Highly-crystallized quaternary chalcopyrite nanocrystals via a high-temperature dissolution-reprecipitation route J. Y. Chane-Ching,* a A. Gillorin, a O. Zaberca, a A. Balocchi b and X. Marie b DOI: 10.1039/c1cc10749f Quaternary chalcopyrite (Cu 2 CoSnS 4 , Cu 2 ZnSnS 4 ) nanocrystals displaying high crystallization and controlled morphology were synthesized via a high-temperature growth regime achieved by dissolution-reprecipitation of tailored ultrafine precursors in the temperature range 400-500 1C.

Nanostructured cells are generally described as third-generation solar cells with the goal to significantly increase device efficiencies. 1 These cells include quantum-dot intermediate band solar cells, 1 hybrid cells 2 and involve the use of semiconducting inorganic nanocrystals. Recently, use of these semiconducting nanocrystals has been extended as inks 3 to replace more expensive non-vacuum techniques to the fabrication of thin film absorbers of second-generation cells. In these applications, surface and bulk properties of these nanocrystals are crucial for improving the efficiency of the nano-crystal-based devices. Regarding the bulk properties, electron-hole recombination which increases current losses, is very sensitive to the presence of defects. Thus, single-crystalline or highly crystallized nano-crystals are highly desirable for these photovoltaic applications. Indeed, a crucial point to increase efficiency of nanostructured solar cells is the ability to control the defect concentration of the semiconductor nanocrystals. 4 However, the limited available information relating to the defect concentration dependence of the photovoltaic properties is mainly due to the difficulty in preparing perfectly crystallized inorganic semiconducting nanocrystals. Therefore, it is important to develop new synthetic routes producing size and morphology controlled nanocrystals which exhibit high crystallization. Among candidate materials, ternary chalcopyrite nanocrystals 5 have attracted great attention because of their potential for use in solar cells. Most recently, quaternary chalcopyrite nanocrystals 6 displaying similar structure and optical properties to CuInS 2 are receiving considerable attention as promising candidates for low-cost absorber layers. 7 Cu 2 ZnSnS 4 is one of the well-known quaternary chalcopyrites, 8 however, few reports describe the synthesis and properties of these nanocrystals. 6 The only reported synthetic routes ,6,9 involve the arrested precipitation in coordinating solvent at temperatures of 200 1C. Nevertheless, UV-Vis absorption spectra recorded on the resulting nanocrystals did not exhibit any well-defined excitonic peak. The lack of a distinct excitonic peak could arise from various factors such as a large variation in size and composition of the quaternary chalcogenide nanocrystals, or from a too large defect concentration existing in these semiconductor nanocrystals, prepared at a relatively low temperature. Herein, we employ a high temperature (450 1C) dissolution-reprecipitation route performed in molten salts to sufficiently increase the crystallization of the nano-crystals such that a distinct excitonic peak can be observed in the UV-Vis absorption spectrum. Critically, the multiplication of nucleation centers resulting from the use of tailored ultrafine Cu 2 MSnS 4 (M = Co, Zn) precursors affords to preserve the nanometric size of the crystals during the high-temperature crystallization stage. Moreover, a high-temperature growth regime was achieved for the size and morphology control of the Cu 2 MSnS 4 nanocrystals via the use of a sulfide-source molten salt such as potassium thiocyanate, KSCN.

In our high-temperature crystallization process, hightemperature growth kinetics is achieved via the selection of a molten salt allowing a fine tuning of supersaturation conditions. Indeed, thermal decomposition of the melt KSCN occurs at temperatures >275 1C, 10 with cyanogen, sulfide and sulfur production. Thus, formation of quaternary chalcopyrite involves oxidation of the sulfur by the cyanide anion, with formation of the sulfide anion. A preliminary set of control experiments aimed at the formation of Cu 2 MSnS 4 (M = Co, Zn) particles was performed using metal or metal oxides as metal precursors in KSCN and thiourea, CS(NH 2 ) 2 . As expected, 11,12 all these experiments yielded only either microcrystalline or fully aggregated crystals. In general, nucleation and growth in dissolution-reprecipitation processes are significantly dependent on precursor dissolution kinetics, permitting size and morphology control of Cu 2 MSnS 4 nanocrystals. Thus, in a first stage, ultrafine precursors displaying Cu 2 MSnS 4 single phase and small primary crystallite size were synthesized from the polycondensation of charged complex species containing Cu + , M n+ , Sn 4+ , CS(NH 2 ) 2 , S 2À and OH À in various solvents such as ethylene glycol, ethanol and isopropanol. A variety of ultrafine precursors was synthesized consisting of 150-250 nm discrete, loose packed aggregates (Fig. 1) and displaying surface charge. Although all these precursors display a single phase as shown from XRD and Raman spectroscopy, slight differences in relative proportions in Raman peak surfaces located in the region 200-400 cm À1 and assigned to secondary and main Cu 2 MSnS 4 vibrations 13 were observed. Further investigations are in progress to understand the origin of these short-range order differences. In a typical experiment, Cu 2 CoSnS 4 ultrafine precursor was synthesized using the following molar ratios: 2.0 CuCl 2 Á 2H 2 O : 1.0 CoCl 2 Á6H 2 O : 1.0 SnCl 4 Á5H 2 O : 10 CS(NH 2 ) 2 : 1.0 TMAOH. The resulting mixture was transferred into a Teflon-line stainless steel autoclave for 16 h at 200 1C. The precipitated solid was washed and ball milled in EtOH using 0.3 mm ZrO 2 balls. After re-dispersion in EtOH, the ultrafine precursor was collected after size selection carried out by centrifugation at 3000 rpm. The particles were aggregates with a mean diameter of 150 nm (Fig. 1) and formed from primary crystallites of 6 nm average size, as calculated from Scherrer's equation using the full width at half maximum (FWHM) of the (112) Cu 2 CoSnS 4 peak. In the second stage, the hightemperature growth stage was carried out in molten KSCN at 450 1C for 6 h. Because the molten salt acts as a sulfide source, a crucial parameter to control growth kinetics is the Cu 2 CoSnS 4 nanocrystal volume fraction, F v nano , F v nano = V nano /(V nano + V MS ), where V nano and V MS denote, respectively, the volume occupied by the nanocrystals and the volume occupied by the molten salt. Indeed, F V nano dictates both the supersaturation level and the interparticle distance of the nanocrystals dispersed in the molten KSCN. Hightemperature growth regimes were investigated for temperatures ranging from 300 to 550 1C and with various F V nano values, 0.045 o F v nano o 0.17. Formation of a Cu 2 CoSnS 4 single phase was achieved up to 450 1C as demonstrated from XRD and Raman spectroscopy investigations. As expected, the high-temperature dissolution-reprecipitation process induces better crystallization as evidenced from the FWHM of the (112) Cu 2 CoSnS 4 XRD peak. Moreover, a decrease of the FWHM (11.5 cm À1 ) of the 327 cm À1 Raman main vibration (Fig. 2) indicates a significant improvement of the short-range order into the nanocrystals. Indeed, large variations in crystal size were observed depending on the chemical properties of the Cu 2 CoSnS 4 precursors. Hence, nanocrystals were only produced with the use of precursors possessing Raman peak surface proportions similar to those determined from the bulk Cu 2 CoSnS 4 spectrum.

Since the various precursors investigated exhibit similar size distributions, differences observed on the corresponding curves describing variation of the crystal size with dissolutionreprecipitation temperatures (Fig. 3) clearly indicate different dissolution kinetics for the Cu 2 CoSnS 4 precursors. A power law increase of nanocrystal size with dissolution-reprecipitation temperature was observed for these highly homogeneous precursors. Our results indicate that the successful preparation of discrete nanocrystals rely on precursor properties such as an ultrafine size and a high short-range order as determined from Raman spectroscopy. A more uniform nanocrystal size distribution was achieved with careful control of F v nano (optimal value = 0.06), temperature and time. Typically, Cu 2 CoSnS 4 solid precursor was mixed with KSCN (Aldrich, 99.99%) in a weight ratio of 1.00 : 5.00 and ground for 10 mn. The mixture was then placed within an alumina crucible which was subsequently heated to 450 1C for 6 h under N 2 atmosphere. After being cooled at room temperature, samples were subsequently washed with deionized water and ethanol. Transmission electron microscopy (TEM) images of the as-synthesized materials prepared using an ultrafine precursor show a uniform distribution of nanocrystals (Fig. 4). The particles were faceted and have a mean size as small as 30 AE 5 nm. From an XRD investigation and using the Scherrer formula, the size of the primary crystallites were determined to be 30 AE 2 nm, demonstrating that our nanocrystals are single crystals. Unlike previous quaternary chalcopyrite nanocrystals reported in the literature, 6 UV-Vis absorption spectra recorded on the Cu 2 CoZnS 4 nanocrystals synthesized at high temperature exhibit a distinct excitonic peak observed at 830 nm (E g = 1.5 eV). The observation of a well-defined absorption peak is the signature of both the high crystalline quality and the homogeneous composition of our nanocrystals.

The adaptability of the synthetic method was shown for the preparation of Cu 2 ZnSnS 4 (CZTS) nanocrystals. A family of CZTS precursors displaying controlled dissolution kinetic was prepared by varying the solvothermal reaction temperature (150-200 1C) and adjusting the [Cu + ] concentration. Hightemperature growth regimes for dissolution-reprecipitation reactions were designed in the 400-500 1C temperature range and F v nano = 0.03-0.18, resulting in discrete CZTS nanocrystals with size ranging from 80 to 250 nm as shown by TEM (Fig. 5). The high crystallization of the nanocrystals is consistent with the features of the Raman spectrum displaying a low FMWH value (6 cm À1 ) of the 335 cm À1 main peak assigned to Cu 2 ZnSnS 4 . 13 Further work involving precursors exhibiting smaller particle size, aimed towards the fabrication of Cu 2 ZnSnS 4 single crystals, is in progress.

In conclusion, a high-temperature process involving the dissolution reprecipitation of Cu 2 MSnS 4 (M = Co, Zn) ultrafine precursors was developed for the preparation of highly crystallized quaternary chalcogenide nanocrystals. Successful synthesis of 30 nm Cu 2 MSnS 4 nanocrystals with controlled morphology was achieved through the fabrication of tailored precursors exhibiting ultrafine size and controlled dissolution kinetic.

We thank Alain Seraphine for very helpful discussions. This research was partly funded by Ecole des Beaux Arts et d'Architecture-Re´gion la Reunion, France.

Notes and references

Fig. 1

 1 Fig. 1 TEM micrographs of (a) Cu 2 CoSnS 4 precursors showing 150 nm discrete nanoparticles and (b) Cu 2 ZnSnS 4 precursors with mean size of 250 nm.

Fig. 2

 2 Fig. 2 (a) XRD pattern of highly crystallized Cu 2 CoSnS 4 nanocrystals. (b) Raman spectrum of highly crystallized Cu 2 CoSnS 4 nanocrystals. Inset: Deconvolution of the 200-400 nm region showing a main peak at 327 cm À1 . Corresponding XRD pattern and Raman spectrum of Cu 2 CoSnS 4 precursors are indicated in dashed curves, highlighting the effect of the crystallization stage.

Fig. 3

 3 Fig. 3 (a) Cu 2 CoSnS 4 precursor Raman spectra. (b) (112) Cu 2 CoSnS 4 XRD peaks plotted as a function of dissolutionreprecipitation temperature. The Raman spectra and XRD peak evolutions correspond to samples displaying after deconvolution different (left figures a 1 and b 1 ) or similar (right figures a 2 and b 2 ) peak surface proportions compared with bulk Cu 2 CoSnS 4 . (c) Inset: XRD crystal size as a function of dissolution-reprecipitation temperature.

Fig. 4

 4 Fig. 4 Highly crystallized Cu 2 CoSnS 4 nanocrystals. (a) TEM image showing discrete well faceted nanocrystals with a mean size of 30 nm. (b) UV-Vis absorption curve exhibiting a excitonic peak at 830 nm (1.5 eV).

Fig. 5

 5 Fig. 5 Highly crystallized Cu 2 ZnSnS 4 nanocrystals. (a) XRD pattern exhibiting a Cu 2 ZnSnS 4 single phase. (b) Raman spectrum showing a major vibration at 335 cm À1 . Note the decrease of the FWHM of this 335 cm À1 peak compared with precursor (dashed curve) demonstrating an increase of the short range order of the nanocrystals. (c) TEM images showing discrete nanocrystals with well faceted shapes and a mean size around 150 mn.

(a) A.Luque and A. Marti, Phys. Rev. Lett., 1997, 78, 5014; (b) A. J. Nozik, Phys. E.,

2002, 14, 115; (c) R. P. Raffaelle, S. L. Castro, A. F. Hepp and S. G. Bailey, Progr. Photovolt.:


Carole Iampietro

Camille Eché

Clément Birbes

Arnaud Di Franco

Andreea Dréau

Claire Kuchly

Christophe C Klopp

Thomas Faraut

Matthias Zytnicki

Denis Erwan

Structural variants detection and de novo genome assembly of a maize line

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Structural variants detection and de novo genome assembly of a maize line

Characterizing the genomic diversity of species is critical to understand the molecular origin of phenotypic variations. Whole genome sequence assemblies at the chromosome scale with low amount of missing data are critical resources for answering such question.

Our aim is to explore combination of technologies to answer this question, and to find the best one. Using a specific Maize line, we have tested different sequencing applications to build a "high quality genome" and identify a large collection of genome variants. Here we demonstrate that the use of HIFI reads combined with Hi-C and Linked-reads generate a chromosome-scale genome assembly with a better contiguity than the reference genome B73. Moreover we show that PacBio CLR reads allow a wide detection of structural variants of the genomes.

Iampietro Carole 1 , Eché Camille 1 , Birbes Clément 2 , Di Franco Arnaud 2 , Dréau Andreea 2 , Kuchly Claire 1 , Klopp Christophe 2 , Faraut Thomas 5 , Zytnicki Matthias 2 , Denis Erwan 1 , Praud Sébastien 4 , Joets Johann 3 , Vitte Clémentine 3 , Charcosset Alain 3 , Gaspin Christine 2 , Milan Denis 1,5 , Donnadieu Cécile 1 .

Sequencing Occitanie Innovation

Workflow for PacBio libraries preparation

Final Assembly statistics Contiguity statistics of our maize assembly versus B73 reference sequence:

The use of HiFi reads allowed us to obtain significantly longer contigs (maize 1) compared to the maize reference sequence (B73 RefGen_v4). With a longer total assembly size (the expected size is ~2.4Gb) and lower gap percentage, our results offer a more complete image of the maize genome. The difference in the number of scaffolds between our assembly and the reference sequence is due to a high number of short scaffolds originated from highly repetitive regions that have not been assembled in the reference. These assembly statistics were done with an old version of the Hifiasm assembler (0.9). The new version (0.15.1) significantly will improve the quality of the assembly: larger contigs and a higher BUSCO score.

Variants detection

Lab workflow optimization details

Optimized genome assembly workflow

PacBio HiFi reads were assembled with Hifiasm v0.9 (default parameters). The assembly was scaffolded using Hi-C reads which were first aligned to the assembly using juicer (default parameters). A pre-scaffolded assembly was generated with 3D-DNA (version 180114) with -r 0 flag (no iterative rounds for mis-join correction). In addition, 10X Chromium reads were aligned on the scaffolds and using tag continuity as 3D-DNA input file was generated to help scaffolding validation. Both 10X chromium and Hi-C links were uploaded in Juicebox to manually review the assembly.

*Hi-C and Chromium 10X (Genome reagent v2) data were sequenced on an Illumina NovaSeq6000 using a paired-end read length of 2x150 pb. The number of genes found in our assembly is similar to the results of the maize reference sequence. A potential cause for the missing genes in our assembly is the variability between different maize lines. All the results, presented here are, have been obtained within the framework of the "Sequencing Occitanie Innovation" project, which aims to acquire expertise on the optimal combination of long fragment sequencing technologies and associated applications to better characterize complex genomes for species with agronomical interest (maize, sheep, cow).

Perspectives

Thanks to the expertise acquired with the assembly of this first Maize genome, we will now apply a similar strategy to gain knowledge on specific Maize lines of interest (Flint and Dent lines) adapted to European regions.

Summary of Variant Calling workflow.

Variants were called using Illumina, CLR and HiFi reads (60, 53 and 28-fold coverage respectively). Reads were first mapped against the reference genome B73v4 and two software were used for calling small variants (SNPs) and large variants (SV). Finally, all VCF (Variant Calling Format) files were filtered for proper variant size (0-50bp for small variants and 50bp+ for SV), only entries with FILTER field equivalent to PASS and useful genotyping information. Illumina reads were mapped with BWA-MEM and variants called using DeepVariant 1.0.0 (WGS model) and Manta 1.6.0. PacBio CLR reads were mapped with PBMM2 1.3.0 and variants called using Longshot 0.4.1 and PBSV 2.4.0. PacBio HiFi reads were mapped with pbmm2 1.3.0 and variants called using DeepVariant 1.0.0 and PBSV 2.4.0. SV from the three sequencing technologies were merged using Jasmine. The correctness of our assembly is indicated by the high quality alignment to the maize reference genome. The first 10 scaffolds represent an almost complete assembly of the maize chromosomes and we obtained also a significant number of short scaffolds originated from highly repetitive regions that have not been assembled in the reference.

B

  DNA Extraction: E.Z.N.A Plant DNA kit; Quality control : Femto-Qubit-Nanodrop ; Shearing : Megaruptor 3 Library preparation (10µg); Library: SMRTbell Express Template Prep Kit 2.0 + Nuclease Treatment of SMRTbell Library (for HIFI only); Size Selection : BluePippin (5µg). De novo assembly More about the SeqOccIn program : https://get.genotoul.fr/seqoccin/ Three majors topics are treated in this project: genome variability analysis, epigenetic mark analysis and metagenome analysis. SeqOccIn projet is carried out by Get-PlaGe and Genotoul Bioinfo platforms and financed by FEDER funds (Programme Opérationnel FEDER-FSE_Midi-Pyrénées et Garonne 2014-2020). The project benefits from the contributions of INRAE research units GenPhySE, MIAT, GABI, GQE. 25 private partners are involved in the project, among them, the AMAIZING project partners (Biogemma, MaïsAdour, KWS, Caussade Semences, Limagrain, RAGT, Euralis, Syngenta).

Figure

  Figure 4 : Busco (v4.0.2) statistics of maize 1 assembly versus B73 reference sequence.

Figure 1 :

 1 Figure 1 : Fragment length distribution in average of SMRTbell Express libraries of maize sample. (Agilent Femto Pulse)

Figure 2 :

 2 Figure 2 : Variant calling comparison between sequencing technologies on Maize1.Overall, PacBio technologies discover far more structural variations than Illumina while the latter performs the best on small variants. A: Count of small variations for Illumina, PacBio CLR and PacBio HiFi. Around 1,300,000 Indels are detected by DeepVariant with both Illumina and HiFi reads. However, no Indel can be detected at the moment with our method for CLR reads. B: Count of large variations for Illumina, PacBio CLR and PacBio HiFi. PacBio reads found far more variants compared to Illumina within each type of variation. HiFi shows hiqh quality results on both small and large variants whereas Illumina excels only on small ones and CLR only on large ones.

  1-INRAE, US 1426, GeT-PlaGe, Genotoul, Castanet-Tolosan, France ; 2-Plateforme Bio-informatique Genotoul, Mathématiques et Informatique Appliquées de Toulouse, INRAE, Castanet-Tolosan, France ; 3-Université Paris-Saclay, INRAE, CNRS, AgroParisTech, GQE -Le Moulon, Gif-sur-Yvette, France. ; 4-Limagrain, Clermont-Ferrand, France ; 5-GenPhySE, Université de Toulouse, INRA, INPT, ENVT, Castanet-Tolosan Cedex, F-31326, France.


Xavier Carpentier

Dominique Bazin
email: dominique.bazin@u-psud.fr

Christelle Combes

Aurélie Mazouyes

Stephan Rouzière

Pierre Antoine Albouy

Eddy Foy

Michel Daudon

Pathobiochemistry High Zn content of Randall's plaque: A -X-ray fluorescence investigation

Keywords:

X-ray diffraction -X-ray fluorescence a b s t r a c t Kidney stone disease, or nephrolithiasis, is a common ailment. Among the different risk factors usually associated with nephrolithiasis are dehydration, metabolic defects (especially with regard to calcium and oxalate). The presence of a mineral deposit at the surface of the renal papilla (termed Randall's plaque) has all been recently underlined. Of note, Randall's plaque is made of the calcium phosphate, carbapatite, and serves as a nucleus for kidney stone formation. The process by which apatite nanocrystals nucleate and form Randall's plaque remains unclear. This paper deals with the possible relationship between trace elements and the formation of this mineral. The investigation has been performed on a set of Randall's plaques, extracted from human kidney stones, through -X-ray diffraction and -X-ray fluorescence analyses in order to determine the chemical composition of the plaque as well as the nature and the amount of trace elements. Our data provide evidence that Zn levels are dramatically increased in carbapatite of RP by comparison to carbapatite in kidney stones, suggesting that calcified deposits within the medullar interstitium are a pathological process involving a tissue reaction. Further studies, perhaps including the investigation of biomarkers for inflammation, are necessary for clarifying the role of Zn in Randall's plaque formation.

Introduction

One of the main challenges for urology in the 21st century is to understand the biochemical mechanisms associated to the formation of Randall's plaque (RP) [START_REF] Randall | An hypothesis for the origin of renal calculus[END_REF]. From an epidemiologic point of view, the proportion of kidney stones (KS) with RP markedly increased from 8.9% in the early eighties to 20.6% between 2000 and 2005 in France [START_REF] Daudon | Stone morphology suggestive of Randall's plaque[END_REF]. A parallel progression of the presence of RP at the tip of the papilla has been recently reported in the United States [START_REF] Low | Endoscopic mapping of renal papillae for Randall's plaques in patients with urinary stone disease[END_REF][START_REF] Matlaga | The role of Randall's plaques in the pathogenesis of calcium stones[END_REF]. Of note, Kim et al. [START_REF] Kim | Stone formation is proportional to papillary surface coverage by Randall's plaque[END_REF] reported that stone formation is proportional to papillary surface coverage by RP. Thus, RP can be at the origin of the significant increase of KS prevalence observed over the past two decades in industrialized countries [START_REF] Hesse | Study on the prevalence and incidence of urolithiasis in Germany comparing the years 1979 vs. 2000[END_REF][START_REF] Yoshida | National trend of the incidence of urolithiasis in Japan from 1965 to 1995[END_REF][START_REF] Trinchieri | Increase in the prevalence of symptomatic upper urinary tract stones during the last ten years[END_REF][START_REF] Stamatelou | Time trends in reported prevalence of kidney stones in the United States: 1976-1994[END_REF]. More precisely, in Europe as well as in the US, 6-18% of the population, depending on the geographical region, are affected by such disease.

Since the seminal work of Randall, numerous studies have been devoted to RP [START_REF] Delatte | New studies on papillary calculi[END_REF][START_REF] Evan | Randall's plaque of patients with nephrolithiasis begins in basement membrane of thin loops of Henle[END_REF][START_REF] Evan | Mechanism of formation of human calcium oxalate renal stones on Randall's plaque[END_REF]. In a recent review, we have underlined the chemical diversity of such biological samples [START_REF] Daudon | Stone morphology suggestive of Randall's plaque[END_REF]. If carbonated calcium phosphate apatite (CA) seems to be the major component of most RP, other mineral phases can be found, such as amorphous carbonated calcium phosphate (ACCP), and less frequently whitlockite or brushite. Other chemical species, namely sodium hydrogen urate and uric acid, are also observed. Such chemical diversity underlines the fact that RP have different origins [START_REF] Daudon | Randall's plaques[END_REF].

Among the possible hypotheses, some trace elements may act as a catalyst. In the literature, different papers underline the fact that some elements such as Mg [START_REF] Oka | Role of magnesium in the growth of calcium oxalate monohydrate crystals[END_REF], Zn [START_REF] Gupta | Role of zinc in nephrolithiasis[END_REF], Al [START_REF] Sutor | Growth studies of calcium oxalate in the presence of various ions and compounds[END_REF][START_REF] Grases | The influence of some metallic ions and their complexes on the kinetics of crystal growth of calcium oxalate[END_REF] or Fe 3+citrate complexes [START_REF] Meyer | Trace metal-citric acid complexes as inhibitors of calcification and crystal growth. II. Effects of Fe(III), Cr(III) and Al(III) complexes on calcium oxalate crystal growth[END_REF] promote or inhibit nucleation and/or crystal growth of mineral or organic species involved in urinary calculi. Also different experimental investigations [START_REF] Pindea-Vargas | Characterization of human kidney stones using micro-PIXE and RBS: a comparative study between two different populations[END_REF][START_REF] Chaudhri | Spatial distribution of major elements and trace elements in bladder and kidney stone[END_REF][START_REF] Rodriguez-Minon Cifuentes | Usual elements in kidney stones[END_REF] have been dedicated to the analysis of the spatial partition of trace elements inside KS by various techniques. In previous studies focused on kidney stones [START_REF] Bazin | Heavy elements in urinary stones[END_REF][START_REF] Bazin | Very first tests on SOLEIL regarding the Zn environment in pathological calcifications made of apatite determined by X-ray absorption spectroscopy[END_REF][24] we have suggested that the presence of trace elements in most cases is not a marker of an active contribution to stone formation but more probably is a consequence of a passive adsorption and/or absorption of these heavy elements respectively on and/or into crystals due to similarity of their charge and radius with calcium. Such a conclusion is in line with the fact that the medical classification [START_REF] Daudon | Urinary calculi: review of classification methods and correlations with etiology[END_REF][START_REF] Daudon | Peculiar morphology of stones in primary hyperoxaluria[END_REF] based on a relationship between the pathology and some structural characteristics of KS ignores the doi:10.1016/j.jtemb.2011.05.004 nature and the amount of trace elements present inside KS. To date, however, only a few experiments have directly addressed the question of whether trace elements may play a role in the formation of RP [START_REF] Bazin | Les techniques de rayonnement synchrotron au service de la caractérisation d'objets biologiques: un exemple d'application, les calculs rénaux[END_REF].

The aim of this paper is to establish whether or not there is a correlation between trace elements and RP. To do so, X-ray diffraction experiments to determine the composition of the RP as well as X-ray fluorescence measurements to identify and evaluate the amount of the trace elements have been performed. Special attention will be paid to a major oligoelement found in stones, namely Zn.

Materials and methods

Samples

RP were extracted using a stereomicroscope from seven human KS. Table 1 summarizes the chemical composition of the kidney stones as determined by Fourier Transform InfraRed (FTIR) spectroscopy. Fig. 1 shows a photo of a Randall's plaque attached to a calculus made of whewellite. All the RP were found to be composed of CA by FTIR.

The KS sample N17105, which is mainly composed of CA, which also is the primary chemical form in RP, was chosen as a reference sample in the present study.

Methods

An FTIR spectrometer Vector 22 (Bruker Spectrospin, Wissembourg, France) was used according to the analytical procedure previously described [START_REF] Estepa | Contribution of F.T. infrared spectroscopy to the identification of urinary stones and kidney crystal deposits[END_REF]. Data were collected in the absorption mode between 4000 and 400 cm -1 with a resolution of 4 cm -1 .

To obtain X-ray diffraction data on very small samples (such as RP), a special device was used. Grazing incidence small-angle X-ray scattering analysis was performed at the Laboratoire de Physique des Solides (Université Paris-Sud, France) with a homebuilt diffractometer using copper K␣ radiation and a microfocus device. Description of the apparatus has been previously reported by Chougnet et al. [START_REF] Chougnet | Substrates do influence the ordering of mesoporous thin films[END_REF]. Diffraction patterns were recorded on photo-sensitive image plates. After a 2D integration, classical /2diffracted intensity representations were obtained and compared to the diffraction data collected for the CA reference kidney stone.

X-ray fluorescence experiments were performed on a RU-200B rotating anode X-ray generator at the Laboratoire Pierre Süe at the Centre d'Etudes Atomiques (LPS-CEA, Saclay, France). Runs were made at 25 mA and 45 kV molybdenum K ␣ radiation with a focal spot size of 30 × 30 m 2 and a dwell time of several hundreds of seconds with a germanium detector. In order to obtain an evaluation of the quantity of Zn in the samples, X-ray fluorescence spectra of a set of reference compounds made with a physical mixture of ZnO and hydroxyapatite were collected.

We use a program for X-fluorescence analysis developed at the European Synchrotron Radiation Facility (ESRF), named PyMCA [START_REF] Solé | A multiplatform code for the analysis of energy-dispersive X-ray fluorescence spectra[END_REF]. First, on the NIST610 material reference and using Ca as internal reference, we found a relative error on the determination of Zn concentration about 8.7% between the Nist value and the calculated one. And then, knowing the Ca concentration deduced from C1, C2 and CA contents (Table 1), we fit the different spectra using the same program and thus we obtain the new set of data for Zn concentration in the samples.

Results and discussion

The Zn content of the different RP made of apatite and extracted from KS made of calcium oxalate is gathered in Table 2. As we can see, the Zn average level (in g/g) measured in the different RP is 5665 ± 490 g/g while the Zn level in CA stones was 1059 ± 934 g/g (p < 0.0001) [START_REF] Bazin | Heavy elements in urinary stones[END_REF]. No correlation was found between the content of trace elements in RP and the chemical composition of the stone as given by FTIR. In spite of the high variability of the Zn level in the biological specimens analysed, the amount of Zn in RP appears dramatically increased by comparison with the reference CA KS. From a medical point of view, pathological calcification refers to two very different entities. One is a concretion defined as a deposition of crystalline material in excretory ducts (KS but also gallstones, salivary stones, etc.) and the other one is referred to as ectopic calcification [START_REF] Giachelli | Ectopic calcification, gathering hard facts about soft tissue mineralization[END_REF], defined as an inappropriate biomineralisation occurring in soft tissue (which can be related to severe pathologies like mammary or testicular cancer). Interestingly, KS belong to the first family while RP to the second one.

Fig. 2 shows that the different X-ray diffraction patterns obtained for RP are clearly similar to the one collected on a KS made of CA which is used as a reference sample in this study (sample N17105). The selected RP samples are made mostly of nanocrystalline apatite exhibiting the usual anisotropy along the c axis. These typical structural characteristics are associated in the X-ray diagram with a relatively fine (0 0 2) diffraction peak (at 2 = 26 • ) and several poorly resolved lines constituting a broad peak between 2 = 30 • and 2 = 35 • . These structural characteristics are shared by numerous biological apatites [START_REF] Vallet-Regı | Calcium phosphates as substitution of bone tissues[END_REF][START_REF] Drouet | Nanocrystalline apatites: from powders to biomaterials[END_REF] associated not only with pathological calcifications but also with normal ones (bone, dentine, etc.).

Of note, significant structural differences may exist among these biological apatites regarding the amount of trace elements. X-ray fluorescence [START_REF] Farquharson | The distribution of trace elements Ca, Fe, Cu and Zn and the determination of copper oxidation state in breast tumour tissue using SRXRF and XANES[END_REF] is a well established and powerful tool for elemental analysis of biomaterials. One important reason for this success is the fact that such experiments are non-destructive. This technique has been successfully used in several investigations of concretions such salivary [START_REF] Zelentsov | The elemental SRXRF analysis and mineral composition of human salivary stones[END_REF] and gallbladder [START_REF] Ekinci | Determination of calcium and iodine in gall bladder stone using energy dispersive X-ray fluorescence spectrometry[END_REF] stones.

In Fig. 3 are shown the X-ray fluorescence emission spectra of the different biological samples. The contribution of Ca 2+ as well as trace elements such as Zn 2+ , Pb 2+ and Sr 2+ are clearly identified by the presence of two main peaks characteristic of each of these elements. Considering our previous study dedicated to KS analysis [START_REF] Bazin | Heavy elements in urinary stones[END_REF] and the experimental conditions (i.e. only photons with energy higher than 3 KeV were detectable), we classified the different elements found in the kidney stone specimen as follows: first, the major element involved in the crystalline mineral phases, i.e. Ca, then trace elements such as Sr (Sr is in the same column of the periodic table as Ca), a group of transition elements including Fe, Cu, and also Zn, and finally pathological elements such as Pb. Most of the Fe present in KS probably results from residual blood at the surface or within layers of the stones (see Fig. 4).

Regarding Sr 2+ , this element follows the same metabolic paths as Ca 2+ . This property has been used to study calcium metabolism in idiopathic hypercalciuria by a strontium oral load test [START_REF] Vezzoli | Study of calcium metabolism in idiopathic hypercalciuria by strontium oral load test[END_REF]. Regarding their location in the body, most of the strontium cations are present in bone. Interestingly, the Sr content of RP is significantly lower than that observed in CA KS (21.8 ± 1.1 g/g vs. 455 ± 364 g/g, p < 0.0001) [START_REF] Bazin | Heavy elements in urinary stones[END_REF]. In the present study, we focused on the analysis of the most abundant trace element, i.e. Zn. It is well known that Zn 2+ is an essential cofactor for the activity of more than 300 enzymes. This element is also involved in other bio- [START_REF] Komaromy-Hiller | Comparison of representative ranges based on U.S. patient population and literature reference intervals for urinary trace elements[END_REF], the zinc concentration is around 15-30 g/g in kidney tissue [START_REF] Caroli | Occupational exposure of goldsmith workers of the area of Rome to potentially toxic metals as monitored through hair analysis[END_REF]. The biological and physical-chemical maturation of biological apatites strongly affects the uptake of trace elements. Forming crystals can easily incorporate trace elements and freshly formed apatites (immature) are less stable and more reactive than older mature mineral deposits [START_REF] Vezzoli | Study of calcium metabolism in idiopathic hypercalciuria by strontium oral load test[END_REF]. In addition, the mineral density in freshly formed areas is also lower than that of the older ones, thus favouring ionic diffusion. Most elements will preferably react with newly formed biological apatites and if these elements remain within the apatite they will be distributed throughout the entire calcification because mineralisation will progress gradually. However, the ability for an element to enter the apatite structure is not a good enough reason to verify its location in the mineral part of biological samples. Trace elements may also be associated with organic matrix, blood or other biological fluids, and cell constituents. Except for a very few elements like fluoride which are known to modify the mineral characteristics, it seems very difficult to determine if a particular trace element belongs to the mineral or to the organic part as most of them do not have any detectable effect on mineral structure and/or composition. Deproteinisation or demineralisation treatments of specimen are not fully reliable techniques to demonstrate the presence of trace elements in mineral or organic parts as some artefacts are possible due to the alteration of mineral or organic component reactivity, composition or structure during these treatments.

From a chemical point of view, biological apatites have been shown to easily incorporate various metals. This may be explained by their nanometer size as well as the similarity between the ion charge and size of Zn 2+ , Sr 2+ and Ca 2+ . Of note, different studies [START_REF] Bigi | Rietveld structure refinements of calcium hydroxyapatite containing magnesium[END_REF][START_REF] Rokita | Substitution of calcium by Sr within selected calcium phosphates[END_REF][START_REF] Sugiyama | Characterization of Ca, Sr, Ba and Pb HAP: X-ray diffraction, photoelectron. Exafs and MAS NMR spectroscopies[END_REF] have shown that the incorporated cations (Mn 2+ , Sr 2+ , Pb 2+ for example) substitute for calcium and have a tendency to occupy preferentially the Ca(II) sites in the apatite structure (vs. the Ca(I) sites).

As noticed by Li et al. [START_REF] Li | Structural characterization of zincsubstituted hydroxyapatite prepared by hydrothermal method[END_REF], the substitution of the Zn 2+ for Ca 2+ cations causes a lower crystallinity of hydroxyapatite. Moreover, a dose-dependence effect has been pointed out by Fujii et al. [START_REF] Fujii | Selective protein adsorption property and characterization of nano-crystalline zinccontaining hydroxyapatite[END_REF] and Ren et al. [START_REF] Ren | Characterization and structural analysis of zincsubstituted hydroxyapatites[END_REF]; the crystallites were smaller and of a more irregular shape as Zn level increased. In addition, chemical analysis of the cations and anions showed that Zn-containing apatites are cation deficient (Ca + Zn)/P atomic ratio < 1.67). Mayer et al. synthesised Zn-substituted carbonated hydroxyapatites and Cuisinier et al. [START_REF] Cuisinier | Microscopie électronique de haute résolution de carbonato-apatites synthétiques enrichies en zinc et en gallium[END_REF] pointed out that Zn 2+ may influence the morphology, the size and the structure (defects) of the crystals even if it is present at low levels in carbonate apatite [START_REF] Mayer | Zinc ions in synthetic carbonated hydroxyapatites[END_REF][START_REF] Ren | Characterization and structural analysis of zincsubstituted hydroxyapatites[END_REF]. Even if the mode of incorporation of Zn in apatite is not clearly understood, type II calcium sites in apatite have been shown to be the most favourable sites for Zn entering the apatite structure. Zn occurs in tetrahedral coor-dination and its incorporation is expected to lead to a decrease of a-lattice parameter and increase of the c-lattice parameter of the apatite [START_REF] Chapell | Zinc substituted hydroxyapatite -a comparison of modelling and experimental data[END_REF][START_REF] Tang | Zinc incorporation into hydroxylapatite[END_REF].

Wu et al. [START_REF] Wu | Differential effects of zinc and magnesium ions on mineralization activity of phosphatidylserine calcium phosphate complexes[END_REF] showed that Zn 2+ ions are potent inhibitors of mineral formation in vitro. This result can be correlated to the in vitro study of Legeros et al. [51] regarding the precipitation of various calcium phosphates of biological interest (amorphous calcium phosphate (ACP), brushite, octacalcium phosphate (OCP), carbonated apatite) from solutions including various concentrations of Zn. They pointed out the dose-dependent effect of Zn 2+ ions on the type and amount of calcium phosphate phases precipitated: a promotion of ACP or Zn-substituted ␤-tricalcium phosphate (␤-TCP) formation was observed for the higher Zn 2+ ions level tested (0.5-2 mM/L) whereas brushite, OCP and apatite formation was inhibited in the presence of a level of Zn 2+ ions in solution as low as 0.1 mM/L. Legeros et al. [START_REF] Legeros | Amorphous calcium phosphates (ACP): formation and stability[END_REF] have also shown that Zn ions are among the ions (carbonate, magnesium and pyrophosphate ions) that most notably inhibit the crystallisation of apatite and brushite and that the combination of some of these ions can have a synergistic effect [START_REF] Wu | Differential effects of zinc and magnesium ions on mineralization activity of phosphatidylserine calcium phosphate complexes[END_REF][START_REF] Legeros | Zinc effect on the in vitro formation of calcium phosphates: relevance to clinical inhibition of calculus formation[END_REF].

Zn 2+ ions can also have an effect on the interaction between the mineral and organic phases. Fujii et al. [START_REF] Li | Structural characterization of zincsubstituted hydroxyapatite prepared by hydrothermal method[END_REF] showed that due to favourable factors such as specific surface area, surface charge and pore size distribution of Zn-containing nanocrystalline apatites, the latter can selectively control the adsorption of ␤2-MG protein, a pathological protein in blood plasma.

To compare the Zn level in RP and in kidney stone made of carbonate apatite (Table 3), reference compounds made of a physical mixing of ZnO and synthetic industrial apatite have been prepared. Their fluorescence emission was collected in the same conditions than those used for the RP specimen analysis in order to determine the Zn amount in the selected samples. The observation of a dramatically increased Zn content in RP by comparison with the reference KS (Table 3) is highly suggestive of a relationship with an inflammatory process. Of note, numerous works have highlighted the link between zinc and inflammation [START_REF] Bao | Zinc supplementation decreases oxidative stress, incidence of infection, and generation of inflammatory cytokines in sickle cell disease patients[END_REF][START_REF] Besecker | The human zinc transporter SLC39A8 (Zip8) is critical in zinc-mediated cytoprotec-tion in lung epithelia[END_REF][START_REF] Haase | Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes[END_REF].

Similarly to carbonated apatite-based kidney stones, RP exhibit higher levels of these three elements than reported in urine and kidney tissue. As observed in Table 3, the content of heavy elements is about 30-1000 times higher in RP than in urine or in kidney tissue. As a consequence, it cannot be asserted that trace elements may play a significant role in the RP formation. As previously reported [START_REF] Grases | The influence of some metallic ions and their complexes on the kinetics of crystal growth of calcium oxalate[END_REF], the spatial distribution of trace elements inside kidney stones was homogeneous. Thus the hypothesis of a passive capture of trace elements by kidney stones was finally more appropriate than a catalytic effect. At this step of discussion, our results from RP lead to a similar explanation regarding the presence of trace elements. However, we cannot confirm by our analysis that trace elements are homogeneously distributed within RP as observed in KS.

A possible way to answer this question should be based on the mapping of trace elements performed on RP obtained from kidney papilla by urological procedures. Already, we have performed an ex vivo structural investigation in order to precise the chemical nature of the RP positioned on the kidney [START_REF] Carpentier | The pathogenesis of Randall's plaque: a papilla cartography of Ca compounds through an ex vivo investigation based on a synchrotron radiation related technique[END_REF].

One obvious limitation of our study is the small number of specimens investigated. Also, further studies, including histological ones and the use of biomarkers of inflammation, are needed to better clarify the role of Zn in RP formation and to understand the mechanisms involved in the genesis of RP.

Conclusion

X-ray fluorescence was used to investigate the possible role of trace elements in the genesis of RP. The nature and the content of the trace elements were obtained through X-ray fluorescence. This set of data thus opens up an interesting possibility for research in urology using X-ray-based analysis techniques related either to laboratory set up or synchrotron radiation facilities. The complete set of data shows that the levels of Zn are significantly increased in carbapatite of RP by comparison to carbapatite in kidney stones. Our results suggest that calcified deposits within the medullar interstitium, despite their high occurrence, are a pathological process.

Fig. 1 .

 1 Fig. 1. Typical Randall's plaque (white area) attached on a calculus made of whewellite (C1).

Fig. 2 .

 2 Fig. 2. Typical X-ray fluorescence spectra collected for a Randall's plaque (counting time 7200s).

Fig. 3 .

 3 Fig. 3. X-ray diffraction diagrams collected for different RP and a KS made of apatite used as a biological apatite reference sample in this study.

Fig. 4 .

 4 Fig. 4. Typical X-ray fluorescence spectra collected for RP and calibration compound NIST610. For RP, we can clearly see the contribution of Ca (EK␣ = 3691 eV, EK␤ = 4012 eV), Zn (EK␣ = 8638 eV, EK␤ = 9572 eV), Pb (EL␣ = 10,551 eV, EL␤ = 12,613 eV) and Sr (EK␣ = 14,165 eV, EK␤ = 15,835 eV). *Peaks corresponding to absorption by the germanium detector of the Compton and Mo Ka elastic peak.

Table 2

 2 Zn and Sr levels in the selected biological samples. Average level of Zn and Sr measured in the reference sample (i.e. KS mainly composed of carbonated apatite).

	Samples	Zn level (g/g)	Sr level (g/g)
	CAinKS a	1539 ± 056	349 ± 181
	RPonN4511	5056 ± 438	24 ± 2
	RP on N7283	4677 ± 405	28.9 ± 0.3
	RPonN10986	9948 ± 862	17 ± 2
	RP on N20872	7939 ± 688	8 ± 1
	RP on N26345	5559 ± 482	9 ± 1
	RP on N29244	4572 ± 396	19.9 ± 0.2
	RPonN42554	1903 ± 165	46 ± 1

a

Table 1

 1 Morphological type and composition of the seven human kidney stones selected for the study.

	Samples	Morphological type	Stone composition
	Reference compound N17105	IVa	89% CA, 6% C2, 3% C1, 2% Prot
	N4511	IIb, Ia, IVa	55% C1, 39% C2, 6% CA
	N7283	Ia, IIb	80% C1, 12% C2, 4% CA, 2% ACCP, 2% Prot
	N10986	Ia, IIb	86% C1, 11% C2, 2% CA, 1% Prot
	N20872	Ia, IIb, IVa	55% C1, 40% C2, 3%CA, 2% Prot
	N26345	Ia, IIb	55% C1, 42% C2, 2% CA, 1% Prot
	N29244	Ia, IIb	84% C1, 11% C2, 3% CA, 2% Prot
	N42554	Ia, IIb	60% C1, 35% C2, 3% CA, 2% Prot

CA, carbonated calcium hydroxylapatite; Prot, protein; C1, whewellite; C2, weddellite; ACCP, amorphous carbonated calcium phosphate.

Table 3

 3 Amount of Zn, Sr and Pb oligoelements in kidney stones, kidney tissue and urine. functions such as nucleic acid metabolism, maintenance of membrane structure and function, hormonal activity, as well as biomineralisation. The body of adult normally contains approximately 2.2 g of Zn2+ . More precisely, Zn 2+ is present in all the tissues and fluid of the body. As shown in Table3

		Zn (g/g)	Sr (g/g)	Pb (g/g)
	CAKS a	1059 ± 1056	349 ± 181	62 ± 39
	RP	5665 ± 490 *	22 ± 13 *	NA
	Kidney tissue b	15-32	0.04-0.12	0.1-0.4
	Urine b	0.002-0.1	0.2	0.012-0.030
	Urine c	0.3 ± 0.2	0.14 ± 0.076	0.0013 ± 0.014
	NA = not available.

a

[START_REF] Bazin | Heavy elements in urinary stones[END_REF]

. b

[START_REF] Kim | Stone formation is proportional to papillary surface coverage by Randall's plaque[END_REF]

(values are ranges observed in large populations). c

[START_REF] Vezzoli | Study of calcium metabolism in idiopathic hypercalciuria by strontium oral load test[END_REF]

. * p < 0.0001 vs. CAKS. logical

Acknowledgements

This work was supported by the physic and chemistry institutes of CNRS and by a ANR-09-BLAN-0120-02 contract.

Disclosure

All authors declared no competing of interests.