Sylvie Labrouche-Colomer

PhD Omar Soukarieh

PhD Carole Proust

Christine Mouton

MD Yoann Huguenin

MD Maguelonne Roux

Céline Besse

Anne Boland

PhD Robert Olaso

PhD Joël Constans

PhD Jean-François Deleuze

Pierre-Emmanuel Morange

PhD Béatrice Jaspard-Vinassa

PhD David-Alexandre Trégouët
email: david-alexandre.tregouet@inserm.fr

A novel rare c.-39C>T mutation in the PROS1 5'UTR causing PS deficiency by creating a new upstream translation initiation codon

Keywords: Protein S deficiency, venous thrombosis, whole genome sequencing, Open Reading Frame, mutation

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

Clinical Perspectives

1: Inherited Protein S deficiency (PSD) is a severe disorder associated with high risk of venous thrombosis. This study aims at characterizing the molecular defect underlying unexplained PSD observed in 7 affected relatives in who clinical sequencing diagnosis was unfruitful.

Introduction

Protein S (PS) is, with Protein C (PC) and Antithrombin (AT), one the three main natural inhibitors of the coagulation cascade and plays thus a key role in the control of blood clot formation. PS is a vitamin-K dependent glycoprotein that acts as a cofactor for PC to inactivate factors Va (FVa) and VIIIa (FVIIIa) and to limit thrombin generation via direct interactions with factor Xa (FXa) and FVa [START_REF] Castoldi | Regulation of coagulation by protein S[END_REF]. In human plasma, PS circulates both under a free and active form (~40%) and an inactive form (~60%) when complexed with C4b-binding protein. Generally, PS plasma concentration can be characterized by antigen measurements of the free and total PS levels or by PS activity. This led to the definition of three clinical subtypes of PS deficiencies: i) Type I refers to deficiency of both free and total PS as well as decreased PS activity, ii) Type II is defined by normal plasma levels but decreased PS activity while iii) Type III shows decreased free PS plasma levels and decreased PS activity but normal total PS plasma levels. Type I and Type III PS deficiencies account for ~95% of PS deficiencies and are considered to be the heterogeneous clinical expression of the same molecular defect [START_REF] Zöller | Evaluation of the relationship between protein S and C4b-binding protein isoforms in hereditary protein S deficiency demonstrating type I and type III deficiencies to be phenotypic variants of the same genetic disease[END_REF].

Autosomal dominant inherited PS deficiency (PSD) (MIM 612336) is a rare disorder caused by private or rare mutations in the structural PROS1 gene. Previous works have shown that complete or partial PSD was associated with increased risk of venous thrombosis (VT) [START_REF] Alhenc-Gelas | GEHT genetic thrombophilia group. PROS1 genotype phenotype relationships in a large cohort of adults with suspicion of inherited quantitative protein S deficiency[END_REF][START_REF] Alhenc-Gelas | Protein S inherited qualitative deficiency: novel mutations and phenotypic influence[END_REF][START_REF] Lijfering | Clinical relevance of decreased free protein S levels: results from a retrospective family cohort study involving 1143 relatives[END_REF][START_REF] Pegelow | Severe protein S deficiency in a newborn[END_REF][START_REF] Pintao | Protein S levels and the risk of venous thrombosis: results from the MEGA case-control study[END_REF].

However, a substantial proportion of PSD cases remain without any definitive molecular diagnosis [START_REF] Alhenc-Gelas | GEHT genetic thrombophilia group. PROS1 genotype phenotype relationships in a large cohort of adults with suspicion of inherited quantitative protein S deficiency[END_REF][START_REF] Hermida | Poor relationship between phenotypes of protein S deficiency and mutations in the protein S alpha gene[END_REF][START_REF] Iwaki | DNA sequence analysis of protein S deficiency--identification of four point mutations in twelve Japanese subjects[END_REF][START_REF] Biguzzi | Molecular diversity and thrombotic risk in protein S deficiency: the PROSIT study[END_REF], up to 40% according to a recent estimate [START_REF] Alhenc-Gelas | GEHT genetic thrombophilia group. PROS1 genotype phenotype relationships in a large cohort of adults with suspicion of inherited quantitative protein S deficiency[END_REF]. The majority of identified PSD causing PROS1 mutations are located in the coding regions of the gene or in their flanking sequences and are mainly missense or nonsense single nucleotide variations [START_REF] Stenson | The Human Gene Mutation Database: towards a comprehensive repository of inherited mutation data for medical research, genetic diagnosis and next-generation sequencing studies[END_REF]. Some intronic splice variants [START_REF] Choi | A rare splicing mutation in the PROS1 gene of a Korean patient with type I hereditary protein S deficiency[END_REF][START_REF] Menezes | PROS1 novel splicesite variant decreases protein S expression in patients from two families with thrombotic disease[END_REF][START_REF] Wang | PROS1 IVS10+5G>A mutation causes hereditary protein S deficiency in a Chinese patient with pulmonary embolism and venous thromboembolism[END_REF] and structural variants [START_REF] Hurtado | First case of protein S deficiency due to a translocation t(3;21)(q11.2;q22)[END_REF][START_REF] Lind-Halldén | Small and large PROS1 deletions but no other types of rearrangements detected in patients with protein S deficiency[END_REF][START_REF] Seo | The genomic architecture of the PROS1 gene underlying large tandem duplication mutation that causes thrombophilia from hereditary protein S deficiency[END_REF] have also been reported.

Note that, compared to exonic variations, very few variations have been described in the promoter region or the 5'UTR of the PROS1 gene [START_REF] Espinosa-Parrilla | Optimization of a simple and rapid single-strand conformation analysis for detection of mutations in the PROS1 gene: identification of seven novel mutations and three novel, apparently neutral, variants[END_REF][START_REF] Hurtado | Functional characterization of twelve natural PROS1 mutations associated with anticoagulant protein S deficiency[END_REF][START_REF] Li | Clinical Manifestation and Mutation Spectrum of 53 Unrelated Pedigrees with Protein S Deficiency in China[END_REF][START_REF] Sanda | An Sp1 binding site mutation of the PROS1 promoter in a patient with protein S deficiency[END_REF][START_REF] Tang | Molecular basis of protein S deficiency in China[END_REF] and even fewer have been functionally characterized. To our knowledge, the c.-168C>T is the sole PROS1 promoter variation that has been experimentally demonstrated to cause inherited PSD by affecting the core binding site of Sp1 transcription factor [START_REF] Sanda | An Sp1 binding site mutation of the PROS1 promoter in a patient with protein S deficiency[END_REF] and no PSD causing mutation has yet been described in the 5'UTR region of PROS1

We here describe a novel mutation in the 5'UTR region of the PROS1 gene that causes PSD in a family with multiple relatives affected with VT. The mutation is a C to T substitution at c.-39 from the natural ATG codon and creates a novel ATG sequence upstream of the main open reading frame that alters the translation machinery of the protein.

Materials and Methods

Recruitment of the family

The studied family was ascertained through a proband addressed to the specialized clinical hematology laboratory of the Pellegrin Hospital (Bordeaux) for a thrombophilia screening in the context of an objectively documented first VT episode early in life (20 years old).

Standard thrombophilia screening revealed a Type I PSD (Fig 1; Supplementary Table 1).

However, no candidate deleterious PROS1 mutation nor large genomic rearrangement were detected by Sanger sequencing of the 15 PROS1 exons and of their flanking intronic regions [START_REF] Labrouche | Protein C and protein S assessment in hospital laboratories: which strategy and what role for DNA sequencing?[END_REF] or by multiplex ligation dependent probe amplification (MLPA) using Salsa MLPA kit (P112 ; MRC-Holland, Amsterdam, the Netherlands), respectively. As the proband reported at least one first degree relative with VT, family members were invited to participate to a genetic exploration upon informed consent agreement according to the Helsinki declaration.

Inherited Type I or Type III PSD was diagnosed in 6 additional relatives (Fig 1 ), a seventh relative (individual IV-4) had very low PS activity but, without information on free and total PS levels, Type I, Type II or Type III PSD could not be objectively distinguished. All PSD patients with DNA available were negative for the presence of PROS1 deleterious coding mutation and for the F5 Leiden (FV Arg506Gln, rs6025), the F2 G20210 (rs1799963) mutations and any other major biological risk factors for venous thrombosis including antithrombin-, PC-deficiency or anti-phospholipid antibodies.

Phenotypic protein S deficiency was diagnosed by measuring APC-cofactor activity, free (FPS) and/or total (TPS) proteins S levels. The cofactor activity of protein S was measured by an activated partial thromboplastin time-based clotting assay using the Staclot protein S kit (Diagnostic Stago, Asnieres, France) or by the IL test protein S kit (Instrument Laboratory Company, Milano,Italy). The concentration of total and free PS antigen were determined by an enzyme-linked immunosorbent assay (Asserachrom total or free protein S; Diagnostica Stago). In normal conditions, TPS levels range from 70% to 150% while normal FPS levels are generally greater than 60% in men, and 50% in pre-menopause women (>55% in menopause women). In the related PSD patients studied here, FPS levels ranged from 18% to 38% and TPS from 51% to 70% (Fig 1). APC-cofactor activity varied between 27% and 32%. Detailed clinical and biological information on family members are given in Supplementary Table 1.

All family members participating to this study signed a written informed consent for genetic investigations, according to the Helsinki declaration.

Whole genome sequencing

With the aim of identifying the hypothesized culprit mutation causing the observed familial PSD, we sequenced the whole genome of 5 PSD patients (individuals III-2, III-8, IV-2, IV-4, IV-5) and 2 unaffected relatives (individuals IV-1 and IV-6) (Fig 1).

Whole genome sequencing was performed at the Centre National de Recherche en Génomique Humaine (CNRGH, Institut de Biologie François Jacob, Evry, FRANCE). After a complete quality control, 1µg of genomic DNA was used for each sample to prepare a library for whole genome sequencing, using the Illumina TruSeq DNA PCR-Free Library Preparation Kit, according to the manufacturer's instructions. After normalisation and quality control, qualified libraries were sequenced on a HiSeqX5 instrument from Illumina (Illumina Inc., CA, USA) using a paired-end 150 bp reads strategy. One lane of HiSeqX5 flow cell was used per sample specific library in order to reach an average sequencing depth of 30x for each sequenced individual. Sequence quality parameters have been assessed throughout the sequencing run and standard bioinformatics analysis of sequencing data was based on the Illumina pipeline to generate FASTQ file for each sample. FastQ sequences were aligned on human genome hg37 using the BWA-mem program [START_REF] Li | Fast and accurate short read alignment with Burrows-Wheeler transform[END_REF]. Variant calling was performed using the GATK HaplotypeCaller (GenomeAnalysisTK-v3.3-0, https://software.broadinstitute.org/gatk/documentation/article.php?id=4148) tool followed by recalibration. Single nucleotide variants (SNVs) that succeeded the "PASS" filter were then annotated using Annovar [START_REF] Wang | ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data[END_REF].

To comply with the autosomal dominant mode of inheritance of the observed PSD, we selected as candidate culprit PSD causing variant any rare variant that has either never been reported in public database or at a very low allele frequency (<1‰) and that were present at the heterozygous states in the 5 whole genome sequenced patients but not present in 2 healthy relatives. Interrogated public databases were dbSNP, GnomAD (https://gnomad.broadinstitute.org/), Ensembl (https://www.ensembl.org/index.html), 1000 genomes (https://www.internationalgenome.org/), ExAC (http://exac.broadinstitute.org/) and FrEx (http://lysine.univ-brest.fr/FrExAC/).

Sanger sequencing validation

Sanger sequencing validation of the identified candidate variant was carried out using primers designed to span all the putative transcription factor binding sites described by de Wolf ( 27) (Supplementary Table 2). PCR amplification of genomic DNA was performed using Phusion™ Green Hot Start II High Fidelity DNA Polymerase with GC Buffer in presence of 10% of DMSO (ThermoFisher). Big dye sequencing chemistry was used to sequence the PCR products in both directions using the ABI 3500xL Genetic analyser (Applied Biosystems, Foster City, CA) and the sequence were analyzed by the SeqScape Software (Applied Biosystems, Foster City, CA).

Plasmid constructs

To assess the impact of the PROS1 c.-39C>T variation, several expression vectors were 3) fused in frame with the myc-His tag into pcDNA3.1; (ii) an elongated mutant PROS1 vector (Elongated mutant PROS1) was obtained from the Natural mutant PROS1 in which the new stop codon induced by the c.-39T allelic variant was suppressed by creating a 1-bp deletion (c.111delT) through rapid-site-directed-mutagenesis using phosphorylated non-overlapping primers (Supplementary Table 3) and Phusion HF DNA Polymerase (ThermoFisher), according to the manufacturer's recommendations. The latter construct was used here as an experimental control to avoid any ambiguity about the detection of the small-size expected fusion protein from the new uORF by western blot. All the recombinant plasmids were verified by Sanger sequencing (Genewiz).

Cell culture and transfection

HeLa cells were cultured in RPMI medium (Gibco-Invitrogen) supplemented with 10% fetal calf serum (Gibco-Invitrogen), 1% penicillin/streptomycin and 1% Hepes. Cells were maintained in T75 flasks in a humidified atmosphere of 95% and 5% CO2 at 37°C. Twenty four hours before the transfection, 6-well plates of HeLa cells were prepared with 4x10 5 cells/well. Transfections were performed with jetPRIME® reagent (Polyplus Transfection) according to the manufacturer's recommendations, with 0.5-1.5 µg/mL of each expression plasmid or empty pcDNA™3.1/myc-His(-) vector at a cell confluence between 60 and 80%. In some experiments, 0,5 μg of pcDNA3.1/myc-His/lacZ was included in each transfection to control the transfection efficiency. Cells were harvested and lysed 48 hours after transfection to extract total RNA and protein.

RNA isolation and RT-qPCR analysis

Total RNA was isolated from the transfected HeLa cells using the RNeasy mini kit (Qiagen) following the manufacturer's instructions. Reverse transcription of 2 µg of total RNA to single-stranded cDNA was performed using High Capacity cDNA kit (Applied Biosystems). To quantify PROS1 transcripts, qPCR were carried out on the generated cDNA (diluted at 1/5) using PowerUp SYBR master mix (Thermofisher Scientific) in a final volume of 10 µl and 40 cycles of amplification on QuantStudio3 Real-Time PCR System (ThermoFisher). Results were analyzed with the QuantStudio design and analysis software. Transcript levels were normalized to the reference RPL32 gene. Relative quantification of PROS1 in different samples was conducted according to threshold cycle (Ct) value based on the ΔΔCT method.

All the primer pairs used in this study (Supplementary Table 4) have a reaction efficiency between 90% and 110% and melting curves were analyzed to check the specificity of each RT-qPCR reaction.

Protein preparation and western blot analysis

Protein extracts from the transfected HeLa cells were prepared by using 140 µL per well of RIPA buffer containing protease inhibitors. After clarification of the extracts by centrifugation, protein concentration was determined by the BCA method (Pierce™ BCA Protein Assay Kit). Equal amounts (50 µg) of total protein suspended in loading buffer were separated by SDS-PAGE using 8% or 4-20% gradient gels and transferred onto PVDF membranes (Immobilon-P or Immobilon PSQ, Merck Millipore) for immunostaining. Then, membrane was divided into two halves and probed with either monoclonal anti Myc Tag antibody (Merck Millipore) or anti-α-tubulin antibody (Sigma-Aldrich). After incubation with goat anti-mouse IgG Alexa Fluor 700 (ThermoFisher), blots were simultaneously scanned on Odyssey Infrared Imaging System (Li-Cor Biosciences) in the 700 channel. α-Tubulin was used as control.

Statistical data analysis

Differential mRNA levels analysis according to experimental conditions were performed with ANOVA followed by Tukey's HSD post-hoc tests for multiple comparisons. A statistical threshold of p <0.05 was used to declare statistical significance.

Results

With the aim of completely ruling out the possibility of PSD causing mutations in the structural gene, we first focused on genetic variations mapping the PROS1 locus, downstream and upstream of the coding sequence. We thus identified a never reported c.-39C>T (variant in the PROS1 5'UTR (Human Genome Variation Society nomenclature: Because of the broad role of uORFs on downstream translation [START_REF] Johnstone | Upstream ORFs are prevalent translational repressors in vertebrates[END_REF][START_REF] Lin | Impacts of uORF codon identity and position on translation regulation[END_REF], we hypothesized that the detected variant could be at the origin of the observed PSD. To validate this hypothesis, we first confirmed the presence of the variant in the five sequenced patients by Sanger sequencing (Supplementary Figure 1) and genotyped it in one additional affected related (individual IV-7) for whom DNA was available. The absence of the the variant in the two healthy relatives with available DNA was confirmed by Sanger sequencing as well.

Then, to experimentally assess the impact of the variant on PROS1 translation, we first generated 2 myc-His tagged molecular constructs (Fig 2B ), the full length PROS1 constructs containing either the wild-type ("WT") or the mutated -39T allele ("Natural mutant"). We observed that transfecting HeLa cells with the wild type or the natural mutant was 5). To go further, we generated an elongated mutant plasmid (see Materials and Methods) allowing the translation of a longer tagged protein from the identified upstream start codon. Western blot analyses showed the presence of a protein at the expected 81.9 kDa weight (Figure 4C), suggesting that a translation from the uAUG and driven by the uORF was initiated. However, the level of expression of the latter tended to be lower than that of the wild type protein, despite similar mRNA levels (Supplementary Table 5). This second set of experiments suggest that the c.-39C>T variant creates a new functional uAUG codon that is able to initiate translation process while inhibiting the translation of the normal form of the Protein S from the main AUG.

Discussion

We here report the first case of inherited PSD due to a never reported mutation in the PROS1 promoter region that creates a premature ATG codon out-of-frame relative to the natural ATG codon, thus generating a premature stop codon.

Following a negative target gene sequencing of the PROS1 exonic sequences and large genomic rearrangement, a whole genome sequencing strategy was adopted to identify the molecular defect responsible for inherited PSD in a French pedigree with 8 PSD affected individuals and 2 unaffected relatives. This led to the identification of the novel c.-39C>T variant, with the rare T allele generating a novel uAUG start codon and a novel overlapping uORF of 51 amino acids. Even if the flanking sequence of the new ATG does not completely match to the consensus predicted Kozak sequence ((G/A)NNAUGG), it seems similar to the one flanking the natural PROS1 ATG, suggesting that the new ATG could be identified by the translational machinery and thus used in the cells [START_REF] Kozak | Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6[END_REF][START_REF] Kozak | Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes[END_REF]. Several works have previously demonstrated that uAUG and uORF are cis-regulatory elements in 5'UTR that are able to control protein expression by altering the translation efficiency and could subsequently be associated with risk of diseases [START_REF] Dvir | Deciphering the rules by which 5'-UTR sequences affect protein expression in yeast[END_REF][START_REF] Orr | Alternative ORFs and small ORFs: shedding light on the dark proteome[END_REF][START_REF] Jones | Translational effects and coding potential of an upstream open reading frame associated with DOPA Responsive Dystonia[END_REF][START_REF] Von Bohlen | A mutation creating an upstream initiation codon in the SOX9 5' UTR causes acampomelic campomelic dysplasia[END_REF]. Many previously ignored uORFs are now known to act as major post-transcriptional regulatory elements or to be translated to produce bioactive peptides or proteins [START_REF] Jones | Translational effects and coding potential of an upstream open reading frame associated with DOPA Responsive Dystonia[END_REF][START_REF] Orr | Alternative ORFs and small ORFs: shedding light on the dark proteome[END_REF].

Interestingly, the relative repression of uORF-containing mRNA was shown to be more pronounced when the uORF overlaps the translation starting site of the CDS [START_REF] Johnstone | Upstream ORFs are prevalent translational repressors in vertebrates[END_REF]. Our experimental data confirm that the c.-39T allele alters the translation of the main ORF leading to low Protein S production consistent with the observed PSD in carriers of the mutation. The c.-39T allele is expected to produce a shorter protein of 51 amino acids with no homology to wild-type Protein S. However, we were not able to detect it through our WB experiments. This prevented us from assessing whether the resulting protein is not stable, or not produced because of a possible degradation of the new mRNA by the nonsense mediated decay (NMD). Unfortunately, we were not able to collect mRNA materials from patients, the main limitation of this study, which prevented us from investigating the aforementioned NMD hypothesis.Would mRNA have been available, we could have validated the presence of the expected uORF in carriers of the identified mutation.

Importantly, none of the popular bioinformatics tools used to annotate variants such as PolyPhen, Sift, CADD [START_REF] Rentzsch | CADD: predicting the deleteriousness of variants throughout the human genome[END_REF], TraP [START_REF] Gelfman | Annotating pathogenic non-coding variants in genic regions[END_REF] or RegulomeDB [START_REF] Boyle | Annotation of functional variation in personal genomes using RegulomeDB[END_REF] were able to predict any deleteriousness for this variant . The PreTIS software (41) dedicated to 5'UTR variants predicted a "moderate" impact of the variant while the DeepSea suite's predictions [START_REF] Zhou | Deep learning sequence-based ab initio prediction of variant effects on expression and disease risk[END_REF][START_REF] Zhou | Predicting effects of noncoding variants with deep learningbased sequence model[END_REF] were neither supportive for a strong functional impact of the variant. While the identified c.-39C>T mutation segregates with PSD, it is also important to emphasize that all but one family members with PSD and complete clinical information experienced one or more VT events, with age of onset of first VT varying between 16 and 38 years old. A part from the PSD patient with missing clinical information (individual IV-4), the sole affected patient carrying the disease mutation that has not (yet) developed a thrombotic event is the youngest one. She is currently 16 yrs old and her free PS levels were 18%. Free PS levels in other relatives with combined PSD & VT ranged from 22% to 38% while such values ranged from 88% to 125% in healthy family members. As no thrombotic event was in non PSD relatives, these observations are in agreement with previous works suggesting genetically induced low free PS levels as a strong risk factor for VT. Indeed, Pintao et al observed that FPS levels lower than 33% was associated with an Odds Ratio (OR) for VT of ~5 [START_REF] Pintao | Protein S levels and the risk of venous thrombosis: results from the MEGA case-control study[END_REF]. In a retrospective family, Lijfering et al. observed an increased OR for VT of ~6 in relatives with FPS levels lower than 41% compared to relatives in the upper quartile of FPS distribution. Similarly, Alhenc-Gelas et al [START_REF] Alhenc-Gelas | GEHT genetic thrombophilia group. PROS1 genotype phenotype relationships in a large cohort of adults with suspicion of inherited quantitative protein S deficiency[END_REF] proposed to define a ~30%-40% cut-off value for FPS levels as a way to identify individuals at high VT risk. All these proposed thresholds were consistent with the FPS levels observed in our studied family. As suggested by Alhenc-Gelas et al, the associated VT risk and the threshold value for FPS levels may be variable and may depend on the characteristics of the underlying causative variant. As an example, Suchon et al. demonstrated that the PROS1 Heerlen mutation was associated with an OR for VT of ~6 but a moderate decrease in free PS levels (~ 70%) [START_REF] Suchon | Protein S Heerlen mutation heterozygosity is associated with venous thrombosis risk[END_REF].

Of note, this family project was set up in the context of the GenMed LABoratory of Excellence (http://www.genmed.fr/index.php/fr/), one of the objectives of which is to propose WGS in Mendelian disorders for which whole exome sequencing and target gene sequencing has been inconclusive. Even though WGS is now more and more affordable, it must be stressed that a less expensive deep sequencing of the PROS1 locus including regulatory regions would have likely permit the identification of the culprit mutation. This illustrates the importance of exploring regulatory regions of known disease gene when searching for molecular diagnostics, a result that goes far beyond PSD but shall generalized to other inherited (hematological) disorders.

Authors ' contributions

Clinical investigations of patients and of their relatives were conducted by SLC, CM, YH and JC. Whole sequencing was performed by CB, AB and RO. MR was in charge of the bioinformatics analysis of the whole genome sequenced data. Experimental investigations were conducted by CP, OS and BJV. The research study was designed by JFD, PEM, BJV and DAT. The manuscript was drafted by SLC, BJV and DAT and further reviewed by all coauthors. (C) Protein S expression was evaluated in HeLa cell lines transfected with 1µg of vectors encoding WT PROS1, Natural mutant PROS1, Elongated mutant PROS1 or empty vector control. To control the transfection efficiency, 0,5 μg of pcDNA3.1/myc-His/lacZ was included in each transfection. Note that the wild-type construct directs expression of full-length protein S. Interestingly, we highlighted the presence of a larger mutant protein with the elongated mutant PROS1 construct, probably resulting from the use of the new AUG.

2 :

 2 We identify the first non coding mutation causing PSD via the creation of a new upstream translation initiation codon that generates an upstream open reading frame (uORF) 3: This work illustrates the importance of better exploring uORF-creating variants in the context of clinical sequencing

  investigated (Fig 2B). The human PROS1 cDNA (GenBank NM_000313.4) was PCR-amplified from HepG2 cDNA and cloned into pcDNA3.1/myc-His(-) plasmid (Invitrogen). The PCR amplification of PROS1 from c.-44 to c.2028 was performed using Phusion High Fidelity (HF) DNA Polymerase (ThermoFisher) and primer pairs designed to be either complementary to the wild-type sequence or carrying the c.-39C>T variant on the forward primer only (Supplementary Table

  [START_REF] Alhenc-Gelas | GEHT genetic thrombophilia group. PROS1 genotype phenotype relationships in a large cohort of adults with suspicion of inherited quantitative protein S deficiency[END_REF]

  ). After restriction enzyme digestion, the wild-type and mutant PCR products were cloned into pcDNA3.1 to fuse the full-length PROS1 coding sequence in-frame with a myc-his tag, generating the wild-type (WT PROS1) and the mutant (Natural mutant PROS1) PROS1 vectors, respectively (Fig 2B). To analyze the translation potential of the novel upstream start codon and overlapping uORF generated by the c.-39 C>T variant, two additional expression vectors were constructed: (i) a truncated mutant PROS1 vector was constructed by PCR-amplification of PROS1 cDNA from c.-44 to c.110 (cloning of the overlapping uORF sequence) using mutagenic primers (Supplementary Table

  5'UTR cDNA : NM_000313.3(PROS1):c.-39C>T ; gDNA: Chr3(GRCh37):g.93692632G>A) present at the heterozygous state in all 5 PSD sequenced patients and absent in the two sequenced healthy relatives. The rare T allele results into a novel upstream translation start codon (uATG) generating a novel overlapping upstream open reading frame (uORF) of 51 codons out-offrame with the normal ORF (Fig 2A).

  homogeneously and significantly associated (p <10 -3 ) with increased PROS1 mRNA levels as compared to controls (Fig 3A and Supplementary Figure 2) without altering the expression of endogeneous PROS1 (Fig 3B). Interestingly, the overexpression was not significantly different (p = 0.38) between WT and Natural mutant. These first results suggest that the c.-39C>T variant does not impact the transcription machinery of the PROS1 transgene. We further investigated the synthesis of the fusion-myc-His construct proteins and confirmed that PROS1 protein was produced in a dose-dependent manner by the wild type plasmid while no PROS1 protein was detected with the natural mutant plasmid (Fig 4A). To better understand the underlying mechanisms, we generated the "truncated mutant" construct, containing the overlapping uORF in frame with the tag (Fig 2B). Our aim was to evaluate the ability of the novel upstream start codon to initiate the translation of an expected 8.3kDa fusion protein (https://www.snapgene.com/snapgene-viewer/). We failed to evidence any fusion protein synthesis in this molecular weight range (Fig 4B) while the expression of PROS1 transgene was confirmed by qPCR (Supplementary Table

  The c.-39C>T variant was further screened in a sample of 150 PS-deficient patients with thromboembolic disease available at the specialized clinical hematology laboratory of the Pellegrin Hospital (Bordeaux) and in sample of 200 VT patients with normal PS plasma levels and sequenced for their whole genome at the Laboratory of Haematology of the La Timone Hospital (Marseille) (bioRxiv 2020.03.29.012054; doi: https://doi.org/10.1101/2020.03.29.012054). No carrier was identified. Nevertheless, in the light of the present finding, it would be interesting to systematically investigate whether such type of non coding mutations that create overlapping uORF in PROS1 could explain PSD cases that have been so far molecularly unsolved.

Figure 1 :Figure 2 :

 12 Figure 1: Pedigree of the studied family affected with Protein S deficiency

Figure 3 :

 3 Figure 3: Impact of the PROS1 c.-39C>T variant on PROS1 RNA expression

4

 4

  ).. Results were normalized to RPL32 mRNA and analyzed by using the comparative Ct Method (ΔΔCt Method). -Ct Values are shown on y-axis.

Figure 4 :

 4 Figure 4: Impact of the PROS1 c.-39C>T variant on the Protein S expression

Acknowledgments

O.S and M.R were financially supported by the GENMED Laboratory of Excellence on Medical Genomics [ANR-10-LABX-0013]. DA T was supported by the «EPIDEMIOM-VTE» Senior Chair from the Initiative of Excellence of the University of Bordeaux. This work was partially supported by the GENMED Laboratory of Excellence on Medical Genomics (ANR-10-LABX-0013) and the French Clinical Research Infrastructure Network on Venous Thrombo-Embolism (F-CRIN INNOVTE), two research programs managed by the National Research Agency (ANR) as part of the French Investment for the Future.


Corrêa Romain David

Pedro Luiz Pizzigatti

Rorie Edmunds

Danton Ferreira Vellenich

Ana Heredia

Jeaneth Machicao

Laurence Mabile

Yasuhiro Murayama

Margaret O'brien

Solange Santos

Pedro Pizzigatti Corrêa

Alison Specht

Shelley Stall
email: sstall@agu.org

Laurence Ma

Challenges in data and software management and sharing at the global level: the case of the PARSEC project

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.

Data Strand in PARSEC

In the PARSEC project, data science experts are partnering with a multi-country synthesis science research team to build relevant tools and processes for better data and software management that integrate into the research lifecycle and are to be shared with the wider research community. In recent months, we have seen important papers and books published that support this larger goal of helping researchers incorporate data and software management during their whole lifecycle, but there is still much work to be done to develop the methods and foster the cultural change needed to implement them in practice across scientific communities. The main challenges identified so far can be grouped into (a) technological ones and (b) cultural ones. We would like to share these considerations and work collaboratively on incremental improvements that would be beneficial for all. The PARSEC project is designed to provide a unique opportunity for data and synthesis scientists to collaborate and exchange in real-time toward the goal of improving research outcomes, data sharing, and data reuse. It will also help pioneer new scientific and data science technologies aimed at improving both the management and conservation of global biodiversity.

We have two interactive teams in this project: a Synthesis Science team and Data Science team.

• The Synthesis Science team is employing artificial intelligence techniques to analyse satellite images and socio-economic information to better predict and mitigate the effect(s) of the creation of Protected Areas that may potentially threaten the livelihoods and health of local (indigenous) communities. Like most researchers who investigate complex environmental problems, the team depends significantly on the availability of high quality, spatially dispersed, multidisciplinary, and time-series data. • The Data Science team of leading environmental data management professionals representative of data communities (RDA, ESIP), societies (AGU), and infrastructures for data attribution (e.g., DataCite and ORCID) are working to develop leading practices on data citation, attribution, credit, and reuse. Using an integrated work with the synthesis science team as a case study of the wider scientific community, the data-science team will provide a review of best practices for data management and stewardship to optimise data access and reuse. The team will also develop and implement a new tool to better track data usage and reuse.

Dealing with international complexity

PARSEC comprises over 30 team members from six countries, speaking 4 languages. There are 4 PIs, and a lead researcher representing each country.

PARSEC team is disciplinarily diverse, with ecologists, statisticians and modellers, economists, spatial scientists, data scientists and experts in artificial intelligence. An overarching challenge for the project is sharing what we learn and develop with the broader research community. Engaging with working groups within the RDA is one way to get additional feedback.

Opportunities to work directly with research teams, such as those funded by our grantor, the Belmont Forum, will be an important first step. This poster articulates the technological challenges prompted by the many aspects of team diversity and physical location as well as the cultural challenges. We are seeking better understanding and we embrace the value of different team dynamics, methods of communication, and preference to research approach.

Planned outcomes

• To be able to determine the influence of natural protected areas on consumption expenditure and asset health of rural communities in a range of countries; • To have refined recommendations for the research data workflow and skills for research teams; • To have developed tools for researchers to view how the data they have deposited are used and cited. This should improve the number of citations to data sets and better attribute them to the data creator;

To have contributed to the promotion of FAIR, CARE and TRUST through authentic examples from an active research project.

PARSEC roadmap proposal to meet data strand objectives

Step 1: Inventory success stories / landscape / good practices / tools / RDA or other fora recommandations

Step 2: Collect the needs of researchers / how to support them

Step 3: Propose practices to researchers / validation by the whole community / training material development

Step 4:Train and support rechearch teams, adapt best tools to researchers needs

Step 5: Asses efficiency to resolve issues and ensure sustainability of applied practices

You reap what you sow!!

A garden that is planned, cultivated and managed well will yield more produce than one where you chose the wrong plants, forgot to mark what seeds were planted where, didn't fertilise or water regularly, and allowed the weeds to take over. Like watering and weeding, a little, consistent work throughout the research life cycle will result in data that are of value to others and able to be shared as part of any research output. In PARSEC, we value scientific data as an important contribution to scholarly work. We are taking careful and measured steps towards ensuring our data are well managed, FAIR and capable of having an enduring role in the scholarly ecosystem beyond the lifetime of our project.

•

  BRAZIL: University of São Paulo -The São Paulo Research Foundation, FAPESP (P. Pizzigatti Corrêa) plus postdoc and technical support (FAPESP) • FRANCE: Foundation for Research on Biodiversity (FRB), University of Toulouse III -Paul Sabatier -ANR (N. Mouquet), ERINHA (FAIR DMP advisor -R. DAVID) • JAPAN: National Institute of Information & Communications Technology (NICT), Research Institute for Humanity and Nature (RIHN) -JST (Y. Murayama) • USA: American Geophysical Union (AGU) -NSF (S. Stall) PARSEC challenges to meet data strand objectives * First data strand PARSEC references: ★ David, R., Mabile, L., Specht, A., Stryeck, S., Thomsen, M., Yahia, M., Jonquet, C., Dollé, L., Jacob, D., Bailo, D., Bravo, E., Gachet, S., Gunderman, H., Hollebecq, J.-E., Ioannidis, V., Le Bras, Y., Lerigoleur, E., Cambon-Thomsen, A. and Research Data Alliance -SHAring Reward and Credit (SHARC) Interest Group, T.R.D., 2020. FAIRness Literacy: The Achilles' Heel of Applying FAIR Principles. Data Science Journal, 19(1), p.32. DOI: http://doi.org/10.5334/dsj-2020-032 RDA's 16th Plenaryremotely, Costa Rica, "Knowledge Ecology", 09 -12 November 2020 PARSEC objectives: Building New Tools for Data Sharing and Reuse through a Transnational Investigation of the Socioeconomic Impacts of Protected Areas

Acknowledgements:

PARSEC is funded by the Belmont Forum through the National Science Foundation (NSF), The São Paulo Research Foundation (FAPESP), the French National Research Agency (ANR), and the Japan Science and Technology Agency (JST).


Caroline Abreu

Yannig Nedellec

Andrew J Gross

Olivier Ondel

Francois Buret

Alan Le Goff
email: alan.le-goff@univ-grenoble-alpes.fr

Michael Holzinger
email: michael.holzinger@univ-grenoble-alpes.fr

Serge Cosnier

Assembly and Stacking of Flow-through Enzymatic Bioelectrodes for High Power Glucose Fuel Cells

Keywords: biofuel cell, glucose, series and parallel stacks, enzymes, carbon nanotubes

Bioelectrocatalytic carbon nanotube based pellets comprising redox enzymes were directly integrated in a newly conceived flow-through fuel cell. Porous electrodes and a separating cellulose membrane were housed in a glucose/oxygen biofuel cell design with inlets and outlets allowing the flow of electrolyte through the entire fuel cell. Different flow setups were tested and the optimized single cell setup, exploiting only 5 mmol L -1 glucose, showed an open circuit voltage (OCV) of 0.663 V and provided 1.03 ± 0.05 mW at 0.34 V. Furthermore, different charge/discharge cycles at 500 Ω and 3 kΩ were applied to optimize long-term stability leading to 3.6 J (1 mW h) of produced electrical energy after 48 h. Under continuous discharge at 6 kΩ, about 0.7 mW h could be produced after a 24 h period. The biofuel cell design further allows a convenient assembly of several glucose biofuel cells in reduced volumes and their connection in parallel or in series. The configuration of two biofuel cells connected in series showed an OCV of 1.35 V and provided 1.82 ± 0.09 mW at 0.675 V, and when connected in parallel, showed an OCV of 0.669 V and provided 1.75 ± 0.09 mW at 0.381 V. The presented design is conceived to stack an unlimited amount of biofuel cells to reach the necessary voltage and power for portable electronic devices without the need for step-up converters or energy managing systems.

■ INTRODUCTION

Fuel cell technology relies on the conversion of chemical energy into electrical energy and has gained much attention as an alternative power generator in the automotive sector. [START_REF] Marx | On the sizing and energy management of an Hybrid Multistack Fuel Cell -Battery System for Automotive Applications[END_REF] The fuel of choice for these applications is hydrogen but its high flammability represents a risk for end-users, requiring sophisticated security measures. [START_REF] Durbin | Review of Hydrogen Storage Techniques for on Board Vehicle Applications[END_REF] Glucose is one of the most important energy sources of many living organisms and can be considered as a safe, easy-to-handle, and even edible fuel. The main difference compared to hydrogen fuel cells is that glucose biofuel cells (GBFC) convert glucose instead of hydrogen at the anode for power production while the principle of the electrocatalytic oxygen reduction at the cathode remains the same. One of the greatest challenges in GBFC research concerns the choice of the catalyst. Noble metal catalysts like platinum are not only expensive but also often inappropriate due to rapid poisoning issues and efficient activities only in acid and alkaline media. [START_REF] Santiago | Review of Implantable and External Abiotically Catalysed Glucose Fuel Cells and the Differences between their Membranes and Catalysts[END_REF][START_REF] Elouarzaki | Electrocatalytic oxidation of Glucose by Rhodium Porphyrin-functionalized MWCNT Electrodes: Application to a fully molecular Catalystbased Glucose/O 2 Fuel Cell[END_REF] Biological catalysts (enzymes) are promising alternatives since most of them are operational at neutral pH and ambient temperature. [START_REF] Minteer | Enzyme-based Biofuel Cells[END_REF] Furthermore, enzymes have a unique selectivity which allows power production in complex media. In the case of glucose oxidase (GOx), this enzyme oxidizes glucose to gluconolactone removing two electrons which are transferred to the cofactor flavin adenine dinucleotide (FAD). The formed FADH 2 usually regenerates by reducing oxygen to hydrogen peroxide which is commonly considered as an unwanted byproduct for biofuel cell applications. [START_REF] Ferri | Review of Glucose Oxidases and Glucose Dehydrogenases: A Bird's Eye View of Glucose Sensing Enzymes[END_REF] To avoid its production, electron mediators are used to regenerate FADH 2 which clearly improve the electron transfer to the electrode. [START_REF] Gregg | Redox Polymer Films containing Enzymes. 2. Glucose oxidase containing enzyme electrodes[END_REF][START_REF] Reuillard | High power Enzymatic Biofuel Cell based on Naphtoquinone-mediated oxidation of Glucose by Glucose oxidase in a Carbon Nanotube 3D matrix[END_REF][START_REF] Milton | Rational design of Quinones for High Power Density Biofuel Cells[END_REF] However, the use of such mediators does not totally prevent H 2 O 2 production and in many cases a further enzyme (catalase) may be incorporated in bioanodes to convert H 2 O 2 to water and oxygen. [START_REF] Zebda | Mediatorless high-power Glucose Biofuel Cells based on compressed Carbon Nanotube-Enzyme Electrodes[END_REF] At the biocathode, multicopper enzymes like laccase and bilirubin oxidase (BOD) are promising candidates since these biocatalysts reduce oxygen to water in a four electron reduction and the catalytic site can be directly regenerated by the electrodes. [START_REF] Goff | Recent Progress in Oxygen-Reducing Laccase Biocathodes for Enzymatic Biofuel Cells[END_REF][START_REF] Lalaoui | Fully Oriented Bilirubin Oxidase on Porphyrin-Functionalized Carbon Nanotube Electrodes for Electrocatalytic Oxygen Reduction[END_REF][START_REF] Lalaoui | Diazonium Functionalisation of Carbon Nanotubes for Specific Orientation of Multicopper Oxidases: Controlling Electron Entry Points and Oxygen Diffusion to the Enzyme[END_REF] With this background, GBFCs are envisioned to produce energy out of body fluids since they contain both glucose and oxygen as fuel and oxidant, respectively. [START_REF] Cosnier | Towards Glucose Biofuel cells implanted in Human Body for powering Artificial Organs: Review[END_REF] As such, GBFCs offer the exotic prospect of providing an autonomous power supply for implantable electronic devices from the human body. Even when the current densities are promising, the cell voltage of individual GBFCs is not high enough to run a device. Szczupak et al. implanted glucose biofuel cells in clams and connected three of them in parallel and in series for charging a capacitor which activated an electric motor to turn. [START_REF] Szczupak | Living Battery -Biofuel Cells operating in vivo in Clams[END_REF] MacVittie et al. connected two "cyborg" lobsters one year later and activated a watch for more than 1 h. [START_REF] Macvittie | From ″Cyborg″ Lobsters to a Pacemaker Powered by Implantable Biofuel Cells[END_REF] Several configurations were tested to connect two GBFCs in series in one lobster (in its back and in its claws) to reach the necessary voltage. However, the low impedance of the lobster's body tissues prevented realization of the desired voltage output. Since it is not realistic to connect two or more individual organisms for sufficient power to run devices, an appropriate design of GBFCs is necessary. Sakai et al. proposed already in 2009 a design with two stacked GBFCs, in a parallel setup, which doubled current densities using individualized inlets and outlets for glucose solutions and air, [START_REF] Sakai | Evolution of Sony's Biofuel Cell[END_REF] but this design is limited to the connection of only two biofuel cells. Miyake et al. used laminar bioelectrodes for a fructose biofuel cell with fructose containing hydrogels as separators and connected three biofuel cells in series. [START_REF] Miyake | Flexible, layered Biofuel Cells[END_REF] Here, the lack of a refueling system and issues concerning drying of the hydrogel led to reduced operational lifetime.

In this respect, the design of scalable and stackable GBFCs with optimized access for both glucose and oxygen is of particular importance. We have designed a novel flow-through concept leading to optimized power outputs, energy yields, and lifetimes. The flow of oxygen and glucose solutions was optimized in especially designed housings which allow the connection of several GBFCs in series and in parallel. While classic biofuel cell designs always privilege two electrodes in an electrolyte or a flow of solution between both electrodes, [START_REF] Reid | Enzymatic Biofuel Cell with a Flow-through Toray Paper Bioanode for Improved Fuel Utilization[END_REF][START_REF] Rincoń | Enzymatic Fuel Cells: Integrating flowthrough Anode and air-breathing Cathode into a Membrane-less Biofuel Cell Design[END_REF][START_REF] Rincoń | Flow-through 3D Biofuel Cell Anode for NAD+-dependent Enzymes[END_REF][START_REF] Du Toit | Continuous Power generation from Glucose with two different Miniature Flow-Through Enzymatic Biofuel Cells[END_REF] we present here the first example of an enzymatic fuel cell in which the electrolyte flows through the whole biofuel cell. This was possible by integrating a porous cathode and anode separated by a porous cellulose membrane. This system has many advantages in terms of ease of fabrication, flexibility, and potential microfabrication. Furthermore, this system allows the easy fabrication of biofuel cells and their integration in more sophisticated circuits. Therefore, the work reported herein is dedicated to the power management of GBFCs to obtain sufficient power to run nomadic low-power devices like a timer or a watch without the need for capacitors or step up converters over long-term periods of several days.

■ MATERIAL AND METHODS

All reagents including glucose oxidase (GOx) from Aspergillus niger and catalase from bovine liver were purchased from Aldrich. Bilirubin oxidase (BOD) from Myrothecium verrucaria was obtained from Amano. Multiwalled carbon nanotubes (MWCNTs) were purchased from Nanocyl (>95% purity, 10 nm diameter, 1.5 μm length). The microporous GDL with 210 μm of thickness, 8 mΩ cm 2 of through plane electrical resistance and through plane air permeability of 70 s, was purchased from Paxitech. The 100% cellulose membrane with 0.13 mm thickness and 64 g m -2 was purchased from FiltraTech. The graphoil came from Panasonic with 0.07 mm thickness and 1000 W/m K thermic conductivity. Enzymes were stored at -20 °C. Distilled water was obtained by water purification to a resistivity of 15 MΩ cm using a Millipore Ultrapure system. High purity oxygen and argon were obtained from Messer. Glucose solutions were left to mutarotate overnight to β-D-glucose prior to use.

For electrochemical characterization of the biofuel cell, the anode was set as the working electrode while the cathode was plugged as the counter-reference electrode. For half-cell testing, cyclic voltammograms (CVs) were recorded with the anode or cathode as the working electrode, a Ag/AgCl (sat. KCl) reference electrode and a Pt wire counter electrode. All experiments were conducted in Mc Ilvaine buffer pH 7. The biofuel cell was connected to the multichannel potentiostat Biologic VMP3 running EC-lab software 10.39. Polarization and power curves were measured after 30 s discharge. All the experiments were performed at room temperature.

Preparation of the Bioanode. The MWCNT pellets were obtained by soft grinding of a mixture of 100 μL of distilled water, 5 mg of 1,4-naphthoquinone, 15 mg of GOx from Aspergillus niger (174 U mg -1 ), 10 mg of catalase from bovine liver (1600 U mg -1 ) and 35 mg of MWCNTs. Catalase was used in combination with GOx in order to catalyze both the decomposition of hydrogen peroxide and the removal of oxygen at the bioanode. The homogeneous paste is then compressed in a hydraulic press to obtain a pellet with a 1.3 cm diameter and 2 mm thickness in a plexiglass holding, as opposed to our classical procedure where a free-standing pellet is obtained. [START_REF] Zebda | Mediatorless high-power Glucose Biofuel Cells based on compressed Carbon Nanotube-Enzyme Electrodes[END_REF] A GDL disc was then connected to one side of the pellet to make the electrical connection with the graphite plate.

Preparation of the Biocathode. A similar procedure was employed using 15 mg BOD from Myrothecium verrucaria (1.96 U mg -1 ) and 35 mg of functionalized MWCNTs. The efficient immobilization and orientation of BOD on MWCNT electrodes by using π-stacked protoporphyrin IX as a direct electron-transfer promoter is used. [START_REF] Lalaoui | Fully Oriented Bilirubin Oxidase on Porphyrin-Functionalized Carbon Nanotube Electrodes for Electrocatalytic Oxygen Reduction[END_REF] Design of the Biofuel Cells. The biofuel cells were designed according to two configurations. First, the flow-through biofuel cell design was developed around a commercial Paxitech housing. It consists of a 5 cm 2 test fuel cell with graphite bipolar plates and a serpentine gas flow design. Our purpose was the alternative utilization of the serpentine for solution and air flow rate management. A 3 mm thick PTFE slice was designed with a hole in the center having a 13 mm diameter. A microporous GDL was then placed at the backside of the PTFE to serve as the electron collector while assuring the diffusion of oxygen and the glucose solution. The CNT-enzyme pellet was compressed in the 13 mm hole onto the GDL. The open side of the pellet was then covered with a cellulose sheet which served as an effective electrolyte reservoir. A further PTFE of 127 μm thickness with a 13 mm hole was placed in between the bioanode and the biocathode as a separator and to prevent any leakage. At the GDL backside of each bioelectrode, a graphite plate with engraved serpentines and inlet and outlet holes, as well as a brass plate for the final current collector, was pressed together to finalize the GBFC design. For standard operation, a peristaltic pump was used to circulate the aqueous solution with a constant 0.15 mL min -1 flow rate. A second biofuel cell design was developed in order to allow the easy fabrication of multiple biofuel cells, using easy-to-handle homemade Plexiglas elements. To provide a constant glucose flow within the cells, either a peristaltic pump or standard glucose perfusion bag, such as those found in hospitals, was explored.

■ RESULTS AND DISCUSSION

Principle and Performances of the Flow-through Biofuel Cell. A complete scheme of the biofuel cell design is displayed in Figure 1. Both the anode and the cathode were made according to our previously described procedure. [START_REF] Reuillard | High power Enzymatic Biofuel Cell based on Naphtoquinone-mediated oxidation of Glucose by Glucose oxidase in a Carbon Nanotube 3D matrix[END_REF] Briefly, the glucose-oxidizing anode is composed of GOx and the mediator naphthoquinone confined in a CNT matrix. The mediator acts as an electron shuttle for the obtained electrons from the enzyme to the CNT electrode. Such mediation is unfortunately not quantitative as unwired GOx reacts by reduction of oxygen to hydrogen peroxide. Thus, catalase is added to consume a maximum amount of the enzymatically generated H 2 O 2 . The oxygen-reducing cathode is composed of protoporphyrin IX functionalized CNTs and BOD. Protoporphyrin IX has been shown to be an efficient electron transfer promoter leading to oriented immobilization of BOD and DET due to its similar structure to the natural substrate bilirubin. [START_REF] Lalaoui | Fully Oriented Bilirubin Oxidase on Porphyrin-Functionalized Carbon Nanotube Electrodes for Electrocatalytic Oxygen Reduction[END_REF] The required electrons for oxygen reduction are therefore directly supplied by the MWCNT matrix. The glucose solution flow was directed from the cathode to the anode to avoid inhibition of the biocathode by any remaining hydrogen peroxide not consumed by the catalase at the anode. Figure S1 in the Supporting Information (SI) displays CV curves corresponding to both the cathode and the anode.

Four flow setups were investigated based on the modular biofuel cell design shown in Figure 1. The different configurations and their associated polarization and power curves are depicted in Figure 2. The configuration A represents a simple biofuel cell in which each electrode is placed in its chamber. The GBFC exhibits an OCV of 0.65 V and delivers a maximum power output of 0.51 ± 0.03 mW (0.38 mW cm -2 , 1.9 mW mL -1 , and 4.43 mW g -1 ) (in 5 mmol L -1 glucose in McIlvaine buffer at pH 7. For all other configurations, a porous cellulose membrane (0.13 mm thickness) was employed in order to separate both electrodes, allow the flow of electrolyte, and minimize electrode distances while ensuring an easy-to-made multilayer fabrication. The configuration B represents the GBFC with an external flow circuit of 5 mmol L -1 glucose flowing through the serpentines at the cathode to the serpentines at the anode. An OCV of 0.55 V with a maximum power output of 0.38 ± 0.02 mW (0.28 mW cm -2 , 1.39 mW mL -1 , and 3.27 mW g -1 ) at 0.33 V was reached. In configuration C, the electrolyte outlet was removed from the cathode, while the inlet was removed from the anode. This allows the air saturated 5 mmol L -1 glucose solution to flow from the cathode to the anode, passing through the cellulose membrane. The measured OCV of this setup is clearly increased and an improved maximum power output of 1.24 ± 0.06 mW (0.93 mW cm -2 , 4.58 mW mL -1 , and 10.76 mW g -1 ) at 0.375 V was obtained. The 3-fold increase for this flow- through design, as compared to configuration A and B, demonstrates that the flow-through electrolyte maximizes the access of substrates to deeply-CNT-entrapped enzymes. This is not the case for configuration A and B where the electrolyte is only in contact with the surface of the CNT electrode.

In spite of the optimized power output with entire wetting and improved glucose/oxygen flow through the pellets, the necessity of the serpentine is an inconvenience of this setup when several GBFCs should be stacked and be connected in parallel or in series. To evaluate the efficiency of our GBFC design for facilitated stacking, while enabling individualized supply of glucose solution, and the possibility for further miniaturization, we fabricated optimized Plexiglas plates as pellet housings in the form of a disc of 36 mm in diameter, 3 mm thickness, and with a 13 mm hole in the center to position each bioelectrode. [START_REF] Goff | Pile àBiocombustible[END_REF] A graphoil disc of 36 mm of diameter is added in each side of the biofuel cell to substitute the graphite plate. A needle was slotted into a drilled hole on the topside of the Plexiglas slice of each bioelectrode to allow complete flowthrough of the air saturated glucose solution from the cathode to the anode with an inlet at the bottom and an outlet at the top of the GBFC (Figure 2, configuration D). This configuration shows slightly better performances than configuration C since the glucose/O 2 solution passes through the entire CNT-enzyme pellet. However, with the aim to stack several biofuel cells in order to connect them in series or in parallel, configuration C is more appropriate.

For configuration D, the glucose biofuel cell exhibits an OCV and maximum power output of 0.663 V and 1.03 ± 0.05 mW (at 0.34 V). This configuration shows slightly lower performances as compared to configuration C. This likely originates from a more homogeneous supply of electrolyte for configuration C, thanks to the presence of the serpentine design at each electrode. Taking into account the surfaces and volumes of the separating membrane and both electrodes, this performance corresponds to power densities of 0.77 mW cm -2 , 3.8 mW mL -1 , and 8.92 mW g -1 . As a control experiment, we also performed power and polarization curves without any flux of glucose; this configuration exhibiting negligible power output (Figure S2).

These values, obtained at 5 mmol L -1 under air and at 25 °C, are among the best performances for a GBFC operating in vitro. This is close to performances of GBFC based on osmium hydrogels (0.74 mW cm -2 at 60 mmol L -1 glucose) [START_REF] Gao | Engineering Hybrid Nanotube wires for high-power Biofuel Cells[END_REF] or our previously developed GBFC based on CNT pellets (1.54 mW cm -2 at 50 mmol L -1 glucose). [START_REF] Reuillard | High power Enzymatic Biofuel Cell based on Naphtoquinone-mediated oxidation of Glucose by Glucose oxidase in a Carbon Nanotube 3D matrix[END_REF][START_REF] Zebda | Mediatorless high-power Glucose Biofuel Cells based on compressed Carbon Nanotube-Enzyme Electrodes[END_REF][START_REF] Reuillard | One-year Stability for a Glucose/ Oxygen Biofuel cell combined with pH Reactivation of the Laccase/ Carbon Nanotube Biocathode[END_REF] Power Management and Lifetime. In order to examine this setup in more detail, in terms of lifetime and practical power management, two operation modes were applied. The use of a peristaltic pump, of course, nullifies the produced energy by the GBFC. In this context, a perfusion bag containing the glucose solution was mounted to replace the pump (Figure 3A). Figure 3B shows a continuous charge-discharge experiment performed during 24 h. A 3 kΩ resistance was applied for the discharge from 0.55 to 0.35 V. This voltage range was chosen for reasonable charge/discharge lengths and improved stability of the setup. When the low voltage value of 0.35 V is reached, the circuit was opened, and the cell voltage was allowed to recharge until the initial 0.55 V (Figure 3Ba black). During this experiment, almost 3.6 J (1 mW h) of energy was accumulated in total after 48 h (Figure 3Bb blue). At constant discharge with a higher applied resistance of 6 kΩ (Figure 3 Bc red), the OCV slowly decreased from around 0.5 V until a plateau was observed at 0.4 V after 18 h. During the first 24 h, about 2.6 J (715 μW h) could be accumulated (Figure 3 Bd green).

Figure 3C displays a close up of the duration of single charge-discharge cycles at 500 Ω and 3 kΩ, at the beginning of the experiment and 48 h later. In addition, a charge-discharge cycle was recorded after 200 h for the 3 kΩ discharge experiment (data not shown). For the 500 Ω discharges, the charge/discharge cycle is quite fast and less than 2 min in the beginning. However, the cycle time increases quite fast until more than 4 min per cycle is required after 48 h. When a 3 kΩ discharge is applied, the time of a single charge/discharge cycle of around 9 min remains constant during 48 h. Since mass transport limitations for this flow setup can be excluded, this increasing cycle time has to be correlated to the reduced electron transfer rates due to the loss of the enzymes' and/or mediators' activity. This indicates that the enzymes lose their activity faster at discharges close to the maximum power output. It is therefore conclusive that during 48 h, 2 times more energy (3.6 J; 1 mW h) could be accumulated at 3 kΩ discharges than at 500 Ω discharges (1.8 J; 0.5 mW h).

It appears to be more appropriate to discharge continuously when high power is required over a short time while discharge/ charge cycles are better for prolonging GBFC lifetime and achieving higher energy yields. To obtain effective energy yields, a perfusion bag containing the glucose solution was mounted to replace the peristaltic pump (Figure 3A). Almost similar performances were observed with this setup which produced 3.6 J (1 mWh) of effective energy after 48 h under discharge/charge cycle mode at 3 kΩ.

Assembly of Two Glucose Biofuel Cells. The circuits used for connecting two stacked GBFCs in series and in parallel are displayed in Figure 4C,D, respectively. Separated inserts were used to supply the bioelectrodes with the air saturated glucose solution while the flow direction was still directed from the cathode to the anode as shown in Figure 4B. The same physiologically relevant 5 mmol L -1 glucose solution was used to supply both GBFCs and was simply separated by T-pieces. Figure 4C shows the polarization and power curves for the system in series. The power curve was plotted from the polarization curve, which was recorded by successive 30 s discharges at constant current (0.1 mA to 5.2 mA). The performances were recorded at pH 7 in the presence of a 5 mmol L -1 air saturated glucose solution flux. For the parallel circuit, the OCV remained constant (0.669 V) while the maximum power output of 1.75 ± 0.09 mW at 0.381 V and current (4.6 mA) increased by a factor of 2 compared to one individual GBFC. The stacked glucose biofuel cell in series (Figure 4C) exhibits an OCV of 1.35 V and a maximum power output of 1.82 ± 0.09 mW (at 0.675 V). Also for the GBFC in series, the generated power and the operational potential is almost two times higher than that observed for one individual GBFC, while the current remains the same. This scholastic doubling of cell voltage current for the connection of two GBFCs, respectively, in series and in parallel, demonstrates the appropriateness of our design for efficient stacking and supply of glucose solution in order to connect several GBFCs in miniaturizable housings without a loss of performance. To demonstrate practical device powering, an electronic timer was run for 17 h, supplied by only two GBFCs connected in series, and without the need for a step up converter or a capacitor (Figure 4A). The OCV of this device remained almost stable and decreased from 1.37 to 1.32 V during the whole experiment (Figure S3).

■ CONCLUSIONS

A new design of a glucose biofuel cell is presented allowing the stacking of two GBFCs and their connection in parallel and in series. Each cell is supplied by an individual air saturated glucose flow which is directed from the cathode through a cellulose membrane to the anode. Under continuous discharge, one individual GBFC generated 2.5 J (0.7 mW h) of power using an air saturated 5 mmol L -1 glucose solution in 24 h. Under charge-discharge conditions, this biofuel cell was stable over 9 days and produced 7.9 J (2.2 mW h). Two stacked biofuel cells provided an almost 2-fold power output when connected either in series or in parallel. The obtained power densities with our stacked GBFC design are comparable to the best performances in the literature [START_REF] Sakai | Evolution of Sony's Biofuel Cell[END_REF][START_REF] Miyake | Flexible, layered Biofuel Cells[END_REF] but the energy yields largely overpass the vast majority of reported performances. Finally, a portable demonstrator device could be operated for 17 h with two GBFCs in series. The stacking and connection of further GBFCs is currently being evaluated. This flow setup represents a step forward toward realistic applications for powering portable or biomedical electronic devices, addressing both voltage and current limitations. The only remaining drawback is the lifetime under continuous operation. Even when our design shows enhanced performances, these power generators can at this stage be envisioned for short-term use devices.

■ ASSOCIATED CONTENT

* S Supporting Information

This material is free of charge via the Internet at The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b06717.

Figures of CV scans

Figure 1 .

 1 Figure 1. Scheme (A) and photo (B) of the flow-through cell used for one individual GBFC. The insets in the scheme represent the components used for the bioanode and the biocathode. The red arrows show the flow direction of the glucose solution.

Figure 2 .

 2 Figure 2. Schemes of the flow designs and corresponding measured performances (power profile (blue) and polarization curve (black)) for (A) the configuration of one biofuel cell where both bioelectrodes were simultaneously supplied with the glucose/O 2 solution; (B) the configuration with an external circuit flowing through the serpentines at the cathode to the serpentines at the anode; (C) the configuration with a glucose solution flowing from the cathode to the anode, passing through the cellulose membrane; and (D) the configuration with a complete flow-through from the cathode to the anode. Each point of the corresponding power profiles represents the value obtained after 30 s discharge between 0.1 and 3.7 mA. All experiments were repeated 3 times under identical conditions.

Figure 3 .

 3 Figure 3. (A) Photograph of the perfusion bag replacing the peristaltic pump for supplying the GBFC with glucose and oxygen. (B) Charge/ discharge cycles between 0.55 and 0.35 V at 3 kΩ during 48 h (a, black) and the produced energy (b, blue); (C) continuous discharge at 6 kΩ during 24 h (a, black) and the produced energy (b, blue); (D) discharge/charge cycle time at 500 Ω (a, black) in the beginning and after 48 h (b, red); and (E) discharge/charge cycle time at 3 kΩ (a, black) in the beginning and after 48 h (b, red).

Figure 4 .

 4 Figure 4. (A) Photograph of two stacked GBFCs connected in series with glucose solution supplied by a perfusion bag and running an electronic timer. (B) Scheme of the flow directions of air saturated 5 mmol L -1 glucose solution for two stacked GBFCs. (C) Applied circuits, corresponding power profile (blue), and polarization curve (black) for the connection of two GBFCs in series (C) and in parallel (D).

  Figures of CV scans, control experiments, and cell voltage evolution over time (PDF)

ACS Appl. Mater. Interfaces XXXX, XXX, XXX-XXX

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The region Auvergne-Rhone-Alpes is acknowledged for the PhD funding (no.: 14-011164-01) of C.A. The authors would also like to thank the mechanical team of LiPhy at Grenoble for the fabrication of the PTFE and Plexiglas plates. The authors wish further to acknowledge the support from the platform Chimie NanoBio ICMG FR 2607 (PCN-ICMG), from the Institute Carnot PolyNat at Grenoble, and from the LabEx ARCANE (ANR-11-LABX-0003-01).