Déborah Le Pennec

Melissa Baravalle-Einaudi

Christian Dupont

Laurent Vecellio

Jean-Christophe Dubus

Cystic fibrosis in the modern therapeutic era: give the shower a think!

Cystic fibrosis (CF) is a genetic disease whose prognosis has been greatly modified, with a considerable increase in life expectancy over the last 50 years [START_REF] Elborn | Report of the European Respiratory Society/European Cystic Fibrosis Society task force on the care of adults with cystic fibrosis[END_REF]. Several factors may explain this such as follow-up of patients in CF centers applying international recommendations for the best CF care, organ transplantation which is nowadays considered as a routine procedure in the CF therapeutic scheme, newborn screening allowing early management and preventing the delay in diagnosis that has affected many families in areas lacking such screening, and availability of CFTR modulators treatments which restore, at least partially, the CFTR protein activity in some specific genetic indications, other modulators being waited for soon [START_REF] Castellani | ECFS best practice guidelines: the 2018 revision[END_REF][START_REF] West | Unmet needs in cystic fibrosis: the next steps in improving outcomes[END_REF] ... At the same time, CF patients continue to receive numerous daily treatments such as chest physiotherapy, pancreatic extracts, nebulizations, and others. Great progress has also been made in alleviating everyday life through reducing the mean 2 hours spent per day for therapy [START_REF] Sawicki | High treatment burden in adults with cystic fibrosis: challenges to disease self-management[END_REF]. For instance, concerning inhaled therapies, which represent 25-50% of total home time of care [START_REF] Baravalle-Einaudi | Vibrating-mesh nebulizer maintenance by CF patients: results from a French survey[END_REF], the time-consuming nebulization may be replaced by a dry powder inhalation for some antibiotics or may be delivered by more sophisticated devices, such as vibrating-mesh nebulizers, with shorter nebulization duration. Of course the daily use of such electronic nebulizers needs careful maintenance as therapeutic efficiency is closely linked to the mesh performance, which may be altered by mesh plugging over time [START_REF] Vecellio | The mesh nebulizer: a recent technical innovation for aerosol delivery[END_REF]. Unfortunately, in a recent study performed in 92 CF patients equipped with an e-Flow rapid® nebulizer (58.7% children; mean 1.8 nebulizations per day) [START_REF] Baravalle-Einaudi | Vibrating-mesh nebulizer maintenance by CF patients: results from a French survey[END_REF], we have shown that mesh maintenance was not adequate, with only 39% of the patients using the specific cleaning system of the mesh, the so-called MeshCare®, which consists of a reverse rinse of the mesh by means of a small shower, thus removing the plugs formed by drug deposition into the mesh holes.

We asked these patients to note daily, for 6 months, their number of nebulizations, the kind of nebulized drug, the application of the maintenance procedure, the occurrence and type of nebulizer incidents. At 6 months, their e-Flow rapid® meshes were collected for in vitro characterization of the delivery of 300 mg tobramycin (Tobi®, Novartis), before and after MeshCare® use (5 ml isotonic saline during 20 min). As only 52 patients completed the 6 months data collection, only 52 meshes were tested with a laser diffractometer (Spraytec®, 15 Lpm aspiration; Malvern, UK). They were characterized, as well as 3 brand new meshes, for residual volume, nebulized volume, nebulization duration (until the bip of end or the aerosol production end), aerosol output, MMAD, and particles < 5 µm. A mesh was considered as "out of order" when producing no aerosol and as "defective" when the variation of the nebulized volume was ≥ 15% in comparison with that obtained with a new mesh [START_REF]pre-authorisation evaluation of medicines for human use. Guideline on the requirements for clinical documentation for orally inhaled products (OIP) including the requirements for demonstration of therapeutic equivalence between two inhaled products for use in the treatment of asthma and chronic obstructive pulmonary disease (COPD) in adults and for use in the treatment of asthma in children and adolescents[END_REF] (Table 1). Three meshes were out of order (5.7%). Mesh properties were clearly altered compared to those of new meshes, with a high variability for all results. Thirty meshes among 49 (61.2%) were defective, 20 generating a too low nebulized volume. No relation was found with the drug used during 6 months (mucoregulators 52%, antibiotics 42%, both 6%), the number of daily nebulizations, and the frequency and kind of nebulizer maintenance. The MeshCare® permitted to statistically improve nearly all tobramycin nebulization parameters, 14 defective meshes (46.6% of the defective meshes) being anew in the expected range of nebulized volume.

Our results consolidate those of a previous work [START_REF] Rottier | Changes in performance of the pari eflow rapid and PariLC plus during 6 months use of CF patients[END_REF] where in vitro evaluation of 15 e-Flow®rapid nebulizers used by CF patients for 3 inhaled tobramycin cycles has showed an increase of the mean nebulization duration from 6.7 to 9.8 min in 6 months, a 36% decrease of the aerosol output, with a total of 8 nebulizers switching off prematurely. After replacing a polluted mesh by a new one, parameters are restored to original values. In our study, after 6 months of daily home use, about 7 meshes out of 10 do not deliver a "clinically validated" amount of tobramycin and may compromise the therapeutic efficiency. Contrary to common ideas [START_REF] Baravalle-Einaudi | Vibrating-mesh nebulizer maintenance by CF patients: results from a French survey[END_REF][START_REF] Rottier | Changes in performance of the pari eflow rapid and PariLC plus during 6 months use of CF patients[END_REF][START_REF] Reychler | Disinfection of devices for nebulization: stakes, difficulties, and improvement proposals[END_REF][START_REF] Reychler | Inhalation equipment hygiene: a Belgian national survey[END_REF], no specific factor correlated with mesh dysfunction, and the mere use of the device alters the mesh performance over time. So, in the absence of key factors predictive of mesh dysfunction and in agreement with the manufacturer's recommendation, we advise a monthly control at home of the nebulization duration with 2.5 ml isotonic saline, and employing the MeshCare® when this duration is increased. We clearly prove for the first time the benefits of this recommendation, allowing the best drug delivery to CF patients. In summary, a mere shower, even in the CF modern therapeutic era, may be sometimes of great help...

Table 1 :

 1 In vitro characteristics of a tobramycin nebulization performed with 3 new meshes and 52 meshes of e-Flow rapid® used for 6 months by cystic fibrosis patients at baseline and after the use of a cleaning system back rinsing the mesh (MeshCare®). A mesh was considered as defective when the variation of the nebulized volume was ≥ 15% in comparison with that obtained with a new mesh

		New meshes	Six months used meshes	Meshes after back rinsing	Mann-Whitney test
		(n=3)	(n=49/52; 3 out of order)	(n=49/52; 3 out of order)	comparing used to back
					rinsed meshes
	Residual volume (ml)	1.5 ± 0.2 [1.3-1.7]	2.4 ± 0.9 [1.1-4.7]	1.9 ± 0.8 [0.9-4.1]	p=0.0011
	Nebulized volume (ml)	3.8 ± 0.3 [3.5-4.6]	2.8 ± 0.9 [0.6-4.2]	3.3 ± 0.8 [1.1-4.2]	p=0.0017
	Nebulization duration (min)	8.1 ± 0.7 [7.33-8.66]	9.5 ± 1.1 [5.5-10.0]	8.9 ± 1.5 [5.16-10.0]	p=0.02
	Aerosol output (ml/min)	0.47 ± 0.01 [0.46-0.48)	0.31 ± 0.14 [0.06-0.71]	0.39 ± 0.15 [0.11-0.74]	p=0.0018
	MMAD (µm)	4.1 ± 0.1 [4.0-4.2]	3.6 ± 0.3 [2.7-4]	3.8 ± 0.3 [2.9-4.5]	p=0.0007
	Particles < 5 µm (%)	66 ± 3 [64-69]	76 ± 6 [68-93]	75 ± 7 [60-92]	NS
	Defective meshes* (n)	0	30 (61.2%)	16 (32.6%)	-

*


Théo Tacail
email: theo.tacail@bristol.ac.uk

Sandrine Le Houedec

Joseph L Skulan

New frontiers in calcium stable isotope geochemistry

Keywords: 33 Calcium isotopes, 34 Vertebrate biology, 35 Palaeobiology, 36 Bioarchaeology, 37 Biomedical research, 38 Bone

Beyond their established uses in Earth and Planetary sciences, calcium (Ca) isotopes 19 have a promising future in the study of the biology of present and past vertebrates, including 20 humans. Early work paved the way to the ongoing research on the potential of Ca isotopes 21 as relevant tools to disciplines other than geology, including palaeobiology, bioarchaeology 22 and biomedical research. In this article, we first review the rationale behind the cycling of Ca 23 isotopes in vertebrate organisms. We then summarize and discuss the use of Ca isotopes as 24 dietary tracers from trophic reconstructions in past vertebrate ecosystems to the tracking of 25 early life dietary transitions. Next, we review and examine the research outcomes on the 26 potential of Ca isotopes as biomarkers of bone loss in physiological and pathological 27 conditions such as bone cancers and osteoporosis. While emphasizing the needs of future 28 research in each of these applications, we suggest new potential uses of Ca isotopes in 29 vertebrate biology. Finally, we identify challenges and barriers faced when developing such 30 interdisciplinary projects and suggest how these can be overcome.

Introduction 80

Stable isotope geochemistry has a rich history of fruitful interdisciplinary collaboration, 81 resulting in the adoption of isotope geochemical techniques and concepts in fields such as 82 ecology, forensics and archaeology. Calcium (Ca) stable isotopes have been found useful in 83 the earth and planetary sciences, for example in reconstructing secular variations in 84 seawater composition (e.g., De La Rocha and DePaolo, 2000;Fantle andDePaolo, 2005, 85 2007;Farkaš et al., 2007aFarkaš et al., , 2007b;;[START_REF] Hinojosa | Evidence for end-Permian ocean acidification from calcium isotopes in biogenic 938 apatite[END_REF][START_REF] Jost | Constraining the cause of the end-Guadalupian extinction with coupled records of 970 carbon and calcium isotopes[END_REF]Le Houedec et al., 86 2017;A. D. Schmitt et al., 2003). However, promising potential applications of calcium 87 isotopes also lie outside of geology. Calcium isotopes have the potential to become an 88 important tool in vertebrate biology and biomedicine, notably if challenges to their 89 widespread application can be overcome.

4

The abundance and widely shared vital functions of Ca in vertebrate organisms 91 makes it a crucial stable isotope system in the evolution and biology of vertebrates, including 92 humans. The appearance in Cambrian marine vertebrates of calcium phosphate mineral 93 tissues, i.e. bioapatite, marks the development of revolutionary strategies in the physiological 94 regulation of Ca and inorganic phosphate, that are now widely shared among vertebrates 95 (e.g., [START_REF] Bouillon | Vitamin D: calcium and bone homeostasis during evolution[END_REF][START_REF] Doherty | Evolutionary Physiology of Bone: 867 Bone Metabolism in Changing Environments[END_REF]. Calcium phosphate minerals have a 96 high preservation potential and are globally more resistant to diagenesis than carbonates. 97

This has resulted in a rich vertebrate fossil record (e.g., [START_REF] Armstrong | Thermal effects on rare earth element 786 and strontium isotope chemistry in single conodont elements[END_REF]Barham et 98 al., 2012;[START_REF] Hinojosa | Evidence for end-Permian ocean acidification from calcium isotopes in biogenic 938 apatite[END_REF][START_REF] Joachimski | Oxygen isotope 966 evolution of biogenic calcite and apatite during the Middle and Late Devonian[END_REF][START_REF] Kohn | Stable Isotope Compositions of Biological Apatite[END_REF]Luz et al., 99 1984;[START_REF] Pucéat | Can crystallinity be used to determine the degree 1057 of chemical alteration of biogenic apatites?[END_REF][START_REF] Wenzel | Comparing oxygen isotope records of 1137 Silurian calcite and phosphate-δ 18 O compositions of brachiopods and conodonts[END_REF]. 100

The pioneering work on Ca isotopes in biology was done by J.L. Skulan in the late 101 1990s [START_REF] Skulan | Biological control of calcium isotopic 1108 abundances in the global calcium cycle[END_REF][START_REF] Skulan | Calcium isotope fractionation between soft and mineralized 1105 tissues as a monitor of calcium use in vertebrates[END_REF][START_REF] Skulan | Calcium isotopes and the evolution of terrestrial reproduction in 1111 vertebrates[END_REF]. Their key finding was 102 that mineralized tissues, including bone, are significantly and constantly depleted in heavy 103 isotopes of Ca when compared to Ca dietary sources. Following this early work, an 104 increasing number of investigators have explored biological and palaeobiological applications 105 of Ca stable isotopes. These explorations have proceeded in two main directions. The first 106 aims at using Ca isotopes to reconstruct the dietary habits of present and extinct vertebrates 107 in order to assess the trophic structures of past ecosystems, or the dietary behaviours of past 108 human individuals and populations. This field of research (that could be referred to as Metal 109

Stable Isotope Palaeobiology or Bioarchaeology) tackles questions relevant to 110 palaeontology, (palaeo-)ecology, archaeology and anthropology. The second topic of 111 ongoing research aims at developing the use of Ca isotopes to better understand and 112 diagnose human metabolic bone disorders, such as bone loss induced by inactivity, 113 microgravity or disease, including osteoporosis and cancers affecting bone. This field of 114 research (that could be referred to as Stable Isotope Metallomics as proposed by Albarède 115 (2015)) is at the crossroads between isotope geochemistry, fundamental physiopathology 116 and applied biomedical research. 117 118 However, beyond the challenges inherent in extending fundamental understanding 119 and developing practical applications, these projects also face difficulties that are common to 120 all interdisciplinary research. More than 15 years have passed by since the first recognition 121 of the potential of Ca isotopes in vertebrate biology, and yet only about 30 articles have been 122

published on the topic during this period (Figure 1). Calcium isotopes remain poorly utilized 123 in scientific communities outside earth and planetary sciences. This raises the following 124 question: what obstacles stand in the way of more widespread applications of Ca stable 125 isotope analysis? 126

In this article, we first briefly summarize the current understanding of the Ca isotope 127 cycling in vertebrates before reviewing and discussing the potential and future of Ca isotopes 128 in palaeobiology and biomedical research. Finally, we identify challenges and barriers related 129 to the collaborative research necessary for the adoption of Ca isotope tools by the wider 130 scientific community. 131 132

The cycling of calcium and its isotopes in vertebrates 133

The biological processing of Ca by vertebrates induces mass dependent isotope 134 fractionations responsible for maintained and observable heterogeneous distributions of Ca 135 isotopes amongst the different compartments of the organisms. Hence, the distribution and 136 biological functions of Ca in vertebrates are central features of the potential of Ca stable 137 isotopes as tools to study their biology. 138 139

Distribution, functions and regulation of Ca in vertebrates 140

In vertebrate organisms, the distribution of Ca 2+ is highly compartmentalized at all 141 anatomical levels (e.g., [START_REF] Valle | Dietary reference intakes for 861 calcium and vitamin D[END_REF][START_REF] Doherty | Evolutionary Physiology of Bone: 867 Bone Metabolism in Changing Environments[END_REF]Peterson and Riggs, 142 2010). Briefly, most body Ca is stored in bone mineral as a major constituent of bioapatite 143 (Ca 10 (PO 4 ) 6 (OH) 2 ). Mineralized tissues account for ca. 99 % of body Ca in vertebrates, while 144 the remaining fraction is found in non-mineralized or so-called soft tissues. The majority of 145 soft tissue Ca (around 90 %) is found inside cells (mainly in organelles), while extracellular 6 fluids including blood contain about 10 % of the soft tissue Ca. However, the free Ca 2+ of 147 extracellular fluids is 10,000 to 20,000 times more concentrated than intracellular cytosolic 148 Ca 2+ [START_REF] Case | Evolution 831 of calcium homeostasis: From birth of the first cell to an omnipresent signalling system[END_REF]. This constitutes a steep and actively maintained chemical gradient 149 between extracellular and intracellular milieus. 150

The heterogeneous and tightly regulated distribution of Ca reflects the adaptation to 151 the vital biological functions to which it contributes (see [START_REF] Berridge | The versatility and universality of calcium 810 signalling[END_REF][START_REF] Bootman | Calcium Signaling[END_REF]152 Brini et al., 2013;[START_REF] Brown | Extracellular calcium sensing and extracellular calcium 821 signaling[END_REF][START_REF] Carafoli | Calcium signaling: A tale for all seasons[END_REF][START_REF] Clapham | Calcium signaling[END_REF]. Calcium 153 interacts with numerous proteins, either for storage and transport or for the regulation of 154 protein activity. In multicellular organisms, Ca 2+ is a ubiquitous intracellular second 155 messenger regulating numerous electrical, chemical, mechanical and genetic cellular 156 responses. From the cell to the whole organism, Ca 2+ is a critical component in many vital 157 functions, including cell differentiation or death, muscle contraction or propagation of 158 electrical signals in some nerve cells. The preservation of these vital functions primarily 159 depends on the fine regulation of Ca 2+ cycle and on the blood Ca 2+ concentrations (Figure 2). 160

In terrestrial tetrapods, the principal source of Ca is the intestinal absorption of dietary 161 Ca, while it is also exchanged with ambient water through gills in bony and cartilaginous 162 fishes and through skin to a certain extent in amphibians [START_REF] Bouillon | Vitamin D: calcium and bone homeostasis during evolution[END_REF]Doherty 163 et al., 2015;[START_REF] Stiffler | Amphibian Calcium Metabolism[END_REF]. Blood and extracellular fluids exchange Ca 2+ with various non-164 excreting reservoirs, such as muscle and more importantly with bone, which acts as a 165 dynamic storage organ, especially in terrestrial vertebrates [START_REF] Bouillon | Vitamin D: calcium and bone homeostasis during evolution[END_REF]. 166

Various sinks balance the incoming Ca 2+ fluxes, with contrasting contributions depending on 167 species and life stages. In mammals, skin, hair and sweat account for some of the Ca 2+ 168 losses but the main loss occurs via urinary excretions and digestive secretions through 169 faeces in most vertebrates. Tissues playing the roles of Ca 2+ dynamic stores (e.g., bone) and 170 interfaces exchanging with the environment (e.g., gills, intestine, kidneys) participate to the 171 regulation of the whole organism Ca cycle, called Ca homeostasis [START_REF] Bouillon | Vitamin D: calcium and bone homeostasis during evolution[END_REF]172 de Matos, 2008;[START_REF] Doherty | Evolutionary Physiology of Bone: 867 Bone Metabolism in Changing Environments[END_REF][START_REF] Flik | Calcium transport in fish gills and intestine[END_REF][START_REF] Peacock | Calcium Metabolism in Health and Disease[END_REF][START_REF] Stiffler | Amphibian Calcium Metabolism[END_REF]. 173

In humans, many physiopathological conditions are associated with disrupted Ca cycles.

7

Chronic perturbation of Ca and phosphate homeostasis may lead to metabolic bone disease 175 (e.g., osteoporosis) or result from disease (e.g., cancers affecting bone, c.f. [START_REF] Peacock | Calcium Metabolism in Health and Disease[END_REF]. 176

In reproductive contexts, Ca 2+ cycles are modified especially in female organisms. In 177 mammals, pregnancy and lactation are associated with modified Ca homeostasis (Del Valle 178 et al., 2011;[START_REF] Doherty | Evolutionary Physiology of Bone: 867 Bone Metabolism in Changing Environments[END_REF][START_REF] Kalkwarf | Hormonal and dietary regulation of changes in bone density during 973 lactation and after weaning in women[END_REF][START_REF] Kovacs | Calcium and bone disorders during pregnancy and 981 lactation[END_REF]. For instance, 179 a significant Ca amount is lost to the foetus skeleton during pregnancy, while breastfeeding 180 involves significant Ca daily output. Vertebrates that lay eggs with heavily mineralized shells 181 may also lose significant fractions of Ca via secretion and formation of shell Ca carbonate as 182 well as yolk and albumen. 183

To conclude, as a major bio-essential metal in vertebrates, calcium lies at the 184 crossroads of important scientific questions relevant to the evolution of biology and ecology 185 of present and past vertebrates as well as to crucial modern health issues in humans. 186 187

Cycling of Ca isotopes in vertebrates 188

The main and first described feature of the Ca isotopes cycle in bony vertebrates is 189 the significantly 44 Ca-depleted isotope composition of bone when compared to dietary Ca 190 [START_REF] Skulan | Calcium isotope fractionation between soft and mineralized 1105 tissues as a monitor of calcium use in vertebrates[END_REF]. Compilation of literature data in 6 species of mammals and one 191 bird (Figure 3) shows that Ca of bone systematically displays lower δ 44/42 Ca values than 192 dietary Ca, by about -0.57 ± 0.10 ‰ (2SE, ~ -1.14‰ in δ 44/40 Ca) [START_REF] Chu | Establishing the 841 potential of Ca isotopes as proxy for consumption of dairy products[END_REF]Heuser et 193 al., 2016;[START_REF] Hirata | 940 Isotopic analysis of calcium in blood plasma and bone from mouse samples by multiple 941 collector-ICP-mass spectrometry[END_REF][START_REF] Skulan | Calcium isotope fractionation between soft and mineralized 1105 tissues as a monitor of calcium use in vertebrates[END_REF][START_REF] Tacail | A simplified protocol for measurement of 1119 Ca isotopes in biological samples[END_REF]. Despite the 194 various represented species in literature, no significant relationship between the extent of this 195 isotope effect and the organism physiology has been identified yet. A diet-bone isotopic 196 offset is well conserved within amniotes, suggesting a phylogenetically shared mechanism 197 resulting in Ca isotope fractionation during biological processing. In teleost and 198 elasmobranch fishes, the amplitude of the offset between diet and mineralized tissues 199 remains to be more thoroughly explored in controlled conditions but is possibly not as 200 marked. Seawater ingestion and osmoregulation involving seawater filtering through gills 201 could possibly attenuate or buffer these effects, as suggested by studies of marine 202 ecosystems [START_REF] Clementz | Are calcium isotopes a reliable monitor of 846 trophic level in marine settings?[END_REF][START_REF] Martin | Calcium isotopes reveal the 1007 trophic position of extant and fossil elasmobranchs[END_REF]. 203

According to the comparison of soft tissue and blood Ca with bone of the same 204 animals [START_REF] Skulan | Calcium isotope fractionation between soft and mineralized 1105 tissues as a monitor of calcium use in vertebrates[END_REF], this 44 Ca-depleted composition of bone primarily 205 results from a major isotope fractionation occurring during mineralisation of bone. On the 206 contrary, Ca loss from bone, during bone remodelling for instance, does not fractionate Ca 207 isotopes. The amplitude of the mineralization isotope effect is thought to be the same as the 208 diet-bone difference [START_REF] Channon | 834 Using natural, stable calcium isotopes of human blood to detect and monitor changes in 835 bone mineral balance[END_REF][START_REF] Heuser | A pilot study on the use of natural calcium isotope 920 (44Ca/40Ca) fractionation in urine as a proxy for the human body calcium balance[END_REF]Morgan et al., 209 2012;[START_REF] Reynard | Calcium isotope ratios in animal 1071 and human bone[END_REF][START_REF] Skulan | Calcium isotope fractionation between soft and mineralized 1105 tissues as a monitor of calcium use in vertebrates[END_REF][START_REF] Skulan | Natural Calcium Isotopic Composition of Urine as a Marker of Bone Mineral 1114 Balance[END_REF], i.e. -0.57‰, 210 although more recent comparison of bone and blood of sheep [START_REF] Tacail | A simplified protocol for measurement of 1119 Ca isotopes in biological samples[END_REF] and pig 211 [START_REF] Heuser | Biological 923 fractionation of stable Ca isotopes in Göttingen minipigs as a physiological model for Ca 924 homeostasis in humans[END_REF] suggest that fractionation during mineralisation could be less 212

pronounced. 213

The processing of Ca by kidneys also induces a significant isotope effect, as 214 observed in various mammals. The Ca excreted in urine is systematically enriched in heavy 215 isotopes when compared to blood Ca, by ca. +1.2‰ in 44/42 Ca (ca. +2.4‰ in 44/40 Ca), in 216 human [START_REF] Channon | 834 Using natural, stable calcium isotopes of human blood to detect and monitor changes in 835 bone mineral balance[END_REF][START_REF] Eisenhauer | Calcium isotope ratios in blood and 873 urine: A new biomarker for the diagnosis of osteoporosis[END_REF][START_REF] Heuser | A pilot study on the use of natural calcium isotope 920 (44Ca/40Ca) fractionation in urine as a proxy for the human body calcium balance[END_REF]217 Skulan et al., 2007), sheep [START_REF] Tacail | A simplified protocol for measurement of 1119 Ca isotopes in biological samples[END_REF] and pig [START_REF] Heuser | Biological 923 fractionation of stable Ca isotopes in Göttingen minipigs as a physiological model for Ca 924 homeostasis in humans[END_REF]Morgan et al., 218 2012). The relationship between the amount of excreted urinary Ca and its isotope 219 composition in human suggests a Rayleigh isotope distillation process resulting from the 220 preferential reabsorption of Ca light isotopes from primary urine to blood along the nephron 221 [START_REF] Heuser | Calcium Isotopes in 927 Human Urine as a Diagnostic Tool for Bone Loss: Additional Evidence for Time Delays 928 in Bone Response to Experimental Bed Rest[END_REF][START_REF] Heuser | A pilot study on the use of natural calcium isotope 920 (44Ca/40Ca) fractionation in urine as a proxy for the human body calcium balance[END_REF][START_REF] Morgan | Rapidly assessing changes in bone mineral balance using natural stable calcium 1029 isotopes[END_REF]. 222 223 Vertebrate reproduction is also associated with significant Ca isotope effects. In 224 mammals, the isotope composition of milk was found to be remarkably 44 Ca-depleted in 225 comparison with that of the mother's diet [START_REF] Chu | Establishing the 841 potential of Ca isotopes as proxy for consumption of dairy products[END_REF]. The amplitude of this difference 226 in δ 44/42 Ca values averages at around -0.6‰ from diet to milk as observed in milk of ewes, 227 cows and humans [START_REF] Chu | Establishing the 841 potential of Ca isotopes as proxy for consumption of dairy products[END_REF][START_REF] Gussone | Biominerals and Biomaterial[END_REF]Heuser, 2016;Tacail, 228 2017). Prolonged milk production by lactating females could also result in a 44 Ca-enriched 229 composition of their bone when compared to males, as suggested by observations in modern 230 sheep [START_REF] Reynard | Calcium isotope ratios in animal 1071 and human bone[END_REF]. In birds, despite limited datasets, Ca isotope compositions of 231 eggs inner parts and carbonate shells were found to display remarkably extreme isotopic 232 patterns (see Skulan and DePaolo, 1999 and Figure 5). While the isotopic composition of 233 eggshell appears to be comparable to diet and significantly enriched in 44 Ca in comparison 234 with bone of egg-laying hens, white albumen, and yolk to a lesser extent, display 235 compositions that are even more enriched in heavy isotopes than for eggshell and diet. Such 236 a 44 Ca-enriched composition of eggshells likely relates to the high demand in Ca for its 237 formation, resulting in high fluxes of poorly fractionated diet-like Ca to the eggshell (Skulan et 238 al., 1997). 239 This understanding of the Ca isotope cycle led to two main hypotheses that have 240 been since then further tested and exploited: 241 (i) as diet directly influences the systematically shifted isotope compositions of mineralized 242 tissues, Ca isotopes can be used to reconstruct dietary behaviours in past vertebrates 243 (ii) as Ca isotopes are fractionated during bone mineralisation, Ca isotope compositions of 244 blood and urine vary with the balance between bone formation and resorption. Calcium 245 isotopes could be used to monitor variations of bone mineral balance in human subjects 246 suffering from bone disorders. 247 248

Future research 249

New research is needed in order to i) further characterize the distribution of Ca isotopes 250 in the various reservoirs of vertebrate organisms, ii) identify factors of variability (e.g., 251 species-specific physiological effects) and iii) improve our understanding of the mechanisms 252 responsible for the major biologically induced isotope fractionations. 253

These remaining aspects would benefit from various approaches. At the scale of the 254 whole organism, the results of controlled feeding experiments done on species from various 255 clades of vertebrates could help further identify physiological factors of variability. This type 256 of approach would help refine the identification of Ca fluxes associated with isotope effects 257 potentially affecting the whole organism Ca isotope distribution (e.g., Balter et al., 2013). 258

Isotope box modelling of whole organism at steady-state and in dynamic conditions will then 259 allow us to test the proposed mechanisms and investigate the potential of Ca isotopes for 260 identified and future applications (e.g., [START_REF] Jaouen | Dynamic 963 homeostasis modeling of Zn isotope ratios in the human body[END_REF]. Various physiological conditions 261 could notably be explored, such as the age-related changes in the balance between bone 262 anabolism and catabolism or the cycling of Ca isotopes between pregnant mammals and 263 their foetus. In order to better understand the cellular and molecular mechanisms at play, it 264 will also be possible to compare the results of experimental approaches on cellular in vitro 265 models with controlled expression of cross-membrane protein transporters (e.g., Cadiou et 266 al., 2017) and estimations of molecular equilibrium fractionation with numerical ab initio 267 calculations (e.g., [START_REF] Moynier | Calcium isotope fractionation between aqueous compounds 1032 relevant to low-temperature geochemistry[END_REF]. All these approaches will greatly benefit both 268 palaeobiology and modern biomedical applications and potentially further reveal other yet to 269 be identified applications. 270 271

Calcium isotopes in palaeobiology and bioarchaeology 272

Stable isotopes and trace elements enable the development of quantitative 273 approaches to reconstruct ecological, physiological and environmental characteristics that 274 otherwise remain unseen. Unravelling the partitioning of resources notably helps to better 275 understand past and present ecosystem dynamics and biodiversity. Geochemical proxies are 276 useful means of complementing inferences of morphofunctional and microwear analyses of 277 osteological remains (e.g., Martin et al., 2017a). The same applies to the study of past 278 human societies, where such tools are valuable to complement the conclusions of 279 archaeology and anthropology [START_REF] Jaouen | What is our toolbox of analytical chemistry for exploring ancient hominin 954 diets in the absence of organic preservation?[END_REF][START_REF] Jaouen | Potential of non-traditional isotope studies for bioarchaeology[END_REF]). Following the 280 example of other more established proxies, such as collagen carbon and nitrogen stable 281 isotopes or Sr/Ca and Ba/Ca elemental ratios [START_REF] Balter | Determination of Sr and Ba partition coefficients between 797 apatite and water from 5 C to 60 C: a potential new thermometer for aquatic 798 paleoenvironments[END_REF][START_REF] Koch | Isotopic Study of the Biology of Modern and Fossil Vertebrates[END_REF]282 Newsome et al., 2010;[START_REF] Peek | Sr/Ca and Ba/Ca variations in environmental and biological 1049 sources: A survey of marine and terrestrial systems[END_REF], Ca stable isotopes of fossil bioapatite 283 have a high potential for the study of past vertebrate ecosystems and human communities 284 for three main reasons. 285 First, as discussed above, the Ca isotope composition of diet primarily determines the 286 composition of vertebrate mineralized tissues. This early finding came along with the 287 hypothesis that such a trophic effect could propagate along food chains. The trophic level 288 index, ranging from primary producer to apex predators and decomposers, describes the 289 position of species within trophic chains and thus allows synthetizing the energetic pathways 290 within ecosystems [START_REF] Polis | Food Web Complexity and Community Dynamics[END_REF]. Early datasets from both marine and terrestrial 291 environments supported this trophic level effect [START_REF] Clementz | Are calcium isotopes a reliable monitor of 846 trophic level in marine settings?[END_REF][START_REF] Depaolo | Calcium isotopic variations produced by biological, kinetic, radiogenic 864 and nucleosynthetic processes[END_REF]292 Skulan et al., 1997;[START_REF] Skulan | Calcium isotope fractionation between soft and mineralized 1105 tissues as a monitor of calcium use in vertebrates[END_REF]. The biological processing of Ca from a 293 trophic level to another would thus lead to a stepwise decrease of the 44/42 Ca values. 294 Second, as a major constituent of a stable mineralogical phase (~ 40 wt.% Ca of 295 bioapatite), the post-mortem diagenetic processes tend not to alter skeletal bioapatite Ca and 296 its isotope compositions especially in the denser and less porous tooth enamel (Heuser et 297 al., 2011;[START_REF] Melin | Calcium and carbon stable 1019 isotope ratios as paleodietary indicators[END_REF]Martin et al., 2017;[START_REF] Martin | Calcium isotopes in enamel of modern 1012 and Plio-Pleistocene East African mammals[END_REF]. The determination of Ca 298 isotope compositions in osteological remains makes it possible to explore deep past 299 ecosystems. So far, studies of vertebrate fossils ecology with Ca isotopes focused on 300 assemblages dating back to Late Devonian [START_REF] Balter | Calcium stable 801 isotopes place Devonian conodonts as first level consumers[END_REF], Triassic to Cretaceous 301 [START_REF] Hassler | 913 Calcium isotopes offer clues on resource partitioning among Cretaceous predatory 914 dinosaurs[END_REF][START_REF] Heuser | Calcium isotopes in fossil bones 931 and teeth -Diagenetic versus biogenic origin[END_REF]Martin et al., 2017b) or Pliocene and Pleistocene 302 [START_REF] Martin | Calcium isotopes in enamel of modern 1012 and Plio-Pleistocene East African mammals[END_REF], 2015, Tacail et al., 2019). Contrastingly, the conservation of pristine 303 nitrogen isotope compositions of bone collagen primarily depends on the preservation of the 304 organic phase, the degradation of which rarely allows investigating on trophic relationships 305 beyond Holocene or Late Pleistocene [START_REF] Koch | Isotopic Study of the Biology of Modern and Fossil Vertebrates[END_REF]. On the other hand, the preservation of 306 biogenic Sr/Ca and Ba/Ca trace element ratios strongly depends on burial conditions (e.g., 307 [START_REF] Martin | Calcium isotopes in enamel of modern 1012 and Plio-Pleistocene East African mammals[END_REF][START_REF] Reynard | Trace elements and their isotopes in bones and teeth: Diet, 1064 environments, diagenesis, and dating of archeological and paleontological samples[END_REF]. 308

Finally, the abundance of Ca in osteological remains makes it possible to sample 309 minute amounts of material (down to 20 µg or less). Such sample sizes allow studying 310 precious fossils, without significantly altering their physical and structural integrity. It also 311 motivates the development of high resolution sampling techniques involving mechanical 312 sampling of tooth enamel by micro-drilling [START_REF] Tacail | Precise Analysis of Calcium Stable Isotope Variations 1122 in Biological Apatites using Laser Ablation MC-ICPMS[END_REF][START_REF] Tacail | 1125 Assessing human weaning practices with calcium isotopes in tooth enamel[END_REF] and laser cutting (Li et 12 al., 2016). In situ laser ablation isotope analyses were shown to be possible in tooth enamel 314 [START_REF] Tacail | Precise Analysis of Calcium Stable Isotope Variations 1122 in Biological Apatites using Laser Ablation MC-ICPMS[END_REF][START_REF] Zhang | In situ calcium isotopic ratio 1150 determination in calcium carbonate materials and calcium phosphate materials using 1151 laser ablation-multiple collector-inductively coupled plasma mass spectrometry[END_REF], although these approaches could greatly benefit 315 from the implementation of technological innovations such as the currently developing use of 316 collision-cell multi-collector plasma mass spectrometry (e.g., Lewis et al., 2018, Zhao and 317 Simon 2019), with potential interference removal. 318

After the first assessments of the utility of Ca isotopes for reconstructions of 319 vertebrate ecology, several studies further explored and developed strategies to reconstruct 320 biological, ecological and behavioural characteristics of past vertebrates and human 321 populations. 322 323

Calcium isotopes and vertebrate ecosystem reconstructions 324

The sensitivity of skeletal Ca isotope compositions to trophic levels was explored and 325 exploited in a series of modern and fossil ecosystems, both in marine and continental 326 environments. 327 328

Marine ecosystems 329

Seawater is the primary source of Ca in most marine ecosystems (~ 420 mg/L, 330 [START_REF] Elderfield | Mid-Ocean Ridge Hydrothermal Fluxes and the Chemical 876 Composition of the Ocean[END_REF]. The Ca isotope composition of seawater is particularly 331 enriched in heavy isotopes (Figure 5) and relatively homogeneous at the scale of the oceans 332 (e.g., [START_REF] Fantle | Calcium isotopes in the global biogeochemical Ca cycle: 885 Implications for development of a Ca isotope proxy[END_REF]. 333

Following the first description of decreasing δ 44/42 Ca values with increasing trophic 334 levels [START_REF] Skulan | Biological control of calcium isotopic 1108 abundances in the global calcium cycle[END_REF], [START_REF] Clementz | Are calcium isotopes a reliable monitor of 846 trophic level in marine settings?[END_REF] reported consistent patterns in bones and 335 teeth of both modern and fossil marine mammals. Species of low trophic levels (feeding on 336 marine vegetation or algae and invertebrates) display 44 Ca-enriched compositions when 337 compared to higher trophic level mammals (feeding on fishes and other marine mammals). 338

This pattern was also later reported in elasmobranch species of diverse ecologies (i.e. 339 cartilaginous fishes, namely sharks and rays, see [START_REF] Martin | Calcium isotopes reveal the 1007 trophic position of extant and fossil elasmobranchs[END_REF]. These findings were 340 finally applied to a fossil marine ecosystem dating back to late Cretaceous (Martin et al., 341 2017b), composed of samples from actinopterygian and elasmobranch fishes, as well as 342

Mosasaurid and Plesiosaurid large marine reptiles. This assemblage shortly predates the 343 end-Cretaceous extinction and sheds light on the vulnerability of this ecosystem, where high 344 trophic level large reptiles were relying on abundant yet poorly diversified resources. The use 345 of Ca isotopes also yielded new constraints onto the ecology and trophic level of extinct 346 Devonian conodonts organisms for which no modern analogue is known [START_REF] Balter | Calcium stable 801 isotopes place Devonian conodonts as first level consumers[END_REF]. 347

These studies identified features that require further dedicated investigation in order 348 to improve the use of Ca isotopes as an indicator of marine ecosystem structures. For 349 instance, data indicates that trophic ecology primarily determines tooth and bone isotope 350 compositions of marine vertebrates, but mammals and fishes (cartilaginous or ray-finned) do 351 not appear to display the same average isotope shifts from one trophic level to another 352 [START_REF] Clementz | Are calcium isotopes a reliable monitor of 846 trophic level in marine settings?[END_REF]Martin et al., 2017b[START_REF] Martin | Calcium isotopes reveal the 1007 trophic position of extant and fossil elasmobranchs[END_REF]. The ingestion of seawater and processing 353 of Ca via gill-mediated osmoregulation in actinopterygian and elasmobranch species is 354 suspected to buffer the diet-skeleton isotope offset. On the other hand, marine mammal 355 organisms are rather closed systems with respect to seawater. The differences in Ca isotope 356 physiology of fishes and mammals should thus motivate dedicated research in order to 357 decipher the physiological and ecological controls on the Ca isotope compositions of their 358 tissues. A better understanding of the isotope systematics of marine mammals would notably 359 be of importance as their ecology makes them useful sentinels of ecosystem changes (e.g., 360 Moore, 2008). 361 362

Continental ecosystems 363

The nature and isotope compositions of primary Ca dietary sources of most terrestrial 364 vertebrate trophic chains are more diversified than in marine ecosystems (see Figure 5). 365 Indeed, in addition to the possible variability of isotope compositions of environmental Ca 366 (e.g., interacting bedrock, soil and water), the plant Ca isotope compositions are highly 367 variable between types, species and organs (e.g., Schmitt, 2016 and Figure 5). Depending 368 on the ecological niches of primary consumers (e.g., in vertebrate herbivores), such 369 variability can propagate throughout the trophic chain and make the interpretation of Ca 370 isotope compositions more complex for trophic relationships reconstructions. 371 [START_REF] Melin | Calcium and carbon stable 1019 isotope ratios as paleodietary indicators[END_REF] explored the bone Ca isotope compositions of various mammals 372 including primates from two tropical forested ecosystems. The authors confirmed the 373 decrease of Ca isotope ratios in large carnivores, but found no significant differences 374 between lower trophic levels, notably small faunivores and insectivores. They however 375 suggest an encouraging complementary use of carbon (C) and Ca stable isotopes. Martin et 376 al. (2017a) reported encouraging bone and tooth enamel Ca isotope compositions of two 377 Western European Pleistocene mammal assemblages. The stable isotope compositions of 378 each taxonomic group (up to 6 individuals) are rather clustered and carnivores tend to 379 display lower δ 44/42 Ca values than herbivores. However, the outlying compositions of some 380 species remain difficult to interpret in terms of trophic inferences, suggesting either species-381 dependent physiological effects or peculiar dietary niches effects. Recently, Martin et al. 382 (2018) reported the results of the systematic study of mammal tooth enamel of modern and 383 fossil Pliocene-Pleistocene ecosystems from East Africa. This study further documents the 384 potential of Ca isotopes as helpful markers for trophic inferences. The combination of C and 385 Ca stable isotopes helps to distinguish between C 3 and C 4 trophic chains structures, in extant 386 and extinct ecosystems. Indeed, the carbon stable isotope compositions of the enamel of 387 mammals ( 13 C) are characteristic of the photosynthetic pathways of the plants at the base of 388 the trophic chain (C 3 or C 4 carbon fixing sugars, c.f. Bender 1971), while Ca isotopes appear 389 to allow discriminating between herbivores and carnivores. This is exemplified by the figure 390 4A, where the carnivores appear depleted in 44 Ca isotopes with respect to their respective 391 inferred preys, being either predominantly C 3 herbivores with low 13 C values (namely 392 browsers) or a mix between the latter and C 4 herbivores with higher 13 C values (namely 393 grazers). Furthermore, when taking into account all available geochemical proxies relevant to 394 feeding and habitat ecology (namely Sr/Ca and Ba/Ca ratios and δ 13 C, δ 18 O, δ 44/42 Ca, see 395 [START_REF] Martin | Calcium isotopes in enamel of modern 1012 and Plio-Pleistocene East African mammals[END_REF], a principal component analysis (Figure 4B) reveals an inferred 396 ecosystem structure that is very similar to what δ 13 C and δ 44/42 Ca alone suggest (Figure 4A). 397

In other words, the coupling of δ 13 C and δ 44/42 Ca appears to efficiently summarize the 398 ecosystem structure. 399

Finally, in the Mesozoic fossil record, despite the fact that rather inconclusive results 400 were obtained from dinosaur faunas on large geographical and chronological scales (Heuser 401 et al., 2011), the systematic study of skeletal remains Ca isotope compositions from a fossil 402 assemblage at regional scale brings new constraints to the resource partitioning between 403 mid-Cretaceous predatory dinosaurs and more precisely to the ecology of spinosaurids 404 [START_REF] Hassler | 913 Calcium isotopes offer clues on resource partitioning among Cretaceous predatory 914 dinosaurs[END_REF]. 405

To conclude, the ongoing exploration at regional scale of the ecological controls over 406 vertebrate Ca isotope compositions in the continental environment is encouraging. Calcium 407 isotopes show a very good potential for the reconstruction of dietary relationships in past 408 vertebrate ecosystems, from Palaeozoic to Quaternary, and allow constraining local 409 ecosystem structures and resource partitioning, especially between herbivores and 410 carnivores. These results motivate the development of new strategies that notably include i) 411 the combination of δ 44/42 Ca with complementary proxies, such as δ 13 C, ii) the study of 412 assemblages composed of taxa with diverse ecologies, iii) at a regional scale, iv) with 413 taxonomic groups comprising multiple individuals. Further research needs to be carried out, 414 notably because some taxa do not entirely fit within this interpretative framework. This could 415 relate to peculiar physiological adaptations or ecological niches (e.g., [START_REF] Martin | Calcium isotopes in enamel of modern 1012 and Plio-Pleistocene East African mammals[END_REF]Martin et al., , 416 2017a)). For instance, the observed isotopic offsets between bone of most secondary or 417 tertiary consumers and their vertebrate preys are likely to primarily rely on the partial 418 consumption of mineralized tissues from the latter. The intake of only 1 wt. % bone in total 419 diet is sufficient to cause a significant trophic effect in Ca isotope compositions (-0.50‰ on 420 the δ 44/42 Ca scale) of consumer's tissues in terrestrial vertebrates [START_REF] Heuser | Calcium isotopes in fossil bones 931 and teeth -Diagenetic versus biogenic origin[END_REF]. 421

Although little is known about muscle and bulk soft tissues isotope compositions, the 422 consumption of energy-rich but Ca-poor soft tissues could result in different isotope offsets 423 between secondary consumers and their vertebrate preys, making trophic level inferences 424 less straightforward. 425 426 3.1.3. Future directions 427 (i.) The development of statistical approaches for diet reconstruction with Ca isotopes could 428 help to refine and make the most of trophic ecosystem reconstructions (such as Bayesian 429 statistical inferences, see for instance [START_REF] Fernandes | Food 899 reconstruction using isotopic transferred signals (FRUITS): A bayesian model for diet 900 reconstruction[END_REF]. The inter-individual variability 430 of Ca isotope compositions within a given taxonomic group could also relate to the degrees 431 of specialisation for various Ca resources (e.g., [START_REF] Martin | Calcium isotopes in enamel of modern 1012 and Plio-Pleistocene East African mammals[END_REF]. By means of statistical 432 methods, Ca isotopes could allow characterizing "isotope niches" and help to document 433 breadths of ecological niches or the specialisation of taxonomic groups (e.g., Yeakel et al., 434 2015). 435 (ii.) Profound changes in the biological management of Ca occurred in the course of 436 vertebrate evolution. A series of major evolutionary steps could potentially be studied with 437 the help of Ca isotopes: the appearance of calcium phosphate mineralized endoskeletons, 438 the ecological and physiological adaptations related to fish-to-tetrapod transitions followed by 439 terrestrialisation, the development of egg-laying and carbonated shells in vertebrates or 440 finally the development of pregnancy and lactation in first mammalians (Bouillon and Suda, 441 2014;[START_REF] Doherty | Evolutionary Physiology of Bone: 867 Bone Metabolism in Changing Environments[END_REF]. Each of these evolutions of vertebrate physiology and ecology 442 likely affected their Ca isotopes cycling, which could in turn be exploited in order to document 443 the evolutionary mechanisms at play. 444 (iii.) Calcium isotope might be useful in other practical applications such as in food forensics. 445

For instance, combined with other stable isotopes, Ca isotopes could help in tracking the 446 consumption of animal meat and bone meals by farmed animals (e.g., cattle, poultry; see 447 [START_REF] Carrijo | Traceability of 828 bovine meat and bone meal in poultry by stable isotope analysis[END_REF]. 448 449

3.2.

Calcium isotopes and dietary reconstructions in hominids and human 450 societies 451

From ecosystems to human societies 452

Major changes in dietary practices in the course of human evolution, being ecologically 453 and/or culturally driven, likely came along with significant changes of their use of Ca 454 resources (e.g., [START_REF] Eaton | Calcium in evolutionary perspective[END_REF]. The changing dietary habits of early Homo or the 455 development of agriculture and livestock farming in the course of Neolithic mark significant 456 modifications of the use of environmental Ca and were associated with major ecological, 457 physiological, genetic or cultural evolutions (e.g., [START_REF] Aiello | The Expensive-Tissue Hypothesis: The Brain and the 779 Digestive System in Human and Primate Evolution[END_REF]Hublin et al., 458 2015;[START_REF] Ségurel | On the Evolution of Lactase Persistence in Humans[END_REF][START_REF] Vigne | The origins of animal domestication and husbandry: A major change in 1134 35 the history of humanity and the biosphere[END_REF]. The utility of Ca isotopes to track these changes 459 has been however poorly explored. While the previously described ecosystem approach 460 using Ca isotopes could help to interpret the evolution of hominid ecologies, the study of 461 bone Ca isotopes in human societies could improve our understanding of their contrasting 462 dietary habits influenced by varied cultural practices. 463

As shown in Figure 5, the major dietary sources of Ca for human have significantly 464 contrasting isotope compositions. Therefore, the culturally driven practices of consumption of 465 particular Ca dietary sources could lead to distinctive distributions of bone 44/42 Ca values in 466 human populations. This could be of use to reconstruct dietary practices of past human 467 societies and characterize their subsistence modes. 468

For instance, dairy products constitute the most 44 Ca-depleted end-member of this 469 isotopic landscape (with modern human milk being the lightest), while eggs and marine 470 resources display the most 44 Ca-enriched compositions. Following these observations, Chu 471 et al. (2006) proposed that Ca isotopes could be used to reconstruct the consumption of 472 dairy products in past human groups, especially during the processes of Neolithization with 473 domestication of dairy producing animals. Indeed, the consumption of significant proportions 474 of Ca from dairy products should induce a global decrease in δ 44/42 Ca values of dietary Ca 475 and thus in bone of human consumers. [START_REF] Reynard | Calcium isotope ratios in animal 1071 and human bone[END_REF][START_REF] Reynard | Calcium isotopes in archaeological 1068 bones and their relationship to dairy consumption[END_REF] tested the potential of Ca 476 isotopes in faunal and human bones as a biomarker of dairy product consumption in several 477 archaeological populations before and after domestication of dairy producing animals. These of the studied archaeological sites after the domestication of milk producing animals. They 480 suggested that controls other than animal milk consumption were likely at play. 481

Nevertheless, further research including actualistic calibrations in human osteological 482 remains could help refine our understanding of the controls of dietary practices over bone Ca 483 isotope compositions. 484 485

Life history reconstructions in individuals 486

Throughout their development and life, humans experience major adaptations of their Ca 487 cycle both in terms of nutrition (e.g., birth or weaning in offspring) or Ca homeostasis (e.g., 488 pregnancy or breastfeeding in females) [START_REF] Humphrey | Weaning behaviour in human evolution[END_REF][START_REF] Kovacs | Calcium and bone disorders during pregnancy and 981 lactation[END_REF]. The 489 characteristics of these events (e.g., timing, frequency) are crucial and informative traits of 490 the life history of individuals, relating to the ecological strategies of species and to cultural 491 behaviours [START_REF] Kramer | When mothers need others: The impact of hominin 984 life history evolution on cooperative breeding[END_REF][START_REF] Lee | Growth and Investment in Hominin Life History Evolution: Patterns, 994 Processes, and Outcomes[END_REF][START_REF] Robson | Hominin life history: reconstruction and evolution[END_REF]. Such 492 changes can in turn influence the Ca isotope distributions, which could be exploited if 493 archived in human remains. 494 So far, the major life history traits explored with Ca isotopes relate to mother's milk 495 consumption by children. The biologically and culturally driven weaning behaviours are 496 central traits of the evolution of mammals and hominins [START_REF] Humphrey | Weaning behaviour in human evolution[END_REF][START_REF] Sellen | Evolution of Infant and Young Child Feeding: Implications for 1100 Contemporary Public Health[END_REF]Van 497 Noordwijk et al., 2013) but these practices remain fairly undocumented in the deep past 498 because biomarkers are lacking. As milk was early recognized as a 44 Ca-depleted material in 499 comparison with diet, [START_REF] Reynard | Calcium Isotopes in 1074 Juvenile Milk-Consumers[END_REF] first tested this hypothesis by analysing bones of 500 past human populations as a function of age of individuals but no significant trends were 501 observed. This hypothesis was later tested on temporary tooth enamel of modern human 502 individuals with known early life histories [START_REF] Tacail | 1125 Assessing human weaning practices with calcium isotopes in tooth enamel[END_REF]. Contrary to bone which 503 continuously remodels, tooth enamel forms incrementally. Provided the sampling techniques 504 allow sufficient spatial resolution, the enamel structure permits the reconstruction of the 505 evolution of Ca isotope compositions over time of formation of tooth crowns. This study 506 demonstrates that weaning practices are recorded within human tooth enamel by Ca 507 isotopes and therefore could allow documenting weaning patterns in past humans and 508 hominids. This method was recently applied to the tooth enamel of Pleistocene early 509 hominins of South Africa [START_REF] Tacail | Bedrest experiments induce bone loss and a decrease of urine and blood 44/42 Ca 1196 values. Changes in Ca isotope compositions (∆ 44/42 Ca) of blood or urine at each date of 1197 experiment are calculated as the difference with average baseline (pre-bedrest) composition 1198 in all subjects of three studies: (A.) ∆ 44/42 Ca in urine of 4 individuals over 17 weeks of bedrest 1199[END_REF]. The results support that early Homo infants 510 were breastfed in significant proportions for longer periods than A. africanus and P. robustus 511 and opens discussion on the evolution of weaning practices in human lineage. 512 513 3.2.3. Future directions 514 (i) Given the reported systematic variability of Ca dietary sources, and provided this 515 variability is further documented, Ca isotopes could be a successful marker of human dietary 516 practices in relation with human cultures and subsistence modes. A more actualistic 517 approach with a focus on modern and historic human populations with documented and 518 contrasting diets would for instance open the way to new archaeological applications, the 519 same way Zn isotopes were shown to be sensitive to consumption of fish and meat (Jaouen 520 et al., 2018). 521 (ii) As biomarkers of weaning practices, Ca isotopes in tooth enamel would benefit from 522 the investigation of its complementarity with other biomarkers of early life dietary transitions 523 such as Ba/Ca ratios in dentin or enamel (e.g., [START_REF] Austin | Barium distributions in teeth reveal early-life 790 dietary transitions in primates[END_REF] or δ 15 N in bone and dentin 524 collagen [START_REF] Tsutaya | Reconstruction of breastfeeding and weaning practices using 1128 stable isotope and trace element analyses: A review[END_REF]. 525 (iii) Beyond tooth enamel, other incrementally growing tissues could be explored for their 526 potential as time resolved archives. Dentin, hair or nail could record Ca isotopic variations 527 relating to weaning practices but also to other physiopathological modifications of Ca 528 homeostasis such as puberty as proposed by [START_REF] Li | Ca isotopic analysis of laser-cut microsamples of 1001 (bio)apatite without chemical purification[END_REF], the same way other isotope 529 systems can be affected (e.g., [START_REF] Ohno | Precise Zn isotopic ratio measurements 1038 of human red blood cell and hair samples by multiple collector-ICP-mass spectrometry[END_REF][START_REF] Tsutaya | Reconstruction of breastfeeding and weaning practices using 1128 stable isotope and trace element analyses: A review[END_REF]. 530 531

Calcium isotopes in biomedical research 532

Stable Isotope Metallomics is the study of normal and disrupted cycles of regulated 533 and essential metals from the perspective of their natural stable isotope compositions. This 534 field of research has developed in the last two decades following seminal works on Ca, Fe, 535 Cu or Zn, aided by the development and widespread application of multicollection inductively 536 coupled plasma mass spectrometry (MC-ICPMS) (see reviews such as [START_REF] Albarède | Medical Applications of Isotope Metallomics[END_REF]537 Costas-Rodríguez et al., 2016). This research has served two main purposes: i) to shed new 538 light on the biological processing of these elements, beyond quantitative description of their 539 elemental distributions (e.g., identify pathways or biochemical mechanisms), ii) to develop 540 new markers for rapid and specific diagnosis or prognosis of diseases affecting these cycles. 541

In this context, the potential of Ca isotopes is important. Because of the significant 542 fractionation occurring during bone mineralisation, Ca stable isotopes are directly indicative 543 of the short-term variations of net bone mineral balance (BMB), i.e. the mass balance 544 between bone resorption and formation. Individuals with positive BMB should thus display 545 blood and urine Ca isotope compositions enriched in heavy isotopes with respect to neutral 546 BMB. On the other hand, individuals with a negative BMB should display 44 Ca-depleted blood 547 and urine. This recognition sparked a vivid interest for the study of Ca isotopes as a marker 548 of disruptions of BMB. 549 Disruptions of bone mineral balance are widespread in modern societies, mainly 550 because of the high incidence of metabolic bone disease, such as osteoporosis in ageing 551 populations [START_REF] Becker | The societal burden of osteoporosis[END_REF][START_REF] Peacock | Calcium Metabolism in Health and Disease[END_REF]. Another related issue is the bone loss 552 induced by microgravity, as experienced by crew members in space missions, for which 553 effective countermeasures are still being investigated [START_REF] Grimm | The impact of microgravity on bone in humans[END_REF]. Clinical tools for 554 predicting and monitoring the evolution of these physiological and pathological conditions 555 resulting from disruptions of Ca metabolism are currently limited to: (i) molecular markers 556 with poor sensitivity or quantitative insight into BMB changes, (ii) X-ray densitometry 557 techniques that only provide a 6-months to yearly resolution of the changes in bone mineral 558 density (e.g., [START_REF] Kuo | Bone biomarker for the clinical assessment of osteoporosis: 987 Recent developments and future perspectives[END_REF]. The potential of Ca isotopes as a quantitative, radiation-559 free and non-invasive biomarker for the measurement of BMB has been investigated in a 560 series of studies over the last twelve years. 561 562

The effects of induced bone loss in bed rest studies 563

Several studies presented evidence of variations in human blood and urine Ca 564 isotope compositions caused by bone loss induced by bed rest [START_REF] Channon | 834 Using natural, stable calcium isotopes of human blood to detect and monitor changes in 835 bone mineral balance[END_REF]565 Heuser et al., 2019;[START_REF] Morgan | Rapidly assessing changes in bone mineral balance using natural stable calcium 1029 isotopes[END_REF][START_REF] Skulan | Natural Calcium Isotopic Composition of Urine as a Marker of Bone Mineral 1114 Balance[END_REF] (see Figure 6). Bed rest 566 provokes significant loss of muscle as well as bone mineral mass in response to inactivity, 567 and mimics the effects of microgravity in spaceflight [START_REF] Pavy-Le Traon | From space to 1044 Earth: Advances in human physiology from 20 years of bed rest studies (1986-2006[END_REF]. Bed rest 568 experiments are helpful in identifying the physiopathological causes of bone mineral loss, 569 and also permit the assessment of drug and physical exercise countermeasures to bone 570 mineral loss. 571 [START_REF] Skulan | Natural Calcium Isotopic Composition of Urine as a Marker of Bone Mineral 1114 Balance[END_REF] first published a report of significant variations of 44/40 Ca values 572 in weekly-pooled urine of individuals submitted to bed rest experiments. The authors 573 reported significant negative departure from baseline in 44 Ca/ 40 Ca ratios during bed rest in 574 the group with no countermeasure to bone loss followed by a return to baseline after the 575 experiment. By contrast, subjects treated with pharmacological or exercise countermeasures 576 to bone loss displayed either no change or positive change of their urine 44/40 Ca values. 577 These results provided a first proof of concept. This work was later augmented by Morgan et 578 al. (2012) with a report of changes in urine Ca isotope compositions during a bed rest 579 experiment conducted with a finer chronological sampling. This study demonstrated that the 580 decrease in 44/42 Ca values of urine occur after one week, indicating a loss of bone in 581 agreement with markers of osteolytic activity (NATX) but long before any changes in bone 582 mineral density could be detected by means of X-ray methods. These results were further 583 confirmed in the blood of the same subjects [START_REF] Channon | 834 Using natural, stable calcium isotopes of human blood to detect and monitor changes in 835 bone mineral balance[END_REF]. The recognition of the 584 isotopic fractionation induced by renal function lead the authors to adjust the mathematical 585 models used to link changes in bone mass and urine Ca isotope compositions (e.g., Heuser 586 andEisenhauer 2010, Morgan et al., 2012). The observed negative offset in urine 44/42 Ca 587 results from loss of Ca from bone but also from an increase in Ca urinary losses in kidneys 588 (i.e. a decrease in Ca reabsorption). Taking this into account, [START_REF] Morgan | Rapidly assessing changes in bone mineral balance using natural stable calcium 1029 isotopes[END_REF] calculated 589 the relative bone loss during the bed rest experiment. The estimated relative bone loss 590 agreed with bone loss predicted on the basis of previous experiments in which bed rest was 591 of a duration long enough to produce changes in BMD resolvable by X-ray densitometry 592 (around 0.25 % of total bone mass, for -0.30‰ decrease of 44/42 Ca values). 593

More recently, in connection with another bed rest experiment, Heuser et al. (2019) 594 proposed a methodology to deconvolute the combined effects of changes in renal Ca 595 excretion and bone loss. Using both the urine Ca isotope compositions and the measured 596 daily Ca urinary excretion rates, the authors distinguish the contribution of bone loss to the 597 variations in urinary Ca isotope compositions while cancelling out the effects of kidney 598 Rayleigh-type isotope distillation. These results stress the need for the development a more 599 thorough understanding of influence of renal function in the variability of urine Ca isotope 600 compositions while emphasizing the potential of 44/42 Ca as a diagnostic biomarker. 601 602

Calcium isotopes as biomarkers of metabolic bone diseases 603

Calcium isotopes are sensitive to other bone related disorders. Their efficiency as a 604 biomarker (i.e. sensitivity and specificity) was tested in two published clinical studies so far. 605 [START_REF] Gordon | Predicting multiple myeloma disease activity by analyzing natural calcium isotopic 905 composition[END_REF] reported significant 44 Ca-depletion of blood serum Ca in subjects 606 affected by active forms of multiple myeloma when compared to subjects with non-active 607 disease. Multiple myeloma is a form of cancer affecting the plasma cells and inducing 608 osteolytic lesions, leading to osteoporosis and bone fracture. While current methods can 609 detect osteolytic lesions only after they have caused significant damage, Ca isotopes could 610 reveal abnormal bone loss, and thus disease activity, long before it can be detected by other 611 means. This preliminary study reported good performances for Ca isotopes as a disease 612 predictor, with significant specificity and sensitivity, regardless of other factors, such as age 613 or gender. 614

Based on those preliminary yet promising first results, Eisenhauer et al. (2019) 615 reported the first clinical trial conducted to assess the potential of Ca isotopes as a diagnosis 616 biomarker for osteoporosis in 80 postmenopausal women. Compared with the outcomes of 617 the gold-standard densitometry diagnosis method, the blood and urine Ca isotope 618 compositions were significantly 44 Ca-depleted in the 14 subjects effectively affected by 619 osteoporosis, when compared to subjects without osteoporosis. Calcium isotopes appear to 620 display very strong sensitivity and good specificity to the osteoporosis condition, and are in 621 good agreement with various classical clinical parameters and chemical biomarkers of bone 622 loss. These results suggest that Ca isotopes could be developed into a stand-alone clinical 623 test for osteoporosis. 624 625 4.3.

Future directions 626

All the work to date has demonstrated that calcium isotopes have characteristics of 627 an efficient isotopic biomarker for bone loss. The clinical applications of Ca isotopes remain 628 in their infancy, but these promising results should stimulate more research to improve 629 diagnostic protocols and develop broad clinical reference databases. 630 (i.) Despite considerable progress, the understanding of how Ca isotopes correlate with 631 epidemiology of metabolic bone diseases could be improved. Continued efforts will be 632 required to advance our understanding of the Ca isotopes cycle in the whole organism both 633 in health and disease. Further characterization of the mechanisms of isotope fractionation at 634 the scale of organs and cells will also greatly benefit to this field of research. 635 (ii.) The reported intra-and inter-individual variability of blood or urine Ca isotope 636 compositions highlights the need for identifying factors of variability other than bone loss. 637 Besides dietary practices varying through time in a given individual and/or from one 638 individual to another, several nutrients and molecules are known to affect Ca homeostasis, 639 and thus potentially intra-and inter-individual variability of their Ca isotope compositions. For 640 instance, dietary Vitamin D has notably been shown to result in changes of urine Ca isotope 641 compositions [START_REF] Rangarajan | Assessing bone mineral changes in response to vitamin D supplementation using 1061 natural variability in stable isotopes of Calcium in Urine[END_REF]. Clearly, strategies need to be implemented in order 642 to take these sources of intra-and inter-individual variability into account and/or cancel them 643 out. 644 (iii.) Other physiopathological conditions affecting Ca homeostasis could be successfully 645 explored with the help of Ca isotopes including diseases or deficiencies that strongly affect 646 the Ca cycle and have high incidence in modern societies [START_REF] Peacock | Calcium Metabolism in Health and Disease[END_REF]Shroff et al., 647 2013). For instance, the progressive disruption of the kidney function in individuals suffering 648 from chronic kidney diseases is likely to affect the distribution of Ca isotopes. Other 649 disturbances of the Ca homeostasis being either induced by drugs or in some digestive 650 disorders could also be explored with Ca isotopes. Dedicated research could also potentially 651 lead to the development of new biomarkers for such conditions of disrupted Ca homeostasis. 652 The use of stable isotopes in palaeobiology and bioarchaeology is not new. From the 660 1970's onwards, palaeontologists, paleoanthropologists and archaeologists (as well as 661 modern ecologists and forensic scientists) used traditional stable isotopes (C, H, O, N) to 662 assess trophic ecology, habitat use, physiology or migration of past and present vertebrates 663 including humans [START_REF] Koch | Isotopic Study of the Biology of Modern and Fossil Vertebrates[END_REF][START_REF] Newsome | Using stable isotope biogeochemistry to 1035 study marine mammal ecology[END_REF]. 664

Beyond the currently investigated scientific questions regarding Ca isotopes, the main 665 leap one could identify lies in the practical and possibly poorly identified differences between 666 these so-called traditional stable isotopes and the developing non-traditional metal stable 667 isotopes. First, while traditional stable isotopes are routinely analysed by means of gas 668 source mass spectrometers, non-traditional stable isotopes, including Ca, require the use of 669 different mass spectrometers (mainly MC-ICP-MS but also possible with TIMS) that are so 670 far predominantly hosted by Earth sciences laboratories and require different analytical skills 671 and laboratory set-ups. These analytical challenges can be overcome, as exemplified by the 672 growing use of other isotope systems requiring these analytical set-ups, such as 87 Sr/ 86 Sr 673 ratios in archaeology. Clear communication on differences between traditional and non-674 traditional stable isotopes is also a key to successful collaborations with communities of 675 palaeobiology and bioarchaeology scientists. For instance, as a major element, Ca allows 676 favourable sampling strategies of osteological remains (small sample sizes, less destructive 677 analyses, high spatial resolution) and grants access to geological periods so far poorly 678 explored with traditional stable isotopes. Also, the elements in the focus of traditional stable 679 isotope systems do not share the same biological cycles and functions in vertebrate 680 organisms as do metal stable isotopes. While the former constitutes the very structure of a 681 variety of soft tissues building blocks, the more specific functional roles of bio-essential 682 metals allow focussing on new aspects of the biology of vertebrates. The developing use of 683 Ca isotopes by these communities can thus count on pre-existing fruitful exchanges and 684 collaborations between isotope geochemists and specialists of these disciplines but also 685 require a communication effort regarding Ca isotopes and their peculiarities. 686 687

Using Ca isotopes from fundamental to applied biomedical research 688

When taking the plunge into biomedical research and aiming at developing diagnosis 689 biomarkers, the challenges are more acute. The natural variations of traditional stable 690 isotope ratios are not routinely used by biomedical research scientists or medical doctors in 691 order to understand element cycling, characterize aetiology of diseases, nor as biomarkers, 692 notably because of their limited specificity. A few groups of isotope scientists and biomedical 693 researchers currently investigate the potential of metal stable isotope systems (e.g., Ca, Fe, 694 Cu or Zn, see reviews such as [START_REF] Costas-Rodríguez | High-precision isotopic analysis 849 of essential mineral elements in biomedicine: Natural isotope ratio variations as 850 potential diagnostic and/or prognostic markers[END_REF][START_REF] Albarède | Medical Applications of Isotope Metallomics[END_REF]. 695 However, this field of research is currently in its infancy. More generally, all involved 696 communities (stable isotope scientists, biomedical scientists and medical doctors) do not 697 have a shared scientific or technical background and may have different goals. Beyond 698 purely scientific questions, there are potential intellectual and cultural barriers that must be 699 overcome in order to implement the truly interdisciplinary collaboration that biomedical stable 700 isotope research demands. 701 702

Initiating successful interdisciplinary research projects 703

As recently reported by [START_REF] Sauzéat | Interdisciplinary Case Study: Geochemistry 1084 Meets the Clinic in Search for a Metal ALS Biomarker[END_REF], interdisciplinary collaboration requires 704 reciprocal curiosity and communication between fields, which in turn requires scientists to 705 understand each other's scientific and technic cultures well enough to identify scientific 706 questions that could be addressed collaboratively. Scientists must endeavour to make their 707 work accessible to people in other fields. For isotope scientists, this implies for instance 708 avoiding unnecessary jargon and preparing clearly written documents, or vade-mecum, that 709 explain key concepts of stable isotope metallomics to non-specialists. 710 Also, the fates of such new frontiers research projects are unknown at first and can be 711 seen as risky. It is important to encourage and support the researchers that develop 712 interdisciplinary careers. While the risk is that such profiles are potentially hard to fit in job 713 descriptions, researchers with multiple backgrounds (e.g., medical doctors or biomedical 714 researcher skilled as isotope scientist) pave the way to the future of this research. In this 715 matter, the support of institutions is particularly valuable, when for example implementing 716 interdisciplinary grant schemes and recruitment campaigns. 717 718

Analytical and methodological challenges 719

Another challenge is to identify the methodological constraints imposed by the various 720 disciplines involved in the project, and to accommodate these constraints in the study design. 721 For example, the extreme care typically required to maintain sterility and integrity of 722 biological samples for molecular biological assays is generally unnecessary for samples 723 destined for isotope analysis. On the other hand, substances commonly used as 724 preservatives in blood and urine samples can be unacceptable contaminants for isotope 725 analysis. Disciplines also have different requirements for experimental controls. In human 726 experiments involving Ca isotopes, this typically requires sampling and analysis of all dietary 727 and pharmacological Ca sources, as well as multiple pre-experimental sampling of all study 728 subjects in order to establish individual baselines. Finally, isotope geochemistry laboratories 729 are not biomedical laboratories. Ethical and health regulations often require formal 730 authorization for the handling of human tissues, as well as adaptation of laboratory practices 731 and proper training of isotope scientists, engineers, technicians or students in the hazards 732 posed by these materials. Also, isotope analyses in isotope geochemistry laboratories tend 733 to be slow when compared to routine biomedical analyses, even when machine time is not 734 limited. For Ca isotopes to become a widely used clinical tool, sample throughputs must be 735 increased by several orders of magnitude, while ensuring a sufficient precision and accuracy 736 of the isotope analysis. This is technically feasible (with approaches such as automated 737 sample preparation, spiking methods, collision-cell instruments, e.g., [START_REF] Romaniello | Fully automated chromatographic purification of Sr and Ca for isotopic analysis[END_REF], 738 Lewis et al., 2018) but will not happen without being spurred by demand. 739 740

Developing a medical biomarker 741

In general, the development of medical biomarkers is codified and standardized, for 742 instance in terms of clinical trials methodology and statistics or ethical and legal aspects. 743

These are constraints for which isotope geochemists, and their host laboratories and 744 institutions, are neither prepared nor able to address on their own. The first challenge is thus 745 to recruit the assistance of medical scientists in the regulatory and administrative aspects of 746 study design. 747

In addition to aforementioned challenges, we suggest that another lies in the differences 748 in technical and scientific cultures in which stable isotope geochemists and medical scientists 749 have evolved. For example, stable isotope geochemists tend to direct their research toward a 750 fundamental understanding of processes while medical scientists and hospital doctors may 751 feel more constrained by the need for practical and immediate tools to achieve clinical goals. 752

This urgency is something geochemists do not often encounter on their home turf. As a 753 result, because geochemists tend to assume that practical application proceeds from 754 theoretical understanding, they could tend to underestimate the current clinical utility of their 755 techniques and undersell them. 756

Finally, medical science is immersed in a biological paradigm that views life as an 757 interaction between complex organic macromolecules (including genes) at the cellular level.

Metal stable isotope analysis, focused as it is on inorganic chemistry, does not mesh well 759 with this paradigm. Apart from the practical clinical tools they can provide, isotope chemists 760 also are in a position to advance a long overdue appreciation of the central role purely 761 inorganic processes play in biology. It is possible to say a great deal about the state of an 762 organism by looking at the distribution of simple ions and isotopes, without reference to cells 763 or macromolecules, in the same way that it is possible to say a great deal about an 764 environmental or geological system without reference to the details of its component parts. 765 -

653 5 .

 5 Challenges of vertebrate Ca isotope geochemistry 654The potential of Ca isotopes is high in the field of vertebrate biology. However the 655 development of new applications and their possible adoption by scientific communities other 656 than Earth scientists faces barriers, and depend on the communities involved.

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authors found however no significant change in the44/42 Ca offset between fauna and human

Acknowledgements 767

The authors wish to thank the Elizabeth Griffith and Matthew Fantle for the invitation to 768