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Retrotransposition is one of the main factors responsible for gene duplication and thus genome evolution. However, the sequences that undergo this process are not only an excellent source of biological diversity, but in certain cases also pose a threat to the integrity of the DNA. One of the mechanisms that protects against the incorporation of mobile elements is the HUSH complex, which is responsible for silencing long, intronless, transcriptionally active transposed sequences that are rich in adenine on the sense strand. In this study, broad sets of human and porcine retrocopies were analysed with respect to the above factors, taking into account evolution of these molecules. Analysis of expression pattern, genomic structure, transcript length, and nucleotide substitution frequency showed the strong relationship between the expression level and exon length as well as the protective nature of introns. The results of the studies also showed that there is no direct correlation between the expression level and adenine content. However, protein-coding retrocopies, which have a lower adenine content, have a significantly higher expression level than the adenine-rich non-coding but expressed retrocopies. Therefore, although the mechanism of HUSH silencing may be an important part of the regulation of retrocopy expression, it is one component of a more complex molecular network that remains to be elucidated. [ABSTRACT FROM AUTHOR]

Abstract Retrotransposition is one of the main factors responsible for gene duplication and thus genome evolution. However, the sequences that undergo this process are not only an excellent source of biological diversity, but in certain cases also pose a threat to the integrity of the DNA. One of the mechanisms that protects against the incorporation of mobile elements is the HUSH complex, which is responsible for silencing long, intronless, transcriptionally active transposed sequences that are rich in adenine on the sense strand. In this study, broad sets of human and porcine retrocopies were analysed with respect to the above factors, taking into account evolution of these molecules. Analysis of expression pattern, genomic structure, transcript length, and nucleotide substitution frequency showed the strong relationship between the expression level and exon length as well as the protective nature of introns. The results of the studies also showed that there is no direct correlation between the expression level and adenine content. However, protein-coding retrocopies, which have a lower adenine content, have a significantly higher expression level than the adenine-rich non-coding but expressed retrocopies. Therefore, although the mechanism of HUSH silencing may be an important part of the regulation of retrocopy expression, it is one component of a more complex molecular network that remains to be elucidated.

Recent years have seen a dramatic increase in the number of canine genome assemblies available. Duplications are an important source of evolutionary novelty and are also prone to misassembly. We explored the duplication content of nine canine genome assemblies using both genome self-alignment and read-depth approaches. We find that 8.58% of the genome is duplicated in the canFam4 assembly, derived from the German Shepherd Dog Mischka, including 90.15% of unplaced contigs. Highlighting the continued difficulty in properly assembling duplications, less than half of read-depth and assembly alignment duplications overlap, but the mCanLor1.2 Greenland wolf assembly shows greater concordance. Further study shows the presence of multiple segments that have alignments to four or more duplicate copies. These high-recurrence duplications correspond to gene retrocopies. We identified 3,892 candidate retrocopies from 1,316 parental genes in the canFam4 assembly and find that ∼8.82% of duplicated base pairs involve a retrocopy, confirming this mechanism as a major driver of gene duplication in canines. Similar patterns are found across eight other recent canine genome assemblies, with metrics supporting a greater quality of the PacBio HiFi mCanLor1.2 assembly. Comparison between the wolf and other canine assemblies found that 92% of retrocopy insertions are shared between assemblies. By calculating the number of generations since genome divergence, we estimate that new retrocopy insertions appear, on average, in 1 out of 3,514 births. Our analyses illustrate the impact of retrogene formation on canine genomes and highlight the variable representation of duplicated sequences among recently completed canine assemblies. [ABSTRACT FROM AUTHOR]

Two transcribed retrocopies of the fibroblast growth factor 4 (FGF4) gene have previously been described in the domestic dog. An FGF4 retrocopy on chr18 is associated with disproportionate dwarfism, while an FGF4 retrocopy on chr12 is associated with both disproportionate dwarfism and intervertebral disc disease (IVDD). In this study, whole-genome sequencing data were queried to identify other FGF4 retrocopies that could be contributing to phenotypic diversity in canids. Additionally, dogs with surgically confirmed IVDD were assayed for novel FGF4 retrocopies. Five additional and distinct FGF4 retrocopies were identified in canids including a copy unique to red wolves (Canis rufus). The FGF4 retrocopies identified in domestic dogs were identical to domestic dog FGF4 haplotypes, which are distinct from modern wolf FGF4 haplotypes, indicating that these retrotransposition events likely occurred after domestication. The identification of multiple, full length FGF4 retrocopies with open reading frames in canids indicates that gene retrotransposition events occur much more frequently than previously thought and provide a mechanism for continued genetic and phenotypic diversity in canids.

Retroposed protein-coding genes are commonly considered to be nonfunctional duplicates. However, they often gain transcriptional capability and have important roles. Amici et al. recently identified novel functions of a retroposed gene. HAPSTR2 , a retrocopy of HAPSTR1 , encodes a protein that stabilizes the HAPSTR1 protein and functionally buffers its loss. [ABSTRACT FROM AUTHOR]

The major advantage of mRNA vaccines over more conventional approaches is their potential for rapid development and large-scale deployment in pandemic situations. In the current COVID-19 crisis, two mRNA COVID-19 vaccines have been conditionally approved and broadly applied, while others are still in clinical trials. However, there is no previous experience with the use of mRNA vaccines on a large scale in the general population. This warrants a careful evaluation of mRNA vaccine safety properties by considering all available knowledge about mRNA molecular biology and evolution. Here, I discuss the pervasive claim that mRNA-based vaccines cannot alter genomes. Surprisingly, this notion is widely stated in the mRNA vaccine literature but never supported by referencing any primary scientific papers that would specifically address this question. This discrepancy becomes even more puzzling if one considers previous work on the molecular and evolutionary aspects of retroposition in murine and human populations that clearly documents the frequent integration of mRNA molecules into genomes, including clinical contexts. By performing basic comparisons, I show that the sequence features of mRNA vaccines meet all known requirements for retroposition using L1 elements—the most abundant autonomously active retrotransposons in the human genome. In fact, many factors associated with mRNA vaccines increase the possibility of their L1-mediated retroposition. I conclude that is unfounded to a priori assume that mRNA-based therapeutics do not impact genomes and that the route to genome integration of vaccine mRNAs via endogenous L1 retroelements is easily conceivable. This implies that we urgently need experimental studies that would rigorously test for the potential retroposition of vaccine mRNAs. At present, the insertional mutagenesis safety of mRNA-based vaccines should be considered unresolved. [ABSTRACT FROM AUTHOR]

Retroduplication variation (RDV), a type of retrocopy polymorphism, is considered to have essential biological significance, but its effect on gene function and species phenotype is still poorly understood. To this end, we analyzed the retrocopies and RDVs in 3,010 rice genomes. We calculated the RDV frequencies in the genome of each rice population; detected the mutated, ancestral and expressed retrogenes in rice genomes; and analyzed their RDV influence on rice phenotypic traits. Collectively, 73 RDVs were identified, and 14 RDVs in ancestral retrogenes can significantly affect rice phenotypes. Our research reveals that RDV plays an important role in rice migration, domestication and evolution. We think that RDV is a good molecular breeding marker candidate. To our knowledge, this is the first study on the relationship between retrogene function, expression, RDV and species phenotype.

Retrocopies, which are considered "junk genes," are occasionally formed via the insertion of reverse-transcribed mRNAs at new positions in the genome. However, an increasing number of recent studies have shown that some retrocopies exhibit new biological functions and may contribute to genome evolution. Hence, the identification of retrocopies has become very meaningful for studying gene duplication and new gene generation. Current pipelines identify retrocopies through complex operations using alignment programs and filter scripts in a step-by-step manner. Therefore, there is an urgent need for a simple and convenient retrocopy annotation tool. Here, we report the development of RetroScan, a publicly available and easy-to-use tool for scanning, annotating and displaying retrocopies, consisting of two components: an analysis pipeline and a visual interface. The pipeline integrates a series of bioinformatics software programs and scripts for identifying retrocopies in just one line of command. Compared with previous methods, RetroScan increases accuracy and reduces false-positive results. We also provide a Shiny app for visualization. It displays information on retrocopies and their parental genes that can be used for the study of retrocopy structure and evolution. RetroScan is available at https://github.com/Vicky123wzy/RetroScan. [ABSTRACT FROM AUTHOR]

The gene encoding the ubiquitous DNA repair protein, Ku70p, has undergone extensive copy number expansion during primate evolution. Gene duplications of KU70 have the hallmark of long interspersed element-1 mediated retrotransposition with evidence of target-site duplications, the poly-A tails, and the absence of introns. Evolutionary analysis of this expanded family of KU70-derived "NUKU" retrocopies reveals that these genes are both ancient and also actively being created in extant primate species. NUKU retrocopies show evidence of functional divergence away from KU70, as evinced by their altered pattern of tissue expression and possible tissue-specific translation. Molecular modeling predicted that amino acid changes in Nuku2p at the interaction interface with Ku80p would prevent the assembly of the Ku heterodimer. The lack of Nuku2p-Ku80p interaction was confirmed by yeast two-hybrid assay, which contrasts the robust interaction of Ku70p-Ku80p. While several NUKU retrocopies appear to have been degraded by mutation, NUKU2 shows evidence of positive natural selection, suggesting that this retrocopy is undergoing neofunctionalization. Although Nuku proteins do not appear to antagonize retrovirus transduction in cell culture, the observed expansion and rapid evolution of NUKUs could be being driven by alternative selective pressures related to infectious disease or an undefined role in primate physiology. [ABSTRACT FROM AUTHOR]

Retrocopies, which are considered “junk genes,” are occasionally formed via the insertion of reverse-transcribed mRNAs at new positions in the genome. However, an increasing number of recent studies have shown that some retrocopies exhibit new biological functions and may contribute to genome evolution. Hence, the identification of retrocopies has become very meaningful for studying gene duplication and new gene generation. Current pipelines identify retrocopies through complex operations using alignment programs and filter scripts in a step-by-step manner. Therefore, there is an urgent need for a simple and convenient retrocopy annotation tool. Here, we report the development of RetroScan, a publicly available and easy-to-use tool for scanning, annotating and displaying retrocopies, consisting of two components: an analysis pipeline and a visual interface. The pipeline integrates a series of bioinformatics software programs and scripts for identifying retrocopies in just one line of command. Compared with previous methods, RetroScan increases accuracy and reduces false-positive results. We also provide a Shiny app for visualization. It displays information on retrocopies and their parental genes that can be used for the study of retrocopy structure and evolution. RetroScan is available at https://github.com/Vicky123wzy/RetroScan.

Abstract Background Different pathogenic germline mutations in the RET oncogene are identified in MEN 2, a hereditary syndrome characterized by medullary thyroid carcinoma (MTC) and other endocrine tumors. Although genetic predisposition is recognized, not all RET mutation carriers will develop the disease during their lifetime or, likewise, RET mutation carriers belonging to the same family may present clinical heterogeneity. It has been suggested that a single germline mutation might not be sufficient for development of MEN 2-associated tumors and a somatic bi-allelic alteration might be required. Here we investigated the presence of somatic second hit mutation in the RET gene in MTC. Methods We integrated Multiplex Ligation-dependent Probe Amplification (MLPA) and whole exome sequencing (WES) to search for copy number alteration (CNA) in the RET gene in MTC samples and medullary thyroid cell lines (TT and MZ-CR-1). We next found reads spanning exon-exon boundaries on RET, an indicative of retrocopy. We subsequently searched for RET retrocopies in the human reference genome (GRCh37) and in the 1000 Genomes Project data, by looking for reads reporting joined exons in the RET locus or distinct genomic regions. To determine RET retrocopy specificity and recurrence, DNA isolated from sporadic and MEN 2-associated MTC (n = 37), peripheral blood (n = 3) and papillary thyroid carcinomas with RET fusion (n = 10) samples were tested using PCR-sequencing methodology. Results Through MLPA we have found evidence of CNA in the RET gene in MTC samples and MTC cell lines. WES analysis reinforced the presence of the CNA and hinted for a retroposed copy of RET not found in the human reference genome and 1.000 Genomes Project. Extended analysis confirmed the presence of a somatic MTC-related retrocopy of RET in both sporadic and hereditary tumors. We further unveiled a recurrent (28%) novel point mutation (p.G548 V) found exclusively in the retrocopy of RET. The mutation was also found in cDNA of mutated samples, suggesting it might be functional. Conclusion We here report a somatic specific RET retroposed copy in MTC samples and cell lines. Our results support the idea that generation of retrocopies in somatic cells is likely to contribute to MTC genesis and progression.

Gene duplication is a major driver of organismal evolution. Gene retroposition is a mechanism of gene duplication whereby a gene's transcript is used as a template to generate retroposed gene copies, or retrocopies. Intriguingly, the formation of retrocopies depends upon the enzymatic machinery encoded by retrotransposable elements, genomic parasites occurring in the majority of eukaryotes. Most retrocopies are depleted of the regulatory regions found upstream of their parental genes; therefore, they were initially considered transcriptionally incompetentgene copies, or retropseudogenes. However, examples of functional retrocopies, or retrogenes, have accumulated since the 1980s. Here, we review what we have learned about retrocopies in animals, plants and other eukaryotic organisms, with a particular emphasis on comparative and population genomic analyses complemented with transcriptomic datasets. In addition, these data have provided information about the dynamics of the different "life cycle" stages of retrocopies (i.e., polymorphic retrocopy number variants, fixed retropseudogenes and retrogenes) and have provided key insights into the retroduplication mechanisms, the patterns and evolutionary forces at work during the fixation process and the biological function of retrogenes. Functional genomic and transcriptomic data have also revealed that many retropseudogenes are transcriptionally active and a biological role has been experimentally determined for many. Finally, we have learned that not only non-long terminal repeat retroelements but also long terminal repeat retroelements play a role in the emergence of retrocopies across eukaryotes. This body of work has shown that mRNA-mediated duplication represents a widespread phenomenon that produces an array of new genes that contribute to organismal diversity and adaptation. [ABSTRACT FROM AUTHOR]

Transposable elements, often considered to be not important for survival, significantly contribute to the evolution of transcriptomes, promoters, and proteomes. Reverse transcriptase, encoded by some transposable elements, can be used in trans to produce a DNA copy of any RNA molecule in the cell. The retrotransposition of protein-coding genes requires the presence of reverse transcriptase, which could be delivered by either non-long terminal repeat (non-LTR) or LTR transposons. The majority of these copies are in a state of "relaxed" selection and remain "dormant" because they are lacking regulatory regions; however, many become functional. In the course of evolution, they may undergo subfunctionalization, neofunctionalization, or replace their progenitors. Functional retrocopies (retrogenes) can encode proteins, novel or similar to those encoded by their progenitors, can be used as alternative exons or create chimeric transcripts, and can also be involved in transcriptional interference and participate in the epigenetic regulation of parental gene expression. They can also act in trans as natural antisense transcripts, microRNA (miRNA) sponges, or a source of various small RNAs. Moreover, many retrocopies of protein-coding genes are linked to human diseases, especially various types of cancer. [ABSTRACT FROM AUTHOR]

Background: The coelacanth is known as a "living fossil" because of its morphological resemblance to its fossil ancestors. Thus, it serves as a useful model that provides insight into the fish that first walked on land. Retrocopies are a type of novel genetic element that are likely to contribute to genome or phenotype innovations. Thus, investigating retrocopies in the coelacanth genome can determine the role of retrocopies in coelacanth genome innovations and perhaps even water-to-land adaptations. Results: We determined the dS values, dN/dS ratios, expression patterns, and enrichment of functional categories for 472 retrocopies in the African coelacanth genome. Of the retrocopies, 85–355 were shown to be potentially functional (i.e., retrogenes). The distribution of retrocopies based on their dS values revealed a burst pattern of young retrocopies in the genome. The retrocopy birth pattern was shown to be more similar to that in tetrapods than ray-finned fish, which indicates a genomic transformation that accompanied vertebrate evolution from water to land. Among these retrocopies, retrogenes were more prevalent in old than young retrocopies, which indicates that most retrocopies may have been eliminated during evolution, even though some retrocopies survived, attained biological function as retrogenes, and became old. Transcriptome data revealed that many retrocopies showed a biased expression pattern in the testis, although the expression was not specifically associated with a particular retrocopy age range. We identified 225 Ensembl genes that overlapped with the coelacanth genome retrocopies. GO enrichment analysis revealed different overrepresented GO (gene ontology) terms between these "retrocopy-overlapped genes" and the retrocopy parent genes, which indicates potential genomic functional organization produced by retrotranspositions. Among the 225 retrocopy-overlapped genes, we also identified 46 that were coelacanth-specific, which could represent a potential molecular basis for coelacanth evolution. Conclusions: Our study identified 472 retrocopies in the coelacanth genome. Sequence analysis of these retrocopies and their parent genes, transcriptome data, and GO annotation information revealed novel insight about the potential role of genomic retrocopies in coelacanth evolution and vertebrate adaptations during the evolutionary transition from water to land. [ABSTRACT FROM AUTHOR]

Gene duplication is a key factor contributing to phenotype diversity across and within species. Although the availability of complete genomes has led to the extensive study of genomic duplications, the dynamics and variability of gene duplications mediated by retrotransposition are not well understood. Here, we predict mRNA retrotransposition and use comparative genomics to investigate their origin and variability across primates. Analyzing seven anthropoid primate genomes, we found a similar number of mRNA retrotranspositions (~7,500 retrocopies) in Catarrhini (Old Word Monkeys, including humans), but a surprising large number of retrocopies (~10,000) in Platyrrhini (NewWorld Monkeys), which may be a by-product of higher long interspersed nuclear element 1 activity in thesegenomes. By inferring retrocopy orthology, we dated most of the primate retrocopy origins, and estimated a decrease in the fixation rate in recent primate history, implying a smaller numbe rof species-specific retrocopies. Moreover, using RNA-Seqdata, we identified approximately 3,600 expressed retrocopies. As expected, most of these retrocopies are located near or within known genes, present tissue-specific and even species-specific expression patterns, and no expression correlation to their parental genes. Taken together, our results provide further evidence that mRNA retrotransposition is an active mechanism in primate evolution and suggest that retrocopies may not only introduce great genetic variability between lineages but also create a large reservoir of potentially functional new genomic loci in primate genomes. [ABSTRACT FROM AUTHOR]

Imprinting of the human RB1 gene is due to the presence of a differentially methylated CpG island (CGI) in intron 2, which is part of a retrocopy derived from the PPP1R26 gene on chromosome 9. The murine Rb1 gene does not have this retrocopy and is not imprinted. We have investigated whether the RB1/Rb1 locus is unique with respect to these differences. For this, we have compared the CGIs from human and mouse by in silico analyses. We have found that the human genome does not only contain more CGIs than the mouse, but the proportion of intronic CGIs is also higher (7.7% vs. 3.5%). At least 2,033 human intronic CGIs are not present in the mouse. Among these CGIs, 104 show sequence similarities elsewhere in the human genome, which suggests that they arose from retrotransposition. We could narrow down the time points when most of these CGIs appeared during evolution. Their methylation status was analyzed in two monocyte methylome data sets from whole-genome bisulfite sequencing and in 18 published methylomes. Four CGIs, which are located in the RB1, ASRGL1, PARP11, and PDXDC1 genes, occur as methylated and unmethylated copies. In contrast to imprinted methylation at the RB1 locus, differential methylation of the ASRGL1 and PDXDC1 CGIs appears to be sequence dependent. Our study supports the notion that the epigenetic fate of the retrotransposed DNA depends on its sequence and selective forces at the integration site. [ABSTRACT FROM AUTHOR]

Gene retrocopies are generated by reverse transcription and genomic integration of mRNA. As such, retrocopies present an important exception to the central dogma of molecular biology, and have substantially impacted the functional landscape of the metazoan genome. While an estimated 8,000-17,000 retrocopies exist in the human genome reference sequence, the extent of variation between individuals in terms of retrocopy content has remained largely unexplored. Three recent studies by Abyzov et al., Ewing et al. and Schrider et al. have exploited 1,000 Genomes Project Consortium data, as well as other sources of whole-genome sequencing data, to uncover novel gene retrocopies. Here, we compare the methods and results of these three studies, highlight the impact of retrocopies in human diversity and genome evolution, and speculate on the potential for somatic gene retrocopies to impact cancer etiology and genetic diversity among individual neurons in the mammalian brain. [ABSTRACT FROM AUTHOR]

Background: RNA-based gene duplicates (retrocopies) played pivotal roles in many physiological processes. Nowadays, functional retrocopies have been systematically identified in several mammals, fruit flies, plants, zebrafish and other chordates, etc. However, studies about this kind of duplication in Caenorhabditis nematodes have not been reported. Findings: We identified 43, 48, 43, 9, and 42 retrocopies, of which 6, 15, 18, 3, and 13 formed chimeric genes in C. brenneri, C. briggsae, C. elegans, C. japonica, and C. remanei, respectively. At least 5 chimeric types exist in Caenorhabditis species, of which retrocopy recruiting both N and C terminus is the commonest one. Evidences from different analyses demonstrate many retrocopies and almost all chimeric genes may be functional in these species. About half of retrocopies in each species has coordinates in other species, and we suggest that retrocopies in closely related species may be helpful in identifying retrocopies for one certain species. Conclusions: A number of retrocopies and chimeric genes exist in Caenorhabditis genomes, and some of them may be functional. The evolutionary patterns of these genes may correlate with their genomic features, such as the activity of retroelements, the high rate of mutation and deletion rate, and a large proportion of genes subject to trans-splicing. [ABSTRACT FROM AUTHOR]

Many enveloped viruses require the endosomal sorting complexes required for transport (ESCRT) pathway to exit infected cells. This highly conserved pathway mediates essential cellular membrane fission events, which restricts the acquisition of adaptive mutations to counteract viral co-option. Here, we describe duplicated and truncated copies of the ESCRT-III factor CHMP3 that block ESCRT-dependent virus budding and arose independently in New World monkeys and mice. When expressed in human cells, these retroCHMP3 proteins potently inhibit release of retroviruses, paramyxoviruses, and filoviruses. Remarkably, retroCHMP3 proteins have evolved to reduce interactions with other ESCRT-III factors and have little effect on cellular ESCRT processes, revealing routes for decoupling cellular ESCRT functions from viral exploitation. The repurposing of duplicated ESCRT-III proteins thus provides a mechanism to generate broad-spectrum viral budding inhibitors without blocking highly conserved essential cellular ESCRT functions. [Display omitted] • Truncated retrocopies of CHMP3 arose independently in New World monkeys and mice • RetroCHMP3 proteins broadly inhibit budding of ESCRT-dependent viruses • RetroCHMP3 proteins evolved to lose cytotoxicity associated with ESCRT inhibition • Delays in ESCRT function inhibit virus budding but spare critical host pathways Truncated retrocopies of ESCRT-III factor CHMP3, which have independently evolved in New World monkey and mouse species, block ESCRT-dependent virus budding with little effect on critical cellular ESCRT processes in human cells. [ABSTRACT FROM AUTHOR]

Abstract Background RNA-based gene duplicates (retrocopies) played pivotal roles in many physiological processes. Nowadays, functional retrocopies have been systematically identified in several mammals, fruit flies, plants, zebrafish and other chordates, etc. However, studies about this kind of duplication in Caenorhabditis nematodes have not been reported. Findings We identified 43, 48, 43, 9, and 42 retrocopies, of which 6, 15, 18, 3, and 13 formed chimeric genes in C. brenneri, C. briggsae, C. elegans, C. japonica, and C. remanei, respectively. At least 5 chimeric types exist in Caenorhabditis species, of which retrocopy recruiting both N and C terminus is the commonest one. Evidences from different analyses demonstrate many retrocopies and almost all chimeric genes may be functional in these species. About half of retrocopies in each species has coordinates in other species, and we suggest that retrocopies in closely related species may be helpful in identifying retrocopies for one certain species. Conclusions A number of retrocopies and chimeric genes exist in Caenorhabditis genomes, and some of them may be functional. The evolutionary patterns of these genes may correlate with their genomic features, such as the activity of retroelements, the high rate of mutation and deletion rate, and a large proportion of genes subject to trans-splicing.

Retroduplication variation (RDV), a type of retrocopy polymorphism, is considered to have essential biological significance, but its effect on gene function and species phenotype is still poorly understood. To this end, we analyzed the retrocopies and RDVs in 3,010 rice genomes. We calculated the RDV frequencies in the genome of each rice population; detected the mutated, ancestral and expressed retrogenes in rice genomes; and analyzed their RDV influence on rice phenotypic traits. Collectively, 73 RDVs were identified, and 14 RDVs in ancestral retrogenes can significantly affect rice phenotypes. Our research reveals that RDV plays an important role in rice migration, domestication and evolution. We think that RDV is a good molecular breeding marker candidate. To our knowledge, this is the first study on the relationship between retrogene function, expression, RDV and species phenotype., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)