Hervé Seligmann

Ganesh Warthi
email: g.warthi6791@gmail.com

Chimeric translation for mitochondrial peptides: regular and expanded codons

Keywords: delRNAs, non-canonical translation, non-canonical transcription, RNA editing, tRNA hopping

Frameshifting protein translation occasionally results from insertion of amino acids at isolated mono-or dinucleotide-expanded codons by tRNAs with expanded anticodons. Previous analyses of two different types of human mitochondrial MS proteomic data (Fisher and Waters technologies) detect peptides entirely corresponding to expanded codon translation. Here, these proteomic data are reanalyzed searching for peptides consisting of at least eight consecutive amino acids translated according to regular tricodons, and at least eight adjacent consecutive amino acids translated according to expanded codons. Both datasets include chimerically translated peptides (mono-and dinucleotide expansions, 42 and 37, respectively). The regular tricodon-encoded part of some chimeric peptides corresponds to standard human mitochondrial proteins (mono-and dinucleotide expansions, six (AT6, CytB, ND1, 2xND2, ND5) and one (ND1), respectively). Chimeric translation probably increases the diversity of mitogenome-encoded proteins, putatively producing functional proteins. These might result from translation by tRNAs with expanded anticodons, or from regular tricodon translation of RNAs where transcription/posttranscriptional edition systematically deleted mono-or dinucleotides after each trinucleotide. The pairwise matched combination of adjacent peptide parts translated from regular and expanded codons strengthens the hypothesis that translation of stretches of consecutive expanded codons occurs. Results indicate statistical translation producing distributions of alternative proteins. Genetic engineering should account for potential unexpected, unwanted secondary products.

Introduction

The low stability of codon-anticodon duplexes does not enable mRNA translation without a ribosome stabilizing this interaction long enough to enable peptide elongation [START_REF] Baranov | Codon size reduction as the origin of trhe triplet genetic code[END_REF]. Ribosomes are very complex molecules [START_REF] Ramakrishnan | The ribosome emerges from a black box[END_REF][START_REF] Noeske | highresolution structure of the Escherichia coli ribosome[END_REF][START_REF] Yusupova | Crystal structure of eukaryotic ribosome and its complexes with inhibitors[END_REF] resulting from complex accretion histories [START_REF] Harish | Ribosomal history reveals origins of modern protein synthesis[END_REF][START_REF] Caetano-Anollés | Commentary: History of the ribosome and the origin of translation[END_REF][START_REF] Farias | Origin and evolution of the peptidyl transferase center from proto-tRNAs[END_REF][START_REF] Caetano-Anollés | a Piecemeal buildup of the genetic code, ribosomes, and genomes from primordial tRNA building blocks[END_REF][START_REF] Caetano-Anollés | Computing the origin and evolution of the ribosome from its structure -Uncovering processes of macromolecular accretion benefiting synthetic biology[END_REF], meaning that tRNA and tRNA-like structures accreted serially, becoming over time modern rRNA. A history derived from classical comparative analyses partly converges with an alternative, structure-based approach [START_REF] Farias | Origin of the 16S ribosomal molecule from ancestor tRNAs[END_REF][START_REF] Petrov | History of the ribosome and the origin of translation[END_REF][START_REF] Gulen | Ribosomal small subunit domains radiate from a central core[END_REF][START_REF] Lanier | Imprint of ancient evolution on rRNA folding[END_REF], from tRNAs [START_REF] Farias | Origin of the 16S ribosomal molecule from ancestor tRNAs[END_REF][START_REF] Bloch | tRNA-rRNA sequence matches from inter-and intraspecies comparisons suggest common origins for the two RNAs[END_REF][START_REF] Bloch | RNA-rRNA sequence homologies: evidence for an ancient modular format shared by tRNAs and rRNAs[END_REF][START_REF] Bloch | tRNA-rRNA sequence homologies: evidence for a common evolutionary origin?[END_REF][START_REF] Bloch | tRNA-rRNA sequence homologies: a model for the origin of a common ancestral molecule, and prospects for its reconstruction[END_REF][START_REF] Caetano-Anollés | Tracing the evolution of RNA structure in ribosomes[END_REF][START_REF] Caetano-Anollés | The natural history of transfer RNA and its interactions with the ribosome[END_REF][START_REF] Barthélémy | Cryptic tRNAs in chaetognath mitochondrial genomes[END_REF] and (perhaps) tRNA dimers [START_REF] Farias | Origin and evolution of the peptidyl transferase center from proto-tRNAs[END_REF][START_REF] Agmon | Could a proto-ribosome emerge spontaneously in the prebiotic world?[END_REF][START_REF] Guimarães | Self-referential encoding on modules of anticodon pairsroots of the biological flow system[END_REF]. Similar accretion histories also apparently produced other types of ancient (protein coding) genes [START_REF] Demongeot | More pieces of ancient than recent theoretical minimal proto-tRNA-like RNA rings in genes coding for tRNA synthetases[END_REF]. Hence some kind of translation presumably occurred before ribosomes evolved, possibly including direct codon-amino acid interactions [START_REF] Pelc | Correlation between coding-triplets[END_REF][START_REF] Pelc | Stereochemical relationship between coding triplets and amino-acids[END_REF][START_REF] Welton | Specificity of the stereochemical relationship between ribonucleic acid triplets and amino-acids[END_REF][START_REF] Yarus | RNA-amino acid binding: a stereochemical era for the genetic code[END_REF][START_REF] De Ruiter | Absolute binding-free energies between standard RNA/DNA nucleobases and amino-acid sidechain analogs in different environments[END_REF][START_REF] Bartonek | mRNA/protein sequence complementarity and its determinants: the impact of affinity scales[END_REF][START_REF] Yarus | The genetic code and RNA-amino acid affinities[END_REF][START_REF] Chatterjee | The origin of prebiotic information system in the peptide/RNA world: a simulation model of the evolution of translation and the genetic code[END_REF].

Expanded codons and anticodons

Ribosome-free translation seems impossible because codon-anticodon duplexes are too weak to enable peptide elongation. A simple potential solution to ancestral ribosome-free translation by codon-anticodon interactions assumes that modern codons are reduced. Ancestral ribosome-free translation presumably resulted from interactions between codons and anticodons consisting of more than three nucleotides [START_REF] Baranov | Codon size reduction as the origin of trhe triplet genetic code[END_REF][START_REF] Landweber | Custom codons come in threes, fours and fives[END_REF][START_REF] Giulio | RNA editing and modifications of RNAs might have favoured the evolution of the triplet genetic code from an ennuplet code[END_REF][START_REF] Giulio | A model for the origin of the first mRNAs[END_REF]. This stabilizes codon-anticodon duplexes long enough to enable ribosomefree peptide elongation (the ribosome also channels the spatial dynamics of aminoacylated tRNA-peptide interactions [START_REF] Seligmann | Genetic code optimization for cotranslational protein folding: codon directional asymmetry correlates with antiparallel betasheets, tRNA synthetase classes[END_REF]). In addition, a code based on a specific subset of tetracodons (codons expanded by a fourth, silent nucleotide) would have self-correcting symmetry properties that could be an adequate ancestor of some modern (mitochondrial) genetic codes (the tessera hypothesis [START_REF] Gonzalez | On the origin of the mitochondrial genetic code: towards a unified mathematical framework for the management of genetic information[END_REF][START_REF] Gonzalez | The non-power model of the genetic code: a paradigm for interpreting genomic information[END_REF]).

Natural tRNA-based translation of tetracodons was observed from the onset of molecular biology [START_REF] Riddle | Frameshift suppression -nucleotide addition in anticodon of glycine transfer-RNA[END_REF][START_REF] Tuohy | Seven, eight and ninemembered anticodon loop mutants of tRNA(2Arg) which cause + 1 frameshifting. Tolerance of DHU arm and other secondary mutations[END_REF][START_REF] Phelps | Translocation of a tRNA with an extended anticodon through the ribosome[END_REF]. Modern ribosomes accommodate tRNAs with expanded anticodons during translation [START_REF] Walker | Recognition and positioning of mRNA in the ribosome by tRNAs with expanded anticodons[END_REF][START_REF] Dunham | Structures of tRNAs with an expanded anticodon loop in the decoding center of the 30S ribosomal subunit[END_REF][START_REF] Maehigashi | Structural insights into +1 frameshifting promoted by expanded or modification-deficient anticodon stem loops[END_REF]. Biotechnological applications use tRNAs with expanded anticodons to introduce non-natural amino acids in proteins [START_REF] Moore | Quadruplet codons: Implications for code expansion and the specification of translation step size[END_REF][START_REF] Magliery | Expanding the genetic code: Selection of efficient suppressors of four-base codons and identification of "shifty" four-base codons with a library approach in Escherichia coli[END_REF][START_REF] Hendrickson | Incorporation of nonnatural amino acids into proteins[END_REF][START_REF] Wang | Systematic evolution and study of UAGN decoding tRNAs in a genomically recoded bacteria[END_REF][START_REF] Arranz-Gibertt | Next-generation genetic code expansion[END_REF]. The antisense sequence of some mitochondrial tRNAs has predicted expanded anticodons [START_REF] Seligmann | a Undetected antisense tRNAs in mitochondrial genomes?[END_REF][START_REF] Seligmann | a Pathogenic mutations in antisense mitochondrial tRNAs[END_REF]. The predicted mitochondrial tRNAs with expanded anticodons coevolve with predicted mitochondrial tetracodon-encoded peptides [START_REF] Seligmann | Overlapping genetic codes for overlapping frameshifted genes in Testudines, and Lepidochelys olivacea as special case[END_REF][START_REF] Seligmann | An overlapping genetic code for frameshifted overlapping genes in Drosophila mitochondria: antisense antitermination tRNAs UAR insert serine[END_REF][START_REF] Seligmann | a Pocketknife tRNA hypothesis: anticodons in mammal mitochondrial tRNA side-arm loops translate proteins?[END_REF][START_REF] Seligmann | a Putative anticodons in mitochondrial tRNA sidearm loops: Pocketknife tRNAs?[END_REF]. Tetracoding in modern organisms seems to be an adaptation to high temperatures where regular codonanticodon interactions become relatively unstable: predicted tetracoding increases with lepidosaurian body temperature [START_REF] Seligmann | Tetracoding increases with body temperature in Lepidosauria[END_REF]. This would be a relict of prebiotic ribosome-free translation and genetic code formation, which likely occurred at high temperatures [START_REF] Giulio | The late stage of genetic code structuring took place at a high temperature[END_REF][START_REF] Giulio | a The universal ancestor was a thermophile or a hyperthermophile: tests and further evidence[END_REF][START_REF] Giulio | The universal ancestor and the ancestor of bacteria were hyperthermophiles[END_REF][START_REF] Akanuma | Experimental evidence for the thermophilicity of ancestral life[END_REF][START_REF]Geological and geochemical constraints on the origin and evolution of life[END_REF]; but see [START_REF] Gutfraind | Error-reducing structure of the genetic code indicates code origin in non-thermophile organisms[END_REF] for some counter-arguments to life's thermophilic origin hypothesis.

Peptides coded by expanded codons

In addition to tetracoding sequences predicted by alignment methods, peptides corresponding in their entireties to the translation of tetra-and pentacodons have been detected in MS data (produced by the medium accuracy Thermo Fisher (Illkirch, France), and the high accuracy Waters (Milford, MA, USA) technologies) from the human mitochondrial peptidome [START_REF] Seligmann | a Codon expansion and systematic transcriptional deletions produce tetra-, pentacoded mitochondrial peptides[END_REF][START_REF] Seligmann | a Translation of mitochondrial swinger RNAs according to tri-, tetra-and pentacodons[END_REF][START_REF] Seligmann | Natural chymotrypsin-like-cleaved human mitochondrial peptides confirm tetra-, pentacodon, non-canonical RNA translations[END_REF][START_REF] Seligmann | Unbiased mitoproteome analyses confirm non-canonical RNA, expanded codon translations[END_REF][START_REF] Seligmann | a Natural mitochondrial proteolysis confirms transcription systematically exchanging/deleting nucleotides, peptides coded by expanded codons[END_REF][START_REF] Seligmann | Reviewing evidence for systematic transcriptional deletions, nucleotide exchanges, and expanded codons, and peptide clusters in human mitochondria[END_REF]. Detections of complete tetracoded peptides differ from occurrences of isolated tetracodons, notably in mitochondrial genomes [START_REF] Beckenbach | Single nucleotide +1 frameshifts in an apparently functional mitochondrial cytochrome b gene in ants of the genus Polyrhachis[END_REF][START_REF] Russell | Recoding of translation in turtle mitochondrial genomes: programmed frameshift mutations and evidence of a modified genetic code[END_REF], which might result from decoding by specific expanded anticodons or as regular codons after deletion of extra nucleotide(s) by post-transcriptional editing [START_REF] Bar-Yaacov | RNA-DNA differences in human mitochondria restore ancestral form of 16S ribosomal RNA[END_REF].

Alternative translations

Translation according to expanded codons by a series of expanded anticodons is not the only known alternative translation. Antitermination, or stop suppression occurs due to decoding by regular near-cognate tRNAs [START_REF] O'connor | tRNA imbalance promotes -1 frameshifting via near-cognate decoding[END_REF][START_REF] Seligmann | Do anticodons of misacylated tRNAs preferentially mismatch codons coding for the misloaded amino acid?[END_REF][START_REF] Seligmann | Error compensation of tRNA misacylation by codonanticodon mismatch prevents translational amino acid misinsertion[END_REF][START_REF] Seligmann | Coding constraints modulate chemically spontaneous mutational replication gradients in mitochondrial genomes[END_REF][START_REF] O'donoghue | Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion[END_REF] or tRNAs with an anticodon matching a stop codon [START_REF] Beier | Misreading of termination codons in eukaryotes by natural nonsense suppressor tRNAs[END_REF][START_REF] Wang | a High frequency of+1 programmed ribosomal frameshifting in Euplotes octocarinatus[END_REF][START_REF] Wang | Large-scale mass spectrometrybased analysis of Euplotes octocarinatus supports the high frequency of +1 programmed ribosomal frameshift[END_REF][START_REF] Massey | The identities of stop codon reassignments support ancestral tRNA stop codon decoding activity as a facilitator of gene duplication and evolution of novel function[END_REF][START_REF] Tharp | tRNAPyl: Structure, function, and applications[END_REF]. Natural stop-suppressor tRNAs have also been adjusted for genetic code expansion to insert non-natural amino acids in proteins in biotechnological applications [START_REF] Gan | Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis[END_REF][START_REF] Brabham | Pyrrolysine amber stop-codon suppression: development and applications[END_REF][START_REF] Tharp | Using amber and ochre nonsense codons to code two different noncanonical amino acids in one protein gene[END_REF]. Natural stop suppression in mitogenes can be predicted based on alignment analyses [START_REF] Seligmann | An overlapping genetic code for frameshifted overlapping genes in Drosophila mitochondria: antisense antitermination tRNAs UAR insert serine[END_REF][START_REF] Seligmann | c Two genetic codes, one genome: frameshifted primate mitochondrial genes code for additional proteins in presence of antisense antitermination tRNAs[END_REF][START_REF] Faure | Probable presence of an ubiquitous cryptic mitochondrial gene on the antisense strand of the cytochrome oxidase I gene[END_REF][START_REF] Seligmann | a Putative mitochondrial polypeptides coded by expanded quadruplet codons, decoded by antisense tRNAs with unusual anticodons[END_REF][START_REF] Seligmann | Overlapping genes coded in the 3'-to-5'-direction in mitochondrial genes and 3'-to-5' polymerization of non-complementary RNA by an 'invertase[END_REF][START_REF] Seligmann | Polymerization of non-complementary RNA: systematic symmetric nucleotide exchanges mainly involving uracil produce mitochondrial RNA transcripts coding for cryptic overlapping genes[END_REF][START_REF] Seligmann | c Systematic asymmetric nucleotide exchanges produce human mitochondrial RNAs cryptically encoding for overlapping protein coding genes[END_REF][START_REF] Seligmann | Triplex DNA:RNA, 3'-to-5' inverted RNA and protein coding in mitochondrial genomes[END_REF][START_REF] Seligmann | Directed mutations recode mitochondrial genes: from regular to stoplessgenetic codes[END_REF], and observed distributions of amino acids inserted at stops [START_REF] Seligmann | a Codon expansion and systematic transcriptional deletions produce tetra-, pentacoded mitochondrial peptides[END_REF][START_REF] Seligmann | a Translation of mitochondrial swinger RNAs according to tri-, tetra-and pentacodons[END_REF][START_REF] Seligmann | Natural chymotrypsin-like-cleaved human mitochondrial peptides confirm tetra-, pentacodon, non-canonical RNA translations[END_REF][START_REF] Seligmann | Unbiased mitoproteome analyses confirm non-canonical RNA, expanded codon translations[END_REF][START_REF] Seligmann | a Natural mitochondrial proteolysis confirms transcription systematically exchanging/deleting nucleotides, peptides coded by expanded codons[END_REF][START_REF] Seligmann | Reviewing evidence for systematic transcriptional deletions, nucleotide exchanges, and expanded codons, and peptide clusters in human mitochondria[END_REF] match genetic code evolution [START_REF] Massey | A comparative genomics analysis of codon reassignments reveals a link with mitochondrial proteome size and a mechanism of genetic code change via suppressor tRNAs[END_REF][START_REF] Seligmann | Phylogeny of genetic codes and punctuation codes within genetic codes[END_REF][START_REF] Seligmann | a Alignment-based and alignment-free methods converge with experimental data on amino acids coded by stop codons at split between nuclear and mitochondrial genetic codes[END_REF] and coding symmetries in the genetic code [START_REF] Seligmann | Bijective codon transformations show genetic code symmetries centered on cytosine's coding properties[END_REF].

Alternative transcriptions

Post-transcriptional editing by systematically deleting every fourth, or every fourth and fifth nucleotide after each transcribed nucleotide triplet could produce noncanonical transcripts whose regular transcription matches noncanonical translation of regular transcripts according to tetra-or pentacodons. Mitochondrial transcripts detected in several independent datasets produced by independent sequencing technologies (Sanger and Illumina) match sequences predicted by systematic deletions after each transcribed nucleotide triplet. These noncanonical transcripts (delRNAs), because of their length corresponding to numerous tricodons separated by deleted mono-or dinucleotides, seem more likely produced by noncanonical transcription systematically deleting mono-or dinucleotides than by posttranscriptional edition, though the latter cannot be excluded [START_REF] Warthi | Transcripts with systematic nucleotide deletion of 1-12 nucleotide in human mitochondrion suggest potential non-canonical transcription[END_REF]. These delRNAs have more than expected homopolymers [START_REF] Warthi | Swinger RNAs in the human mitochondrial transcriptome[END_REF] that frequently induce frameshifting [START_REF] Houmami | Evolution of nucleotide punctuation marks: from structural to linear signals[END_REF].

Note that the human mitogenome, assuming systematic deletion of mono-or dinucleotides after each transcribed nucleotide triplet, includes more palindromes than random sequences with the same length and nucleotide content [START_REF] Seligmann | Localized context-dependent effects of the "ambush" hypothesis: more off-frame stop codons downstream of shifty codons[END_REF].

A different type of noncanonical transcription exists, consisting of systematic exchanges between nucleotides, producing swinger RNAs that do not resemble their template DNA unless considering the transformation rule that produced the noncanonical RNA. Nine systematic nucleotide exchanges are symmetric (X<->Y, example G<->T [START_REF] Seligmann | Overlapping genes coded in the 3'-to-5'-direction in mitochondrial genes and 3'-to-5' polymerization of non-complementary RNA by an 'invertase[END_REF][START_REF] Seligmann | Polymerization of non-complementary RNA: systematic symmetric nucleotide exchanges mainly involving uracil produce mitochondrial RNA transcripts coding for cryptic overlapping genes[END_REF][START_REF] Seligmann | Triplex DNA:RNA, 3'-to-5' inverted RNA and protein coding in mitochondrial genomes[END_REF], and fourteen are asymmetric (X->Y->Z->X, example C->T->G->C [START_REF] Seligmann | c Systematic asymmetric nucleotide exchanges produce human mitochondrial RNAs cryptically encoding for overlapping protein coding genes[END_REF]). Empirical genomic coverages by swinger RNAs are replicable across independent datasets [START_REF] Warthi | Swinger RNAs in the human mitochondrial transcriptome[END_REF] and sequencing techniques (Sanger and Illumina, human mitogenomes [START_REF] Seligmann | a Translation of mitochondrial swinger RNAs according to tri-, tetra-and pentacodons[END_REF]; 454 and SOLID, Mimivirus [START_REF] Seligmann | Stem-loop RNA hairpins in giant viruses: invading rRNA-like repeats and a template free RNA[END_REF]). The human mitogenome, assuming systematic nucleotide exchanges, includes more palindromes forming stem-loop hairpins than random sequences with the same length and nucleotide contents [START_REF] Seligmann | d Swinger RNA self-hybridization and mitochondrial noncanonical swinger transcription, transcription systematically exchanging nucleotides[END_REF]. Systematic nucleotide exchanges conserve error-correcting properties of the genetic code and its embedded circular code regulating ribosomal translation frame [START_REF] Michel | Bijective transformation circular codes and nucleotide exchanging RNA transcription[END_REF][START_REF] Fimmel | On dichotomic classes and bijections of the genetic code[END_REF][START_REF] Fimmel | a Circular codes, symmetries and transformations[END_REF][START_REF] Fimmel | Dinucleotide circular codes and bijective transformations[END_REF].

Chimeric RNAs and peptides

Note that swinger DNA has also been reported [START_REF] Seligmann | Species radiation by DNA replication that systematically exchanges nucleotides?[END_REF][START_REF] Seligmann | Mitochondrial swinger replication: DNA replication systematically exchanging nucleotides and short 16S ribosomal DNA swinger inserts[END_REF], in one case with abrupt switches between the regular and the swinger-transformed part of the mitogenome [START_REF] Seligmann | Sharp switches between regular and swinger mitochondrial replication: 16S rDNA systematically exchanging nucleotides A<->T+C<->G in the mitogenome of Kamimuria wangi[END_REF]. Chimeric RNAs, partly corresponding to regular transcription of the human mitogenome, and partly corresponding to swinger-transcription of adjacent parts of the mitogenome, also occur, including abrupt switches between these two parts [START_REF] Seligmann | d Swinger RNAs with sharp switches between regular transcription and transcription systematically exchanging ribonucleotides: Case studies[END_REF]. These either result from regular transcription of genomic swinger DNA, or from swinger transcription of part of the template mitogenome. Chimeric peptides corresponding to translation of adjacent parts of regular and swinger-transformed RNA exist in mitochondrial proteomic data, including peptides whose regular part corresponds to mitochondrion-encoded proteins, for example CytB [START_REF] Seligmann | f Chimeric mitochondrial peptides from contiguous regular and swinger RNA[END_REF].

These chimeric molecules are strong evidence for swinger phenomena. First because if they reflected unknown artefacts, these would not have produced the regular parts of the RNA and peptide sequences. Chimeric RNAs and peptides show that unknown phenomena producing variants of known RNAs and proteins exist. In addition, the regular parts of the chimeric RNA/peptide are natural matched positive controls for adjacent noncanonical parts. This strengthens the confidence in the biological reality of these noncanonical phenomena.

In the context of long stretches of translation according to expanded codons, chimeric peptides corresponding in part to regular translation, and in adjacent parts to translation according to expanded codons (Figure 1), would also consist strong evidence for translation of stretches of expanded codons, and indicate that variants of known proteins including parts encoded by expanded codons exist. Hence here we present analyses of two mitochondrial MS datasets (one produced by Thermo Fisher and one by Waters technologies) that explore for chimeric peptides resulting from translation according to adjacent stretches of regular and expanded codons.

These analyses presume that translation produces a distribution of alternative protein products, some might present functional advantages, for example by having functional optima at conditions that differ from those of known canonical proteins (for example temperature). The approach implies caution: genetic engineering should account for potential unwanted byproducts resulting from little known and/or unknown alternative transcriptions and translations: unlike engineered genes, natural genes adapted to avoid or minimize disruptive effects by proteins resulting from rarer noncanonical transcriptions and/or translations.

Materials and Methods

The data and analytical methods are identical to those previously used for chimeric swinger peptides [START_REF] Seligmann | f Chimeric mitochondrial peptides from contiguous regular and swinger RNA[END_REF]. The human mitogenome (NC_012920, length 16569 basepairs) was downloaded in its entirety.

Predicted peptides

The difference with previous analyses consists in the fact that analyses compare observed MS/MS data with hypothetical peptides that result in part from canonical, and from noncanonical translations for consecutive, adjacent stretches of amino acids. The design of these hypothetical chimeric peptides uses two sizes of running windows: one for tetracodons (codons expanded by one nucleotide) and one for pentacodons (codons expanded by two nucleotides). Each running window codes for 30 amino acids according to regular tricodons, 30 consecutive amino acids coded according to noncanonical codons expanded by a mono-or dinucleotide, and another consecutive 30 amino acids coded according to regular tricodons. Hence each hypothetical chimeric peptide consists of 30 canonically coded amino acids at each its 5' and 3' extremities, and 30 noncanonical codons, each expanded by a mono-or dinucleotide. This produces window sizes of (3x30)+(4x30)+(3x30) = 90+120+90=300 nucleotides for tetracodons and 90+150+90=330 nucleotides for pentacodons. Windows move by steps of single nucleotides along the complete genome, producing 2x16569 theoretical chimeric tetra-, and 2x16569 chimeric pentacoded peptides, for + and -mitogenome strands.

For hypothetical chimeric peptides, the length of 30 consecutive amino acids with the same translation modus was chosen because previous experience shows that most detected peptides are shorter than 30 amino acids. Adopting shorter lengths, for example 15 amino acids, for the different parts of the hypothetical chimeric peptide, would not detect chimeric peptides with canonical and/or noncanonical parts longer than 15 amino acids.

For each hypothetical peptide, the relevant proteome analysis software predicts a theoretical mass spectrometry distribution. This distribution is compared with observed MS/MS data.

Translation of stop codons

Peptides translated from sequences including stop codons are included 19 times in the pool of theoretical peptides, each time inserting at all stop codons the same amino acid (there are 20 amino acids, but leucine and isoleucine have equal masses and hence cannot be distinguished by MS/MS techniques, resulting in 19 alternative peptides where a different amino acid species is inserted at all stops). Hence analyses consider the possibility that each amino acid can translate stop codons. Approximately 2x16569x19=629622 chimeric peptides exist for each tetra-and pentacoded chimeric translations.

Medium accuracy data searches

For the MS/MS data from [START_REF] Gueugneau | Proteomics of muscle chronological ageing in post-menopausal women[END_REF], consensus searches between observed and predicted MS/MS data were handled with the Sequest (Thermo Fisher Scientific, Illkirch) algorithm with the following mass tolerances: Parent = 1 Da and Fragment = 0.5 Da (monoisotopic masses). Fixed carbamidomethyl (C) and variable Oxydation (M) modifications were activated, as well as the lysine → pyrrolysine modification, and only one missed trypsin cleavage was allowed. False discovery rate was estimated against a reverse decoy database using the Percolator algorithm. No protein grouping was allowed since the database only contained non-redundant entries. Peptides with false discovery rate q < 0.05 and score Xcorr > 1.99 were considered identified. The score Xcorr is a likelihood of match between expected and observed MS/MS data unaffected by peptide length. The false discovery rate q is adapted to populations of detected peptides [START_REF] Käll | Posterior error probabilities and false discovery rates: two sides of the same coin[END_REF].

Previous analyses show that searches of the trypsinized proteome produced by [START_REF] Gueugneau | Proteomics of muscle chronological ageing in post-menopausal women[END_REF] detect much more peptides when analyzing data separately for peptides cleaved at K, and separately for those cleaved at R, and when searches assume cleavage at the amino extremity of these amino acids rather than the carboxyl extremity at which trypsin cleaves proteic chains. These searches mainly detect peptides ending at the carboxyl extremity of K or R, corresponding to the experimental trypsin cleavage by assuming one missed cleavage. This increased efficiency of searches remains unexplained, but is not due to artifacts, because noncanonical peptides detected by all searches (assuming cleavage at any amino acid and at any (carboxyl and amino) extremities) map with comparable rates on corresponding, detected noncanonical RNAs, and correspond in their overwhelming majorities to trypsinized peptides as expected by the experimental parameters (addition of trypsin) [START_REF] Seligmann | a Natural mitochondrial proteolysis confirms transcription systematically exchanging/deleting nucleotides, peptides coded by expanded codons[END_REF].

High accuracy data searches

The same pool of theoretical chimeric peptides was used for PLGS searches of another human mitoproteome dataset [START_REF] Alberio | Mitochondrial proteomics investigation of a cellular model of impaired dopamine homeostasis, an early step in Parkinson's disease pathogenesis[END_REF], extracted by a higher accuracy method (Waters, Milford, MA). Mass peak estimates are more accurate (5 ppm for data extracted by [START_REF] Alberio | Mitochondrial proteomics investigation of a cellular model of impaired dopamine homeostasis, an early step in Parkinson's disease pathogenesis[END_REF], versus 0.5 Da for data by [START_REF] Gueugneau | Proteomics of muscle chronological ageing in post-menopausal women[END_REF]). Precise comparison of accuracies between these techniques is unfeasible: Sequest (Thermo Fisher Scientific, Illkirch) uses fixed cutoffs; PLGS adapts cutoffs to masses of detected peptide: 0.5 Da in the latter sample would occur for peptides with mass 5x10 6 x0.5 Da.

The twelve samples from [START_REF] Alberio | Mitochondrial proteomics investigation of a cellular model of impaired dopamine homeostasis, an early step in Parkinson's disease pathogenesis[END_REF] were processed using ProteinLynx Global Server version 3.0.1 (Waters, Saint-Quentin En Yvelines, France). Processing parameters were 250 counts for the low energy threshold, 100 counts for the elevated energy threshold and 750 counts for the intensity threshold. Hits are considered significant according to standard criteria, with PLGS peptide score 6.49. This score is compared to a decoy database to estimate FDR, as done for Xcorr from the dataset produced by [START_REF] Gueugneau | Proteomics of muscle chronological ageing in post-menopausal women[END_REF], and peptides with q<0.05 are retained.

Each sample was searched separately for peptides 38 times, each search assuming cleavage at a different extremity (carboxyl or amino) of each amino acid species (merging L and I, 2x19=38).

Minimal size of detected chimeric peptides

Detected peptides are further filtered so as to retain only peptides with at least eight consecutive amino acids coded according to regular codons, and at least eight consecutive amino acids coded according to noncanonical expanded codons. This size was determined so that each regular-and noncanonically-encoded parts of the chimeric peptide have an approximate maximal e value 0.0014 (629622x1/19 8 ).

Results and discussion

Chimeric peptides with a tetra-or pentacoded part

Tables 1 and 2 present 28 chimeric peptides detected in the MS/MS data published by [START_REF] Gueugneau | Proteomics of muscle chronological ageing in post-menopausal women[END_REF] and 14 chimeric peptides detected in the MS/MS data published by [START_REF] Alberio | Mitochondrial proteomics investigation of a cellular model of impaired dopamine homeostasis, an early step in Parkinson's disease pathogenesis[END_REF], with at least eight amino acids coded by tricodons and eight adjacent amino acids coded by tetracodons. Tables 1 and2 also present 19 chimeric peptides detected in the MS/MS data published by [START_REF] Gueugneau | Proteomics of muscle chronological ageing in post-menopausal women[END_REF] and 18 chimeric peptides detected in the MS/MS data published by [START_REF] Alberio | Mitochondrial proteomics investigation of a cellular model of impaired dopamine homeostasis, an early step in Parkinson's disease pathogenesis[END_REF], with at least eight amino acids coded by tricodons and eight adjacent amino acids coded by pentacodons.

In about half the cases, the noncanonical part of the peptide is on the 5' extremity of the peptide, not for tetra-and not for pentacoded parts, nor for any dataset. The noncanonical part can either be at the 5'-or the 3'-encoded extremity of chimeric peptides, with no apparent bias for one of these extremities in the current results.

Chimeric peptides integrated in regular mitogenome-encoded proteins

Most peptides include amino acids inserted at stop codons, in each regular-encoded and noncanonical parts. The regular-encoded part of a total of seven chimeric peptides corresponds to one among the 13 classical mitogenome-encoded proteins. Six among these have adjacent tetracoded parts, and one an adjacent pentacoded part. The proteins are: AT6, CytB, ND1, ND2 (two different peptides), ND5 (tetracoded) and ND1 (pentacoded). These small numbers do not enable to test whether biases exist in terms of which proteins tend to include more or less noncanonical parts, nor in relation to their position on the mitogenome, as these genes are scattered across the whole mitochondrial operon. The hypothesis that noncanonical peptides result from mitochondrial polymorphisms and heteroplasmy [START_REF] Wallace | Mitochondrial DNA sequence variation in human evolution and disease[END_REF][START_REF] He | Heteroplasmic mitochondrial DNA mutations in normal and tumour cells[END_REF][START_REF] Avital | Mitochondrial DNA heteroplasmy in diabetes and normal adults: role of acquired and inherited mutational patterns in twins[END_REF][START_REF] Ramos | Frequency and pattern of heteroplasmy in the complete human mitochondrial genome[END_REF][START_REF] Ross | Germline mitochondrial DNA mutations aggravate ageing and can impair brain development[END_REF][START_REF] Ye | Extensive pathogenicity of mitochondrial heteroplasmy in healthy human individuals[END_REF] is unlikely: their exact correspondence to sequences predicted by translation of expanded codons excludes this option. As a group, they result from regular mitogenomic DNA variability.

Noncanonical transcription or translation?

Above results confirm that tetra-and pentacoded of amino acid stretches occur, conjugated with regular encoded stretches of amino acids. The alternative, that chimeric peptides originate from regular translation of chimeric RNAs produced in part by regular transcription, and in part by noncanonical transcription systematically deleting mono-or dinucleotides after each transcribed trinucleotide, cannot be excluded. The data at hand don't enable to test between these two alternatives potentially producing identical peptides. We tentatively presume that both mechanisms are at work because expanded codons and anticodons have been previously reported, and because noncanonical transcripts corresponding to transcription systematically deleting mono-and dinucleotides also exist.

Adaptive diversity

Amino acid stretches encoded by noncanonical codons (or resulting from noncanonical transcription) might be integrated in regular mitogenome-encoded proteins, as suggested by their association with stretches of tricodon-encoded amino acids clearly corresponding to regular membrane-bound mitochondrial proteins. The possibility that these chimeric peptides are part of functional proteins cannot be excluded. The existence of chimeric peptides suggests that natural protein diversity can be increased by mixing types of decoding processes, such as regular tricodons and noncanonical codons expanded by one or two nucleotides. This diversity might have unknown adaptive/functional components, including widening ranges of functionally optimal conditions at which some metabolic activities might occur.

Results also stress that natural translation of expanded codons is not extremely rare. The hypothesis that expanded codons (but not systematic deletions) are adaptive at high temperatures could be tested by comparing abundances of detected peptides coded by expanded codons at different temperatures, expecting more translation according to expanded codons at higher temperatures. Other analyses searching for peptides corresponding to codons expanded by more nucleotides (>2) will also contribute to our understanding of these noncanonical transcriptions and translations that increase the coding potential of sequences.

Unwanted effects of genetic engineering

Experiments and analyses exploring for which genome regions undergo the different noncanonical transcriptions and translations in which cell types and under which conditions would deepen our understanding of cell metabolism, implying likely biomedical applications. Results also stress that genetic engineering should explore potential effects of proteins produced from noncanonical transcripts and/or by noncanonical translations, to avoid undesirable effects from discarded noncanonical processes such as swinger and del-transcriptions, and translation of stop codons and according to expanded codons.

Acknowledgement

Figure 1. RNA sequence and its chimeric translation according to regular tricodons and tetra-and pentacodons. Sequences corresponding to 90 codons (two groups of 30 regular tricodons, each at the 5' and 3' extremity of a group of 30 noncanonical codons expanded by mono-or dinucleotides (tetra-and pentacodons)) form running windows of 90+120+90=300 nucleotides (tetracodons) and 90+150+90=330 nucleotides (pentacodons). Hence for each of the 16569 positions along the human mitogenome, chimeric peptides are translated from 30 regular, 30 noncanonical and 30 regular codons. These hypothetical peptides (lengths truncated in Figure 1 for presentation purposes) are compared with actual MS mitoproteomic data.

Table 1 .

 1 Chimeric peptides transcribed in part according to regular, and in part according to expanded codons (tetra-and pentacodons) from the human mitogenome, detected in MS data from Guegneau et al (2014). Columns are: 1. Regular tricodon translation frame (positive strand, 1-3; negative strand, 4-6), tetra-and pentacoded parts indicated by T and P; 2. Position of regular tricoded part on translated human mitogenome; 3. S, amino acid inserted at stop codons; 4. Detected peptide sequence, minor letters indicate translated stops, "|" separates regular tricoded from other part, underlined parts are tetra-and pentacoded. Ambiguous limits between tricoded and other part are also indicated when occurring, ambiguous part is considered parsimoniously as tricoded. 5. Xcorr between expected and observed MS; 6. PSM, counts observed MS matching expected MS; 7. q, false discovery rate; 8. PEP, posterior error probability, peptide specific; 9. Position-specific amino acid modifications; 10. Positions in regular mitogenome-encoded proteins matching regular tricoded part of detected chimeric peptide.

	Tetracodons
	1 ATGACCCCAATACGCAAAACTAACCCCCTAATA|AAATTAATTAACCACTCATTCATCGACCTCCCCACC|CCATCCAACATCTCC
	M T P	M R K T N	P	L M |K	*	*	H	H	H	D	S	H	|P	S	N	I	S
	2 TGACCCCAATACGCAAAACTAACCCCCTAATAA|AATTAATTAACCACTCATTCATCGACCTCCCCACCC|CATCCAACATCTCCG
	W P	Q Y A K	L T P	*	*	|N	N	N	T	I	I	T	P	T	|H	P	T S	P
	Pentacodons
	1 ATGACCCCAATACGCAAAACTAACCCCCTAATA|AAATTAATTAACCACTCATTCATCGACCTCCCCACCCCATCCAAC|ATCTCC
	M T P	M R K T N	P	L M |K	N	T		S	H	T	P		P	P	|I S
	2 TGACCCCAATACGCAAAACTAACCCCCTAATAA|AATTAATTAACCACTCATTCATCGACCTCCCCACCCCATCCAACA|TCTCCG
	W P	Q Y A K	L T P	*	*	|N	I	P		H	I	P	P		P	Q	|S P

Table 2 .

 2 Chimeric peptides transcribed in part according to regular, and in part according to expanded codons (tetra-and pentacodons) from the human mitogenome, detected in MS data from[START_REF] Alberio | Mitochondrial proteomics investigation of a cellular model of impaired dopamine homeostasis, an early step in Parkinson's disease pathogenesis[END_REF]. PLGS is the score estimating goodness of fit between observed and expected MS in the PLGS peptide detection software. Δ ppm is the difference between expected and observed MS total mass. Cl indicates cleavage expected by the MS/MS search that detected the specified peptide, C indicates cleavage at the carboxyl-, and N the amino-end of the acid. Chym and elas indicate cleavage by chymotrypsin elastase.

	T Pos	S Peptide

This work was supported by the A*MIDEX project (no ANR-11-IDEX-0001-02) funded by the « Investissements d'Avenir » French Government program, managed by the French National Research Agency (ANR).


Y El Khessaimi

Y El Hafiane

A Smith

Examination of ye'elimite formation mechanisms

Keywords: Ye'elimite, Calcium aluminate, Calcium sulfoaluminate cement, Avrami model, BSE-SEM, EDS mapping

Ye'elimite is the main constituent of calcium sulfoaluminate (CSA) cement and one of the major constituents of belite sulfoaluminate or belite sulfoaluminate ferrite cements. The main objective of this work is to describe precisely the formation mechanisms of ye'elimite by solid-state reaction.

Mineralogical composition development was monitored using XRD analysis, while microstructural monitoring was conducted using BSE-SEM coupled to EDS analysis. The results show that CaAl2O4 and CaAl2O7 are the main intermediate products during ye'elimite formation. At the microstructural scale, ye'elimite forms on calcium aluminate phases. Finally, Avrami's model was suggested to discuss the ye'elimite formation rate according to sintering temperature and duration.

Introduction

Concrete is the most widely used construction material in the world. Ordinary Portland Cement (OPC) has traditionally been used as the binder material in concrete [START_REF] Taylor | Cement chemistry[END_REF]. However, the production of OPC cements contributes of approximately 5% of global anthropogenic CO2 emissions [START_REF] Hendriks | Emission reduction of greenhouse gases from the cement industry[END_REF]. The cement industry is making strong effort to reduce the CO2 footprint in basically three directions:

improvement of the fabrication process, development of low CO2 cements, use of Supplementary Cementitious Materials (SCM) to mix with cement. Belite-CalciumSulfoaluminate-Ferrite cements -2-(BCSAF) or Calcium SulfoAluminate cements (CSA) are potential alternative cementitious binder for OPC cements [START_REF] Hanein | Production of belite calcium sulfoaluminate cement using sulfur as a fuel and as a source of clinker sulfur trioxide: pilot kiln trial[END_REF][START_REF] Quillin | Performance of belite-sulfoaluminate cements[END_REF]. The production of calcium sulfoaluminate clinkers involves lower embodied energy and CO2 emissions compared to the Ordinary Portland Clinker, because they require less limestone, lower grinding energy and lower clinkering temperatures than OPC clinker [START_REF] Gartner | Alternative cement clinkers[END_REF].

Ye'elimite (Ca4Al6O16S or C4A3S̅ ) * is a significant component in BCSAF and CSA cements [START_REF] Gartner | Alternative cement clinkers[END_REF]. The detailed understanding of ye'elimite formation during the thermal treatment may be the key for solving burnability † encountered during the production of CSA type clinkers. Table 1 shows the cement phases present in CSA clinkers compositions given in the literature [START_REF] Winnefeld | Hydration of calcium sulfoaluminate cements-experimental findings and thermodynamic modelling[END_REF][START_REF] Telesca | A hydration study of various calcium sulfoaluminate cements[END_REF][START_REF] Pelletier-Chaignat | Beneficial use of limestone filler with calcium sulphoaluminate cement[END_REF][START_REF] Martin | Contribution of limestone to the hydration of calcium sulfoaluminate cement[END_REF][START_REF] Bizzozero | Expansion mechanisms in calcium aluminate and sulfoaluminate systems with calcium sulfate[END_REF][START_REF] Zhou | An alternative to Portland cement for waste encapsulation-the calcium sulfoaluminate cement system[END_REF][START_REF] Pelletier | The ternary system Portland cement-calcium sulphoaluminate clinker-anhydrite: hydration mechanism and mortar properties[END_REF]. It is absolutely clear that on top of ye'elimite CSA clinker products contain various calcium aluminate phases as minor phases. Similar observations were made during pure ye'elimite powder preparation by solidstate reaction methods [START_REF] Winnefeld | Hydration of calcium sulfoaluminate cements-experimental findings and thermodynamic modelling[END_REF][START_REF] Winnefeld | Calorimetric and thermogravimetric study on the influence of calcium sulfate on the hydration of ye'elimite[END_REF][START_REF] Chitvoranund | Comparison of different preparation methods for ye'elimite clinker synthesis[END_REF][START_REF] Hargis | Early age hydration of calcium sulfoaluminate (synthetic ye'elimite, C4A3S̅ ) in the presence of gypsum and varying amounts of calcium hydroxide[END_REF][START_REF] Cuesta | Structure, atomistic simulations, and phase transition of stoichiometric yeelimite[END_REF][START_REF] Cuesta | Pseudocubic crystal structure and phase transition in doped ye'elimite[END_REF][START_REF] Álvarez-Pinazo | Rietveld quantitative phase analysis of Yeelimite-containing cements[END_REF][START_REF] Khessaimi | Solidstate synthesis of pure ye'elimite[END_REF] or by low temperature chemical routes [START_REF] Khessaimi | Ye'elimite synthesis by chemical routes[END_REF][START_REF] Song | Direct synthesis and hydration of calcium aluminosulfate (Ca4Al6O16S)[END_REF], where the final product contained CaO, CaAl2O4 and CaAl4O7 as main impurities when the sintering temperature was below 1300°C. Hence, successful preparation of highly pure ye'elimite powder by solid-state reactions required multiple milling, pelleting and firing cycles at 1300°C. However, the question of how these successive processing steps increased the ye'elimite content was not raised. Table 2 represents the possible reactions involved during ye'elimite formation in different temperature ranges [START_REF] Khessaimi | Solidstate synthesis of pure ye'elimite[END_REF]. Solid-state formation of ye'elimite from pure raw materials starts at 1000°C and it continues until 1300°C through reactions between calcium aluminate phases (CA, CA2) and CaSO4 until 1300°C. In this temperature range, CA and CA2 can be qualified as 'intermediate' compounds since they form between 1000 and 1100°C. From 1300°C, ye'elimite can decompose into calcium aluminate phases (CA and C12A7), O2 gas and SO2 gas. Li et al. [START_REF] Li | Kinetics of calcium sulfoaluminate formation from tricalcium aluminate, calcium sulfate and calcium oxide[END_REF] studied the formation of ye'elimite from C3A and gypsum; they reported that the optimal conditions for C4A3S̅ formation were 1150 to 1350°C for 1 h to 6 h. Ca 2+ and SO4 2-were the diffusive species in both the different reactions. Nonetheless, there is no suitable study at microstructural scale involving ye'elimite formation from pure Al2O3, CaO and CaSO4.2H2O phases.

As shown by reactions R6 and R7 (table 2), ye'elimite formation involves calcium aluminate phases (CA and CA2). The microstructural changes happening during the formation of calcium aluminates are well described in literature (which is not the case for ye'elimite). For example, Iftekhar et al. [START_REF] Iftekhar | Phase formation of CaAl2O4 from CaCO3-Al2O3 powder mixtures[END_REF] studied the formation of CA from a mixture of CaCO3-Al2O3 powders in a temperature range between 1300 and 1500°C. The authors noted that Ca 2+ ions diffuse through calcium aluminates and react with alumina, the dense layer of CA2 formed around the A/CA2 contact region acts then as a diffusion barrier. The high calcium concentration observed at the pore surface may be an indication that Ca 2+ diffusion during phase formation occurs at the grain surface. Other authors do not confirm this last hypothesis about the formation of phases in the direction of the interface but confirm the fact that Ca 2+ diffuse through the calcium aluminates, from calcium-rich aluminates to calcium-poor aluminates [24] [25]. De Bilbao et al. [START_REF] De Bilbao | Time-resolved high-temperature X-ray diffraction for studying the kinetics of corrosion of high-alumina refractory by molten oxides[END_REF] studied the corrosion kinetics of high-alumina refractories by a binary slag (Al2O3 50 wt.% CaO 50 wt.%) at 1650°C for 30 min. It was observed via scanning electron microscopy that CA6/CA2 layers formed around the alumina aggregates. The formation of these layers has been explained by the diffusion of Ca 2+ and O 2-ions, and the existence of each layer depends on the temperature. Mercury et al. [START_REF] Mercury | Synthesis of CaAl2O4 from powders: Particle size effect[END_REF] studied the formation of CA phase from a mixture of Al2O3 and CaCO3 milled by attrition and from a Al(OH)3 and CaCO3 powder mix. From 900°C, the formation of mayenite C12A7 occurs at the contact between the CaO grains and the alumina particles.

At higher temperatures, Ca 2+ ions of calcium-rich phases diffuse through the C12A7 phase layer to then form the CA phase and so on for the formation of the CA2 phase. Once these reactions are complete, the densification starts from 1250°C onwards. Scian et al. [START_REF] Scian | High alumina cements. Study of CaO.Al2O3 formation II. Kinetics[END_REF] also studied the formation of the calcium aluminte phase CA, and identified two steps in the process of its formation. During the first stage (short duration and low temperature), the reaction between CaO and Al2O3 produces a layer of calcium aluminate at a controlled rate by an interface process (chemical reaction, nucleation and product growth). Then, inside this interface of reaction, separating the two reagents, the calcium (free or combined), diffuses to react with the alumina rich phases and diffusion was identified as the limiting step of the reactions [START_REF] Mohamed | Kinetics and mechanism of formation of tricalcium aluminate, Ca3Al2O6[END_REF].

As far as we learned from the literature [START_REF] Winnefeld | Calorimetric and thermogravimetric study on the influence of calcium sulfate on the hydration of ye'elimite[END_REF][START_REF] Cuesta | Structure, atomistic simulations, and phase transition of stoichiometric yeelimite[END_REF][START_REF] Cuesta | Pseudocubic crystal structure and phase transition in doped ye'elimite[END_REF][START_REF] Álvarez-Pinazo | Rietveld quantitative phase analysis of Yeelimite-containing cements[END_REF][START_REF] Khessaimi | Solidstate synthesis of pure ye'elimite[END_REF][START_REF] Song | Direct synthesis and hydration of calcium aluminosulfate (Ca4Al6O16S)[END_REF][START_REF] Rungchet | Hydrothermal synthesis of calcium sulfoaluminate-belite cement from industrial waste materials[END_REF][START_REF] Chitvoranund | Synthesis and hydration of alitecalcium sulfoaluminate cement[END_REF][START_REF] Halstead | The composition and crystallography of an anhydrous calcium aluminosulphate occurring in expanding cement[END_REF][START_REF] Ikeda | Structure refinement of calcium sulfoaluminate, C4A3S ̄ with emphasis to oxygen deficiency[END_REF][START_REF] Calos | Structure of calcium aluminate sulfate Ca4Al6O16S[END_REF], ye'elimite-rich cements present high compressive strength at early age of hydration. Industrial producers of this type of cements seek to obtain a clinker with high ye'elimite amounts. The ideal situation is to convert all the raw materials into ye'elimite during a thermal treatment. However, in most cases, the clinker product contains calcium aluminate phases (CA, CA2) and small quantities of raw materials (Table 1). The presence of cementitious phases other than ye'elimite in calcium sulfoaluminate cements change the hydration properties (workability, early strength properties, expansion and durability) [START_REF] Bizzozero | Expansion mechanisms in calcium aluminate and sulfoaluminate systems with calcium sulfate[END_REF][START_REF] Hargis | Calcium sulfoaluminate (Ye'elimite) hydration in the presence of gypsum, calcite, and vaterite[END_REF][START_REF] Bullerjahn | CSA raw mix design: effect on clinker formation and reactivity[END_REF][START_REF] Bullerjahn | Effect of raw mix design and of clinkering process on the formation and mineralogical composition of (ternesite) belite calcium sulphoaluminate ferrite clinker[END_REF][START_REF] Palou | Effects of sulphate, calcium and aluminum ions upon the hydration of sulphoaluminate belite cement[END_REF][START_REF] Boisvert | Hydration of calcium sulfate hemihydrate (CaSO4•1/2H2O) into gypsum (CaSO4•2H2O). The influence of the sodium poly (acrylate)/surface interaction and molecular weight[END_REF][START_REF] Adams | Volume Stability of Calcium Aluminate Cement and Calcium Sulfoaluminate Cement Systems, Factors Influ. Convers[END_REF]. In this context, the phenomenological description of ye'elimite formation could help calcium sulfoaluminate cement producers to improve the burnability of the raw materials during the clinkering process and it could also help academic researchers to synthesize lab-made calcium sulfoaluminate cement for crystallographic characterizations and hydration studies. The purpose of the current work is to investigate the ye'elimite formation mechanisms by solid-state reactions from pure raw materials: calcium carbonate (CC̅ ), alumina (A) and gypsum (CS̅ H2). This study was conducted using X-ray diffraction and scanning electron microscopy to get information about mineralogical and microstructural assemblages at different synthesis temperatures. Avrami's model was suggested to highlight the effect of synthesis conditions on ye'elimite kinetic formation.

-5-

Experiments 2-1-Materials

The starting materials used in this work were high purity CaCO3, Al2O3 and CaSO4.2H2O all from Sigma-Aldrich. Table 3 shows the chemical analysis and physical characteristics of these raw materials. The samples were prepared in the form of pellets from stoichiometric mixtures (SM)

according to the protocol mentioned in [START_REF] Khessaimi | Solidstate synthesis of pure ye'elimite[END_REF]. Each mixture (102 g) was first homogenized in a Turbula © shaker for 15 min. Then it was crushed and homogenized again at 120 rpm for 1 h in planetary ball mills, using a 250 ml corundum jar and fifty corundum balls of 10 mm diameter. The homogenized mixture was uniaxially pressed under a load of 1 ton into pellets using a 20 mm cylindrical die. The compacted green body was subsequently placed in a platinum crucible and thermally treated. Table 4 shows the sample identification names and the corresponding thermal cycles (one or two cycles) for each prepared sample. The choice of 1300°C for isothermal study is based on the results of our previous work [START_REF] Khessaimi | Solidstate synthesis of pure ye'elimite[END_REF] on pure ye'elimite synthesis since we found that 1300°C was the optimal ye'elimite synthesis temperature. The heat treatment from 1100 to 1300°C

were performed in a platinum crucible and in an elevator-furnace to ensure air quenching. To avoid the degradation of the elevator-furnace heating elements, experiments at 1000°C were carried out in muffle furnace without air quenching.

2-2-Characterization techniques

The scanning electron microscope (SEM, JSM-IT300, JEOL, Japan) used for this study was equipped with an Energy Dispersive Spectrometer (EDS, Oxford, UK). rpm in order to increase particle statistics. The diffractometer was equipped with energy-dispersive LYNXEYE XE-T detector for filtration of fluorescence and Kβ radiation. Table 6 shows ICSD (Inorganic Crystal Structure Database) collection codes used for XRD phase identification [START_REF] Calos | Structure of calcium aluminate sulfate Ca4Al6O16S[END_REF][START_REF] Hörkner | Zur kristallstruktur von CaAl2O4[END_REF][START_REF] Ponomarev | Crystal structure of calcium dialuminate, CA2[END_REF][43][START_REF] Cheng | The crystal structure of anhydrite (CaSO4)[END_REF].

The particle size distribution (PSD) of the raw powders was obtained using a Mastersizer 2000 laser size analyzer (Malvern). Prior to this measurement, the powder (approximately 80 mg) was mixed in 20 ml of absolute ethanol and the suspension was sonicated 1 min in order to break the coarse agglomerates. The BET specific surface area was estimated from N2 adsorption experiments using a Micromeritics model Tristar®II 3020 analyzer. Prior to measurements, a degassing step of the sample powder was carried out at 80°C overnight in order to achieve a pressure equal to 60 mTorr.

Chemical analyses of the raw materials powders were carried out using X-ray fluorescence spectrometer, PANalytical, Zetium model. Samples were prepared as fused beads using glass discs prepared by melting about 1 ±0.001 g of sample powder with 10 ±0.001 g of lithium tetra borate flux. The Loss on Ignition (LOI) was calculated for samples treated at 1050°C for 1 h. The oxide contents are expressed in weight percent (wt.%).

Results and discussion

3-1-Mineralogical analysis

3-2-Microstructural observation

The observed microstructural changes during ye'elimite formation are shown in fig. 3 5b). Four regions can be distinguished: the first is Al2O3 rich (blue ellipse), the second is close to CA2 (red ellipse), the third is close to CA (green ellipse) and the last one is close to C4A3S̅ (pink ellipse). Thus, CA2 phase tends to form on Al2O3 grains, then CA phase tends to form on CA2 grains and C4A3S̅ forms on CA grains according to solid-state reaction R5 (table 2).

3-3-Discussion

Based on the XRD analysis and the microstructural observations, ye'elimite amount increases as the sintering temperature goes from 1000 to 1300°C for 3 h. However, it is still not pure at 1300°C since some CA, CA2, CaO, Al2O3 and CaSO4 remain. The presence of these phases could be explained by an incomplete solid-state reaction between the different reactants.

Fig. 6a summarizes the possible solid-state diffusion during ye'elimite formation. Fig. 6b and6c give a schematic representation of the microstructure transformations as a function of temperature for a given duration (fig. 6b) and as a function of dwell time at 1300 °C (fig. 6c). Based on the literature [START_REF] Li | Kinetics of calcium sulfoaluminate formation from tricalcium aluminate, calcium sulfate and calcium oxide[END_REF], we assume that the diffusive species are Ca 2+ and SO4 2-. Ca 2+ ions coming from CaO can reach the CA/CA2 interface and react with CA2 to produce CA (reaction (a), fig. 6a); the consequence is a growth of CA grain and a consumption of CA2 (the movement of the CA/CA2 interface is schematized by arrow 1, fig. 6a). Alternatively, CA can decompose into CaO -thus producing Ca 2+ ions -and CA2 (reaction (b), fig. 6a); the result is a growth of CA2 grain and a decrease of CA (the displacement of the CA/CA2 interface is schematized by arrow 2, fig. 6a). The Ca 2+ ions can also reach the next interface CA2/Al2O3 and consume alumina to form CA2 (reaction (c) with a growth of CA2 and a decrease in size of Al2O3, arrow 3). For the outer layer which contains C4A3S̅ phase, its growth depends upon the contact between CA phase and CaSO4 phase. Ca 2+ and S VI+ may diffuse from CaSO4 grains to C4A3S̅ /CA and/or to C4A3S̅ /CA2 to form C4A3S̅ (reactions R6 and R7, table 2). Avrami's kinetic model based on geometrical contraction of reactant grains, can be qualitatively applied to our case. Quantitative calculations cannot be done because the grains geometry is more complex than simple spheres. Nervertheless, Avrami's This model enables us to identify the parameters which influence upon the microstructural changes as a function of time or temperature.

The rate of a solid-state reaction is described by equation ( 4) [START_REF] Khawam | Solid-state kinetic models: basics and mathematical fundamentals[END_REF]:

= k f(σ) Eq.( 4
)
where σ is the conversion fraction given by equation ( 5), f(σ) is the function of the reaction model and k is the reaction rate constant, k is usually given by the Arrhenius equation (equation 6) [START_REF] Kingery | Introduction to ceramics[END_REF]:

σ = Eq.( 5
)
m0 is the initial reactant weight, m is the reactant weight at sintering time t, and m is the final weight which is generally approximated to be equal to 0 (complete reaction). k = A. e

Eq.( 6)

A is the frequency factor, R is the gas constant, T is the sintering temperature and Ea is the activation energy. In the terminology of homogeneous kinetics, the activation energy (Ea) is usually identified as the energy barrier that must be overcome to enable the occurrence of the bond redistribution steps required to convert reactants into products. The pre-exponential term, or frequency factor (A), gives

Fig. 1

 1 Fig.1represents X-Ray diffractograms of the samples heated between 1000 to 1300°C for 3 h. For temperatures ranging from 1000 to 1200°C, the diffraction peaks with the main intensities correspond to CaO (C), Al2O3 (A) and CaSO4 (CS̅ ). The peaks for ye'elimite (C4A3S̅ ) and krotite (CA) are much smaller. The appearance of CaSO4 and CaO phases is attributed to the decomposition of gypsum and CaCO3, respectively (reactions R1 and R2 in table 2). At 1100°C, CA2 diffraction

  and fig. 4. The colored EDS mapping of back-scattered electron images for the samples sintered at different temperatures (from 1000 to 1300°C) for 3 hours are shown in fig. 3. The following descriptions can be made: samples treated at 1000°C present a heterogeneous microstructure with the presence of three materials, namely Al2O3, CaO and CaSO4. CaO and CaSO4 come respectively from CaCO3 decarbonation and CaSO4.2H2O dehydration. After sintering at 1100°C, small amounts of CA and CA2 are formed around fine alumina grains. Similar observations can be made for the sample sintered at 1200°C. By increasing the temperature to 1250°C, the CA layer around alumina aggregates thickens while alumina grains decrease in size. We also see C4A3S̅ grains formed thanks to the reaction between CA and CaSO4 grains. As the temperature increases to 1300°C, the CA phase almost totally surrounds alumina grains whose size reduces considerably. These observations are in accordance with the XRD results shown in fig. 1.After the examination of the microstructural changes as a function of heating temperature, it is interesting to look at the microstructural changes as a function of dwell time for a given temperature.Colored EDS mapping of back-scattered electron images for the samples sintered at 1300°C from 15 to 180 min are shown in Fig.5. After 15 min heat treatment at 1300°C, ye'elimite surrounds CA and CA itself surrounds Al2O3 grains (fig.4a). Some lime grains are present. For the sample sintered for 30 min, we notice the presence of a core of Al2O3 grains surrounded by CA2, CA and an external layer of C4A3S̅ (fig.4b). For the sample sintered for longer time (60 and 120 min, fig.4c and 4d), C4A3S̅ becomes dominant compared to CA and Al2O3. Lastly, after 180 min, large C4A3S̅ grains are formed and no Al2O3, CaO and CaSO4 are left (fig.4e). It clearly shows that the formation of ye'elimite is almost complete for sintering duration above 120 min.

Fig. 5a corresponds

  Fig. 5a corresponds to EDS data of atomic percentage for calcium, aluminum and sulfur elements as a function of the position on the scan line for the sample heated at 1300 °C for 30 min (see the red line on fig. 4b). We can obviously identify four zones in fig. 5a. To identify the compositions corresponding to each zone, the data are plotted in atomic percentage in the ternary composition diagram Al-Ca-S (fig. 5b). Four regions can be distinguished: the first is Al2O3 rich (blue ellipse), the

Fig. 6b and

  Fig. 6b and fig. 6c give a schematic representation of the microstructural evolutions. As the temperature increases (fig. 6b), the Al2O3 grains progressively disappear and are replaced by CA and CA2. These two compounds themselves eventually transform into ye'elimite. From microstructural observations of samples treated at 1300°C for different durations (fig. 4), the schematic representation on fig. 6c represents what happens: Al2O3 grains then calcium aluminate phases present at 1300°C are progressively consumed to give C4A3S̅ -rich regions.

Figure 1 X

 1 Figure 1X-Ray diffractograms of samples treated from 1000 to 1300°C for 3 h.

Figure 2 X

 2 Figure 2X-Ray diffractograms of samples treated at 1300 °C from 0 to 180 min.

Figure 3 EDS

 3 Figure 3 EDS mapping of elements present in the samples treated for 3 h between 1000°C (a), 1100°C (b), 1200°C (c), 1250°C (d) and 1300°C (e). Magnification of x150 was used of all images (Bars represent 250 μm). The corresponding EDS point analysis are shown in Appendix A.

Figure 4 EDS

 4 Figure 4 EDS mapping of elements present in the samples treated at 1300°C for different durations: 15 min (a), 30 min (b), 60 min (c), 120 min (d) and 180 min (e). Magnification of x500 was used of all images (Bars represent 50 μm). The red line of the sample treated at 30 min (Fig. 3(a)) represents EDS line-scan, the results of the line scan are shown in Fig. 4 and the corresponding EDS point analyses are shown in Appendix A.

Figure 5 (

 5 Figure 5 (a) Composition profile obtained by EDS along the red line for the sample treated at 1300°C for 30 min (The EDS line-scan is represented by the red line in Fig. 3), (b) Representation of EDS line scan data in ternary atomic percentage diagram of Al-Ca-S (the red square, represents theoretical atomic composition of CA2, the green square represents theoretical atomic composition of CA and the purple square represents theoretical atomic composition of C4A3S̅ . The black triangles are associated to the collected EDS data.

Figure 6 (

 6 Figure 6 (a) Schematic representation of solid-state diffusion during ye'elimite formation. (b) Schematic representation of microstructural development of ye'elimite formation according to the sintering temperature. (c) Schematic representation of microstructural development of ye'elimite formation according to the sintering duration.

Figure 7

 7 Figure 7Simple schematic model of reaction-product layers forming on alumina grain.Appendix A

Figure. 1 Figure. 2 EDS

 12 Figure. 1

Figure. 7

 7 Figure. 7

  In this respect, the impregnation of the sample (under the form of a pellet) is done with a low-viscosity epoxy resin (IP, PRESI France). The hardening time of the resin is about 24 h at room temperature. Once the sample is removed from the mold, the top side of the embedded sample is polished using SiC paper and absolute ethanol as a lubricant. Water should not be used at any time as it could lead to hydration of anhydrous grains. Regular observation of the surface under a light microscope is essential to check the surface aspect after each polishing step. Polishing was performed using Minitech 233 type machine (Presi, France). Details of the polishing protocol are given in table5.

The microscope was operated at 15 kV accelerating voltage. Prior to any observation, a 15 nm Au-Pd or Carbone coating was deposited in order to ensure suitable electronic conduction at the surface of samples. The specimens were stored in desiccators filled with silica gel prior to and after examinations to prevent hydration or carbonation. Colored Backscattered Electron (BSE) images reveal the different phases because of the atomic number contrast. EDS mapping and compositional profile along a line were made to localize each formed phase. For reliable observations, the essential prerequisite is a wellpolished sample. Polished sections can provide representative images of a cross section of the samples microstructure. X-Ray Diffraction (XRD) data were collected at room temperature in the Bragg-Brentano geometry using a Bruker D8 Advance X-ray diffractometer with CuKα radiation (λCu= 1.54056 Å, without monochromator) at a step scan of 0.02° with a time counting per step of 0.45 second operated at a voltage 40 kV and an electric current 40 mA. The sample was rotated during data collection at 15

  ). At 1100°C, CA2 diffraction peaks appear, while those related to CaO and Al2O3 are attenuated. Grossite (CA2) forms by the solid-state reaction between CaO and Al2O3 (reaction R5 in table 2)[START_REF] Mercury | Synthesis of CaAl2O4 from powders: Particle size effect[END_REF]. From 1100 to 1200°C, the diffraction intensities of CaO and Al2O3 are attenuated while those of CA and CA2 peaks are slightly more intense; this variations in peak intensities can be related to the crystallisation of calcium

	The XRD results can be summarized as follows: (i) ye'elimite starts forming at 1000°C; (ii) CA and
	CA2 are intermediate phase formed between 1000 and 1100°C; (iii) above 1100°C, CA (resp. CA2)
	progressively react with CS̅ through R6 (table 2), (resp. C and CS̅ through R7, table 2) to convert
	into ye'elimite.
	Hence, the reaction of ye'elimite formation is well activated by increasing sintering temperature,
	especially from 1200 to 1300°C.
	Fig. 2 shows the XRD patterns of the samples heated at 1300°C for various durations, from 15 to 180
	min. CaO, CaSO4, Al2O3, CA, CA2 and ye'elimite were identified in the sample heated at 1300°C
	and immediately quenched (dwell time at 1300°C : 0 min). By increasing the dwell time from 0 to
	180 min, CA diffraction peaks become more intense, while those related to CaO, Al2O3 and CaSO4
	are attenuated. Simultaneously, the intensity of the peaks related to ye'elimite phase increases with
	increasing the dwell time with a simultaneous decrease in calcium aluminate peak intensities. This
	confirms the conversion of calcium aluminate phases to ye'elimite phase according to the reactions
	R6 and R7 (table 2). Hence, the reaction of ye'elimite formation is well activated by increasing the
	dwell time at 1300°C.
	-8-

aluminates (reactions R3 and R5, Table

2

)

[START_REF] Mercury | Synthesis of CaAl2O4 from powders: Particle size effect[END_REF]

. Beyond 1200°C, the peak intensities of CA and CA2 decrease while the peak intensities of ye'elimite increase; this last phase can form through solid-state reaction between calcium aluminates and calcium sulphate reactions R6 and R7 (table 2)

[START_REF] Khessaimi | Solidstate synthesis of pure ye'elimite[END_REF]

.

Table captions Table 1

 captions1 Some CSA clinkers compositions found in literature.

Table 2

 2 Possible reactions involved during ye'elimite formation in different temperature ranges

	[19]. The cementitious notations are the following: C= CaO, A = Al2O3, S̅ = SO3, C̅ =
	CO2, H = H2O

Table 3

 3 Chemical analysis and physical characteristics of the raw materials.

Table 4

 4 Samples identification and corresponding thermal cycle.

Table 5

 5 Polishing steps.Table 6 ICSD collection codes used for XRD phase identification.

				Table 1
	Reference			Composition Wt.%
		Ye'elimite	CA	CA2	C12A7	C3A	Other phases
	[6]	50.0	8.0	-	-	-	42.0
	[7]	52.1	-	-	1.2	9.4	37.3
	[8]	62.8	8.1	3.1	-	-	26.0
	[9]	68.1	3.2	0.7	1.4	-	26.6
	[10]	50.0	-	-	-	< 8.0	42.0
	[11]	57.0	15.0	-	1.0	-	27.0
	[12]	68.1	7.8	1.2	-	3.4	19.5

Table 5

 5

	Polishing	SiC paper	Lubricant	Polishing	Force	Rotation speed of the
	Steps			time (s)	(daN)	polishing disc (rpm)
	1	P240	Ethanol (*)	30	0.25	150
	2	P600	Ethanol	180	0.25	150
	3	P1200	Ethanol	270	0.25	150
	4	P2400	Ethanol	360	0.25	150
	5	P4000	Ethanol	450	0.25	150

(*) Absolute ethanol was used as polishing lubricant to avoid sample hydration reactions.

Table 6

 6

	Phase name	Formula	ICSD codes	Ref.
	Orthorhombic ye'elimite orth-C4A3S̅	80361	[34]
	Krotite	CA	260	[41]
	Grossite	CA2	34487	[42]
	Lime	C	52783	[43]
	Anhydrite	CS̅	15876	[44]

SM(1000;3) SM(1100;3) SM(1200;3) SM(1250;3) SM(1300;3) SM(1300;15) SM(1300;30) SM(1300;60) SM(1300;120) SM(1300;180)

an indication of the frequency of occurrence of the reaction situation. It is usually dependent upon the vibration frequency of the reaction co-ordinate [START_REF] Galwey | Application of the Arrhenius equation to solid state kinetics: can this be justified?[END_REF]. Fig. 7 is a schematic representation of the successive layers formed on the surface of Al2O3 grain. In the present case, the rate of Al2O3 grain consumption is supposed to be controlled by the progress of the interface reaction towards the middle of the grain. The radius (r) at sintering time t is given by equation ( 7) [START_REF] Khawam | Solid-state kinetic models: basics and mathematical fundamentals[END_REF]: r = r0 -kt Eq. [START_REF] Telesca | A hydration study of various calcium sulfoaluminate cements[END_REF] where r0 is the radius at time t0 and t is the solid-state reaction time.

If we make a simple assumption that Al2O3 grains are spherical with a volume of 4πr 3 /3 and a density ρ, equation ( 5) becomes: σ = Eq.( 8) which reduces to equation ( 9):

1σ = Eq.( 9) By combining equation [START_REF] Telesca | A hydration study of various calcium sulfoaluminate cements[END_REF] with equation ( 9) we have:

Eq. [START_REF] Bizzozero | Expansion mechanisms in calcium aluminate and sulfoaluminate systems with calcium sulfate[END_REF] By combining equation ( 4) with equation ( 6) and [START_REF] Bizzozero | Expansion mechanisms in calcium aluminate and sulfoaluminate systems with calcium sulfate[END_REF] we can write:

The suggested model expressed by equation [START_REF] Zhou | An alternative to Portland cement for waste encapsulation-the calcium sulfoaluminate cement system[END_REF] highlights qualitatively the effect of the synthesis conditions on ye'elimite formation using a simple 3D-geometrical contraction model. To go further, i.e. to obtain quantitative values for Ea and A, it would be in principle feasible, but their values would be meaningless and would not describe precisely the complex solid-state formation of ye'elimite. In fact, there are many limitations to discuss quantitatively the application of the proposed model. First, the starting point of theoretical explanation of Arrhenius behavior in homogeneous reactions is the Maxwell-Boltzmann energy distribution function, although this model is inapplicable to immobilized constituents of a solid [START_REF] Galwey | Application of the Arrhenius equation to solid state kinetics: can this be justified?[END_REF]. Furthermore, the formation process of ye'elimite involves many reactions that occur sometimes simultaneously. Thirdly, the suggested model assumes one value of alumina particle radii. In reality, there is a distribution of alumina particle size and the particles sometimes agglomerate. Since no realistic alternative is capable of expressing the formation rate versus temperature during complex solid-state reactions [START_REF] Galwey | Application of the Arrhenius equation to solid state kinetics: can this be justified?[END_REF], Avrami's model based on the Arrhenius equation has been widely accepted and successfully applied by many authors to describe numerous reactions involving solids.

In the present case, the proposed sequence is the following: Al2O3 converts into CA2 which in turn transforms into CA. Then CA reacts with CaSO4 to form C4A3S̅ . So ye'elimite formation depends on Al2O3 consumption. Referring to Avrami's kinetic model mathematically expressed by equation ( 11), Al2O3 conversion rate increases with increasing sintering temperature (T), increasing sintering time (t) and/or reducing the size of the starting Al2O3 grain (r0). This could explain qualitatively the high thickness of C4A3S̅ -rich and CA-rich regions around the smallest Al2O3 grains. It should be noted that there is some extreme values for T and t that should not be exceeded due to the sulfate instability above 1300°C as synthesis temperature and/or 4 h duration [START_REF] Khessaimi | Solidstate synthesis of pure ye'elimite[END_REF].

Conclusion

The reaction sequence by which ye'elimite forms through solid-state reactions from pure raw materials (alumina, lime and gypsum) was studied. First, mineralogical development during ye'elimite formation from 1000 to 1300°C for 3 hours and from 15 to 180 min at 1300°C, shows that ye'elimite content increases with increasing sintering temperature and duration. These results were confirmed by microstructural observations using colored EDS mapping of BSE micrographs. The results reported herein deepen the understanding of the phenomenological description of ye'elimite solid-state formation, which could help cement producers to improve the burnability of raw materials during the clinkering process of calcium sulfoaluminate cements (ye'elimite-rich cements) and to minimize the presence of secondary phases like calcium aluminate phases. Lastly, the present study could also help academic researchers to synthesize lab-made calcium sulfoaluminate cements for crystallographic characterizations and hydration studies.