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.