Cécile Bétry
email: cecile.betry@yahoo.fr

Emmanuelle Meugnier

Marie Pflieger

Guillaume Grenet

Serge Hercberg

Pilar Galan

Emmanuelle Kesse-Guyot

Hubert Vidal

Martine Laville

David Greenfield

Cécile Bétry David

High expression of CPT1b in skeletal muscle in metabolically healthy older subjects

Keywords: Ageing, Calorimetry, Glucose clamp technique, Insulin resistance, Sirtuins

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

Introduction

Ageing is a complex multifactorial and continuous process [START_REF] Aunan | Molecular and biological hallmarks of ageing[END_REF]. Given the dramatic increase in average lifespan, healthy ageing and autonomy are now regarded as both a personal and a public health challenge. To that extent, frailty, which may be a reversible step before loss of autonomy, has generated much research interest [START_REF] Ng | Nutritional, Physical, Cognitive, and Combination Interventions and Frailty Reversal Among Older Adults: A Randomized Controlled Trial[END_REF]. Frailty is defined as an age-related condition with reduced functional reserves. It is intertwined with disability, sarcopenia and various disorders, including metabolic diseases like type 2 diabetes mellitus (T2DM) [START_REF] Fried | Frailty in older adults: evidence for a phenotype[END_REF]. On the other hand, some robust people maintain good metabolic health throughout their lives, thereby highlighting the diversity of phenotypes of ageing [START_REF] Barbieri | Metabolic aspects of the extreme longevity[END_REF].

In recent years, a large bulk of research has focused on skeletal muscle ageing. Growing old is not only associated with loss of skeletal muscle mass, but also with functional alterations in skeletal muscle such as insulin resistance, and both are involved in the onset of frailty [START_REF] Barzilai | The Critical Role of Metabolic Pathways in Aging[END_REF]. Nevertheless, most of our knowledge of skeletal muscle and ageing-for example, development of insulin resistance, reduced fat-free mass, reduced mitochondrial capacity and quantity, and increased intramyocellular lipid contents-results from studies comparing older and younger subjects [START_REF] Johannsen | Ectopic Lipid Accumulation and Reduced Glucose Tolerance in Elderly Adults Are Accompanied by Altered Skeletal Muscle Mitochondrial Activity[END_REF][START_REF] Crane | The Effect of Aging on Human Skeletal Muscle Mitochondrial and Intramyocellular Lipid Ultrastructure[END_REF]. Thus, important differences in the way every individual faces growing old are rarely considered.

With the Implication of Metabolic and Genomic Modifications in Elderly Subjects

(COMPALICLAMP) study, the aim was to determine the metabolic characteristics associated with frailty and robustness in healthy older subjects. For this purpose, wholebody substrate oxidation and insulin sensitivity were assessed to provide new insights at a molecular level into the expression of a set of candidate genes in skeletal muscle. Subjects were recruited from the French cohort of the Supplémentation en Vitamines et Minéraux Antioxydants 2 (SU.VI.MAX 2) Study.

Materials and methods

SU.VI.MAX and SU.VI.MAX 2

These two studies have been well described in previous reports [START_REF] Hercberg | .VI.MAX Study: a randomized, placebo-controlled trial of the health effects of antioxidant vitamins and minerals[END_REF][START_REF] Kesse-Guyot | Adherence to nutritional recommendations and subsequent cognitive performance: findings from the prospective Supplementation with Antioxidant Vitamins and Minerals 2 (SU.VI.MAX 2) study[END_REF]. In brief, all participants in the SU.VI. those in the first tertile were assigned to the frail group, while those in the third tertile were assigned to the robust group.

The COMPALICLAMP study

From among the SU.VI.MAX 2 study participants, subjects from both the robust and frail groups were recruited, in the Lyon region (France), to take part in the COMPALICLAMP study. Inclusion criteria were age > 60 years, body mass index (BMI) score ≥ 18 kg.m -2 but < 35 kg.m -2 and stable weight for the past 3 months. Participants were also non-smokers, and free of diabetes and digestive, cardiovascular, renal, liver or tumour diseases.

Ultimately, 26 subjects (15 frail, 11 robust) were included (see 'Subject inclusion' below).

The COMPALICLAMP study was conducted according to Declaration of Helsinki guidelines and approved by the local ethics committee. Written informed consent was obtained from all subjects. The study was registered at clinicaltrials.gov as NCT00951392.

Study design

After the inclusion visit, each participant attended the Research Centre for one day of comprehensive metabolic exploration after a 10-h overnight fast. All subjects underwent percutaneous biopsy of the vastus lateralis muscle, as previously described [START_REF] Laville | Acute regulation by insulin of phosphatidylinositol-3-kinase, Rad, Glut 4, and lipoprotein lipase mRNA levels in human muscle[END_REF]. This skeletal muscle tissue (62 ± 28 mg wet weight) was taken under local anaesthesia (2% lidocaine) and immediately frozen in liquid nitrogen, then stored at -80° C. The biopsy was performed before the clamp study to avoid the confounding effect of insulin infusion on gene expression.

Euglycaemic-hyperinsulinaemic clamp

Cannulae were inserted into both antecubital veins for blood sampling, and for glucose and insulin administration. After a 1-h baseline period, the euglycaemic-hyperinsulinaemic clamp was performed for 2 h. Exogenous insulin was administered as a primed continuous infusion at a rate of 1 mIU.kg -1 .min -1 , while the variable glucose infusion rate was adjusted to maintain blood glucose concentration at the target level of 5.5 ± 0.5 mmol/L. Venous blood samples were collected every 10 min for measurement of glucose concentration using the hexokinase method (Olympus analyzer kit; bioMérieux SA, Marcy l'Etoile, France), and also at seven time points (T-20, T-10, T0, T90, T100, T110, T120 min) for measurement of plasma insulin (BI-INS immunoradiometric assay kit; CIS Bio International, Gif-sur-Yvette, France) and fatty acids (NEFA-C kit, Wako Chemicals, Neuss, Germany). The M value was determined during the final 30 min of the 2-h clamp.

Indirect calorimetry

These measurements were taken with a Deltatrac metabolic monitor (Datex-Ohmeda Oy, Helsinki, Finland) during the basal period (T-40 to T0 min) and at the end of the clamp (T60 to T120 min). Oxygen consumption (VO2 in L/min) and carbon dioxide production (VCO2 in L/min) were recorded every minute. Lipid and carbohydrate oxidation rates and resting metabolic rates were calculated using Ferrannini's equation [START_REF] Ferrannini | The theoretical bases of indirect calorimetry: a review[END_REF].

Respiratory quotient (RQ) was assessed as the VCO2/VO2 ratio. To calculate protein oxidation, urine samples were collected twice a day (at T0 and T120 min) for measurement of urinary nitrogen using a chemiluminescence method (Antek 7000; Alytek, Juvisy, France).

Body composition

This was determined using a bioelectrical multifrequency bioimpedance analyzer (Spar 50, Spengler SAS, Aix-en-Provence, France). The fat-free mass index (FFMI) was calculated by dividing fat-free mass (in kg) by height (in m) squared.

mRNA expression

In skeletal muscle Tissue samples were pulverized in liquid nitrogen, and total RNA was prepared using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions. RNA concentrations were analyzed using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). mRNA levels were measured by reverse transcription followed by real-time polymerase chain reaction (PCR). Reverse transcription was performed with 500 ng of total RNA using SuperScript II and RNAse H treatment (Thermo Fisher Scientific), as previously described [START_REF] Alligier | Subcutaneous adipose tissue remodeling during the initial phase of weight gain induced by overfeeding in humans[END_REF]. Real-time PCR was performed with a Rotor-Gene 6000 cycler (QIAGEN N.V., Venlo, The Netherlands) on a final volume of 20 µL, containing 5 µL of a 60-fold dilution of synthesized complementary DNA (cDNA) and 15 µL of reaction buffer (ABsolute Blue QPCR Mix, SYBR Green Plus ROX; Thermo Fisher Scientific), and 10.5 pmol of forward and reverse primers [START_REF] Alligier | Subcutaneous adipose tissue remodeling during the initial phase of weight gain induced by overfeeding in humans[END_REF]. A standard curve was systematically generated with eight different amounts of purified cloned cDNA, and each assay was performed in duplicate. Values were normalized against hypoxanthine phosphoribosyltransferase 1 (HPRT1), a reference gene that is similar across samples.

Based on the obtained results for lipid oxidation and whole-body insulin action (see 'Results' below), and the available data in the literature regarding alterations in skeletal muscle with increasing age [START_REF] Krishnan | Impaired substrate oxidation in healthy elderly men after eccentric exercise[END_REF][START_REF] Levadoux | Reduced whole-body fat oxidation in women and in the elderly[END_REF], our genomic study focused on the expression of a subset of genes encoding key enzymes of lipid metabolism [CPT1B and CPT2, acetylcoenzyme A (CoA) acyltransferase 2 (ACAA2), acetyl-CoA carboxylase beta (ACACB), malonyl-CoA decarboxylase (MLYCD)] and glucose metabolism [glycogen synthase 1 (GYS1)], and a key regulator of lipid oxidation [peroxisome proliferator-activated receptorgamma coactivator 1-alpha (PPARGC1A) and pyruvate dehydrogenase kinase 4 (PDK4)].

In addition, the expression of seven sirtuin genes (SIRT1-SIRT7) were also investigated, as these sirtuin proteins have been demonstrated to be major actors in the metabolism of ageing [START_REF] Yu | The Role of Sirtuins in the Control of Metabolic Homeostasis[END_REF]. The primer sequences used are listed in Table S1 (see supplementary materials associated with this article online).

In peripheral blood leucocytes

Fresh blood samples were collected in PAXgene Blood RNA System tubes (PreAnalytiX, QIAGEN) and stored at -80° C until needed for processing. Total RNA was isolated using PAXgene Blood RNA Kits (QIAGEN) as per manufacturer's instructions: 500 ng of total RNA underwent reverse transcription, followed by real-time PCR as described above.

Statistical analyses

Values are presented as either means ± standard deviation (SD) when pertaining to the entire cohort or as means ± standard error of mean (SEM) after adjustment by group (frail or robust). Variables were considered to not be normally distributed using the Shapiro-Wilk test [START_REF] Royston | Algorithm AS 181: The W Test for Normality[END_REF]. On inclusion, comparisons between the robust and frail groups were performed using Student's t test and the Wilcoxon non-parametric test when necessary. Fisher's exact test for categorical variables was applied to compare gender ratios. Similar statistical analyses were also performed to compare the biopsy group with the entire group.

In addition, given the difference in age between the two groups, subsequent comparisons were also assessed by multiple linear regression analyses adjusted for age. For each analysis, the residuals were normally distributed. Dixon's Q test was used to exclude single outliers in the mRNA analysis. Also, as CPT1b mRNA levels were not normally distributed, correlations between CPT1b and SIRT mRNA levels were determined by Spearman's correlation coefficients (rho) with bootstrap 95% confidence intervals (CIs). A value of P < 0.05 was considered significant.

All statistical analyses and plots were performed using RStudio 1.0.136 software [START_REF]RStudio: Integrated Development Environment for R[END_REF].

The RVAideMemoire package with the 'spearman.ci' function was used for the bootstrap CIs, and the outliers package with the 'dixon.test' function for excluding an outlier. Scatterplots were fitted with the 'scatter.smooth' function.

Results

Subject inclusion

A flow chart of participants' inclusion into our study is shown in Fig. 1. One of the eight frail subjects was subsequently excluded from mRNA analysis because the mRNA levels of SIRT1, SIRT2 and SIRT3 were all outliers (P < 0.001, P < 0.05 and P < 0.001, respectively, by Dixon's Q test; Fig. S1; see supplementary materials associated with this article online). Nevertheless, significance was not altered when the analysis was run with and without this subject (data not shown). Furthermore, anthropometric and metabolic characteristics at the time of screening did not differ between the biopsy group (n = 16) and the entire group (n = 26; Table S2; see supplementary materials associated with this article online).

Subject characteristics

Participants' characteristics when screening for frail and robust subjects are presented in Table I. Their average age was 67 ± 4 years, with a gender ratio of 14 men to 12 women and a mean BMI of 24.5 ± 2.9 kg.m -2 . As expected with subjects free of diabetes, their mean fasting glycaemia was 5.3 ± 0.5 mmol/L and mean HbA1c was 5.6 ± 0.3%. Given their inclusion on the basis of the ageing indicator, this was significantly lower in the frail compared with the robust group. Furthermore, frail subjects were 5 years older than robust subjects (P < 0.01); given this difference, all subsequent comparisons were adjusted for age. Otherwise, the general anthropometric and metabolic characteristics were similar between both groups.

Body composition

There were no differences in terms of body composition between the two groups, with a fat-free mass of 51.1 ± 2.4 kg vs 53.0 ± 3.3 kg, and a fat mass of 15.3 ± 1.0 kg vs 16.1 ± 1.3 kg, for frail vs robust subjects, respectively (all P > 0.6). FFMI was also similar (frail: 18.5 ± 0.5 kg.m -2 ; robust: 19.1 ± 1.0 kg.m -2 ; P > 0.6).

Whole-body substrate oxidation

As shown in Table II, resting energy expenditure (REE) did not differ between frail and robust subjects. Both groups displayed significant differences in whole-body substrate oxidation at baseline, with markedly higher lipid oxidation in robust subjects (P < 0.05). As a result, the RQ tended to be lower in that group (P = 0.07). However, during the euglycaemic-hyperinsulinaemic clamp, no differences were found, although robust subjects displayed significantly higher non-oxidative glucose metabolism (P < 0.01).

Insulin sensitivity

During the euglycaemic-hyperinsulinaemic clamp, no differences were found in glycaemia (frail: 6.1 ± 0.2 mmol/L vs robust: 6.0 ± 0.2 mmol/L), insulinaemia (frail: 37.0 ± 2.6 mIU/L vs robust: 37.5 ± 3.4 mIU/L), fatty acids (frail: 39 ± 6 μmol/L vs robust: 30 ± 6 μmol/L) or fatty acid suppression (frail: 92.1 ± 1.1% vs robust 93.7 ± 1.4%; P > 0.35 for all). The M value, an index of insulin sensitivity, was significantly higher in robust subjects (frail: 7.4 ± 0.6 mg.kg -1 .min -1 vs robust: 8.2 ± 0.8 mg.kg -1 .min -1 ; P < 0.01) even after normalization for fat-free mass (frail: 10.0 ± 0.7 mg.kg -1 .min -1 vs robust: 10.9 ± 1.2 mg.kg -

Expression of metabolic genes in skeletal muscle

The mRNA levels of eight genes involved in skeletal muscle lipid and glucose metabolism are presented in Fig. 2. The level of CPT1b mRNA was around four times higher in robust subjects (P < 0.05). Moreover, it correlated with the ageing indicator (rho: 0.734, 95% CI: 0.343-0.881; P < 0.01). Otherwise, mRNA levels did not significantly differ between the two groups.

Expression of sirtuin genes

Skeletal muscle expression levels of the seven SIRT genes (SIRT1-SIRT7) are reported in Fig. 3 (except for SIRT4, which was undetectable); SIRT2 and SIRT6 mRNA levels were lower in robust than in frail subjects (P < 0.05 and P < 0.01, respectively).

Sirtuin gene expression was also measured in peripheral blood leucocytes to potentially determine circulating biomarkers able to discriminate robust from frail subjects. However, SIRT1, SIRT2 and SIRT6 mRNA levels did not differ between the two groups when measured in peripheral blood leucocytes (Table S3; see supplementary materials associated with this article online).

Correlation between CPT1b and sirtuin gene expression

As revealed in Fig. 4, mRNA expression levels of CPT1b strongly correlated with SIRT1 (rho: 0.675, 95% CI: 0.122-0.935; P < 0.01) and SIRT6 (rho: -0.814; 95% CI: -0.936, -0.512; P < 0.001) in skeletal muscle. Using multiple linear regression analysis with CPT1b as the dependent variable, and SIRT1 or SIRT6, age, gender and BMI as independent variables, both SIRT1 and SIRT6 remained significantly correlated (P < 0.01 and P < 0.05, respectively; data not shown).

Discussion

With increasing age, age-related changes in skeletal muscle mass and function are at least partly responsible for a wide range of disabilities and metabolic diseases, including T2DM. Until now, physiological and metabolic studies in the field of skeletal muscle ageing have largely been based on comparisons between older and younger subjects. However, phenotypes in older individuals are heterogeneous, and a better understanding of the metabolic differences among older people may help to identify relevant targets for promoting healthy metabolic ageing.

Our present study compared robust and frail, but otherwise disease-free, older individuals to better understand the potential factors involved in metabolic health with older age. The most striking result to emerge from our data was that robust subjects had a fourfold higher level of CPT1b mRNA than did frail subjects. In good agreement with such overexpression, those subjects also oxidized more lipid in a fasting state. These findings support preclinical studies in which experimental overexpression of CPT1b enhanced fatty acid oxidation and, more importantly, partially reversed the skeletal muscle alterations associated with ageing [START_REF] Bruce | Overexpression of carnitine palmitoyltransferase-1 in skeletal muscle is sufficient to enhance fatty acid oxidation and improve high-fat diet-induced insulin resistance[END_REF][START_REF] Henique | Increased mitochondrial fatty acid oxidation is sufficient to protect skeletal muscle cells from palmitate-induced apoptosis[END_REF]. It may also be assumed that the overexpression of CPT1b accounts for the greater insulin sensitivity of robust subjects due to their lower intramyocellular lipid contents. This hypothesis is consistent with a previous clinical study showing that levels of metabolites derived from intramyocellular lipids is the same in older insulin-sensitive and younger subjects, whereas it is higher in older insulin-resistant subjects [START_REF] Chee | The relative contribution of intramyocellular lipid to whole body fat oxidation is reduced with age, but subsarcolemmal lipid accumulation and insulin resistance are only associated with overweight individuals[END_REF]. Moreover, in that same study, levels of CPT1b mRNA transcripts were higher, albeit not significantly so, in older insulin-sensitive subjects than in older insulinresistant subjects [START_REF] Chee | The relative contribution of intramyocellular lipid to whole body fat oxidation is reduced with age, but subsarcolemmal lipid accumulation and insulin resistance are only associated with overweight individuals[END_REF]. In the present study during the euglycaemic-hyperinsulinaemic clamp, our robust subjects also displayed higher levels of non-oxidative glucose metabolism, which is associated with higher levels of glucose storage in muscle and has been related to better insulin sensitivity [START_REF] Vaag | Decreased insulin activation of glycogen synthase in skeletal muscles in young nonobese Caucasian first-degree relatives of patients with non-insulin-dependent diabetes mellitus[END_REF]. These findings are in agreement with preclinical data showing that the overexpression of CPT1b in skeletal muscle induces an increase in glycogen content [START_REF] Hénique | Increasing mitochondrial muscle fatty acid oxidation induces skeletal muscle remodeling toward an oxidative phenotype[END_REF].

Our present data also suggest that sirtuin proteins and, more particularly, SIRT1 and SIRT6 could be involved in the overexpression of CPT1b during ageing. Although SIRT1 expression did not significantly differ between our two groups, SIRT6 and SIRT2 expression was lower in our robust subjects. In line with this observation, SIRT2 expression was higher in insulin-resistant skeletal muscle cells, whereas inhibition of SIRT2 improved insulin sensitivity in a model in vitro [START_REF] Arora | SIRT2 negatively regulates insulin resistance in C2C12 skeletal muscle cells[END_REF]. The lower expression of SIRT6 in our robust subjects was also consistent with a previous preclinical study showing an increase of SIRT6 mRNA level with increasing age that can be reversed by exercise [START_REF] Koltai | Exercise alters SIRT1, SIRT6, NAD and NAMPT levels in skeletal muscle of aged rats[END_REF].

However, our research has a few limitations. First, frailty assessment in geriatric populations remains controversial. While the initial score as per Fried criteria is based only on physical aspects, other scores have since been further developed for multidimensional evaluations [START_REF] Fried | Frailty in older adults: evidence for a phenotype[END_REF][START_REF] Clegg | Diagnostic test accuracy of simple instruments for identifying frailty in community-dwelling older people: a systematic review[END_REF]. In fact, the present study used a new composite ageing indicator based equally on cognitive assessments and physical tests to determine our subgroups of frail and robust subjects. Unexpectedly, the frail subjects were older than the robust subjects, thereby requiring adjustment for age in further analyses. Nevertheless, such a discrepancy has been reported in previous studies based on other frailty scores, highlighting the difficulty of assessing frailty independently of age [START_REF] Watanabe | Relationship Between Frailty and Oral Function in Community-Dwelling Elderly Adults[END_REF][START_REF] Lin | Association between Physical Fitness and Successful Aging in Taiwanese Older Adults[END_REF]. Furthermore, both of our groups displayed similar metabolic and anthropometric profiles at screening, including comparable levels of fasting glucose, fasting lipid, HbA1c and blood pressure. This suggests that our new ageing indicator is able to identify subjects at an early stage before the onset of significant metabolic changes [START_REF] Cederholm | Diagnostic criteria for malnutrition -An ESPEN Consensus Statement[END_REF]. Second, the number of subjects was relatively low in each of our groups. Yet, despite this, significant differences were demonstrated in both metabolic parameters and gene expression levels, although smaller differences, especially the level of SIRT1 expression, may not have been detected in our study. Finally, only gene expression was measured and, thus, there is the possibility that the increase in CPT1b expression was not associated with an increase in levels of the protein itself. However, functional measurement of lipid oxidation by indirect calorimetry was in accordance with greater activity of CPT1b in our robust subjects.

The present study represents a first step in our understanding of the factors involved in metabolic health with increasing age. The results are promising and need to be validated by a larger sample size; our findings also suggest that the expression of CPT1b should now be assessed in longitudinal studies. Such studies could confirm that CPT1b expression increases with age to limit both intramyocellular lipid deposition and the development of insulin resistance. As previous preclinical studies have shown that physical activity stimulates CPT1b expression [START_REF] Morifuji | Dietary soya protein intake and exercise training have an additive effect on skeletal muscle fatty acid oxidation enzyme activities and mRNA levels in rats[END_REF][START_REF] Niu | Aerobic exercise's reversal of insulin resistance by activating AMPKα-ACC-CPT1 signaling in the skeletal muscle of C57BL/6 mice[END_REF], further work should also aim to determine whether the amount of physical activity is directly related to CPT1b expression in older subjects.

Moreover, it has been shown that physical training can improve muscle mitochrondrial quantity and oxidative capacity in older people [START_REF] Irving | Combined training enhances skeletal muscle mitochondrial oxidative capacity independent of age[END_REF]. In this case, a physical training programme might reverse the metabolic abnormalities associated with frailty at an earlier stage. Also, the putative effects of sirtuin histones on CPT1b gene expression need to be explored in more detail in further experimental investigations.

In conclusion, our present study has demonstrated that robust subjects are more insulin-sensitive, oxidize more lipid in a fasting state and store more glucose during a euglycaemic-hyperinsulinaemic clamp test compared with frail subjects. The overexpression of CPT1b in robust subjects could at least partly explain their better metabolic profile and might even limit the development of subsequent metabolic changes.

In addition, our research may help to determine strategies to reverse frailty at an early stage and to promote metabolic health with increasing age. the writing of this article (grant number 40184). These funding bodies had no involvement in the design/conduct of the research, in data analysis/interpretation or in writing/approving the manuscript.

Legends to figures

  MAX 2 Study were invited to undergo a medical checkup, including an overall clinical examination. They also underwent the Mini-Mental State Examination (MMSE) and Dubois five-word test (5WT) for assessment of cognitive function, as well as the self-reported Center for Epidemiologic Studies Depression Scale (CES-D) and Cognitive Difficulties Scale (CDS). Furthermore, a muscle performance evaluation was carried out, including the Timed Up & Go (TUG) Test, Chair Stand Test, and tests of walking speed and of balance over a 30-sec interval. A score ranging from 0, the worst, to 3, the best, was subsequently assigned to each test/scale. From this assessment, a composite ageing indicator was computed as the sum of the quartile of the six tests-namely, the MMSE, 5WT, TUG Test, Chair Stand Test, walking speed and balance tests, and CES-D (reversed) and CDS. Thus, their potential ageing indicator scores ranged from 0 to 24. Participants were grouped into tertiles according to this ageing indicator:

Fig. 1 .

 1 Fig. 1. Flow chart of participant inclusion in the study.

Fig. 2 .

 2 Fig. 2. Skeletal muscle expression of eight genes involved in skeletal muscle metabolism-

Fig. 3 .

 3 Fig. 3. Skeletal muscle expression of sirtuin (SIRT) genes in frail (n = 7) and robust (n = 8)

Fig. 4 .

 4 Fig. 4. Scatterplots illustrating the relationship between carnitine palmitoyltransferase 1b

.min -1 ; P < 0.05).

Acknowledgments

We are grateful to Aisling Burns for English proofreading.

Funding:

The SU.VI. MAX 2 Study was funded by the French National Research Agency (grant number ANR-05-PNRA-010) and French Ministry of Health. C.B. was funded by the Fondation pour la Recherche Médicale (FRM; Foundation for Medical Research) during

Conflicts of interest: none

Appendix supplementary material

Supplementary materials (Fig. S1, Tables S1-S3) associated with this article can be found at http://www.scincedirect.com at doi . . .

Supplementary figure legend


T Bertin

D Mondelain

E V Karlovets

S Kassi

V I Perevalov

A Campargue
email: alain.campargue@univ-grenoble-alpes.fr

High sensitivity cavity ring down spectroscopy of N2O near 1.74 µm

Keywords: Nitrous oxide, 14 N2 16 O, CRDS, Vibration-rotational transitions, Line positions, Line intensities

In spite of being a greenhouse gas with a large global warming potential, the absorption spectrum of nitrous oxide in the near infrared is insufficiently characterized. In the present work, the spectral region near 1.74 µm (5695-5910 cm -1 ) is investigated by high sensitivity cavity ring down spectroscopy (CRDS).The noise level of the CRDS spectra corresponds to a typical minimum detectable absorption, αmin, below 10 -10 cm -1 . 3326 transitions are measured and rovibrationally assigned to 50 bands of five nitrous oxide isotopologues ( 14 N2 16 O, 14 N 15 N 16 O, 15 N 14 N 16 O, 14 N2 18 O and 14 N2 17 O) in natural isotopic abundance. The weakest lines assigned have an intensity below10 -28 cm/molecule. For comparison, only three 14 N2 16 O bands are included in the HITRAN database in the region, with a 2×10 -25 cm/molecule intensity cut off. The rovibrational assignments of the spectra were performed by comparison with predictions performed for each isotopologue in the frame of the effective operator approach.

The overall quality of the predictions is satisfactory for line positions. Deviations larger than 0.1 cm -1 are nevertheless noted for 14 N2 16 O and 14 N2 18 O.

The spectroscopic parameters of the upper level of the observed bands were derived from the standard band-by-band fit of the measured line positions. A significant number of bands were found to be perturbed by local rotational perturbations and in some cases, extra lines due to an intensity transfer could be assigned. The interaction mechanisms and the perturbers were univocally identified on the basis of the effective Hamiltonian model. In particular, interpolyad couplings were evidenced indicating that the polyad version of the effective Hamiltonian has to be extended to include Coriolis and interpolyad anharmonic interactions. No satisfactory modeling of the N2O line intensities is yet available in the region.

The CRDS intensity values derived in this work provides a solid set of measurements for future empirical intensity modeling in the region.

Introduction

Nitrous oxide is a powerful greenhouse gas with a Global Warming Potential about 300 times stronger than that of carbon dioxide. Over the last century, the N2O atmospheric concentration has experienced a steep increase from about 270 ppb to 330 ppb mainly due to anthropogenic sources. The present contribution takes part in a long standing effort to better characterize the absorption spectrum of nitrous oxide in the near and short wavelength infrared. In the last years, high sensitivity absorption spectra were recorded by cavity ring down spectroscopy (CRDS), extending importantly previous observations by Fourier Transform Spectroscopy (FTS). The large 5900-8334 cm -1 range (1.7-1.2 µm) was covered using a series of about one hundred fibered distributed feed-back (DFB) laser diodes [START_REF] Liu | High sensitivity CW-cavity ring down spectroscopy of N2O near 1.5l μm (I)[END_REF][2][START_REF] Liu | High sensitivity CW-cavity ring down spectroscopy of N2O near 1.5 μm (III)[END_REF][START_REF] Liu | High sensitivity CW-Cavity Ring Down Spectroscopy of N2O near 1.28 μm[END_REF][START_REF] Lu | High Sensitivity CW-Cavity Ring Down Spectroscopy of N2O between 6950 and 7653 cm -1 (1.44-1.31 µm): I. Line positions[END_REF][START_REF] Karlovets | High sensitivity CW-Cavity Ring Down Spectroscopy of N2O between[END_REF] and an external cavity diode laser (ECDL) [START_REF] Karlovets | High sensitivity Cavity Ring Down Spectroscopy of N2O near 1.22 µm: (I) Rovibrational assignments and band-by-band analysis[END_REF][START_REF] Tashkun | High sensitivity Cavity Ring Down Spectroscopy of N2O near 1.22 µm: (II) 14 N2 16 O line intensity modeling and global fit of 14 N2 18 O line positions[END_REF]. In the present study, we extend the studied spectral interval to lower energy by covering the 5695-5910 cm -1 range using a newly purchased set of DFB laser diodes. The high sensitivity of the recordings (typical noise equivalent absorption of the spectra on the order of αmin~ 8×10 -11 cm -1 ) allowed us for measuring weak lines with intensities below 10 -28 cm/molecule. Overall, more than 4300 lines were measured in the studied region. The rovibrational assignments were performed on the basis of the predictions of the effective Hamiltonian models developed for each isotopologue of nitrous oxide present in natural isotopic abundance in the studied sample. As a result more than 3300 transitions belonging to 50 bands could be assigned to five isotopologues ( 14 N2 16 O, 14 N 15 N 16 O, 15 N 14 N 16 O, 14 N2 18 O and 14 N2 17 O).

After the description of the data acquisition and of the line list construction in the next section, the rovibrational assignments are presented in Section 3. Section 4 is devoted to the band-by-band derivation of the spectroscopic constants of the upper levels and to the analysis of the evidenced rovibrational perturbations. Finally, we discuss in Section 5 the comparison with the line lists calculated in the frame of the effective operator approach.

Experimental setup

The reader is referred to Ref. [START_REF] Kassi | Cavity ring down spectroscopy with 5×10 -13 cm -1 sensitivity[END_REF] for general description of the CRDS setups developed in our group. The spectra recordings were performed with the CRD spectrometer used recently for investigating the 1.75 µm transparency window of carbon dioxide [START_REF] Čermák | High sensitivity CRDS of CO2 in 1.74 µm transparency window. A validation test for the spectroscopic databases[END_REF][START_REF] Karlovets | Analysis and theoretical modelling of the 18 O enriched carbon dioxide spectrum by CRDS near 1[END_REF][START_REF] Karlovets | The 13 CO2 absorption spectrum by CRDS near 1.74 µm[END_REF]. It is of the same type as the DFB setup used in our previous investigations of nitrous oxide at higher energy [START_REF] Liu | High sensitivity CW-cavity ring down spectroscopy of N2O near 1.5l μm (I)[END_REF][2][START_REF] Liu | High sensitivity CW-cavity ring down spectroscopy of N2O near 1.5 μm (III)[END_REF][START_REF] Liu | High sensitivity CW-Cavity Ring Down Spectroscopy of N2O near 1.28 μm[END_REF][START_REF] Lu | High Sensitivity CW-Cavity Ring Down Spectroscopy of N2O between 6950 and 7653 cm -1 (1.44-1.31 µm): I. Line positions[END_REF][START_REF] Karlovets | High sensitivity CW-Cavity Ring Down Spectroscopy of N2O between[END_REF]. Using a set of eight DFB laser diodes (from Eblana Photonics) as light sources, the 5695-5900 cm -1 spectra interval is now accessible for our CRDS recordings. Each laser diode tuning ranges about 20 cm -1 by temperature tuning from -5°C to 55°C allowing for the coverage of the 5693-5850 cm -1 range with only a few spectral gaps: 5714.71-5722.06, 5742.72-5745.47, 5773.38-5778.54, 5847.59-5858.01, 5882.35-5883.52 cm -1 . The finesse of the cavity varied from 130,000 at 5850 cm -1 to 200,000 at 5700 cm -1 resulting in measured ring down (RD) times from 200 μs to 300 μs, respectively. About 30 to 100 RD events were averaged per spectral point.

Fig. 1 CRDS spectrum of natural nitrous oxide near 5810 cm -1 . The sample pressure was 1.0 Torr. Four successive enlargements illustrate the high dynamics of the recordings giving access to absorption coefficients from 10 -6 cm -1 to the noise level αmin~ 5×10 -11 cm -1 .

The cell was filled with N2O (Alphagaz N40, purity>99.99%) in normal isotopic abundance at a pressure of 1.0 Torr. The cell temperature was measured with a temperature sensor (TSic 501, IST-AG, 0.1 K accuracy) fixed on the cell surface, covered by an external blanket of foam for thermal isolation. During the recordings reported here, the cell temperature varied between 293.4 and 295.1 K. The gas pressure was continuously measured by a capacitance gauge (MKS Baratron, 10 Torr, 0.25 % accuracy of the reading).

During the spectrum recording, a wavemeter measured the wavenumber of the laser emission at each spectral step of typically 0.002 cm -1 giving a first frequency calibration of the spectrum. This calibration was then refined using accurate N2O line positions provided by the HITRAN2016 database [13]. This leads to an estimated uncertainty of 1×10 -3 cm -1 on the line center determination of unblended lines.

The minimum detectable absorption coefficient (evaluated as the rms of the base line fluctuation), αmin, varies between 1×10 -10 cm -1 and 4×10 -11 cm -1 depending on the RD time. As a result, line intensities spanning four orders of magnitude (from 5×10 -25 to 5×10 -29 cm/molecule) could be retrieved from the measured spectra. The successive enlargements of the spectra presented in Fig. 1 illustrate the sensitivity and high dynamic range of the recordings and the noise level on the order of αmin~ 5×10 -11 cm -1 . The observed high spectral congestion made the rovibrational assignment particularly laborious. Fig. 2 Fragment of the CRDS (top panel) and simulated (middle panel) room temperature spectra of N2O recorded at 1 Torr near 5795 cm -1 . The residuals between measured and simulated spectra are given in the bottom panel. The stick spectrum in the middle panel corresponds to the retrieved CRDS line list.

Fig. 3

Overview of the spectrum of N2O in natural isotopic abundance in the 5695-5910 cm -1 region. Upper panel: CRDS line list (black circles) obtained in this work. The HITRAN line list which is superimposed consists of bands of 14 N2 16 O (green) and 14 N2 18 O (red). Narrow spectral gaps in the CRDS line list correspond to interval inaccessible with the laser sources at disposal. Lower panel: Line list predicted within the effective operator approach for the various N2O isotopologues. Calculated intensities are rough estimations useful for rovibrational assignments (see Text).

The line centres and line intensities were obtained by using a homemade interactive least squares multi-lines fitting code written in LabVIEW. A Voigt profile with the width of the Gaussian component fixed to the calculated Doppler broadening was adopted for each line. At the pressure of the recordings (1 Torr), the self collisional width is less than 10 -3 cm -1 (HWHM) [13]. This value is significantly smaller than the Doppler broadening of 5.3× 10 -3 cm -1 . For the weak lines, the Lorentzian width was fixed by default to HITRAN value but for the medium and strong lines, significant residuals were observed and the quality of the spectra allowed refining the collisional width. It is difficult to attach individual error bar to the retrieved line intensities. In the case of unblended lines with intensity larger than 3×10 -26 cm/molecule, we estimate the intensity uncertainty to be better than 5 %. This value can reach 50% for weaker lines in particular when they are blended.

The global line list was obtained by gathering line parameters retrieved from the different recordings. It counts 4337 lines presented in the upper part of Fig. 3 which also includes the HITRAN2016 line list for comparison. HITRAN line list consists in the strongest bands of the main isotopologue, 14 N2 16 O and of the strongest bands of the fourth abundant isotopologue, 14 N2 18 O. As shown on Fig. 3, part of the HITRAN lines of 14 N2 16 O are absent from our list. This is due to the fact that they induce a large absorption which results in a very weak transmitted signal by the cavity making the measurement of the ring down time inaccurate. Note that, as a result of the 3.6×10 -4 isotopic abundance of 14 N2 18 O, all the bands included in the HITRAN list for this isotopologue have line intensities below the HITRAN intensity cut off of the main isotopologue 14 N2 16 O (2×10 -25 cm/molecule). As a result, the HITRAN line list is thus largely incomplete above the intensity cut off of the 14 N2 18 O isotopologue (10 -29 cm/molecule).

Rovibrational assignments

In a first step, the presence of impurity lines in the line list, in particular water vapor lines, was searched. By comparison with the HITRAN database [13], 27 water lines were identified and discarded. The rovibrational assignments were performed on the basis of predictions performed for each isotopologue using the effective operator approach. Predicted line lists were generated using the effective Hamiltonians (EH) and effective dipole moment (EDM) operators. The EH has a polyad structure resulting from the approximate relations ω3≈ 2ω1≈ 4ω2 between the harmonic normal mode frequencies. The diagonal blocks are characterized by the polyad number, P, given by P= 2V1+V2+ 4V3 where V1, V2, and V3 denote the normal mode vibrational quantum numbers. The EH used for the different isotopologues are presented in the following references: Ref. [START_REF] Perevalov | Global modeling of the line positions of 14 N2 16 O within the framework of the polyad model of effective Hamiltonian[END_REF] ( 14 N2 16 O), Ref. [START_REF] Ni | Fourier-transform spectroscopy of 14 N 15 N 16 O in the 3800-9000 cm -1 region and global modelling of its absorption spectrum[END_REF] ( 14 N 15 N 16 O), Ref. [START_REF] Tashkun | Global fittings of 14 N 15 N 16 O and 15 N 14 N 16 O vibrational-rotational line positions using the effective Hamiltonian approach[END_REF] ( 15 N 14 N 16 O) and Ref. [START_REF] Tashkun | High sensitivity Cavity Ring Down Spectroscopy of N2O near 1.22 µm: (II) 14 N2 16 O line intensity modeling and global fit of 14 N2 18 O line positions[END_REF] ( 14 N2 18 O). In the case of the 14 N2 17 O isotopologue, a preliminary set of EH parameters fitted to very restricted number of the line positions was used [START_REF] Vlasova | Global fittings of the line positions of the rare isotopic species of the nitrous oxide molecule[END_REF].

In the studied region, all but three bands belong to ΔP= 10 series of transitions. Three 14 N2 16 O bands (3310-0000, 3420-0110 3400-0110 near 5722.8, 5744.7 and 5784.5 cm -1 , respectively) belong to the ΔP= 9 series. In principle, the calculations of the line intensities within the effective operator approach require EDM parameters for each ΔP series and for each isotopologue. This is particularly true in the case of N2O where interpolyad couplings strongly impact the band intensities and differ importantly between the different isotopologues. In the present case, the required sets of ΔP= 9 and 10 EDM parameters are only available for the main isotopologue [START_REF] Daumont | Line intensity measurements in 14 N2 16 O and their treatment using the effective dipole moment approach. I. The 4300-5200 cm -1 region[END_REF][START_REF] Daumont | Line intensity measurements in 14 N2 16 O and their treatment using the effective dipole moment approach. II. The 5400-11,000 cm -1 region[END_REF] which makes the predictions of the line intensities of the minor isotopologues quite hazardous. Nevertheless, although not accurate, intensity predictions are needed for rovibrational assignments based on the comparison of the experiment to the predictions. This is why, the EDM parameters of 14 N2 16 O were used to compute line intensities for all the isotopologues.

The overview of the resulting predicted line list for N2O in natural isotopic abundance is compared to the CRDS line list in Fig. 3. The level of agreement observed at the scale of the overview does not hold at a detailed scale, in particular for line intensities. Nevertheless, in spite of their rough accuracy, the line intensity criterion was crucial in the assignment process.

The assignments of the 14 N 15 N 16 O and 15 N 14 N 16 O lines were made easier by the fact that the line position predictions are accurate as they reproduce FTS measurements obtained with enriched species [START_REF] Ni | Fourier-transform spectroscopy of 14 N 15 N 16 O in the 3800-9000 cm -1 region and global modelling of its absorption spectrum[END_REF][START_REF] Song | Fourier-transform spectroscopy of 15 N 14 N 16 O in the 3500-9000 cm -1 region[END_REF] which include our observations. Fig. 4 Example of spectrum interval involving the contribution of four N2O isotopologues in natural isotopic abundance.

At the final step of the analysis, a total of 3326 transitions could be rovibrationally assigned to 50 bands of the first five nitrous oxide isotopologues, leaving unassigned 984 weak or super weak lines. A sample of spectra involving contributions of four isotopologues is presented in Fig. 4. Table 1 presents the statistics of the assignments. The assigned line list is provided as Supplementary Material. In the case of the principal isotopologue, 14 N2 16 O, 30 bands were assigned. All but five are newly reported while the observations are significantly extended towards high J values for the bands previously reported by FTS (see Fig. 3). The four 14 N2 18 O and two 14 N2 17 O bands are the first ones of these species reported in the studied region. Fig. 5 shows an overview comparison between the experimental and predicted line lists for each isotopologue. The list of the observed bands is given in Table 2. Previous observations in the studied region were all performed by Fourier transform spectroscopy (FTS). The three strongest 14 N2 16 O bands (1401-0000, 2311-0110 and 2201-0000 near 5762.37, 5873.3 and 5888.1 cm -1 , respectively) are those included in the HITRAN database (see Fig. 3). They were first analyzed by Amiot and Guelachvili from spectra recorded with a 60 m absorption path length [START_REF] Amiot | Vibration-rotation bands of 14 N2 16 O: 1.2 micron-3.3 micron region[END_REF]. The HITRAN line parameters of these bands are those retrieved by Toth using absorption path lengths up to 433 m [START_REF] Toth | Line positions and strengths of N2O between 3515 and 7800 cm -1[END_REF]23]. In addition, Toth reported the 4001-1000 weaker band centered near 5929.8 cm -1 which is not included in the HITRAN database. Finally, the 2311-0110 hot band centered at 5737.5 cm -1 was analyzed by Wang et al. on the basis of an FTS spectrum recorded with 105 m path length [START_REF] Wang | Fourier transform spectroscopy of N2O weak overtone transitions in the 1-2 μm region[END_REF]. In the considered region, the 3001-0000 band of 14 N 15 N 16 O and 15 N 14 N 16 O are the only bands of minor isotopologues detected by FTS of natural N2O [START_REF] Toth | Line positions and strengths of N2O between 3515 and 7800 cm -1[END_REF]. Nevertheless, using samples with a very high isotopic enrichment (>97%) and a path length on the order of 100 m, Ni et al. and enriched samples, did not provide observations in our spectral region. Fig. 5 Overview comparison between the CRDS observations in the 5695-5910 cm -1 spectral region and the predictions within the framework of the method of effective operators for the five N2O isotopologues contributing to the spectrum. Note that there are a few spectral gaps in the experimental line list which could not be covered with the diode lasers at disposal. On the upper panel, the HITRAN line list of 14 N216 O (pink circles) is superimposed to the corresponding CRDS line list.

Band-by-band analysis and rovibrational perturbations

Let us first emphasize that in our region nitrous oxide band assignment in terms of quantum numbers of the harmonic oscillators is ambiguous. There are a number of strong anharmonic and Coriolis resonance interactions in this molecule which lead to a delocalisation of the eigenfunctions in the basis of the harmonic oscillators eigenfunctions.

For a given eigenfunction, several harmonic oscillator basis functions can have a large contribution with similar amplitude, leading to ambiguities in the normal mode vibrational labelling. In addition, two or even three eigenfunctions may correspond to the same dominant basis function leading to different vibrational bands with the same normal mode labelling.

Moreover the contributions can change strongly with the J rotational quantum number. For the unambiguous assignment of a vibrational state, the {P ℓ2 i} triplet is used, where P is a polyad number, ℓ2 is vibrational angular momentum quantum number. The integer index, i, increasing with energy, numbers the energy levels with the same P and ℓ2 values, starting with

i= 1.
The standard expression of the vibration-rotation energy levels was used for the bandby-band fit of the spectroscopic parameters:

3 3 2 2 ) 1 ( ) 1 ( - ) 1 ( ) ( + + + + + = J J H J J D J J B G J F v v v v v (1)
where Gv is the vibrational term value, Bv is the rotational constant, Dv and Hv are the centrifugal distortion constants, J is the angular momentum quantum number. The lower state constants were constrained to the values obtained by Toth [START_REF] Toth | Line positions and strengths of N2O between 3515 and 7800 cm -1[END_REF]23]. As the e and f sub-bands may be perturbed in a different way, they were fitted independently. The derived constants are listed in Table 2. The rms values of the differences between the fitted and calculated positions are smaller than 10 -3 cm -1 , which is consistent with our claimed accuracy on line positions.

The detailed results of the band-by-band fit of the spectroscopic parameters are provided as Supplementary Materials. For the bands with rotational structure extending above the high energy border of the investigated region (5910 cm -1 ), the set of line positions used in the fit was complemented with CRDS measurements from Ref. [2] which covers the 5905-6300 cm -1 range. In absence of CRDS values (spectral gaps or too strong absorption), we also completed our set of line positions with HITRAN values for the three strongest 14 N2 16 O bands [13,23].

The added HITRAN line positions (279 in total) are indicated in the Supplementary Material.

Fig. 6

Resonance coupling in two 14 N2 16 O sub-bands leading to local perturbation of line positions and line intensities: 0821f-0220f and 2201e-0000e (left and right hand, respectively). The 0821f upper level (P= 12) is coupled with the 5110f level (P=11) through an interpolyad Coriolis resonance interaction with an energy crossing around Jcross= 16 [2]. The 2201e upper level (P= 10) is coupled with the 1820e level (P= 10) through an intrapolyad anharmonic + ℓ-type resonance interaction with an energy crossing around Jcross= 56. From top to bottom: (i) Predictions of the effective model of 14 N2 16 O [START_REF] Perevalov | Global modeling of the line positions of 14 N2 16 O within the framework of the polyad model of effective Hamiltonian[END_REF], (ii) CRDS line list including a number of extra lines (red circles) due to an intensity transfer to the dark state (A number of the strongest lines of the 2201e-0000e band were taken from the HITRAN database), (iii) Differences between the experimental and EH predicted values of the line positions. As the extra lines of the 0821f-0220f are absent in the effective operator model, the deviations were calculated using the EH predicted positions of the corresponding 0821f-0220f lines, (iii) Differences between the experimental line positions and the corresponding values computed using the spectroscopic parameters (SP) of the upper state derived in this work (Table 2). The plotted shift of the extra lines is relative to the line position calculated with the bright state SP.

A significant number of bands were found to be affected by a local perturbation induced by a resonance vibration-rotation interaction. In those cases, the perturbed line positions were excluded from the fit of the spectroscopic parameters. Table 3 lists the perturbed bands together with the corresponding perturbers and interaction mechanisms identified on the basis of the effective Hamiltonian model. The value (Jcross) of the angular momentum quantum number at which the energy level crossing between bright and dark perturber states takes place is given in the table.. When the coupling involves vibrational states belonging to the same polyad (intrapolyad interaction), the perturbation may be satisfactorily predicted by the EH model but in two cases, an interpolyad coupling was found responsible of the perturbation. For three bands (0821-0220 and 2201-0000 of 14 N2 16 O and 3111-0110 of 14 N2 18 O), a few extra lines could be identified around the energy crossing lines, using lower state combination relations. Fig. 6 compares the observations to the EH predictions for the 0821-0220 and 2201-0000 bands of 14 N2 16 O affected by inter-and intrapolyad interaction, respectively. As expected, around the energy crossing, the interpolyad interaction induces important shifts of the line positions compared to both the EH predictions and the spectroscopic parameters (SP) calculations. At the opposite, in the case of the intrapolyad interaction of the 2201-0000 band, the EH positions of the perturbed lines are very close to the measurements even for the extra lines. b Previous reference in which the perturbation of the upper state was observed.

Discussion

Fig. 7 shows an overview comparison between the CRDS positions values and the corresponding EH model predictions [START_REF] Tashkun | High sensitivity Cavity Ring Down Spectroscopy of N2O near 1.22 µm: (II) 14 N2 16 O line intensity modeling and global fit of 14 N2 18 O line positions[END_REF][START_REF] Perevalov | Global modeling of the line positions of 14 N2 16 O within the framework of the polyad model of effective Hamiltonian[END_REF][START_REF] Ni | Fourier-transform spectroscopy of 14 N 15 N 16 O in the 3800-9000 cm -1 region and global modelling of its absorption spectrum[END_REF][START_REF] Tashkun | Global fittings of 14 N 15 N 16 O and 15 N 14 N 16 O vibrational-rotational line positions using the effective Hamiltonian approach[END_REF]. As only few EH parameters of the 14 N2 17 O isotopologue were fitted to very restricted number of the line positions [START_REF] Vlasova | Global fittings of the line positions of the rare isotopic species of the nitrous oxide molecule[END_REF], this isotopologue is excluded from the comparison.

As expected, the agreement is very good for the 14 N 15 N 16 O and 15 N 14 N 16 O species for which the EH model relies on extensive measurements by FTS of highly enriched samples [START_REF] Ni | Fourier-transform spectroscopy of 14 N 15 N 16 O in the 3800-9000 cm -1 region and global modelling of its absorption spectrum[END_REF][START_REF] Song | Fourier-transform spectroscopy of 15 N 14 N 16 O in the 3500-9000 cm -1 region[END_REF]. The average and rms values of the (EH-CRDS) position differences are 1.2×10 -3 and 1.8×10 -3 cm -1 for 14 N 15 N 16 O, respectively and 6×10 -4 and 1.6×10 -3 cm -1 for 15 N 14 N 16 O, respectively. Considering the frequent blending and the weakness of the absorption lines of these minor isotopologues in natural abundance in the CRDS spectra, these values are satisfactory. In the case of the main isotopologue, the largest deviations (up to about 0.2 cm -1 ) are due to the interpolyad perturbation of the 0821f-0220f sub band. Deviations up to 0.035 cm -1 are observed for high J transitions of the 2311f-0110f sub-band which is also perturbed by interpolyad Coriolis resonance interaction. Excluding 5 % of the measured line positions, the average and rms values of the (EH-CRDS) position differences of 14 N2 16 O are 3.2×10 -4 and 4×10 -3 cm -1 , respectively. In the case of 14 N2 18 O, the set of EH parameters of Ref. [START_REF] Tashkun | High sensitivity Cavity Ring Down Spectroscopy of N2O near 1.22 µm: (II) 14 N2 16 O line intensity modeling and global fit of 14 N2 18 O line positions[END_REF] was fitted against a scarce set of measured line positions consisting of FTS data below 2435 cm -1 and CRDS data in the 6007-8230 cm -1 range. The present data provides then a test of the interpolation capabilities of this model. The largest deviations (up to 0.3 cm -1 ) concern the highly perturbed 1511-0110 hot band near 5780.2 cm -1 . The HITRAN line list of 14 N2 18 O reproduces the predictions of the effective model [START_REF] Tashkun | High sensitivity Cavity Ring Down Spectroscopy of N2O near 1.22 µm: (II) 14 N2 16 O line intensity modeling and global fit of 14 N2 18 O line positions[END_REF]. It appears that for a significant part of the measured lines, the deviations largely exceed the HITRAN uncertainty attached to the 14 N2 18 O positions (between 0.001 and 0.01 cm -1 ).

Fig. 7

Differences between the line positions predicted using the effective Hamiltonians [START_REF] Tashkun | High sensitivity Cavity Ring Down Spectroscopy of N2O near 1.22 µm: (II) 14 N2 16 O line intensity modeling and global fit of 14 N2 18 O line positions[END_REF][START_REF] Perevalov | Global modeling of the line positions of 14 N2 16 O within the framework of the polyad model of effective Hamiltonian[END_REF][START_REF] Ni | Fourier-transform spectroscopy of 14 N 15 N 16 O in the 3800-9000 cm -1 region and global modelling of its absorption spectrum[END_REF][START_REF] Tashkun | Global fittings of 14 N 15 N 16 O and 15 N 14 N 16 O vibrational-rotational line positions using the effective Hamiltonian approach[END_REF] and the measured values, for the first four N2O isotopologues.

The intensity modeling of the 14 N2 16 O spectrum in the region was performed in Ref.

[19] on the basis of intensity measurements from Fourier transform spectra. The lines with intensities higher than 5×10 -26 cm/molecule at 296 K were used to fit the ΔP= 10 EDM parameters. The vibration-rotation eigenfunctions needed to calculate the transition moments corresponded to an old set of EH [START_REF] Perevalov | Global fittings of rovibrational line positions of nitrous oxide[END_REF]. The EDM fit of Ref. [START_REF] Daumont | Line intensity measurements in 14 N2 16 O and their treatment using the effective dipole moment approach. II. The 5400-11,000 cm -1 region[END_REF] reached the experimental uncertainties of the line intensities of a few %. Since that time a lot of new experimental line positions were published by CRDS [START_REF] Liu | High sensitivity CW-cavity ring down spectroscopy of N2O near 1.5l μm (I)[END_REF][2][START_REF] Liu | High sensitivity CW-cavity ring down spectroscopy of N2O near 1.5 μm (III)[END_REF][START_REF] Liu | High sensitivity CW-Cavity Ring Down Spectroscopy of N2O near 1.28 μm[END_REF][START_REF] Lu | High Sensitivity CW-Cavity Ring Down Spectroscopy of N2O between 6950 and 7653 cm -1 (1.44-1.31 µm): I. Line positions[END_REF][START_REF] Karlovets | High sensitivity CW-Cavity Ring Down Spectroscopy of N2O between[END_REF][START_REF] Karlovets | High sensitivity Cavity Ring Down Spectroscopy of N2O near 1.22 µm: (I) Rovibrational assignments and band-by-band analysis[END_REF][START_REF] Tashkun | High sensitivity Cavity Ring Down Spectroscopy of N2O near 1.22 µm: (II) 14 N2 16 O line intensity modeling and global fit of 14 N2 18 O line positions[END_REF] and FTS [START_REF] Wang | Fourier transform spectroscopy of N2O weak overtone transitions in the 1-2 μm region[END_REF]. Part of them (Refs. [START_REF] Liu | High sensitivity CW-cavity ring down spectroscopy of N2O near 1.5l μm (I)[END_REF][2][START_REF] Liu | High sensitivity CW-cavity ring down spectroscopy of N2O near 1.5 μm (III)[END_REF][START_REF] Liu | High sensitivity CW-Cavity Ring Down Spectroscopy of N2O near 1.28 μm[END_REF][START_REF] Lu | High Sensitivity CW-Cavity Ring Down Spectroscopy of N2O between 6950 and 7653 cm -1 (1.44-1.31 µm): I. Line positions[END_REF][START_REF] Karlovets | High sensitivity CW-Cavity Ring Down Spectroscopy of N2O between[END_REF][START_REF] Wang | Fourier transform spectroscopy of N2O weak overtone transitions in the 1-2 μm region[END_REF]) were used to refine the EH parameters of 14 N2 16 O [START_REF] Perevalov | Global modeling of the line positions of 14 N2 16 O within the framework of the polyad model of effective Hamiltonian[END_REF]. The new set of EH parameters contains a number of additional resonance interaction parameters. Consequently, the expansion coefficients of the eigenfunctions corresponding to the old set [START_REF] Perevalov | Global fittings of rovibrational line positions of nitrous oxide[END_REF] and to the new set [START_REF] Perevalov | Global modeling of the line positions of 14 N2 16 O within the framework of the polyad model of effective Hamiltonian[END_REF] of EH parameters differ considerably. This is why line intensities computed using the EDM parameters of Ref. [START_REF] Daumont | Line intensity measurements in 14 N2 16 O and their treatment using the effective dipole moment approach. II. The 5400-11,000 cm -1 region[END_REF] and the new eigenfunctions [START_REF] Perevalov | Global modeling of the line positions of 14 N2 16 O within the framework of the polyad model of effective Hamiltonian[END_REF] may be inaccurate. This is illustrated in Fig. 6 where the strong 2201-0000 band near 5762 cm -1 is predicted more than two times smaller than measured while it was satisfactorily reproduced in Ref. [START_REF] Daumont | Line intensity measurements in 14 N2 16 O and their treatment using the effective dipole moment approach. II. The 5400-11,000 cm -1 region[END_REF]. In the future, the line intensities measurements relative to the ΔP=10 series [START_REF] Daumont | Line intensity measurements in 14 N2 16 O and their treatment using the effective dipole moment approach. I. The 4300-5200 cm -1 region[END_REF][START_REF] Daumont | Line intensity measurements in 14 N2 16 O and their treatment using the effective dipole moment approach. II. The 5400-11,000 cm -1 region[END_REF][START_REF] Toth | Line positions and strengths of N2O between 3515 and 7800 cm -1[END_REF] will be collected and a new set of EDM parameters will be derived using the eigenfunctions of Ref. [START_REF] Perevalov | Global modeling of the line positions of 14 N2 16 O within the framework of the polyad model of effective Hamiltonian[END_REF].

As mentioned above, the line intensity predictions were performed for the sake of the line assignments. The 14 N2 18 O predicted line list [START_REF] Tashkun | High sensitivity Cavity Ring Down Spectroscopy of N2O near 1.22 µm: (II) 14 N2 16 O line intensity modeling and global fit of 14 N2 18 O line positions[END_REF] included in the HITRAN2016 database has line intensities computed using the ΔP= 10 EDM parameters of the principal isotopologue [START_REF] Daumont | Line intensity measurements in 14 N2 16 O and their treatment using the effective dipole moment approach. II. The 5400-11,000 cm -1 region[END_REF]. In Fig. 8, the comparison of our measured 14 Ratios of the 14 N2 18 O line intensities measured by CRDS to those from HITRAN2016 in the 5695-5910 cm -1 region.

Concluding remarks

The knowledge of the absorption spectrum of nitrous oxide has been extended by high sensitivity cavity ring down spectroscopy near 1.72 µm. Line parameters were derived for more than 4000 lines with intensities as small as 10 -28 cm/molecule at 296 K. The line assignment was performed using the predictions within the framework of the method of effective operators. The polyad models of EH were used for the line position and intensity calculations. Overall, 3326 lines were assigned to 50 bands of five nitrous oxide isotopologues ( An overall good agreement between the predicted and observed line positions is observed except for two bands -0821-0220 and 2311-0110 of 14 N2 16 O -which are perturbed by interpolyad Coriolis resonance interactions (these interactions are not taken into account by polyad model of EH) and the 3111-0110 band of 14 N2 18 O which is perturbed by very strong intrapolyad anharmonic resonance interaction. These results demonstrate once again the importance of interpolyad couplings which make necessary to develop a non-polyad EH model accounting for all interpolyad anharmonic and Coriolis resonance interactions in the 14 N2 16 O molecule.

As concerns line intensities, the present study has shown that a new modeling of the line intensities for the ΔP= 10 series of transitions has to be performed. It will be the subject of a separate contribution. The use of an old set of EDM parameters [START_REF] Daumont | Line intensity measurements in 14 N2 16 O and their treatment using the effective dipole moment approach. II. The 5400-11,000 cm -1 region[END_REF] combined with the eigenfunctions corresponding to the most recent EH modeling (that of Ref. [START_REF] Perevalov | Global modeling of the line positions of 14 N2 16 O within the framework of the polyad model of effective Hamiltonian[END_REF]) leads to inaccurate line intensity predictions. In general, transition moments have to be computed using consistent sets of EDM parameters and eigenfunctions. This was not the case in the predicted intensities computed in this work in order to help for the rovibrational assignments.

Note that, in principle, this issue applies to all the ΔP< 10 series of the Nitrous Oxide Spectroscopy Databank (NOSD) [START_REF] Tashkun | NOSD-1000, the high-temperature nitrous oxide spectroscopic databank[END_REF] which should be reconsidered.

Table 2. Spectroscopic parameters (in cm -1 ) for the different bands of the N2O isotopologues assigned in the CRDS spectrum between 5695 and 5910 cm -1 .

The bands are ordered in increasing order of the upper level vibrational term. Lower state constants were constrained to the values of Refs. [START_REF] Toth | Line positions and strengths of N2O between 3515 and 7800 cm -1[END_REF]23] Band a (P l2 i) b Gv(cm -1 ) Bv (cm -1 ) Dv(10 -7 cm -1 ) Hv (10 -12 cm -1 ) ν0 c (cm -1 ) a V1V2ℓ2 V3correspond to the maximum value of the modulo of the expansion coefficients of the eigenfunction. V2 is given between parentheses when it is larger than 10. * The asterisk is used to distinguish the different upper vibrational states with the same labelling in terms of the harmonic oscillators quantum numbers b Cluster labeling notation: (P=2V1+V2+4V3, ℓ2,i): i is the order number within the cluster, increasing with the energy. c ν0 is the band center d Observed branch with the maximum value of the total angular momentum quantum number.

Jmax d P/Q/R
e n is number of transitions included in the fit; N is number of assigned transitions.

f Root Mean Squares of residuals of the spectroscopic parameters fit is given in 10 -3 cm -1 .

g Notes:

1 -Interpolyad Coriolis resonance interaction (12 2 12) ↔ (11 1 11) (energy levels crossing at e: J=10; f: J= 16)

  N2 18 O line intensities to HITRAN2016 values is presented. As one can see from this figure, the average ratios of the line intensities vary from 0.4 to 1.6. In spite of the low 14 N2 18 O natural abundance in our sample (all the measured intensities are smaller than 2×10 -26 cm/molecule) the present line intensity measurements of 14 N2 18 O can be used for the improvement of the line intensity modeling in the ΔP= 10 series of transitions of this isotopologue.

Fig. 8

 8 Fig.8

Table 1

 1 Summary of the number of transitions and bands of different N2O isotopologues reported in the literature and obtained in this work by CRDS in the 5695 -5910 cm -1 region.

	Isotopologue HITRAN notation Abundance [13]	Number of bands This work Literature	Number of transitions
	14 N2 16 O	446	0.990333	31	5 [19,21-25]	2193
	14 N 15 N 16 O	456	3.64093×10 -3	5	7 a [15]	344
	15 N 14 N 16 O	546	3.64093×10 -3	8	13 a [20]	434
	14 N2 18 O	448	1.98582×10 -3	4	0	310
	14 N2 17 O	447	3.6928×10 -4	2	0	45
			Total	50		3326
	Note

a Observations using a sample highly isotopically enriched

Table 3

 3 Observed perturbations of the N2O bands between 5695 -5910 cm-1 Isotopologue Band affected Center (cm -1 ) Resonance interaction mechanism Jcross a Ref.b

				Intrapolyad anharmonic+ℓ-type
		2201e-0000e 5762.3723	(10 0 5)↔ (10 2 7)	56
				Extra lines: P56 R54 R55
		2221e-0000e 5772.6227	Intrapolyad anharmonic (10 2 9)↔ (10 2 7)	72
		2311e-0110e 5737.4930	Interpolyad Coriolis (11 1 5)↔ (10 2 20)	smooth
		1402e-0001e 5774.8275	Intrapolyad anharmonic (14 0 8)↔ (14 0 7)	29	[7]
		3600e-0200e 5861.7095	Intrapolyad anharmonic (12 0 9)↔ (12 0 8)	smooth
	14 N2 16 O	3221e-0220e 5859.2559 3221f-0220f 5859.2543	Intrapolyad anharmonic (12 2 15)↔ (12 2 16)	smooth [5]
		3620e-0220e 5860.4318 3620f-0220f 5860.4317	Intrapolyad anharmonic (12 2 16)↔ (12 2 15)	smooth [5]
		0821e-0220e 5714.0247	Interpolyad Coriolis
				(12 2 12) ↔ (11 1 11)	10
		0821f-0220f 5714.0255	Extra lines: P10 P11 P12 R10 Interpolyad Coriolis		[2]
				(12 2 12) ↔ (11 1 11)	16
				Extra lines: P14-P17 R11-R16
		0911e-0310e 5840.3476 0911f-0310f 5840.3488	Intrapolyad anharmonic (13 1 8)↔ (13 1 9)	smooth [5]
	14 N 15 N 16 O	1511e-0110e 5794.6863 1511f-0110f 5794.6889	Intrapolyad anharmonic (11 1 6)↔ (11 1 7)	smooth [15]
	15 N 14 N 16 O	0711e-0110e 0711f-0110f	5683.5629 5683.5627	Intrapolyad anharmonic+ℓ-type (11 1 5)↔ (11 3 7)	45 51
				Extra lines: P17 P18 R15 R16
		3111e-0110e 5780.2161	Intrapolyad anharmonic	smooth
	14 N2 18 O			Extra line: P19 Extra lines: P17 P18 R15 R16
		3111f-0110f 5780.21832	Intrapolyad anharmonic	smooth
				(11 1 6)↔ (11 1 7)
	Notes

a Value of the angular momentum quantum number at which the energy level crossing takes place.

  14 N2 16 O, 14 N 15 N 16 O, 15 N 14 N 16 O, 14 N2 18 O and 14 N2 17 O). 31 bands are newly reported for 14 N2 16 O, 14 N2 18 O and 14 N2 17 O.

N 14 N

O[START_REF] Song | Fourier-transform spectroscopy of 15 N 14 N 16 O in the 3500-9000 cm -1 region[END_REF], respectively. The low isotopic abundance of these isotopologues in our natural sample (about 3.6×10 -3 ) prevented the detection of new bands. As concerned the 14 N2

O and 14 N2

O species, the FTS investigation performed by Amiot[START_REF] Amiot | Vibration-rotation bands of 15 N2 16 O-14 N2 18[END_REF] with highly

Acknowledgements

This work is jointly supported by CNRS (France) and RFBR (Russia) in the frame of the International Associated Laboratory SAMIA.


Evelyne Poli

Amandine Magnaudeix

Chantal Damia

Fabrice Lalloué

Vincent Chaleix

Eric Champion

Vincent Sol

Advanced protocol to functionalize CaP bioceramic surface with peptide sequences and effect on murine pre-osteoblast cells proliferation

Keywords:

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

Bioorganic & Medicinal Chemistry Letters

Calcium phosphate hydroxyapatite (HA), is a bioceramic material commonly used as bone graft substitute. It is highly biocompatible, osteoconductive and bioactive 1 . Silicon is known to be involved in early-stage bone formation 2 and for several years a new silicon-substituted hydroxyapatite (SiHA) has been investigated. [3][4][5][6] However, HA or Si-HA ceramics do not exhibit intrinsic osteoinductive ability. This limitation could be overcome by the addition of biologically active molecules (i.e. peptides or proteins) involved in bone formation to the calcium phosphate (CaP) apatite. In this way, active biomolecules targeting cell adhesion and proliferation are of interest to improve the biological properties of the material. The interaction of bone cells with proteins belonging to the extracellular matrix (ECM) is a key point for the first steps of bone healing. 7 Numerous works have shown that the RGD (Arg-Gly-Asp) sequence is the smallest active peptide able to induce a cellular response. 8,9 Several research teams have highlighted the influence of linear or cyclic RGD-containing pentapeptide on biomaterials surface to improve cell attachment and they have shown that the presence of c-(DfKRG) induces a better and stronger adhesion in the early times (3 h) after cell seeding. 10,11 Ceramic functionalization by peptides can be processed by two routes: adsorption of the peptides on the implant surface, or covalent binding. Because of its easy implementation, adsorption of RGD peptides on HA has been chosen by several groups. 12,13 As reviewed by Carragee et al. in the case of bone morphogenetic protein BMP2 , the major drawbacks of the adsorption methods are the poor control of both the adsorption process and the biomolecule delivery (kinetics and actually released amounts). 14 In order to better control these processes, the covalent grafting of RGD sequences onto various surfaces (titanium, 15,16 polyethylene terephthalate (PET), 17 cellulose, 18 glasses 19 and apatites such as hydroxyapatite 11 or apatite-wollastonite 20 ) has been developed. Nevertheless, a series of three successive steps of chemical reactions is classically used in order to functionalize the material surface: (i) silanization of the material surface, (ii) immobilization of an organic cross-linker agent and finally (iii) the biomolecule attachment. These successive chemical steps can be challenging with the potential absorption of chemical reactants or byproducts on the material surface. Moreover, the initial silanization leads usually to the formation of a continuous polymer coating on the ceramic surface, thus decreasing the biological properties of the latter.

In this context, the present work is devoted to surface functionalization of SiHA ceramic by covalent grafting of cyclo-RGD at an optimized low-density distribution to improve the biological properties. The cyclo-RGD, known to primarily enhance cell adhesion as described above, was chosen to evaluate the efficiency of the grafting method. The low density of immobilized peptides will keep most of the substrate surface free from RGD to maintain the interactions between the CaP substrate and the cells (i.e. osteoconduction). To this end, low density SiHA surface silanization by 3-aminopropyltriethoxysilane (APTES, noted T) was first performed so as to prevent the formation of a polymer layer that would cover the whole ceramic surface. Second, a spacer bearing cyclo-(DfKRG) prepared separately was covalently grafted onto SiHA-T. This grafting strategy consists of only a two-step protocol, which allows for a better control of the purity of the reaction products and ruling out unwanted contamination of the surface by adsorption of organic reactants or reaction side-products. The functionalized biomaterial was characterized by means of mass spectrometry, UV-visible spectroscopy, X-ray photoelectron spectroscopy and confocal microscopy. Finally, it was evaluated in vitro in order to check the activity of bonded peptide on cell attachment and proliferation (1, 4 and 7 days). SiHA (Ca10(PO4)5.6(SiO4)0.4(OH)1.6) powder was synthesized by an aqueous precipitation method. 4 SiHA ceramic pellets (Ø = 10 mm) were sintered at 1200 °C for 30 min in a Super Khantal furnace under air atmosphere and then they were mirror polished. 6 Silanization of pellets was realized by heating them for 3 h, under reflux, in a solution of APTES in anhydrous toluene under argon atmosphere (scheme 1, see also supplementary materials). After reaction, the pellets were washed successively with 10 mL of toluene, ethanol diethylether under sonication (10 min). Finally, the pellets were dried in a ventilated oven 100 °C for 1 h and stored in a desiccant.

A R T I C L E I N F O A B S T R A C T

Primary amine sites of SiHA-T pellets were quantified by UV-Vis photometric methods. 22 To check the distribution of the organosilane on the ceramic pellet surface, a fluorescent dye (5fluorescein isothiocyanate isomer I (FITC)) was reacted with the primary amine function of SiHA-T. In order to discriminate the fluorescence from FITC possibly adsorbed on the ceramic surface, a bare (un-silanised) SiHA pellet was used as a negative control. The remaining FITC in the labeling solution and in the washing solution was quantified by spectrofluorometry. For an initial FITC solution of 5×10 -6 M, after immersion of the SiHA-T pellets, only a remaining concentration of 2.6×10 -8 M was measured in the supernatant. After the first washing of the SiHA-T-FITC pellets with absolute ethanol and under mild ultrasonic treatment, the concentration decreased to 3.0×10 -9 M and after the second one to 1.2×10 -10 XPS analyses of the ceramic surface are given in Table 1. As expected, Ca, P and Si elements were detected at the surface of the bare SiHA pellet. SiHA-T ceramic clearly showed the presence of the nitrogen coming from the APTES. 1 22.5 50.9 8.0 13.4 0.7 2.2 trace 2 1 SiHA-T-PEG6-S-c-(DfKRG) pellet analysed after two rounds of 7 days of cell culture. 2 Sulphur detected in amount below the significant quantification limit.

Immobilization of the c-(DfKRG) peptide at the surface of silanised ceramics was obtained by the addition of SiHA-T pellets and c-(DfKRG)-S-PEG6-NHS into a PBS 10X pH 7.4 solution (5 mL) (Scheme 1). The mixture was vigorously planetary stirred (231 rpm) at RT for 48 h under argon. Then the ceramic pellets were washed with 3 mL of fresh PBS 10X pH 7.4 and sonicated for 5 min in order to eliminate potential organic molecules adsorbed onto the ceramic surface. Finally, the peptide functionalized SiHA-T-PEG6-Sc-(DfKRG) pellets were lyophilized and stored in desiccant at RT.

The potential enhancement of cell adhesion and proliferation on the surface of functionalized SiHA ceramic with the adhesive c-(DfKRG) peptide was investigated in vitro using murine preosteoblast cells MC3T3-E1. This cell line is commonly used to study biocompatibility, and osteoconductive properties of various biomaterials designed for bone engineering. [23][24][25] The cell behavior was evaluated at three different time points: 1, 4 and 7 days. In a first instance, the cytocompatibility of the SiHA-T-PEG6-S-c-(DfKRG) was assessed. As all the terminal amines of APTES may not have reacted during the step of c-(DfKRG) immobilization (scheme 1), the cell viability was also checked on the SiHA-T to ensure that the possible unreacted APTES remaining on the ceramic surface was not deleterious. Whatever the time course point, the cell morphology is not affected by any of the chemically modified biomaterials (Fig 2A ), which is consistent with previous works. 26,27 The cell population density (i.e. the number of cells by unit surface) was determined after 1 day and 4 days (Fig 2B &2C). After one day of growth on the different materials (Fig 2B), the cellular density was significantly lower on glass coverslip when compared to the ceramics (0.9×10 4 on glass and from 1.8×10 4 to 2.0×10 4 on ceramics, p<0.0001). When the ceramic pellets were compared to each other, there was no significant difference. Thus, SiHA is very efficient as a support surface for cell attachment and adhesion. Moreover, the surface modifications due the functionalization do not decrease this ability. Four days after seeding (Fig 2C), the cell density on pellets increased to reach values between 1.1×10 5 and 2.1×10 5 , about ten times those measured after 1 day. In brief, cell population is growing on all ceramics. However, cells were significantly less dense on the c-(DfKRG) functionalized pellet in comparison with both the SiHA and SiHA-T pellets, p<0.0001. This could be due to the inhibitory effect of RGD peptide associated to apatite on cell density that has been reported in the literature. 28,29 This effect could relate to the peptide immobilization settings: the surface density of the c-(DfKRG), the nature the the peptide (aminoacids flanking RGD), the cross-linker chain and the material substrate itself. 30 The cell density on glass coverslips was close to those observed on SiHA or SiHA-T pellets showing that the presence of APTES had clearly no influence on cell tissue growth.

The proliferation rate of MC3T3-E1 cells seeded onto the bare SiHA, SiHA-T and SiHA-T-PEG6-S-c-(DfKRG) pellets was assessed by studying the incorporation of EdU into their DNA during DNA replication (S-phase) just before cell division (Fig 2D). After four days, the cell proliferation rate on SiHA-T-PEG6-S-c-(DfKRG) was nearly twice that found on SiHA samples (respectively 7.2% vs. 13.7%, ***p = 0.0161) whereas the proliferation rate was much lower for cells growing on glass coverslips (1.9%). The number of replicating cells counted on SiHA-T was not significantly higher than the number of replicating cells found on SiHA (9.1% vs 7.2%, p = 0.7304). This confirms the results of cell density. In conclusion, APTES, in low concentration at the surface of CaP ceramics, has no influence on cell behavior.

After 4 days, cells reach confluency whatever the conditions are (Fig 2A). Lower proliferation observed on SiHA and SiHA-T at day 4 can be due to the higher cell density when compared with SiHA-T-PEG6-S-c-(DfKRG), which causes a proliferation arrest in vitro. As cell density was lower on the latter substrate surface (Fig 2C ), cells keep on proliferating (Fig 2D). Thus, a delayed cell proliferation may be considered on SiHA-T-PEG6-S-c-(DfKRG) due to the peptide.

In order to assess the robustness of the covalent bonding of the c-(DfKRG) peptide and APTES to the ceramic, the cells were removed from the different SiHA pellets, which were further cleaned, sterilised and used again for cell culture of MC3T3-E1 preosteoblasts. The same biological tests as the previous ones were done for 4 and 7 days (Fig 3).

No toxicity was recorded during any of the repeated experiments with a viability rate close to 99%. After 4 days, the cell density was significantly increased on SiHA-T and SiHA-T-PEG6-S-c-(DfKRG) in comparison with bare SiHA pellet (p<0.0001 in each case) (Fig. 3A) and the proliferation rates were increased on SiHA-T-PEG6-S-c-(DfKRG) when compared with the other substrates (Fig. 3C). After 7 days, the cell density was still significantly higher on SiHA-T-PEG6-S-c-(DfKRG) (Fig. 3B). After 4 days, the proliferation rate was significantly higher on SiHA-T-PEG6-S-c-(DfKRG) than on SiHA or SiHA-T (Fig. 3C). After 7 days, the proliferation rates were globally lower when compared to those obtained at day 4 (Fig. 3D). This is probably because of a phenomenon of contact inhibition due to cell confluence. As a result the proliferation rate was not significantly higher for SiHA-T-PEG6-S-c-(DfKRG) when compared with the SiHA control (p=0.0605) or SiHA-T. On the SiHA-T substrate, the proliferation diminished more slowly between 4 and 7 days, resuming a rate comparable to that measured on SiHA-T-PEG6-S-c-(DfKRG) (Fig. 3C and Fig. 3D). A third round of cell culture on these SiHA pellets was done after a second cleaning cycle and gave the same variations in terms of cell density and proliferation rate at 4 and 7 days as those observed after the first reuse (data not shown). The XPS analysis of SiHA-T-PEG6-S-c-(DfKRG) ceramic surface after two successive rounds of cleaning and cell culture of seven days still shows the presence of nitrogen, C-O and C=O contributions proving the sturdiness of the organic-peptide modified surface (Table 1 and Fig. S2 in supplementary materials).

In this work, an organic-inorganic materials associating siliconsubstituted hydroxyapatite and c-(DfKRG) peptide has been developed, thanks to a new advanced protocol of functionalization allowing the covalent bonding in low amount of an organic molecule containing c-(DfKRG) on the SiHA ceramic surface. The c-(DfKRG)-S-PEG6-NHS peptide was synthesized before being bonded to an APTES anchor previously bonded to the SiHA ceramic surface. The final SiHA-T-PEG6-S-c-(DfKRG) organic-inorganic bone substitute was biologically tested and showed a superior biological behavior. Two successful consecutive reuses proved the preservation of both its structural integrity and its biological activity. This approach of the functionalization of bone substitutes allows better results in comparison to previous works reported in the literature. Indeed, using this new protocol, we demonstrated that it is possible to:

(i) limit contamination by adsorbed products due to successive in situ synthesis steps of classically used protocols, (ii) enhance the biological behavior of CaP ceramic substrates with a low dose-efficient amount of peptide while preventing its burst release by strong covalent immobilization, (iii) use organic molecules in sufficiently low density to preserve the CaP surface partially free of organic molecules and thus the bioactive properties of this material.

Using the same strategy, the grafting of other peptides, proteins or growth factors of interest in bone tissue engineering will be performed.

  j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m Advanced protocol to functionalize CaP bioceramic surface with peptide sequences and effect on murine pre-osteoblast cells proliferation. Evelyne Poli, a Amandine Magnaudeix, a Chantal Damia, a* Fabrice Lalloue, b Vincent Chaleix, c Eric Champion, a Vincent Sol c a Université de Limoges, CNRS, IRCER UMR 7315, F-87000 Limoges, France. b Université de Limoges, CAPTuR, EA3842, F-87000 Limoges, France c Université de Limoges, Laboratoire PEIRENE, EA 7500, F-87000 Limoges, France

Scheme 1

 1 Scheme 1 Synthesis of the spacer bearing c-(DfKRG)-S-PEG6-NHS peptide and its grafting on silanised SiHA-T ceramic. A commercial c-(DfKRG) peptide (American peptide) was chemically modified on its lysine by the 2-iminothiolane hydrochloride (Traut's reagent) to produce a c-(DfKRG)-SH peptide providing a clickable thiol function (Scheme 1). After purification by Sephadex® size exclusion chromatography, only one peak at 11.36 min corresponding to pure c-(DfKRG)-SH was observed. Then, the c-(DfKRG)-SH peptide was functionalized on its thiol function by NHS-PEG6-Maleimide according to the thiol-ene click reaction strategy. 21 The reaction was realized in pH 7.4 PBS solution and was monitored by TLC. The pure product (white powder with 79.9% of reaction yield) was lyophilized. The purity of the product was confirmed by ESI-MS/MS analysis (see Fig S1 in supplementary materials).SiHA (Ca10(PO4)5.6(SiO4)0.4(OH)1.6) powder was synthesized by an aqueous precipitation method.4 SiHA ceramic pellets (Ø = 10 mm) were sintered at 1200 °C for 30 min in a Super Khantal furnace under air atmosphere and then they were mirror polished.6 Silanization of pellets was realized by heating them for 3 h, under reflux, in a solution of APTES in anhydrous toluene under argon atmosphere (scheme 1, see also supplementary materials). After reaction, the pellets were washed successively with 10 mL of toluene, ethanol diethylether under sonication (10 min). Finally, the pellets were dried in a ventilated oven 100 °C for 1 h and stored in a desiccant.Primary amine sites of SiHA-T pellets were quantified by UV-Vis photometric methods.22 To check the distribution of the organosilane on the ceramic pellet surface, a fluorescent dye (5fluorescein isothiocyanate isomer I (FITC)) was reacted with the primary amine function of SiHA-T. In order to discriminate the fluorescence from FITC possibly adsorbed on the ceramic surface, a bare (un-silanised) SiHA pellet was used as a negative control. The remaining FITC in the labeling solution and in the washing solution was quantified by spectrofluorometry. For an initial FITC solution of 5×10 -6 M, after immersion of the SiHA-T pellets, only a remaining concentration of 2.6×10 -8 M was measured in the supernatant. After the first washing of the SiHA-T-FITC pellets with absolute ethanol and under mild ultrasonic treatment, the concentration decreased to 3.0×10 -9 M and after the second one to 1.2×10-10 M. Pictures obtained by confocal fluorescence microscopy of SiHA-T pellets and un-silanised SiHA pellet (used as control) after reaction with FITC are presented in Figure 1. The control surface seems homogeneous with no FITC (Fig 1B). Only a weakly uniform green color due to auto-fluorescence of the pellet is observed. SiHA-T-FITC surface shows good and uniform dispersion of the fluorescent

  M. Pictures obtained by confocal fluorescence microscopy of SiHA-T pellets and un-silanised SiHA pellet (used as control) after reaction with FITC are presented in Figure 1. The control surface seems homogeneous with no FITC (Fig 1B). Only a weakly uniform green color due to auto-fluorescence of the pellet is observed. SiHA-T-FITC surface shows good and uniform dispersion of the fluorescent dye and consequently of the grafted organosilane (Fig 1A). This confirms the absence of a continuous polymer layer covering the ceramic surface, as expected from the silanization settings.

Figure 1

 1 Figure 1 Confocal fluorescence microscopy: distribution of FITC on the surface of (A) silanised SiHA-T pellet and (B) un-silanised bare SiHA pellet. Images are squares of 90×90 µm (lens ×100).

Figure 2

 2 Figure 2 MC3T3-E1 murine pre-osteoblast cells behavior. (A) Living cell morphologies 4 days after seeding. Cells were stained with 10 µM calcein AM and nuclei by 20 µM Hoescht 33343 (scale bar length is 20 µm). (B) Cell density after 1 day (B) and (C) 4 days of culture. (D) Cell proliferation rate after 4 days of culture. Percentage of cells incorporating EDU was calculated from microscopy images by taking into account the number of cells positive for EDU versus the total number of nuclei. *p<0.05 was considered as significant (n.s. non-significant; **p ≤ 0.01; ***p ≤ 0.001).

Figure 3

 3 Figure 3 MC3T3-E1 murine pre-osteoblast cells behaviour after cultivation on SiHA, SiHA-T and SiHA-T-PEG6-S-c-(DfKRG) pellets reused after a first cell seeding. Cell density at (A) 4 days and (B) 7 days after the cell seeding on the substrate. Proliferation rate evaluated after (C) 4 days and (D) 7 days of culture. p<0.05 was considered as significant (n.s. non-significant; * p≤0.05; ** p≤0.01; ***p≤0.001).

Table 1

 1 XPS atomic composition (in mass percent) of the ceramics surface.

	Sample	C	O	P	Ca	Si	N	S
	SiHA	26.3 46.4	10.2	36.3	1.4	/	/
	SiHA-T	58.3 30.8	4.7	6.7	2.6	1.4 /
	SiHA-T-
	PEG6-S-c-
	(DfKRG)

Acknowledgments

The authors thank the French Région Limousin and the French Agence Nationale de la Recherche in the scope of the LabEx SigmaLim (ANR-10-LABX-0074-01) and the "CeramOs" project (ANR-13-BSV5-0002) for their financial support. The authors are grateful to Emilie Pinault, Claire Carrion and the core facility Biologie Integrative Santé Chimie Environnement (BISCEm) of Limoges University for the mass spectrometry analyses and confocal microscopy imaging respectively; and Etienne Laborde and the Service commun de Caractérisation des Matériaux de Limoges (CARMALIM) for XPS analyses. The authors are also grateful to Pr Michel Guilloton for critical editing.

Supplementary data

Supplementary data and experimental procedure for the synthesis associated with this article can be found in the online version, at

Graphical Abstract