Grégory Eot-Houllier

Géraldine Fulcrand

Laura Magnaghi-Jaulin

Christian Jaulin
email: christian.jaulin@univ-rennes1.fr

Histone deacetylase inhibitors and genomic instability

Keywords: Histone deacetylase inhibitors, genomic instability, chromosome segregation, DNA damage and repair

Histone deacetylase inhibitors and genomic instability.

Introduction:

Although the presence of acetyl groups on histones and their role in transcription was first reported more than forty years ago [1; 2], the function of protein acetylation/deacetylation remained a relatively neglected field over the following thirty years. Since the identification of the first histone acetyltransferases (HATs) and histone deacetylases (HDACs) and their respective functions in regulation of transcription in the mid-90s [START_REF] Brownell | Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation[END_REF][START_REF] Brownell | An activity gel assay detects a single, catalytically active histone acetyltransferase subunit in Tetrahymena macronuclei[END_REF][START_REF] Bannister | The CBP co-activator is a histone acetyltransferase[END_REF][START_REF] Ogryzko | The transcriptional coactivators p300 and CBP are histone acetyltransferases[END_REF][START_REF] Rundlett | HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription[END_REF][START_REF] Taunton | A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p[END_REF][START_REF] Yang | Transcriptional repression by YY1 is mediated by interaction with a mammalian homolog of the yeast global regulator RPD3[END_REF], interest in previously overlooked protein post-translational modifications has been renewed [START_REF] Ehrenhofer-Murray | Chromatin dynamics at DNA replication, transcription and repair[END_REF][START_REF] Bhaumik | Covalent modifications of histones during development and disease pathogenesis[END_REF][START_REF] Kouzarides | Chromatin modifications and their function[END_REF][START_REF] Li | The role of chromatin during transcription[END_REF]. There is now a growing body of evidence that protein acetylation is involved in a large number of cell signaling pathways, reminiscent of protein phosphorylation [START_REF] Yang | Lysine acetylation and the bromodomain: a new partnership for signaling[END_REF]. Protein acetylation is catalyzed by HATs, which transfer an acetyl group from acetyl-CoA to the ε-amino group of a lysine; the reverse reaction, involving the deacetylation of acetylated lysines, is mediated by HDACs. Acetylation and deacetylation by HATs and HDACs were first described for histones. However, these processes occur in bacteria [15; 16], which do not have histones.

Indeed, a rapidly growing number of non-histone proteins have been found to be regulated by acetylation/deacetylation [17; 18].

There are four classes of HDAC in humans: class I HDACs (HDAC1, 2, 3 and 8) share sequence similarity with the yeast RPD3 deacetylase, are ubiquitously expressed and localized in the nucleus. Class II HDACs (HDAC4, 5, 6, 7, 9 and 10) are homologous to the yeast Hda1 deacetylase, are mainly cytoplasmic (or both nuclear and cytoplasmic) and restricted to certain tissues [START_REF] Thiagalingam | Histone deacetylases: unique players in shaping the epigenetic histone code[END_REF]. Class II HDACs are subdivided into two subclasses, class IIa (HDAC4, 5, 7 and 9) and class IIb (HDAC6 and 10). Class III HDACs are represented by sirtuins (SIRT1 to SIRT7), a family of seven HDACs sharing homology with yeast silent information regulator 2 (Sir2) [START_REF] Denu | The Sir 2 family of protein deacetylases[END_REF]. In addition to their deacetylase activity, some sirtuins are able to transfer ADP-ribose from NAD+ to an acetylated protein target. Class IV has only one member, HDAC11, which shares conserved residues with both class I and II HDACs [START_REF] Gregoretti | Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis[END_REF].

Class I, II and IV HDACs activity requires Zn 2+ and can be inhibited by a variety of different pharmacological HDAC inhibitors (HDACIs) [START_REF] Dokmanovic | Histone deacetylase inhibitors: overview and perspectives[END_REF], whereas class III enzymes are NAD+dependent deacetylases with only a limited number of inhibitors described [20; 23]. Class I, II and IV HDAC-specific inhibitors have antiproliferative activity and several are currently being tested in clinical trials, alone or in combination with other therapies, for their antitumor properties [24; 25]. There is now accumulating evidence of anticancer activity in myeloid malignancies and solid tumors for several HDACIs (reviewed in [26; 27]) and the first HDACI to obtain FDA approval for cancer therapy was suberoylanilide hydroxamic acid (SAHA, vorinostat), which was effective for the treatment of cutaneous manifestations of cutaneous T-cell lymphoma in clinical trials [START_REF] Mann | FDA approval summary: vorinostat for treatment of advanced primary cutaneous T-cell lymphoma[END_REF]. Although HDACIs represent a new class of very promising anticancer drugs, their pleiotropic effects on a variety of cell functions make it difficult to identify the precise mechanism(s) underlying their antiproliferative properties. The most obvious target for HDACIs is gene expression; indeed, previous studies suggest that HDACI treatment (valproic acid, trybutirine, sodium butyrate [START_REF] Rocchi | p21Waf1/Cip1 is a common target induced by short-chain fatty acid HDAC inhibitors (valproic acid, tributyrin and sodium butyrate) in neuroblastoma cells[END_REF] and SAHA [START_REF] Richon | Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation[END_REF]) leads to the reexpression of silenced tumor-suppressor genes, such as the p21(WAF1/CIP1)-encoding gene, in cancer cells. Some other specific targets for particular HDACIs have also been described. For example, treatment with valproic acid, but not trichostatin A (TSA), selectively induce proteasome dependent HDAC2 degradation [START_REF] Kramer | The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2[END_REF]. However, potential downstream targets of HDACIs include various aspects of the cell cycle, DNA recombination and repair, extrinsic and intrinsic apoptosis pathways, angiogenesis, autophagy and senescence, among others; and it is not clear whether transcription mediates HDACI action in these processes [START_REF] Xu | Histone deacetylase inhibitors: molecular mechanisms of action[END_REF]. Only a relatively small proportion of genes are up-or down-regulated following treatment with the HDACIs TSA [START_REF] Mariadason | Genetic reprogramming in pathways of colonic cell maturation induced by short chain fatty acids: comparison with trichostatin A, sulindac, and curcumin and implications for chemoprevention of colon cancer[END_REF][START_REF] Glaser | Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines[END_REF][START_REF] Chiba | Cell growth inhibition and gene expression induced by the histone deacetylase inhibitor, trichostatin A, on human hepatoma cells[END_REF], SAHA and MS-275 [START_REF] Glaser | Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines[END_REF] and non-transcriptional targets have been proposed [36; 37].

A substantial number of studies have demonstrated a synergistic effect of conventional and HDACI-based therapies. In particular, HDACI treatments sensitize cycling cells to irradiation and DNA-targeting drugs, possibly resulting from HDACI-induced chromatin remodeling.

Additionally, HDACIs seem to impair a variety of cellular processes that ensure genome stability, such as the control of DNA damage and repair, the regulation of reactive oxygen species (ROS) concentration and the progression into mitosis (relevant HDACIs and their known effects on genomic instability are listed on Table 1). Targeting genome integrity in rapidly cycling cells has always been a preferred strategy in cancer therapy; thus, in this review, we will focus on the different aspects of genome instability induced by pharmacological inhibition of Zn 2+ -dependent HDACs.

HDACIs and DNA damage and repair.

There is no evidence suggesting that HDACIs have direct mutagenic or clastogenic properties.

However, histone hyperacetylation induced by HDAC inhibition causes profound changes in chromatin structure. These changes may expose sections of DNA that are normally protected from damage in tightly packed chromatin. HDAC inhibition may also affect the expression of DNA repair genes and disrupt their finely-tuned expression timing, leading to improper cellular responses to DNA damage and accumulation of strand breaks and modified bases.

Moreover, the activity of certain DNA repair proteins may be directly regulated by acetylation, resulting in a shift of the cellular acetylation/deacetylation balance towards hyperacetylation, and impaired DNA repair function. Thus, HDAC inhibition may indirectly exert clastogenic and mutagenic effects through DNA exposure and abnormal regulation of the DNA repair machinery.

Sensitization to exogenous DNA damage by HDACI.

Early observations indicated that the HDACI sodium butyrate enhances cell radiosensitivity in vitro [START_REF] Prasad | Sodium ascorbate potentiates the growth inhibitory effect of certain agents on neuroblastoma cells in culture[END_REF][START_REF] Arundel | Enhancement of radiation injury in human colon tumor cells by the maturational agent sodium butyrate (NaB)[END_REF][START_REF] Nackerdien | Chromatin decondensed by acetylation shows an elevated radiation response[END_REF]. Since these findings and the development of HDACIs as anticancer drugs, the number of studies reporting synergistic action between ionizing irradiation (IR) and HDACIs in cancer cell lines or animal pre-clinical models has grown exponentially. Various HDACIs, belonging to different chemical classes, appear to sensitize cultured cancer cells to IR (reviewed in [START_REF] Karagiannis | The paradox of histone deacetylase inhibitor-mediated modulation of cellular responses to radiation[END_REF]). In mouse xenograft models, the combination of HDACI treatment (MS-275 [START_REF] Camphausen | Enhancement of xenograft tumor radiosensitivity by the histone deacetylase inhibitor MS-275 and correlation with histone hyperacetylation[END_REF], valproic acid [START_REF] Camphausen | Enhancement of in vitro and in vivo tumor cell radiosensitivity by valproic acid[END_REF], LBH589 [START_REF] Geng | Histone deacetylase (HDAC) inhibitor LBH589 increases duration of gamma-H2AX foci and confines HDAC4 to the cytoplasm in irradiated non-small cell lung cancer[END_REF], LAQ824 [START_REF] Cuneo | Histone deacetylase inhibitor NVP-LAQ824 sensitizes human nonsmall cell lung cancer to the cytotoxic effects of ionizing radiation[END_REF]and AN-9 [START_REF] Entin-Meer | In vivo efficacy of a novel histone deacetylase inhibitor in combination with radiation for the treatment of gliomas[END_REF]) with radiation therapy, results in a greater delay in tumor growth than that accounted for by a simple additive effect of the corresponding individual treatments. HDAC inhibition by LBH589 [START_REF] Geng | Histone deacetylase (HDAC) inhibitor LBH589 increases duration of gamma-H2AX foci and confines HDAC4 to the cytoplasm in irradiated non-small cell lung cancer[END_REF], TSA [START_REF] Karagiannis | The histone deacetylase inhibitor, Trichostatin A, enhances radiation sensitivity and accumulation of gammaH2A.X[END_REF], depsipeptide [START_REF] Zhang | Histone deacetylase inhibitors enhance phosphorylation of histone H2AX after ionizing radiation[END_REF] and SAHA [START_REF] Munshi | Vorinostat, a histone deacetylase inhibitor, enhances the response of human tumor cells to ionizing radiation through prolongation of gamma-H2AX foci[END_REF] stabilizes and enhances IR-induced phosphorylated histone H2AX nuclear foci, classical markers of DSBs, suggesting that HDACIs inhibit DSB repair and/or render DNA more susceptible to IR-induced damage. HDACIs facilitate radiation-induced killing in tumor cells and at the same time appear to protect healthy tissues from cutaneous radiation syndrome. Although this hinders our understanding of the mechanisms involved, it will be a significant benefit for future therapies [START_REF] Chung | Antitumor histone deacetylase inhibitors suppress cutaneous radiation syndrome: Implications for increasing therapeutic gain in cancer radiotherapy[END_REF].

HDACIs such as HC-toxin, MS-275, SAHA and TSA also promote the induction of cell death by a variety of DNA-targeting drugs such as mitomycin C, cisplatin, bleomycin, topotecan, doxorubicin, etoposide, 5-fluorouracil and Ara-C [51; 52]. An HC-toxin induced increase in phosphorylated H2AX foci was observed for all drugs that directly, or indirectly, cause DSBs (mitomycin C, cisplatin, bleomycin, topotecan, doxorubicin and etoposide) [START_REF] Ozaki | Histone deacetylase inhibitors enhance the chemosensitivity of tumor cells with crossresistance to a wide range of DNA-damaging drugs[END_REF]. This further supports the notion that HDAC inhibition stabilizes DSBs and/or increases DNA sensitivity to DSBs. In fission yeast, strains mutated for components of HDAC complexes are highly sensitive to genotoxic agents, with a massive accumulation of DSBs [START_REF] Nicolas | Distinct roles of HDAC complexes in promoter silencing, antisense suppression and DNA damage protection[END_REF]. The same study also demonstrated that these strains increase antisense transcripts, suggesting that DSBs could be triggered by the presence of RNA-DNA hybrids, structures known to induce genomic instability [START_REF] Li | Cotranscriptional processes and their influence on genome stability[END_REF].

HDACI and double strand break repair machinery.

Spontaneous endogenous DNA double-strand breaks occur at the surprisingly high rate of 50 DSBs per cell and per generation in normal cells [START_REF] Vilenchik | Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer[END_REF]. Given that going through mitosis with a single unrepaired DSB is lethal in most cases, DSB repair mechanisms have to be remarkably efficient. Most DSBs are repaired by non homologous end joining (NHEJ) during the G1 phase of the cell cycle, while the availability of a sister chromatid following DNA replication allows homologous recombination (HR) repair in S and G2 phases [START_REF] Couedel | Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells[END_REF]. HDACIs alone do not induce DSBs but nevertheless induce a DNA-damage response [START_REF] Bakkenist | DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation[END_REF]. Generating unstable DNA structures or making DNA more accesible to genotoxic agents may not be the only mechanism by which HDACIs sensitize cells to DNA damaging agents: histone acetylation/deacetylation also appears to be important for the DNA-damage response. In the yeast S. cerevisiae, HR triggers the transient acetylation of histones H3 and H4 N-terminal tails flanking a DSB. HDACs Rpd3, Sir2 and Hst1 are involved in the following deacetylation step, and RPD3 mutants show sensitivity to DSBs [START_REF] Tamburini | Localized histone acetylation and deacetylation triggered by the homologous recombination pathway of double-strand DNA repair[END_REF]. In addition, RPD3 deacetylates lysine 16 on histone H4 in the vicinity of a DSB that can only be repaired by NHEJ. Consequently, RPD3 mutants also disrupt NHEJ, indicating that HDAC activity is also involved in the NHEJ-mediated DSB repair [START_REF] Jazayeri | Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair[END_REF]. Time-course chromatin immunoprecipitation data suggest that HDACs are involved in restoring chromatin to its original conformation during the final steps of the DSB repair process [START_REF] Utley | Regulation of NuA4 histone acetyltransferase activity in transcription and DNA repair by phosphorylation of histone H4[END_REF]. Changes in the global chromatin configuration induced by HDACI treatment also promote DNA-damage signaling pathways, reflecting the importance of the HAT/HDAC balance in determining an appropriate cellular response to DNA damage [57; 61]. In yeast, the DNA-damage response may be mediated by histone H3 acetylation at lysine 16 (H3K16) by the HAT, Rtt109 [62; 63]. The sirtuins Hst3 and Hst4 appear to be involved in the regulation of this H3K16-dependent DNA-damage response pathway [START_REF] Maas | Cell cycle and checkpoint regulation of histone H3 K56 acetylation by Hst3 and Hst4[END_REF]. In addition to their action on chromatin structure during DSB repair, HDACs can also affect the expression or activity of repair machineries. Treatments with the HDACIs TSA, SAHA, MS-275 and OSU-HDAC42 leads to acetylation of Ku70, a key NHEJ component, and sensitizes prostate cancer cells to DNA-damaging agents. This study showed that Ku70 acetylation does not affect the formation of a complex with its natural partner, Ku80; but, when the authors mimicked acetylation by replacing key lysines with glutamines, the ability of Ku70 to bind DNA extremities was suppressed [START_REF] Chen | Histone deacetylase inhibitors sensitize prostate cancer cells to agents that produce DNA double-strand breaks by targeting Ku70 acetylation[END_REF]. HDACIs can also affect the expression of genes encoding HR components, as reported by two recent studies. Cell treatment with TSA induces down-regulation of a panel of genes involved in DNA repair, such as ATR (Ataxia telangiectasia-related, involved in DNA-damage signaling), BLM (Bloom's syndrome helicase, involved in collapsed replication fork repair), BRCA1 and -2 (Breast Cancer 1 and -2, involved in HR) and NBS1 (Nijmegen breakage syndrome 1, involved in both DSB sensing and repair) [START_REF] Zhang | Attenuated DNA damage repair by trichostatin A through BRCA1 suppression[END_REF]. In this study, TSA did not sensitize BRCA1null cells to irradiation, whereas a cell line complemented with a BRCA1 wild-type allele showed a TSA-dependent enhanced susceptibility to IR, suggesting that down-regulation of BRCA1 is a key event in HDACI-induced radiosensitization [START_REF] Zhang | Attenuated DNA damage repair by trichostatin A through BRCA1 suppression[END_REF]. However, the picture is likely to be much more complex since this study did not detect a change in gene expression for the key strand transfer protein RAD51, whereas another group found that it was downregulated to one third of its normal levels following treatment with PCI-24781, another broadspectrum HDACI [START_REF] Adimoolam | HDAC inhibitor PCI-24781 decreases RAD51 expression and inhibits homologous recombination[END_REF]. Consistent with HDACI-induced RAD51 depletion, treated cells exhibited HR repair defects and NHEJ mutant cells were hypersensitive to PCI-24781 [START_REF] Adimoolam | HDAC inhibitor PCI-24781 decreases RAD51 expression and inhibits homologous recombination[END_REF].

As recentely reported, deletion of a particular HDAC, HDAC3, in mouse embryonic fibroblasts affects S phase progression and causes DNA breaks, perhaps by disrupting DNA repair [START_REF] Bhaskara | Deletion of histone deacetylase 3 reveals critical roles in S phase progression and DNA damage control[END_REF]. Consequentely, HDAC3 may represent an important target for HDACIs in terms of genomic instability. Thus, HDAC inhibition seems to promote the generation and stabilization of DNA DSBs by a pleiotropic mechanism. It sensitizes DNA to exogenous genotoxic damage, impairs the "DNA-damage histone code" function and down-regulates the activity of DNA repair machinery, either by acting on the expression of the corresponding genes or by directly inactivating the components by acetylation.

HDACI-induced oxidative stress.

Reactive oxygen species (ROS), such as superoxide anion, hydrogene peroxide or hydroxyl radicals, represent a major source of endogenous DNA damage. Normal cells control ROS concentrations through the actions of enzymatic and non-enzymatic ROS scavengers.

However, cells with particularly high ROS concentrations, or with defective ROS scavengers, enter a state of genotoxic oxidative stress which often leads to apoptosis. The most widely studied mechanism of ROS-induced mutagenesis is base oxidation. Oxidized bases are normally removed by glycosylases. The resulting abasic site is then recognized by an APendonuclease, which makes an incision on the DNA strand, generating a single-strand break (SSB) (reviewed in [START_REF] Sancar | Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints[END_REF]). SSBs, which can also result from the direct oxidative damage of the nucleotide's sugar component [START_REF] Demple | Dynamics and diversions in base excision DNA repair of oxidized abasic lesions[END_REF], are normally efficiently repaired to prevent their conversion into potentially lethal DSBs during DNA replication or transcription [START_REF] Caldecott | Mammalian single-strand break repair: mechanisms and links with chromatin[END_REF].

A number of studies have reported that HDAC inhibition by several HDACIs such as SAHA, MS-275, and NCH-51 leads to accumulation of ROS [START_REF] Ruefli | The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species[END_REF][START_REF] Rosato | The histone deacetylase inhibitor MS-275 promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21CIP1/WAF1 1[END_REF][START_REF] Ungerstedt | Role of thioredoxin in the response of normal and transformed cells to histone deacetylase inhibitors[END_REF][START_REF] Xu | Intrinsic apoptotic and thioredoxin pathways in human prostate cancer cell response to histone deacetylase inhibitor[END_REF][START_REF] Sanda | Proteome analyses of the growth inhibitory effects of NCH-51, a novel histone deacetylase inhibitor, on lymphoid malignant cells[END_REF]. Furthermore, the resulting with ROS scavengers such as N-acetylcysteine decreases HDACI-induced cell death [72; 73; 76]. SAHA treatment upregulates TBP2, an inhibitor of the ROS scavenger thioredoxin, possibly accounting for HDACI-induced accumulation of ROS [75; 77]. Notably, 8oxoguanine-DNA glycosylase 1 (OGG1) is regulated by acetylation. This enzyme is responsible for the removal of 8-oxoguanine, a common oxidative base lesion. OGG1 activity is stimulated by its acetylation and it associates with class I HDACs, which may be involved in its deacetylation [START_REF] Bhakat | Acetylation of human 8oxoguanine-DNA glycosylase by p300 and its role in 8-oxoguanine repair in vivo[END_REF]. Consequently, HDACI treatment may enhance oxidative DNA

HDACIs and control of chromosome segregation.

Mitosis is a common target in anticancer therapy, as illustrated by the success of microtubule targeting drugs such as vinca alkaloids or taxanes. Inhibition of the main mitotic segregation control mechanism, the spindle assembly checkpoint (SAC), using RNA interference, leads to cell death in a few generations [START_REF] Kops | Lethality to human cancer cells through massive chromosome loss by inhibition of the mitotic checkpoint[END_REF]. Thus, forcing rapidly cycling cells towards aneuploidy by damaging chromosome segregation control mechanisms may represent a novel therapeutic strategy. It now clear that HDAC inhibition also impairs the control of mitotic progression, supporting the potential development of novel HDACI-based anticancer therapy targeting mitosis.

HDACI effects on mitosis.

Cells treated in culture with HDACI often display mitotic defects. One of the most striking effects of HDACI treatment is the disruption of pericentric heterochromatin associated with chromosomal segregation defects. Long-term incubation in TSA leads to loss of the heterochromatin-specific factor HP1 and to the appearance of lagging chromosomes [START_REF] Taddei | Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases[END_REF].

HP1 proteins normally bind to a site encompassing histone H3 tri-methylated at lysine 9 by the histone methyl-transferase Suv39h [81; 82]. In fission yeast, HP1 and Suv39h orthologs are required for centromeric chromatid cohesion [83; 84]. It is thus possible that TSA-induced segregation defects result from abnormal acetylation of histone H3 on lysine 9, which would prevent Suv39h tri-methylation and inhibit HP1binding. Failure to bind HP1 proteins would in turn hamper heterochromatin formation and sister chromatid cohesion. However, a recent study demonstrated that, in contrast with fission yeast, the Suv39h-HP1 pathway is not required for chromatid cohesion in mammalian cells [START_REF] Koch | The Suv39h-HP1 histone methylation pathway is dispensable for enrichment and protection of cohesin at centromeres in mammalian cells[END_REF], suggesting that TSA-induced heterochromatin disruption and segregation defects may be unrelated.

TSA-treated cells also exhibit chromosome condensation defects. Short-term treatment with high doses of TSA induces heterogeneity in chromosome condensation in all mitotic stages, associated with a delay in prophase progression and the occurrence of lagging chromosomes and chromatid bridges in anaphase [START_REF] Cimini | Histone hyperacetylation in mitosis prevents sister chromatid separation and produces chromosome segregation defects[END_REF]. In a Xenopus oocyte maturation system commonly used to mimic mitosis, TSA treatment impedes chromosome condensation without preventing meiosis progression [START_REF] Magnaghi-Jaulin | Histone deacetylase activity is necessary for chromosome condensation during meiotic maturation in Xenopus laevis[END_REF]. Chromosome condensation is an essential step to eliminate DNA catenation at prophase and prevent chromosome missegregation at anaphase. The condensation defects observed following HDAC inhibition may thus contribute to the segregation defects reported.

HDAC inhibition may also disrupt mitosis by acting on mitotic kinases or their substrates.

The class I histone deacetylase, HDAC3, targets chromosomes and associates with the mitotic kinase, Aurora B, during mitosis. Selective down-regulation of HDAC3 leads to a decrease of Aurora B-dependent phosphorylation of histone H3 on serine 10, and causes G2/M delay and segregation defects. The involvement of HDAC3 in providing a hypoacetylated histone H3 Nterminal tail, the preferred substrate for Aurora B, has been suggested [START_REF] Li | A novel histone deacetylase pathway regulates mitosis by modulating Aurora B kinase activity[END_REF]. HDAC3 also seems to be involved in microtubule-kinetochore attachment and, may therefore have critical functions in different functional subcompartments during mitosis [START_REF] Ishii | Histone deacetylase 3 localizes to the mitotic spindle and is required for kinetochore-microtubule attachment[END_REF]. However the picture may be more complex since a recent study reported that, although HDAC3 knockout leads to embryonic lethality, no mitosis defects were observed in HDAC3 -/-embryonic fibroblasts while these cells appear to be defective in DSB repair. [START_REF] Bhaskara | Deletion of histone deacetylase 3 reveals critical roles in S phase progression and DNA damage control[END_REF]. Another mitotic kinase often overexpressed in cancer cells, Aurora A, may also represent a target for HDACIs. A recent study showed that the HDACIs LAQ824 and SK-7068 induced tumor-selective mitotic toxicity by depleting Aurora A. Treated cells had spindle defects and the mechanism by which Aurora A was down-regulated following treatment may involve stimulation of proteasomedependent degradation of Aurora A [START_REF] Park | Inhibitors of histone deacetylases induce tumor-selective cytotoxicity through modulating Aurora-A kinase[END_REF]. Mitotic spindle defects have also been reported using other HDACIs such as SBHA, SAHA, sodium butyrate and TSA [START_REF] Stevens | Histone deacetylase inhibitors induce mitotic slippage[END_REF].

The spindle assembly checkpoint.

Among their many effects, HDACIs appear to be potent pharmacological inhibitors of an essential segregation control mechanism: the mitotic spindle assembly checkpoint (SAC). The SAC maintains cells in mitosis with attached sister chromatids by preventing cyclin B and securin proteasome-dependent degradation until all kinetochores have been captured by spindle microtubules and tension is established between sister kinetochores (reviewed in [START_REF] Musacchio | The spindle-assembly checkpoint in space and time[END_REF]). The SAC can be kept artificially activated by treating cells with the microtubuledisrupting drug, nocodazole. Nocodazole-treated cells accumulate cyclin B and securin and can remain in a prometaphase-like state during several hours. Exit from mitosis in the presence of microtubule-disrupting drugs requires either loss of function of SAC or slow degradation of cyclin B in the presence of an active SAC ("mitotic slippage") [START_REF] Brito | Mitotic Checkpoint Slippage in Humans Occurs via Cyclin B Destruction in the Presence of an Active Checkpoint[END_REF].

Nocodazole-arrested cells subsequently treated with HDACIs (SBHA or TSA) fail to accumulate cyclin B and associated cdk1 activity, suggesting that HDAC inhibition can induce exit from mitosis, despite nocodazole-induced SAC activation [94; 95]. Accordingly, in mitosis-arrested cells, TSA or SBHA treatment induces chromosome decondensation, nuclear membrane reformation, loss of phosphorylation on serine 10 of histone H3 and decreases the proportion of cells with 4 N DNA content [91; 95; 96]. Furthermore, key SAC proteins, such as Bub1, BubR1 and Mad2, fail to localize properly at the kinetochores following TSA treatment of nocodazole-arrested cells, suggesting a failure to maintain an active SAC when HDAC functions are disrupted [START_REF] Magnaghi-Jaulin | Histone deacetylase inhibitors induce premature sister chromatid separation and override the mitotic spindle assembly checkpoint[END_REF][START_REF] Dowling | Mitotic spindle checkpoint inactivation by trichostatin a defines a mechanism for increasing cancer cell killing by microtubule-disrupting agents[END_REF][START_REF] Shin | Inhibition of histone deacetylase activity increases chromosomal instability by the aberrant regulation of mitotic checkpoint activation[END_REF]. In line with these findings, HDACIs (TSA, apicidin and sodium butyrate) induce premature sister chromatid separation [START_REF] Magnaghi-Jaulin | Histone deacetylase inhibitors induce premature sister chromatid separation and override the mitotic spindle assembly checkpoint[END_REF], a phenotype consistently observed when the SAC is inhibited by RNA interference or genetic means [START_REF] Michel | Complete loss of the tumor suppressor MAD2 causes premature cyclin B degradation and mitotic failure in human somatic cells[END_REF][START_REF] Michel | MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells[END_REF][START_REF] Kienitz | Partial downregulation of MAD1 causes spindle checkpoint inactivation and aneuploidy, but does not confer resistance towards taxol[END_REF]. Securin proteasome-dependent degradation is also observed in TSA treated mitotic cells and most likely accounts for premature sister chromatid separation [START_REF] Magnaghi-Jaulin | Histone deacetylase inhibitors induce premature sister chromatid separation and override the mitotic spindle assembly checkpoint[END_REF]. A recent study reported that SBHA treatment results in failure to properly recruit the chromosomal passenger complex (CPC) at centromeres. Given that survivin and Aurora B, two essential CPC componants, control SAC protein localization [START_REF] Carvalho | Survivin is required for stable checkpoint activation in taxol-treated HeLa cells[END_REF][START_REF] Lens | Survivin is required for a sustained spindle checkpoint arrest in response to lack of tension[END_REF][START_REF] Morrow | Bub1 and aurora B cooperate to maintain BubR1-mediated inhibition of APC/CCdc20[END_REF], it was proposed that the HDACI-induced overriding of SAC results from CPC mistargeting [START_REF] Stevens | Histone deacetylase inhibitors induce mitotic slippage[END_REF]. Consistent with a role for HDACI in inhibiting the SAC, a synergistic effect of a combination of microtubule-targeting drugs with TSA has been reported [START_REF] Dowling | Mitotic spindle checkpoint inactivation by trichostatin a defines a mechanism for increasing cancer cell killing by microtubule-disrupting agents[END_REF]. Together, these findings demonstrate that HDAC inhibition leads to SAC dysfunction, providing a possible explanation for the previous observations that HDACIs treatments induce segregation defects and polyploidy [80; 86; 97; 104].

Conclusions and future directions.

HDACIs appear to affect genome stability by targeting various cellular mechanisms of genome surveillance (outlined on Figure 1). This pleiotropic effect may explain why combinations of HDACIs and other genome-targeting agents, with different mechanisms of action, often show synergetic effects. The variety of potential genomic HDACI targets and the other effects of HDAC inhibition on cellular functions should be considered when optimizing HDACI-based anticancer therapies. Accordingly, the order in which drugs are administered can be important for combined treatments involving HDACIs. For example, cotreatment with SAHA or TSA and the topoisomerase II (Topo II) inhibitors VP-16 (Etoposide) only has a synergistic effect if HDACIs are administrated before VP-16; indeed, the effect of treatment with these drugs in the reverse order has no greater effect than with each drug alone, suggesting that HDACI-induced chromatin relaxation is required for increased efficacy of Topo II inhibitors [START_REF] Kim | Inhibition of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA[END_REF]. Similarly, targeting mitosis and the SAC with HDACIs to cause gross aneuploidy in cancer cells is predicted to be effective only if the cells are first allowed to reach mitosis. However, many cells respond to HDACI treatment by upregulation of p21(WAF1/CIP1) and activation of the p38 MAPK checkpoint pathway, resulting in G1 and G2 arrest, respectively [29; 30; 105; 106]. Therefore, HDACI treatment aimed at overriding the SAC to cause a mitotic catastrophe, may require pretreatment with microtubule-targeting drugs, such as vinca alkaloids or taxanes, to accumulate cells in mitosis.

Targeting chromosome segregation to fight cancer may seem an attractive strategy but it must be remembered that forcing cells to chromosome missegregation is a double-edged sword since it appears to be both oncogenic and anti-oncogenic. As a matter of fact, mice heterozygous for the SAC components MAD1 and MAD2 are more susceptible to spontaneous tumors [99; 107], while down regulation of SAC component leads to proliferative cell death in an in vitro assay [START_REF] Kops | Lethality to human cancer cells through massive chromosome loss by inhibition of the mitotic checkpoint[END_REF]. It was proposed that moderate chromosome missegregation could be tumorigenic while massive missegregation would lead to tumor suppression [START_REF] Weaver | Aneuploidy: instigator and inhibitor of tumorigenesis[END_REF]. A recent and elegant study reports that mice heterozygous for the SAC component CENP-E had an increase incidence of spleen lymphomas and lung adenomas.

However, the same animals had a decreased incidence of spontaneous liver cancers and of cancers chemically or genetically induced [START_REF] Weaver | Aneuploidy acts both oncogenically and as a tumor suppressor[END_REF]. Thus, within the same organism, chromosome missegregation can act both as a tumor promoter and a tumor suppressor, depending on the cell type and the genetic and environmental contexts. This dual potential should be kept in mind when optimizing therapeutic protocols intended to target chromosome segregation for cancer therapy.

Thus, the pleiotropic effect of nonspecific HDAC inhibition may hamper treatment targeting a given cellular function such as transcription, DNA repair or mitosis. As a consequence, the identification of the individual HDACs involved in these functions and the design of specific HDACIs that target single, or a small subset of, HDAC(s) remains a priority.

  damage by both causing ROS accumulation and disrupting mechanisms involved in the repair of oxidative DNA lesions. Thus, by acting on chromatin conformation, disrupting the intracellular redox balance and deregulating repair mechanisms, HDACIs may induce extensive oxidative DNA damage. It is possible that the resulting accumulation of SSBs saturates repair mechanisms and that residual SSBs are converted to DSBs following DNA duplex unwinding during replication or transcription. The resulting chromosome fragmentation would then contribute to induction of apoptosis by HDACI.

Figure legend : Figure 1 :

 :1 Figure legend:

Table 1 .

 1 HDAC inhibitors and their action on genomic instability. Data from {Kim, 2006 #456}{Rasheed, 2007 #453}; **Data from {Rasheed, 2008 #452; Rasheed, 2007 #453}. DDR : DNA damage and repair; OS: Oxidative stress; MD: Mitosis defects.

	Strutural class	HDACI	HDACs	Clinical trials**	Effect on genomic
			targeted*	(phase)	instability
	Hydroxamic acid-	TSA	Classes I and II		DDR, MD
	Derived compounds	SAHA	Classes I and II	FDA approval	DDR, OS, MD
		LAQ824	Classes I and II	I	DDR, MD
		LBH589	Classes I and II	I/II	DDR
		PCI-24781			DDR
		SBHA			MD
	Short-chain fatty acids	Sodium butyrate	Classes I and IIa		MD
		Valproic acid	Classes I and IIa	I	DDR
		AN-9		I/II	DDR
		OSU-HDAC42			DDR
	Benzamides	MS-275	Class I (HDAC1,2 and 3)	I/II	DDR, OS
	Cyclic peptides	Depsipeptide	(HDAC1,2, 4 and 6)	I/II	DDR
		Apicidin			MD
		HC-toxin			DDR
	Thiolate	NCH-51			OS

*

Acknowledgments:

We apologize to all authors whose work was not cited in this article due to space and knowledge limitations. Our laboratory is supported by La Ligue Contre le Cancer (comités du Languedoc-Roussillon et de l'Aveyron) and by the Fondation Pour la Recherche Médicale. G. E.-H. is supported by INCa-Lilly fellowship; G. F. is supported by a Ligue Contre le Cancer Research PhD scholarship (comité des Côtes d'Armor). C.J. is an investigator of the Institut National de la Santé et de la Recherche Médicale (INSERM).

.


Hicham Farsi

Driss Harti

Mohamed Rachid Achaâban

Mohammed Piro

Véronique Raverot

Béatrice Bothorel

Mohammed Ouassat

Etienne Challet

Paul Pévet

Khalid El

Khalid El Allali
email: khalid_elallali@yahoo.fr

Melatonin rhythm and other outputs of the master circadian clock in the desert

Keywords: Goat, entrainment, body temperature, melatonin, locomotor activity, ambient temperature, circadian clock

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

Introduction

In mammals, the circadian organization of physiological and behavioural functions is under the control of the master clock, located within the hypothalamic supra-chiasmatic nuclei (SCN), and of secondary clocks within peripheral organs [START_REF] Refinetti | Variability of diurnality in laboratory rodents[END_REF][START_REF] Challet | Entrainment of the suprachiasmatic clockwork in diurnal and nocturnal mammals[END_REF][START_REF] Dibner | The mammalian circadian timing system: organization and coordination of central and peripheral clocks[END_REF] . The light-dark (LD) cycle was found to be the most powerful zeitgeber for the SCN (For review see [START_REF] Challet | Entrainment of the suprachiasmatic clockwork in diurnal and nocturnal mammals[END_REF][START_REF] Dibner | The mammalian circadian timing system: organization and coordination of central and peripheral clocks[END_REF] ). How ambient light synchronizes the circadian system is now well documented [START_REF] Reppert | Molecular analysis of mammalian circadian rhythms[END_REF][START_REF] Challet | Interactions between photic and nonphotic stimuli to synchronize the master circadian clock in mammals[END_REF][START_REF] Takahashi | The genetics of mammalian circadian order and disorder: implications for physiology and disease[END_REF][START_REF] Mohawk | Central and peripheral circadian clocks in mammals[END_REF] . However, little is known about the ability of ambient temperature (Ta) cycles to entrain circadian rhythms. Such entrainment is well established for the circadian system in non-mammalian vertebrates (For review see [START_REF] Rensing | Temperature effect on entrainment, phase shifting, and amplitude of circadian clocks and its molecular bases[END_REF] ). In laboratory mammals, despite the responsiveness of peripheral clocks to the synchronizing effects of ambient temperature cycles [START_REF] Brown | Rhythms of mammalian body temperature can sustain peripheral circadian clocks[END_REF][START_REF] Tsuchiya | Temperature compensation and temperature resetting of circadian rhythms in mammalian cultured fibroblasts[END_REF][START_REF] Buhr | Temperature as a universal resetting cue for mammalian circadian oscillators[END_REF] , experiments showed either a complete absence of entrainment of the master clock in some species [START_REF] Buhr | Temperature as a universal resetting cue for mammalian circadian oscillators[END_REF][START_REF] Hoffmann | Die relative Wirksamkeit von Zeitgebem[END_REF] or a partial entrainment of locomotor activity (LA) rhythm in only some individuals [START_REF] Tokura | Effects of Temperature on the Circadian Rhythm of PigTailed Macaques Macaca nemestrina[END_REF][START_REF] Aschoff | Circadian activity rhythms in squirrel monkeys: entrainment by temperature cycles[END_REF][START_REF] Francis | Ambient Temperature Cycles Entrain the Free Running Circadian Rhythms of the Stripe-Faced Dunnart, Sminthopsis macroura[END_REF][START_REF] Rajaratnam | Entrainment of activity rhythms to temperature cycles in diurnal palm squirrels[END_REF][START_REF] Pálková | Effect of ambient temperature on the circadian activity rhythm in common marmosets, Callithrix j. jacchus (primates)[END_REF][START_REF] Refinetti | Entrainment of circadian rhythm by ambient temperature cycles in mice[END_REF] . In such conditions, it would be very difficult to conclude of a real synchronization of the master circadian clock since, as reported [START_REF] Francis | Ambient Temperature Cycles Entrain the Free Running Circadian Rhythms of the Stripe-Faced Dunnart, Sminthopsis macroura[END_REF][START_REF] Rajaratnam | Entrainment of activity rhythms to temperature cycles in diurnal palm squirrels[END_REF][START_REF] Pálková | Effect of ambient temperature on the circadian activity rhythm in common marmosets, Callithrix j. jacchus (primates)[END_REF][START_REF] Refinetti | Entrainment of circadian rhythm by ambient temperature cycles in mice[END_REF] , such entrainment of LA rhythm could be a result of a masking effect of Ta. Accordingly, Ta cycles are assumed to have a weak effect on the master circadian clock at least in endothermic mammals (For review see [START_REF] Rensing | Temperature effect on entrainment, phase shifting, and amplitude of circadian clocks and its molecular bases[END_REF] ). Within mammals, a group of several species including camel, display heterothermy phenomenon, which means that under special conditions (e.g. dehydration and exposure to high Ta), Tb can follow Ta [START_REF] Bouaouda | Daily regulation of body temperature rhythm in the camel (Camelus dromedarius) exposed to experimental desert conditions[END_REF] . Moreover, it should be noted that the circadian system of heterothermic animals was shown to be more sensitive to environmental Ta [START_REF] Lindberg | Thermoperiodic Entrainment of Arousal from Torpor in the Little Pocket Mouse, Perognathus longimembris[END_REF][START_REF] Erkert | Differences in Temperature Sensitivity of the Circadian Systems of Homoeothermic and Heterothermic Neotropical Bats[END_REF][START_REF] Pohl | Temperature Cycles as Zeitgeber for the Circadian Clock of Two Burrowing Rodents, the Normothermic Antelope Ground Squirrel and the Heterothermic Syrian Hamster[END_REF] . Thereby, in a previous study [START_REF] El Allali | Entrainment of the circadian clock by daily ambient temperature cycles in the camel (Camelus dromedarius)[END_REF] , our team has shown that, in the camel, the Ta cycles are a strong zeitgeber for the circadian clock in the SCN. Two outputs of the master clock, plasma melatonin (Mel) and body temperature (Tb) rhythms were indeed found to be synchronized by Ta cycles. To our knowledge, this finding was the first demonstration in mammals of such non-photic entrainment to Ta.

In camels under heat stress and dehydration, heterothermy corresponds to a switching from a perfect endothermic state to a heterothermic state [START_REF] Bouaouda | Daily regulation of body temperature rhythm in the camel (Camelus dromedarius) exposed to experimental desert conditions[END_REF][START_REF] Schmidt-Nielsen | Body temperature of the camel and its relation to water economy[END_REF] . The Ta synchronization of the circadian

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This article is protected by copyright. All rights reserved system described in the camel could be related to this specific thermoregulation process. Recently, such heterothermy has been also shown in other desert ungulates, including oryx and goat [START_REF] Cain | Mechanisms of thermoregulation and water balance in desert ungulates[END_REF][START_REF] Mengistu | Mechanisms of water economy in lactating Ethiopian Somali goats during repeated cycles of intermittent watering[END_REF][START_REF] Al-Tamimi | Renal efficiency underlies adaptive heterothermy of heat-stressed hypohydrated goats[END_REF] . Thus, the raised question is whether the observed entrainment of the circadian clock by Ta cycles is specific to camel or is a mechanism shared by other desert large species.

To test this hypothesis, we have studied three outputs of the master clock, namely: Tb, LA and Mel rhythms, in the desert goat.

Experimental procedures

Animals

Seventeen healthy adult male desert goats (Capra hircus) weighing 26±0.3 kg and aged around 3.5 years were used in this study. Animals were housed in specific facilities at the Hassan II Agronomy and Veterinary Medicine Institute of Rabat (Latitude: 34°01′N, Longitude: 6°50′W).

Males were used instead of females to avoid eventual effects of oestrous and gestation on circadian rhythms.

This study was carried out in accordance with the International Ethical Standards [START_REF] Touitou | Ethics, standards and procedures in human and animal research in chronobiology[END_REF] and the national recommendations for animal farm husbandry, experimentations and surgeries of the Hassan II Agronomy and Veterinary Medicine Institute of Rabat.

Experimental Design

Experiment 1 was intended to verify if Ta cycles could entrain the Tb and Mel rhythms in the goat.

To avoid the masking effect of LA on the Tb rhythm [START_REF] El Allali | Entrainment of the circadian clock by daily ambient temperature cycles in the camel (Camelus dromedarius)[END_REF][START_REF] Refinetti | The circadian rhythm of body temperature[END_REF] , bucks (n=10) were maintained in forced inactivity but allowed an easy standing, moving around and sitting in sternal and lateral recumbency. Animals received a complete compound food (Maraa  , Alf-Sahel, Morocco) and hay 4 times a day to avoid a possible entrainment by feeding [START_REF] El Allali | Entrainment of the circadian clock by daily ambient temperature cycles in the camel (Camelus dromedarius)[END_REF][START_REF] Piccione | The circadian rhythm of body temperature of the Horse[END_REF] . Water was ad libitum. The air relative humidity during this experiment was around 23%. This experiment was carried on five stages as follows:

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Stage 1 (5 days): is an adaptive period to indoor conditions with an artificial LD cycle (12L-12D) and uncontrolled Ta (17.00°C-25.50°C).

Stage 2 (21 days): this step was designed to demonstrate the existence in the goat of a freerunning rhythm of Tb. Animals were placed under constant conditions of total darkness (DD) and constant ambient temperature (CTa) of 23±0.37°C to avoid any temporal perception from the environment. This CTa was chosen as thermo-neutral Ta for goats.

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This article is protected by copyright. All rights reserved Stage 5 (23 days): corresponds to a return to the previous conditions of stage 2. Details of these experimental procedures are detailed in Figure 1.

Temperature measurements

The rhythm of Tb was measured during experiment 1 by using data logger, Ibuttons® (DS1922L, Dallas Semi-Conductor, USA). The devices were programmed for 16-bit with a resolution of 0.0625°C and to record Tb every 12 min. Then, the Ibuttons were protected with silicone elastomer (Rhodorsil RTV141) before their surgical implantation in the abdominal cavity of bucks. This site has been widely used to record Tb in several species [START_REF] Lovegrove | Modification and miniaturization of Thermochron iButtons for surgical implanation into small animals[END_REF][START_REF] Mccafferty | Challenges of measuring body temperatures of freeranging birds and mammals[END_REF][START_REF] Adam | Surgical implantation of temperature-sensitive transmitters and data-loggers to record body temperature in koalas (Phascolarctos cinereus)[END_REF] .

Locomotor activity measurements:

The LA rhythm of bucks was recorded during experiment 2 using the validated video-locomotion scoring technique (For more details, see [START_REF] Farsi | Validation of locomotion scoring as a new and inexpensive technique to record circadian locomotor activity in large mammals[END_REF] ) which is based on a visual attribution of scores to activity, where 0: animal on a sitting position and not moving; 1: animal on a sitting position and moving lightly its limbs beneath the body; 2: animal on standing position without movements; 3: animal on standing position moving its limbs but without locomotion; 4: animal walking and exploring the environment; 5: animal in intense locomotor activity or agitated.

The LA rhythm was measured separately in each animal using a time set of 5 minutes. This time interval is imposed by the used software which provides activity as actograms. The recorded video sequences were visually analysed on a computer screen by two well-trained evaluators who assigned a score to each activity state (For details, see [START_REF] Farsi | Validation of locomotion scoring as a new and inexpensive technique to record circadian locomotor activity in large mammals[END_REF] ).

Melatonin assay

To determine the plasma concentration of Mel under various conditions of experiment 1, blood was sampled every 2-hours during the last 24-hours of stages 3, 4 and 5. Blood was collected in 10 ml lithium heparin BD-Vacutainer tubes by puncturing alternatively the right and left external jugular veins. To minimize stress, bucks were accustomed to handling and sampling procedures for several weeks. The blood sampling was performed under complete darkness without any source of light by well-trained operators. Samples were immediately centrifuged at 1760 g for 30 min and then plasma was stored at -20°C for subsequent Mel assay.

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This article is protected by copyright. All rights reserved Mel concentrations were measured using an in-house radioimmunoassay after extraction from plasma with diethylether [START_REF] Brun | Effect of modafinil on plasma melatonin, cortisol and growth hormone rhythms, rectal temperature and performance in healthy subjects during a 36 h sleep deprivation[END_REF] . Duplicated extracted samples were mixed with in-house antibody and labelled 125Iodomelatonin (Perkin Elmer NEX2360), incubated overnight at 4°C. The radioactivity of the pellet obtained after precipitation by anti-rabbit γ-globulin solution was counted. The intra-assay CV was 9.5% at levels 39 and 57ng/L. The inter-assay CV was 12.7% at levels 50 ng/L and 12.4% at 100 ng/L. The quantification limit was of 3.7 ng/L.

Data Analysis

Temperature data. Tb data was presented as means ± Standard Errors of the Means (SEM) and plotted in simple curves using the SigmaPlot ® software (Systat. Sigmaplot v12.0, Systat, Chicago, IL). The period, acrophase, mesor, amplitude and the robustness of Tb rhythm were calculated by nonlinear least squares regression using Acro ® , Cosinor ® and LSP ® softwares (Refinetti R,

Circadian

Rhythm Laboratory, University of South Carolina, http://www.circadian.org/softwar.html). Cosinor analysis was performed according to the equation:

f=y0+a*cos[2π*(t-φ)/]

Where f is Tb at time t, y0 is the mesor, a is the amplitude, φ is the acrophase and τ the period. For each parameter, a confidence interval of 95% probability is given. Similarly, the degree of significance of the regression is calculated. The One-Way ANOVA test was used to determine if there are statistical differences in the periods and the acrophases of Tb between the different stages of experiment 1, mainly between NRT, RT and NRTr conditions.

Locomotor activity data. LA rhythm data was presented as individual actograms using the Actogram Plotter ® software (Refinetti R, Circadian Rhythm Laboratory, University of South Carolina, http://www.circadian.org/softwar.html). As for Tb, the rhythm parameters of LA were calculated by the same Cosinor analysis equation.

Similarly, a one-way ANOVA test was also used to compare periods and acrophases of LA rhythm between stages of experiment 2 and also, to verify the equality of means of the daily activity onsets and offsets around the daily Ta transition.

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This article is protected by copyright. All rights reserved Melatonin data. Results data of Mel were presented as means ± SEM and plotted using the SigmaPlot ® software. The Mel concentrations under different Ta treatments were evaluated separately for each individual and then for the mean of all animals. Accordingly, the Mel rhythm secretion for stages 3, 4 and 5 were fitted using a nonlinear regression of the following exponential mathematical equation [START_REF] Vivien-Roels | Environmental control of the seasonal variations in the daily pattern of melatonin synthesis in the European hamster, Cricetus cricetus[END_REF][START_REF] Bothorel | In the rat, exogenous melatonin increases the amplitude of pineal melatonin secretion by a direct action on the circadian clock[END_REF][START_REF] El Allali | Seasonal variations in the nycthemeral rhythm of plasma melatonin in the camel (Camelus dromedarius)[END_REF] :

f=y0+(ymax/((1+exp(slope1*(phi1-x)))*(1+exp(slope2*(x-phi2)))))
Where f is the concentration of Mel variation at time x, y0 is the basal level, ymax is the amplitude of the Mel peak concentration, phi1 and phi2 are the inflection points and represent the time points at which 50% of the maximal increase and 50% of the decline were reached respectively.

Slope1 and slope 2 are the slopes of the onset and the offset (inflection points at half the peak height) and were assigned with arbitrary constant values. The equation was chosen because it minimizes the dispersion of the abscissas of the inflection points and their collinearities with other parameters. The algorithm provides an ANOVA for the mathematical regression which was tested using F-test. It also provides an asymptotic estimate (by excess) of the standard error for each parameter. Shapiro-Wilk's test was used to check the homogeneity of residuals and Bartlett's test for the homogeneity of variances [START_REF] El Allali | Entrainment of the circadian clock by daily ambient temperature cycles in the camel (Camelus dromedarius)[END_REF] . The following step was designed to compare the obtained phi1 and phi2 between NRT, RT and NRTr conditions of Ta cycles corresponding respectively to stages 3, 4 and 5 of experiment 1. A paired t-test was performed twice to determine the phi1 and phi2 statistical differences, first between NRT (stage 3) and RT (stage 4) conditions and then between RT (stage 4) and NRTr (stage 5) conditions. P ≤0.05 was considered statistically significant in all statistical tests.

Results

Under stage 1 conditions, Tb showed a clear daily rhythm (Figure 2) characterized by a robustness of 40±2.7%, a mesor of 38.74±0.12°C, an amplitude of 0.69±0.36°C, an acrophase at 17:23±0.28h and a period of exactly 24.0h (Table 1). Tb was lowest around 08h00 am, started then its ascending phase during the early morning achieving high value in the early evening. After

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This article is protected by copyright. All rights reserved Light/Dark transition, Tb dropped across the scotophase achieving its lowest value at Dark/Light transition.

Under similar environment (stage 1, experiment 2), LA rhythm showed intense daily activity during the photophase (Figure 3). Cosinor regression of LA rhythm shows that the robustness was of 32.0±1.8%, the acrophase occurred at the middle of the photophase, exactly at 12:33±0.30h, the period was exactly of 24.0h and the mesor and the amplitude were respectively 1.59±0.08 and 1.7±0.09 (Table 2).

The existence of circadian rhythms in goat was inferred by stage 2 of both experiments 1 and 2.

We observed that both Tb and LA rhythms (24.0h period) first shown under a cyclic indoor environment of stage 1, persisted when goats were transferred under constant conditions (stage 2)

with a period different from 24.0h (Figures 2 and3; Tables 1 and2 A slight reduction of the shape regularity of the Tb and LA curves has been noticed during constant conditions (stage 2: Figures 2 and3). Moreover, animal activity on double-plot LA actograms appears to be daily shifted indicating a free-running state of LA. In fact, the two rhythms were shifted since acrophases were delayed after transferring animals from cyclic environment to constant conditions (Tables 1 and2). The acrophase was shifted by 21.5h for Tb and by 22.4h for LA during DD and CTa. Under free-running conditions, the robustness also changed, decreasing significantly from 40.0% to 27.1% for Tb and from 32% to 7.3% for LA (Tables 1 and2). These results, together with the persistence of rhythmicity under constant conditions, confirm the existence of a circadian clock that drives these two rhythms.

The next step of this study was to verify if daily Ta cycles are able to synchronize the two outputs of the master circadian clock, the Tb and LA rhythms. This investigation was carried out under DD and Ta cycles of stages 3, 4 and 5 of experiment 1 and stages 3 and 4 of experiment 2. Under DD conditions, the Ta cycles of stage 3 were able to generate regular rhythms of Tb and LA (Figures 2 and3). The period changed from 25.3h (Tb) and 25.0h (LA) during constant conditions

Accepted Article

This article is protected by copyright. All rights reserved to become exactly 24.0h, indicating that Ta daily cycle entrained both Tb and LA rhythms. Indeed, an increase of the robustness was observed ranging from 27.1% and 7.3% under constant conditions to 58.3% and 15.3% under cycling Ta, respectively for Tb and LA rhythms (Tables 1 and2).

In any case, when Ta rose, Tb gradually increased and its acrophase occurred during the heat phase, (Figures 4A-B, 5A-1 and 5B-1). For LA rhythm, actograms showed also high activity during heat phase while inactivity coincides with the cooling phase of Ta cycle (Figure 3, stage 3 and 4). Acrophases occurred at 15:39±0.21h for Tb rhythm and at 13:12±0.12h for LA rhythm (Tables 1 and2).

The entrainment by the Ta cycles was confirmed by the fact that a 12-h phase advance of its cycle (stage 4) led to a significant phase advance of the Tb and LA rhythms (Figures 3,4A, 5A-2 and 5B-2). All the ten bucks of experiment 1 showed a clear entrainment of Tb rhythm by Ta cycles while in experiment 2 which was performed on 7 bucks, six exhibited an entrainment of LA rhythm by Ta cycles as can be shown in Figure 3; while only one animal displayed a weak response to the entrainment effect by this cue.

The shifting of the rhythm was only within 6±0.26 days for Tb (Figures 5A-2 and 5B-2) while it took 10±0.26 days for the LA (Figure 3). The final pattern of LA rhythm was completely synchronized to the new Ta cycle within 12.5±1.07 days, having therefore a peak of activity during heat-phase at the subjective night. Accordingly, acrophase of LA occurred at 02:49±0.27h and that of Tb at 02:20±0.16h (Tables 1 and2). The one-way ANOVA confirmed the existence of a significant (Tb F (5.40) = 1114; P <0.0001 and LA F (2.50) = 874,9; P <0.0001) shifting in both acrophases.

When NRT and RT phases were separately analysed, LA onsets and offsets did not show statistical differences between successive days. This indicates a significant equality of the means around each Ta cycle transition (Figure 6). As for acrophase, the onset and the offset of LA were shifted and seemingly following the Ta cycle conditions (Figure 6). Indeed, the two parameters coincide respectively with the cold-heat and heat-cold transitions, whatever the nature of the Ta cycle.

Accepted Article

This article is protected by copyright. All rights reserved When the goats returned to the former Ta cycle with maximum of heat during subjective day (stage 5, experiment 1), (Figures 4 B, 5A-3 and 5B-3), the Tb rhythm was delayed and went back to its initial pattern observed in stage 3. Acrophase was delayed by 12.56h to occur at 14:54±0.26h

(Table 1). The ascendant and descendant phases of Tb follow those of the Ta cycles as previously seen in stage 3 (Figure 4 and 5-A).

Concerning LA rhythm, a return from an inversed Ta cycle (stage 4) to the constant conditions (stage 5) induced a free-running state showing a daily delay of its phase (stage 5: Figure 3).

Excluding the Ta cycling under DD conditions, the period becomes significantly different (F (4.30) = 39.23; P <0.0001) from 24.0h, precisely of 24.9±0.12h. The acrophase was also shifted to occur at 03:35±0.32h. Moreover, the regularity of the rhythm decreased with a robustness that also dropped to 9.2±0.48% (Table 2).

All together, these changes emphasize once again the entrainment effect of Ta cycles.

From experiments 1 and 2, Ta seemed to be a zeitgeber capable of synchronizing the circadian clock of the goat. Even if two outputs of the clock (Tb, LA) are driven by Ta, as demonstrated previously, it remains critical to ascertain that these results correspond to a real entrainment of the circadian clock and not to a masking effect. So, we verified if Ta is able to entrain the endogenous rhythm of Mel, one of the most robust outputs of the clock activity of SCN. An endogenous rhythm of Mel under DD and constant Ta (stage 2) was observed in all animals (data not shown).

No major changes were noticed in the Mel rhythm when Ta cycle with heating period during the subjective day was applied (stage 3). However, when daily Ta cycle was 12-h phase advanced (Maximum of Ta at the end of subjective night, Stage 4), the Mel rhythm was shifted (phase delay, Figure 7). A Mel phase advance was then observed when animals returned to the former Ta cycle with maximum of Ta during the subjective day (stage 5, Figure 7).

The paired t-test showed that Ta treatment induced significant phase differences for both onsets (Phi1) and offsets (Phi2) of Mel rhythm, except for offset in the return from RT (stage 4) to NRTr (stage 5) (Table 3). Under all these conditions, Mel rhythm peaked when the Ta cycle was in the cold-phase.

Accepted Article

Discussion

The daily variations of Tb and LA in goat were previously recorded in several thermoregulatory [START_REF] Finch | Why black goats in hot deserts? Effects of coat color on heat exchanges of wild and domestic goats[END_REF][START_REF] Shkolnik | Water economy, energy metabolism and productivity in desert ruminants[END_REF][START_REF] Maia | Thermoregulatory responses of goats in hot environments[END_REF] or chrono-biological [START_REF] Piccione | Circadian rhythms of body temperature and liver function in fed and food-deprived goats[END_REF][START_REF] Piccione | Thermal chronobiology of domestic animals[END_REF][START_REF] Piccione | Locomotor activity and serum tryptophan and serotonin in goats: Daily rhythm[END_REF][START_REF] Piccione | Daily locomotor activity in five domestic animals[END_REF] studies. They were clearly identified, but their circadian nature was not established yet, until we did in the present study. Indeed, our results demonstrate that both rhythms persist under constant conditions with a period close to 24.0h. This indicates that in the goat, as in other mammals [START_REF] El Allali | Entrainment of the circadian clock by daily ambient temperature cycles in the camel (Camelus dromedarius)[END_REF][START_REF] Aschoff | Human circadian rhythms in activity, body temperature and other functions[END_REF][START_REF] Stephan | Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions[END_REF][START_REF] Wever | Internal interactions within the human circadian system: the masking effect[END_REF][START_REF] Pickard | Splitting of the circadian rhythm of body temperature in the golden hamster[END_REF][START_REF] Kuwabara | Circadian, sleep and brain temperature rhythms in cats under sustained daily light-dark cycles and constant darkness[END_REF][START_REF] Lehman | Circadian rhythmicity restored by neural transplant. Immunocytochemical characterization of the graft and its integration with the host brain[END_REF] , Tb and LA comply with the rules defining circadian rhythms.

Under their desert biotope, goats are frequently exposed to a very wide range of daily Ta cycles.

Such variations are suspected to strongly affect several systems and circuits in the brain, particularly, the circadian system as demonstrated in the camel [START_REF] El Allali | Entrainment of the circadian clock by daily ambient temperature cycles in the camel (Camelus dromedarius)[END_REF] . Based on these specific findings, we have hypothesized that in the desert goat, cyclic fluctuations of Ta could also synchronize its master circadian clock. Our results clearly show that, under 24.0h applied DD, Ta cycle entrains the Tb and LA rhythms to exactly 24.0h. A 12-h switch of the daily Ta cycle induced a clear shift of both rhythms. Similarly, the return to the former Ta cycle induced a few days later a return to the initial phase (phase delay) of the Tb rhythm. Such result raises a pertinent question regarding the interpretation of the observed effect: is it a real entrainment of the master clock or a masking effect of Ta on Tb and LA rhythms?

Zeitgebers are known to act on the period and phase of the rhythm [START_REF] Refinetti | Entrainment of circadian rhythm by ambient temperature cycles in mice[END_REF] , while masking cues strengthen or weaken the rhythm and affect generally the amplitude and the shape of the overt rhythm [START_REF] Refinetti | The circadian rhythm of body temperature[END_REF][START_REF] Hanneman | Measuring circadian temperature rhythm[END_REF][START_REF] Kelly | Body temperature variability (Part 2): Masking influences of body temperature variability and a review of body temperature variability in disease[END_REF] . A masking effect of Ta on Tb is unlikely since the observed Tb peak in the goat, as in the camel [START_REF] El Allali | Entrainment of the circadian clock by daily ambient temperature cycles in the camel (Camelus dromedarius)[END_REF] , is delayed from that of Ta. This delay indicates that there is no important radiant heat load (radiant, conductive and convective) occurring at the moment of Ta peak. Tb is also modified by evaporative water loss, thermal conductance and metabolic heat production.

Moreover, in our study any significant heat production from locomotion during the experiment 1 is very unlikely since the LA of animals was restrained. The animals were also in perfect endothermy and any mechanisms of evaporative water loss, exogenous heat loading and dissipation are unlikely to occur.

When applying Ta cycle in experiment 2, a transient and progressive period before final synchronization of the LA rhythm is seen after several days. This transition is typical of an

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This article is protected by copyright. All rights reserved entraining effect on the circadian clock. Moreover, the observed changes in the period and phase led to infer that Ta does not induce the LA rhythm directly, but entrained it through a circadian clock. In natural conditions, the goat is a diurnal species [START_REF] Farsi | Validation of locomotion scoring as a new and inexpensive technique to record circadian locomotor activity in large mammals[END_REF][START_REF] Piccione | Locomotor activity and serum tryptophan and serotonin in goats: Daily rhythm[END_REF][START_REF] El Allali | Smartphone and a freely available application as a new tool to record locomotor activity rhythm in large mammals and humans[END_REF] , showing its maximal daily activity during the heat phase (the day). During Ta entrainment, bucks were also active during the heat period, regardless of Ta cycle applied. The phase relationship of activity with Ta cycle, as seen in natural conditions, is therefore maintained under Ta entrainment. In all species studied until now, in which an entrainment of LA rhythm was shown, animals keep their phase relationship with Ta cycle, nocturnal species being active during the cold phase of Ta while diurnal species being active during the warm phase [START_REF] Tokura | Effects of Temperature on the Circadian Rhythm of PigTailed Macaques Macaca nemestrina[END_REF][START_REF] Pálková | Effect of ambient temperature on the circadian activity rhythm in common marmosets, Callithrix j. jacchus (primates)[END_REF][START_REF] Refinetti | Entrainment of circadian rhythm by ambient temperature cycles in mice[END_REF][START_REF] Lindberg | Thermoperiodic Entrainment of Arousal from Torpor in the Little Pocket Mouse, Perognathus longimembris[END_REF][START_REF] Pohl | Temperature Cycles as Zeitgeber for the Circadian Clock of Two Burrowing Rodents, the Normothermic Antelope Ground Squirrel and the Heterothermic Syrian Hamster[END_REF][START_REF] Fritsche | Circadian Activity Rhythm and Entrainment in the Subterranean Common Mole-Rat (Cryptomys hottentotus)[END_REF] .

The temperature range used in the present study (from cold: 16°C to heat: 32°C) is seemingly strong enough to provide a temporal information for synchronizing LA rhythm of the goat.

Similarly, close oscillations of Ta were able to induce an entrainment of LA rhythm in rodents [START_REF] Refinetti | Entrainment of circadian rhythm by ambient temperature cycles in mice[END_REF] . It is also established that the thermo-neutral zone in the goat is between 13°C and 21°C [START_REF] Martini | Fisiologia degli animali domestici Martini[END_REF] . We believe that in desert goat breeds, adapted to their biotope high temperatures, this interval would be higher. As previously reported, the used range of Ta cycle between 16° to 32°C induced a stronger entrainment of LA rhythm. However, narrower ranges would probably provide a weaker zeitgeber. No attempt was made till now to investigate such intensity-response curve for temperature entrainment.

In mammals, Mel secretion from the pineal gland is known to be the most powerful phase-marker of the master circadian clock. It depends much more on LD than on Ta cycles [START_REF] Rensing | Temperature effect on entrainment, phase shifting, and amplitude of circadian clocks and its molecular bases[END_REF] . In goat, as in the camel [START_REF] El Allali | Entrainment of the circadian clock by daily ambient temperature cycles in the camel (Camelus dromedarius)[END_REF] , under DD conditions, plasma Mel follows Ta cycle and its peak occurred only in the cryophase. Analysis of plasma Mel combined to Tb and LA under DD conditions has, therefore, confirmed that the daily Ta cycle entrain three outputs of the master circadian clock, which precludes any masking effect. Thus, Ta cycle is a true zeitgeber in the goat. Beside the camel, this finding constitutes the second demonstration of such clear entrainment of the circadian system by Ta cycles in mammalian species. How the non-photic information of Ta reaches the SCN through central and/or peripheral thermo-receptors, and how it could presumably change its molecular machinery still need further investigation.

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Conclusion

The present work demonstrates that daily Ta cycle, a strong environmental cue in the desert habitat, is able to synchronize the central clock of the goat by entraining the Tb, LA and Mel rhythms. Until now, a similar effect has been described in only two mammalian species, namely the camel and herein the goat. This finding raises enquiry on whether such phenomenon represents common process of adaptation of the circadian system in heterothermic species to the desert or it is specific to these two species only. Accordingly, further investigations in other desert species are needed to elucidate the prevalence of this peculiar thermoregulatory feature in mammals.

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Stage 3 (Stage 4 (

 34 21 days): the DD conditions were maintained and a 24-h artificial daily Ta cycle (NRT) with a heating-period applied during the subjective day (maximum 31.93°C, minimum 16.17°C). Stage 4 (29 days): correspond to the same DD conditions but a 12-h phase advance of Ta cycle (RT) (maximum 32.47°C, minimum 16.30°C) was realized with a heating-period shifted to occur during the subjective night. Stage 5 (21 days): a return to the former conditions of stage 3 with Ta cycle peaking under the subjective day (NRTr). Experiment 2 was conducted on 7 bucks and designed to check if Ta cycles could synchronize the LA rhythm. Each buck was able to move freely in an individual controlled sheltered stable of 36m 2 . The same feed diet of experiment 1 and water were provided ad libitum; while the relative humidity was around 21%. To avoid the possible masking effect of the social behaviour of bucks on LA rhythm, experiment was conducted individually and lasts each 125 days. Each individual experiment consisted of the following stages: Stage 1 (5 days): is an adaptive period to indoor conditions with an artificial LD cycle (12L-12D) and uncontrolled Ta cycle (19.05°C-24.60°C). Stage 2 (26 days): This stage was designed to demonstrate the existence in the goat of a freerunning rhythm of LA rhythm. Constant conditions of DD and CTa (23±0.40°C) were applied. Stage 3 (27 days): The DD conditions were maintained and a 24-h artificial Ta cycle (NRT, maximum: 31.38°C and minimum: 15.98°C) with a heating-period during the subjective day was applied. 44 days): Animals were kept under the same conditions of stage 3 but a 12-h phase advance of Ta cycle (RT, maximum 32.68°C and minimum 15.01°C) was realized, with a heatingperiod during the subjective night.

  ): exactly of 25.3±0.09h for Tb rhythm and 25.0±0.10h for LA rhythm. The one-way-ANOVA showed that the periods of constant conditions (stage 2) were statistically different (Tb F (4.45)= 53.36; P <0,0001; and LA F (4.30)= 39.23; P <0.0001) from those under indoor cyclic conditions (stage 1).

Table 1 :

 1 Parameters of the Tb rhythm during different stages of experiment 1. : One-way ANOVA analysis was used for the calculation of statistical differences between periods and acrophases across different stages of the experiment 1. Different superscript lowercase letters indicate significant differences between different stages (p≤0.05). Similar superscript lowercase letters indicate no statistically significant differences between compared stages (p>0.05).

	Experiment 1	Period		Acrophase	Mesor	Amplitude Robustness
	Stage 1: LD uncontrolled Ta	24.0±0.09 h	a 17:23±0.28 h	a 38.74±0.12°C	0.69±0.36°C	40.0±2.7%
	Stage 2: DD+CTa	25.3±0.09 h	b 14:55±0.14 h	b 38.82±0.06°C	0.23±0.18°C	27.1±1.8%
	Stage 3: DD+NRT	24.0±0.02 h	a 15:39±0.21 h	c 39.18±0 .10°C	0.43±0.24°C	58.3±3.0%
	Stage 4: DD+RT	24.0±0.02 h	a 02:20±0.16 h	d 39.34± 0.15°C	0.55±0.38°C	55.7±2.3%
	Stage 5: DD+NRTr	24.0± 0.03 h	a 14:54 ±0.26 h		c 39.18± 0.18°C	0.50±0.29°C	57.3±1.9%

Note

Table 2 :

 2 Parameters of the LA rhythm during different stages of experiment 2.Note: One-way ANOVA analysis was used for the calculation of the statistical differences between periods and acrophases across different stages of the experiment 2. Different superscript lowercase or uppercase letters indicate significant differences between different stages (p≤0.05). Similar superscript lowercase or uppercase letters indicate no statistically significant differences between compared stages (p>0.05).

	Experiment 2	Period	Acrophase	Mesor	Amplitude	Robustness
	Stage 1: LD uncontrolled Ta	24.0±0.04 h	a 12:33±0.30 h		a	1.59±0.08°C 1.70±0.09°C	32.0±1.8%
	Stage 2: DD+CTa	25.0±0.10 h	b 10:58±0.30 h		aA	1.20±0.90°C 0.60±0.02°C	07.3±0.3%
	Stage 3: DD+NRT	24.0±0.05 h	a 13:12±0.12 h		aAb	1.60±0.06°C 1.00±0.03°C	15.3±1.3 %
	Stage 4: DD+RT	24.0±0.03 h	a 02:49±0.27 h	bAcB 1.53±0.10°C 0.86±0.07°C	16.4±1.1 %.
	Stage 5: DD+CTa	24.9±0.12 h	b 03:35±0:32 h	cAdB 1.31±0.06°C 0.47±0.10°C	09.2±0.5 %

Table 3 :

 3 Mean values ± S.E.M. of individual phi1 and phi2 estimated with the nonlinear regressions applied for each animal in NRT, RT and NRTr conditions of Ta cycle respectively during stages 3, 4 and 5.Note: Paired t-test analysis for the estimated Phi1 and Phi2 were performed for NRT and RT and then for RT and NRTr treatments: 1) Different superscript lowercase letters indicate significant differences between NRT and RT treatment for both Phi1 and Phi2 (P ≤0.05). 2) Different superscript uppercase letters indicate significant differences between RT and NRTr treatment in Phi1 (P ≤0.05). 3). Similar superscript uppercase letters indicate no statistically significant differences between

	Phi 1

Acknowledgments

The authors are grateful to Hassan II Agronomy and Veterinary Medicine Institute (Rabat, Morocco), the Institute of Cellular and Integrative Neurosciences (Strasbourg, France), the Laboratory of Hormonology (Lyon, France) and the Moroccan AVPN Association (Association de Vulgarisation et Promotion des Neurosciences) for supporting this study.

Funding

This study was funded by the International Foundation for Science (IFS: Research Grant Agreement No. I-3-B-6082-1).

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This article is protected by copyright. All rights reserved experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data and drafted the manuscript.

Conflict of Interest Statement

The authors declare no actual or potential conflict of interest.

Author Contributions