Claudia Crimella

Orazio Cantoni

Andrea Guidarelli

Chiara Vantaggiato

Andrea Martinuzzi

Mara Fiorani

Catia Azzolini

Genny Orso

Nereo Bresolin

PhD Maria Teresa Bassi
email: mariateresa.bassi@bp.lnf.it.

A novel nonsense mutation in the APTX gene associated with delayed DNA

Keywords: APTX, AOA1, DNA-SSB Repair, Hydrogen Peroxide, Methyl methane sulphonate APTX, AOA1, DNA-SSB Repair, Hydrogen Peroxide, Methyl methane sulphonate

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

ABSTRACT: APTX is the gene involved in ataxia with oculomotor apraxia type 1 (AOA1), a recessive disorder with early-onset cerebellar ataxia, oculomotor apraxia and peripheral neuropathy. The encoded protein, aprataxin, is a DNA repair protein processing the products of abortive ligations, 5'-adenylated DNA. We describe a novel nonsense mutation in APTX, c.892C>T (p.Gln298X), segregating in two AOA1 patients and leading to the loss of aprataxin protein in patient's cells. These cells, while exhibiting reduced catalase activity, are not hypersensitive to toxicity elicited by H 2 O 2 exposure at either physiologic or ice-bath temperature. On the other hand, the rate repair of DNA single-strand-breaks (SSBs) induced in both conditions is always significantly slower in AOA1 cells. By using the alkylating agent methyl methane sulphonate (MMS) we confirmed the association of the APTX mutation with a DNA repair defect in the absence of detectable changes in susceptibility to toxicity. These results, while consistent with a role of aprataxin in the repair of SSBs induced by H 2 O 2 , or MMS, demonstrate that other mechanisms may be recruited in AOA1 cells to complete the repair process, although at a slower rate. Lack of hypersensitivity to the oxidant, or MMS, also implies that delayed repair is not per se a lethal event.

INTRODUCTION

Ataxia with oculomotor apraxia type 1 (AOA1) (MIM# 208920) is an autosomal recessive disorder characterized by early-onset cerebellar ataxia, oculomotor apraxia, and peripheral neuropathy often associated with hypoalbuminemia and hyperlipidemia (Le [START_REF] Ber | Cerebellar ataxia with oculomotor apraxia type 1: clinical and genetic studies[END_REF]Tranchant et al., 2003). Additional features, such as cognitive impairment and involuntary movements can also be present overall leading to a wide variability in clinical impact and/or severity of the disease. The disease is due to mutations in the APTX gene (MIM# 606350) [START_REF] Date | Early-onset ataxia with ocular motor apraxia and hypoalbuminemia is caused by mutations in a new HIT superfamily gene[END_REF][START_REF] Moreira | The gene mutated in ataxia-ocular apraxia 1 encodes the new HIT/Zn-finger protein aprataxin[END_REF], encoding aprataxin, a 342 aa protein, member of the histidine triad (HIT) superfamily of nucleotide hydrolases and/or transferases. These proteins are involved in the DNA-strand-breakrepair machinery [START_REF] Moreira | The gene mutated in ataxia-ocular apraxia 1 encodes the new HIT/Zn-finger protein aprataxin[END_REF]. Aprataxin can remove adenosine-monophosphate (AMP) from various AMP-linked substrates in vitro [START_REF] Seidle | Disease-associated mutations inactivate AMP-lysine hydrolase activity of Aprataxin[END_REF] and also from the 5'-terminus of DNA single-or double-strand breaks (SSBs and DSBs) thus promoting efficient ligation [START_REF] Ahel | The neurodegenerative disease protein aprataxin resolves abortive DNA ligation intermediates[END_REF][START_REF] Caldecott | Single-strand break repair and genetic disease[END_REF][START_REF] Daley | Genetic interactions between HNT3/Aprataxin and RAD27/FEN1 suggest parallel pathways for 5' end processing during base excision repair[END_REF].

The forkhead-associated (FHA) region at the N-terminus of aprataxin interacts with DNA repair proteins like XRCC1 and XRCC4 among the others [START_REF] Sano | Aprataxin, the causative protein for EAOH is a nuclear protein with a potential role as a DNA repair protein[END_REF], forming multiprotein complexes of the SSB and DSBrepair machinery respectively (SSBR and DSBR) [START_REF] Clements | The ataxia-oculomotor apraxia 1 gene product has a role distinct from ATM and interacts with the DNA strand break repair proteins XRCC1 and XRCC4[END_REF][START_REF] Gueven | Aprataxin, a novel protein that protects against genotoxic stress[END_REF][START_REF] Hirano | DNA single-strand break repair is impaired in aprataxin-related ataxia[END_REF][START_REF] Caldecott | Single-strand break repair and genetic disease[END_REF].

The C-terminal region is a zinc-finger (ZF) domain required for DNA/RNA interaction [START_REF] Gibson | A model for the tertiary structure of the 28 residue DNA-binding motif ('zinc finger') common to many eukaryotic transcriptional regulatory proteins[END_REF]. HIT and ZF domains cooperate to form a high-affinity complex that locks the enzyme onto adenylated DNA lesions (Takahashi et al., 2007;[START_REF] Rass | Molecular mechanism of DNA deadenylation by the neurological disease protein aprataxin[END_REF]. Most of the mutations identified in AOA1 patients (available at http://www.hgmd.cf.ac.uk) fall within the HIT domain or just upstream the C-terminal ZF (see [START_REF] Caldecott | Single-strand break repair and genetic disease[END_REF] for review).

In spite of that, attempts to demonstrate a defect in DNA-SSB repair in AOA1 resulted conflicting for some time. SSBR is known to be impaired in aprataxin knocked-down cells [START_REF] Hirano | DNA single-strand break repair is impaired in aprataxin-related ataxia[END_REF] and a defect in cellular SSBR has also been reported in some AOA1 patient's derived cells [START_REF] Mosesso | The novel human gene aprataxin is directly involved in DNA single-strand-break repair[END_REF][START_REF] Gueven | A subgroup of spinocerebellar ataxias defective in DNA damage responses[END_REF]. Aprataxin is indeed recruited at sites of SSBs in an XRCC1-dependent manner and the targeting is mediated by PARP-1, a protein interacting with both XRCC1 and aprataxin and working as key player in SSBs detection [START_REF] Harris | Aprataxin, poly-ADP ribose polymerase 1 (PARP1) and apurinic endonuclease 1 (APE1) function together to protect the genome against oxidative damage[END_REF].

Lymphoblast cell lines from an AOA1 patient carrying the T739C mutation were hypersensitive in terms of induction of chromosomal aberrations by CPT (camptothecin), an inhibitor of DNA topoisomerase I causing DNA single-strand breakage [START_REF] Mosesso | The novel human gene aprataxin is directly involved in DNA single-strand-break repair[END_REF]. By contrast, no evidence for such a defect was obtained in other studies [START_REF] Gueven | Aprataxin, a novel protein that protects against genotoxic stress[END_REF]Reynolds et al., 2009a). However, cells from AOA1 patients with different mutations (689insT/840delT, P206L/P206L, P206L/V263G) were hypersensitive to agents that cause SSBs (methyl methane sulfonate, MMS, and hydrogen peroxide, H 2 O 2 ), thereby suggesting a protective role of aprataxin against genotoxic stress [START_REF] Gueven | Aprataxin, a novel protein that protects against genotoxic stress[END_REF].

Here we describe a novel aprataxin mutation, p.Gln298X, truncating the protein just upstream the ZF domain and leading to loss of protein expression and activity in the patient's derived lymphoblast cell line (Takahashi et al., 2007;Reynolds et al., 2009b).

These cells display slower rates of resealing of SSBs induced by either H 2 O 2 or MMS. However, no hypersensitivity to toxicity induced by these agents was detected in these cells. Implications of these findings are discussed.

F

METHODS

Clinical Findings

The family pedigree in Figure 1 shows 2 affected siblings: the proband (II-2) and her brother (II-3) both displaying features of AOA1 disease with associated hypoalbuminemia. Detailed clinical description is provided as supplementary material.

Mutation analysis and lymphoblastoid cell lines generation

Blood specimens were obtained from affected and unaffected available members of the family and from control subjects under a protocol of informed consent approved by the ethical committee of E. Medea Institute. Genomic DNA from peripheral blood leukocytes was prepared with the standard high salt extraction method. APTX genomic regions are available at the Human Genome Browser Gateway (http://genome.ucsc.edu/cgibin/hgGateway). We amplified and directly sequenced all 7 exons of the longest transcript using primers designed on the flanking intronic sequences. Sequence and mutation nomenclature is based on RefSeq NM_175073.1 (considering the A of the ATG as nucleotide 1) and on RefSeq NC_000009.11 for APTX cDNA and genomic sequences respectively, and follows the guidelines of the Human Genome Variation Society (http://www.hgvs.org/mutnomen/). Sequences were performed with a Big Dye Terminator Sequencing Kit (version 3.1 Applied Biosystem Foster City, CA, USA), run on an Applied Biosystems ABI 3130 XL Genetic Analyzer (Applied Biosystems). The identified mutation was confirmed on a second PCR performed on a second aliquot of patient DNA. Two hundreds healthy individuals from the Italian population were used as controls. Lymphoblast cells lines from patients and control were established by EBV infection with standard protocol and cultured in RPMI 1640 medium (Invitrogen Life Science, Carlsbad, CA, USA) containing 20% FCS (Hyclone Celbio, Milano, Italy) and supplemented with 2 mM Glutamine and 100U/ml penicillin/streptomycin (Invitrogen).

Aprataxin protein levels and deadenylation activity in AOA1 lymphoblast cell lines SDS-PAGE and Western Blot were performed as previously described [START_REF] Panzeri | The first ALS2 missense mutation associated with JPLS reveals new aspects of alsin biological function[END_REF] with total extracts from two controls (Cr 26 and Cr 48) and EM223/07 lymphoblast cell lines. Aprataxin was revealed with antiaprataxin Ab (raised against the N-terminal of the protein, residues 1-177) and normalized with anti-actin Ab (both from Abcam, Cambridge, UK). The anti-aprataxin Ab specifically recognizes a single band at 39 kDa as a fulllength aprataxin isoform. Deadenylation activity of aprataxin was performed as described (Takahashi et al., 2007;Reynolds et al., 2009b). Details are provided as supplementary material.

Cytotoxicity assays

The cells (3 x 10 5 ) were exposed for 15 (4°C) or 30 (37°C) min to H 2 O 2 in 2 ml of saline A (8.182 g/l NaCl, 0.372 g/l KCl, 0.336 g/l NaHCO 3 and 0.9 g/l glucose). Treatments with MMS were performed at physiologic temperature in 2 ml of RPMI 1640 culture medium supplemented as detailed above. After treatments, the cells were washed with saline A and re-suspended in pre-warmed complete culture medium, plated into 35 mm tissue dishes and finally incubated at 37°C for up to three days. The number of viable cells was estimated 24, 48 and 72 h after treatments with a hemocytometer, after dilution of the cell suspension (1:1) with 0.4% trypan blue. Under these conditions, cultures were characterised by the presence of viable cells (i.e., excluding trypan blue), cells presenting features of apoptosis (i.e., blebbing, apoptotic bodies) and by the absence of necrotic cells (i.e., taking up trypan blue). In other experiments, however, dead cells were largely detected in the absence of apoptotic cells after a 30 min post-treatment incubation with high concentrations of H 2 O 2 .

Measurement of DNA single-strand breakage

The cells (3 x 10 5 ) were exposed for 15 min (4°C or 37°C) to increasing concentrations of H 2 O 2 in 2 ml of saline A. In other experiments, the cells were treated for 1 h (37°C) with increasing concentrations of MMS in 2 ml of RPMI 1640 culture medium supplemented as detailed above. Cells were then analyzed for DNA damage either immediately or after increasing time-intervals of incubation in pre-warmed complete culture medium. SSBs were determined using the alkaline-halo assay developed in our laboratory [START_REF] Cantoni | Indirect mechanisms of DNA strand scission by peroxynitrite[END_REF]. Details on the processing of fluorescence images and on the calculation of the experimental results are as reported [START_REF] Cantoni | Indirect mechanisms of DNA strand scission by peroxynitrite[END_REF]. SSBs were quantified by calculating the nuclear spreading factor value, representing the ratio between the area of the halo (obtained by subtracting the area of the nucleus from the total area, nucleus + halo) and that of the nucleus, from 50 to 75 randomly selected cells/experiment/treatment condition. Results are expressed as relative nuclear spreading factor values calculated by subtracting the nuclear spreading factor values of control cells from those of treated cells.

SSBs were also determined using the comet assay, following the procedure detailed by [START_REF] Singh | A simple technique for quantitation of low levels of DNA damage in individual cells[END_REF], with minor modifications [START_REF] Cantoni | Indirect mechanisms of DNA strand scission by peroxynitrite[END_REF]. The extent of DNA strand scission is expressed as tail/nucleus ratio values, calculated according to the method of [START_REF] Müller | Comet assay studies of radiation-induced DNA damage and repair in various tumour cell lines[END_REF]. Data are typically collected from 50-75 randomly selected cells/experiment/treatment condition.

Non-protein thiol assay

Cellular non-protein thiol content was determined as described by Beutler [START_REF] Beutler | Red cell metabolism[END_REF]. In brief, cells (5 x 10 6 ) were washed three times with saline A, centrifuged and the pellet was subsequently re-suspended in 150 µl of a metaphosphoric acid solution [1.67% (v/v) metaphosphoric acid /0.2% EDTA/30% (w/v) NaCl]. After a 5 min incubation in an ice-bath, the cell suspension was centrifuged for 5 min at 10,000 g. The non-protein thiol content was measured spectrophotometrically in the supernatant, at 412 nm, using 5,5'-dithiobis(2-nitrobenzoic acid) (ε 412 = 13600 M -1 . cm -1 ).

Catalase, glutathione peroxidase and superoxide dismutase activities

Cells were washed twice with saline A, re-suspended in the same medium at a density of 5 x 10 6 cells/ml and finally sonicated three times on ice with a Branson sonifier operating at 20 W for 15 sec. The resulting homogenates were centrifuged for 5 min at 18,000 x g at 4°C. Catalase and glutathione peroxidase [START_REF] Beutler | Red cell metabolism[END_REF] and superoxide dismutase [START_REF] Paoletti | A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts[END_REF] activities were determined spectrophotometrically in the supernatants.

Statistical analysis

The results are expressed as means ± SD. Statistical differences were analyzed by one-way ANOVA followed by Dunnett's test for multiple comparison or two-way ANOVA followed by Bonferroni's test for multiple comparison. A value of p < 0.05 was considered significant.

RESULTS

Genetic analysis and 5'-AMP SSB repair in mutant lymphoblastoid cell line

The proband (EM223/07) of the AOA1 family was screened for mutations in the APTX gene and a novel homozygous nonsense mutation (c.892C>T) in exon 7 was identified leading to a premature protein interruption, p.Gln298X (Fig. 1A). The mutation was also found in the affected brother, while the two healthy brothers (II-1, II-4) were heterozygous carriers. The mutation was not found in 200 controls. The parents were not available for analysis. The mutation is predicted to cause complete loss of the aprataxin zinc-finger domain, important for DNA/RNA interaction (Fig. 1C). Aprataxin protein levels were analyzed by Western Blot in controls and EM223/07 lymphoblast cell lines with an anti-aprataxin Ab raised against the N-terminal of the protein (Fig. 1B). While control cells (Cr 26 and Cr 48) expressed the 39 kDa full-length aprataxin isoform, EM223/07 cells failed to express the 34 kDa truncated p.Gln298X protein. This suggests that the p.Gln298X mutation results in loss of aprataxin protein. We also checked the presence of any residual aprataxin deadenylation activity in EM223/07 lymphoblastoid cell lines, incubating increased concentration of Cr 48 and EM223/07 cell extracts with a duplex substrate carrying a 5'-AMP end SSB for 1 hour. While the control (Cr 48) efficiently repairs the SSBs, EM223/07 extract does not show any residual activity (Fig. 1S).

Lymphoblast cell lines sensitivity to hydrogen peroxide and methyl methane sulphonate

In order to investigate the impact of the p.Gln298X mutation on SSB repair and on toxicity induced by H 2 O 2 , preliminary studies were performed to determine possible confounding factors, which may affect the above responses independently of aprataxin expression. Changes in the redox status of the cells, regardless of their dependence on the mutation, may indeed provide misleading results.

Cells from the AOA1 patient EM223/07 were therefore analysed for their glutathione peroxidase and superoxide dismutase activities, as well as for their non-protein-thiol pool, obtaining results comparable with those derived from a normal age-matched control (Cr 48) (Table 1S). Further analyses, however, provided evidence for a significantly reduced activity of catalase, the major H 2 O 2 -detoxifying enzyme.

In order to assess whether loss of aprataxin delays SSB repair, we employed a strategy minimising the contribution of catalase activity in the accumulation of the initial damage induced by H 2 O 2 . For this purpose, cells were treated for 15 min with increasing concentrations of H 2 O 2 in an ice bath, i.e., at a temperature remarkably slowing down all enzymatic activities. Under these conditions (Fig. 2A), the dose-response curves relating the degree of DNA strand scission to the concentration of H 2 O 2 were virtually superimposable in normal and aprataxin-deficient cells. This notion was established using two separate techniques for the assessment of DNA damage, the alkaline-halo and comet assays, based on different principles but nevertheless characterised by identical sensitivities [START_REF] Cantoni | Indirect mechanisms of DNA strand scission by peroxynitrite[END_REF]. Both techniques involve the use of an alkaline pH, converting alkali labile sites in frank DNA SSBs. EM223/07 and Cr 48 cells were next treated on ice with 50 µM H 2 O 2 and subsequently incubated at 37°C to investigate the rate of SSB resealing. Importantly, data obtained in these experiments (Fig. 2B), represent the observed net frequency, i.e., the result of a dynamic balance between the breaks introduced by repair endonucleases in response to various types of DNA damage and the sealing of all types of breaks by polymerisation and ligation. In both cells types, a time-dependent increase in the rate of SSB removal was consistently observed, thereby suggesting that the expected exponential kinetics are masked by an early formation of endonuclease-dependent DNA SSBs restricted to the first 15-30 minutes of incubation at 37°C. The rate of SSBR was however significantly slower in EM223/07 cells (t1/2 = 25.8 min) as compared to Cr 48 cells (t1/2 = 17 min). On the other hand, SSBs were nevertheless completely resealed in both cell types. Importantly, the results presented in Fig. 2B were obtained using the alkaline-halo as well as the comet assays, with identical outcomes.

There was no evidence of toxicity in either of the cell types under investigation, as assessed by visual inspection as well as by release of lactate dehydrogenase or trypan blue uptake (data not shown). We also assessed the proliferation rate of these cells over three days of growth after H 2 O 2 treatment on ice: the kinetics were superimposable for both cell types (Fig. 2C andD), regardless of the treatment received (vehicle vs H 2 O 2 ). Hence, the observed delay in SSB repair does not appear to be dependent on non-specific toxic effects of the oxidant and is most likely attributable to lack of aprataxin expression.

We next addressed the question of whether the repair defect was associated with delayed hypersensitivity to the toxic effects of H 2 O 2 . For this purpose, the cells were treated with concentrations of the oxidant higher than those employed in DNA repair studies. The proliferation rates of normal or AOA1 cells were however similarly affected by H 2 O 2 (Fig. 2C andD) so that identical toxicity curves were obtained for the two cell types (Fig. 2E). It is important to note that these experiments measure the number of viable cells, which decreases after exposure to H 2 O 2 as a result of both growth inhibition and cell killing, taking place by apoptosis, as determined by visual inspection (cell blebbing and chromatin condensation, not shown) and activation of caspases (not shown). It should be emphasised that these results were obtained in cells exposed to H 2 O 2 at ice bath temperature and should therefore reflect the exclusive action of chemical/free-radical processes. However, the biological effects of H 2 O 2 at 4°C or 37°C may not be necessarily mediated by the same mechanisms. At 37°C, biochemical/enzymatic processes (which are not operative at 4°C) may became important for H 2 O 2 metabolism and the mechanisms of toxicity may be altered.

We therefore decided to perform the experiments at 37°C; consistently with the observed reduced catalase activity (Table 1S), we obtained results indicating that AOA1 cells are hypersensitive to H 2 O 2 -induced DNA strand scission induced at physiologic temperature (Fig. 2F). Note that treatments were for 15 min and that, under these conditions, remarkably lower concentrations of the oxidant were necessary to produce SSBs levels comparable to those obtained at ice-bath temperature, as assessed with both the alkaline-halo and comet assays. The results illustrated in Fig. 2G indicate that equivalent levels of SSBs induced by H 2 O 2 exposure at 37°C in control (10 µM) and AOA1 (5 µM) cells are repaired with identical kinetics, superimposable on those observed in cells oxidativelyinjured at ice-bath temperature.

These findings therefore indicate that loss of aprataxin expression delays the rate of SSB repair, regardless of whether the initial damage was induced at 37° or 4°C. As previously noted for experiments involving treatments at 4°C, there were no signs of toxicity under the conditions of exposure at 37°C (data not shown), thereby emphasizing the specificity of the repair defect, most likely dependent on the lack of aprataxin expression. A significant toxicity, also characterised by biochemical and morphological features of apoptosis (not shown), was however observed by doubling the exposure time to 30 min and post-incubating the cells in fresh culture medium for up to 72 h (Fig. 2H andI). The resulting toxicity curves were identical for control and AOA1 cells (Fig. 2L), in contrast to AOA2 EM518 cells, remarkably more sensitive to H 2 O 2 [START_REF] Airoldi | Characterization of two novel SETX mutations in AOA2 patients reveals aspects of the pathophysiological role of senataxin[END_REF].

Taken together, these results lead to the conclusion that, despite their hypersensitivity to DNA strand scission (probably due to reduced catalase activity) and slow rate of SSB removal (likely due to lack of aprataxin expression), EM223/07 cells are not hypersensitive to toxicity elicited by H 2 O 2 exposure at physiologic temperature. This notion was also established by studies in which cells were incubated for 30 min (37°C) with high concentrations of H 2 O 2 and subsequently analysed for viability with the trypan blue assay after a 30 min incubation in fresh culture medium (Fig. 2S). By contrast, the AOA2-EM518 cell line was hypersensitive also under these conditions. It is interesting to note that the results illustrated in Fig. 2S provide an estimate of the number of dead cells, trypan blue positive, and are therefore indicative of necrosis, as expected, since toxicity arises very rapidly in response to high concentrations of the oxidant. There was no evidence of apoptosis in any of the cell types employed.

These results confirm and extend the above findings, indicating the existence of a dichotomy between the repair defect and sensitivity to toxicity, by showing identical outcomes even under conditions in which massive SSB induction is associated with a necrotic mode of death.

The final set of experiments involved the use of another SSB inducer, the alkylating agent MMS. The results obtained are in line with those previously described in cells treated with H 2 O 2 at 4°C. Control and AOA1 cells were indeed equally sensitive to DNA strand scission (Fig. 3A) and toxicity (Fig. 3C-E) induced by increasing concentrations of MMS but nevertheless displayed remarkably different rates of SSB rejoining (Fig. 3B). As previously noted for H 2 O 2 , also MMS-induced DNA cleavage was repaired more slowly in EM223/07 cells than in Cr 48 cells.

Collectively, our results indicate that aprataxin-deficient cells display reduced rates of repair of MMS-or H 2 O 2induced SSBs. These data demonstrate also that this event is not associated with hypersensitivity to toxicity.

DISCUSSION

A large variety of proteins is involved in the repair of DNA SSBs, the commonest lesions arising in cells from different sources [START_REF] Caldecott | Single-strand break repair and genetic disease[END_REF]. Most of these lesions are repaired by a rapid-global-SSB-repair process, SSBR. Additional pathways exist all considered sub-pathways of SSBR, with an extensive overlap between the enzymes used [START_REF] Caldecott | Single-strand break repair and genetic disease[END_REF]. Two of the proteins involved in the end-processing step are APTX and TDP1 [START_REF] Caldecott | Single-strand break repair and genetic disease[END_REF], involved in neurodegenerative disease, AOA1 [START_REF] Date | Early-onset ataxia with ocular motor apraxia and hypoalbuminemia is caused by mutations in a new HIT superfamily gene[END_REF][START_REF] Moreira | The gene mutated in ataxia-ocular apraxia 1 encodes the new HIT/Zn-finger protein aprataxin[END_REF]SCAN1 (Takashima et al., 2002), respectively. As previously shown for TDP1 (Takashima et al., 2002), recent studies indicate that aprataxin catalyzes the hydrolytic release of DNA end-blocking lesions with APTX being specific for end-blocking 5'-adenylate and TDP1 for 3'-tyrosyl termini [START_REF] El-Khamisy | Defective DNA singlestrand break repair in spinocerebellar ataxia with axonal neuropathy-1[END_REF]Rass et al., 2007a;Rass et al., 2007b;[START_REF] Rass | Molecular mechanism of DNA deadenylation by the neurological disease protein aprataxin[END_REF]. A clear defect in cellular SSB repair, however, has only been reported for mutations in TDP1, as the attempts to demonstrate a similar defect in AOA1 failed to give consistent results. While aprataxin knock down appears to delay resealing of SSBs accumulated under conditions of GSH depletion [START_REF] Hirano | DNA single-strand break repair is impaired in aprataxin-related ataxia[END_REF], other studies failed to detect slower repair kinetics of lesions caused by H 2 O 2 in AOA1 cells carrying different APTX mutations [START_REF] Gueven | Aprataxin, a novel protein that protects against genotoxic stress[END_REF] or in APTX knock out chicken DT40 cells, APTX -/mouse fibroblasts, AOA1 human fibroblasts and AOA1 lymphoblast cell lines (Reynolds et al., 2009a). It was thus hypothesized that lack of consistent defects in SSB repair in aprataxin-defective cells, may reflect either the low abundance of 5'-AMP breaks or the availability of other factors for repair of these types of lesions (El-Khamisy et al., 2009). More recently evidence for both direct and indirect modulating functions for aprataxin on base excision repair were provided [START_REF] Harris | Aprataxin, poly-ADP ribose polymerase 1 (PARP1) and apurinic endonuclease 1 (APE1) function together to protect the genome against oxidative damage[END_REF].

To gather more information on the repair of SSBs induced by different agents in aprataxin-deficient cells, we employed an AOA1 patient's derived lymphoblast cell line carrying a novel nonsense mutation in aprataxin, leading to loss of aprataxin expression and activity. These cells exhibited a delayed rate of SSB rejoining (Figs 2B,2G and 3B). In order to appreciate the significance of our findings, it is important to note that the above phenotype was not associated with poor viability, as experiments comparing DNA repair in normal and AOA1 cells involved the use of exposure paradigms devoid of intrinsic toxicity. In addition, the genotoxic agents employed, H 2 O 2 and MMS, are two well-characterised DNA-damaging agents widely used as reference compounds in DNA repair studies. Conditions were also used producing identical levels of initial damage (Fig. 2A and3A), falling within a range in which two different techniques accurately measure the extent of DNA strand scission. As a final note, exposure to H 2 O 2 was performed at ice-bath temperature, a condition allowing the accumulation of identical levels of DNA lesions in response to the same concentration of the oxidant in the two cell types (Fig. 2A), despite the reduced catalase activity expressed by AOA1 cells. This technical aspect is critical to validate the outcome of results which are potentially affected by a variety of mutation-unrelated factors.

The results obtained lead to the univocal conclusion that the mutation associated with loss of aprataxin expression delays resealing of SSBs induced by H 2 O 2 or MMS. The same conclusion is reached from studies in which cells were treated with H 2 O 2 at physiologic temperature. Under these conditions, EM223/07 cells were hypersensitive to SSB induction but nevertheless the repair kinetics were identical in AOA1 cells and in the normal counterpart (Fig. 2F andG). While the different sensitivity to the initial damage is most likely dependent on reduced catalase activity expressed by AOA1 cells, it is also important to observe that the repair kinetics of SSBs induced by H 2 O 2, at 4° (Fig. 2B) or 37°C (Fig. 2G), in either of the two cell types, are also identical. This is an unexpected finding, as we previously detected remarkably slower repair kinetics for the DNA lesions induced by the oxidant at 37°C vs 4°C in Chinese Hamster Ovary cells [START_REF] Cantoni | Effect of 3-aminobenzamide on DNA strand break rejoining and cytotoxicity in CHO cells treated with hydrogen peroxide[END_REF].

A second important point emerging from our studies is that SSBs, although with slower rates, were nevertheless completely resealed in AOA1 cells, thereby implying that aprataxin is dispensable for SSB resealing. (Figs. 2B,2G and 3B). A recent study indeed provided results consistent with the notion that long-patch repair compensates for defective short-patch repair in AOA1 cells (Reynolds et al., 2009a).

The third relevant point we can make from our experiments is that delayed removal of SSBs is not associated with hypersensitivity to toxicity. Using intrinsically toxic concentrations of H 2 O 2 at 4°C (up to 10-fold greater than those employed in DNA repair studies), we indeed obtained identical toxicity curves in normal and AOA1 cells (Fig. 2E). The same outcome was provided by studies using significantly lower H 2 O 2 concentrations (Fig. 2L), however given to the cells at physiological temperature, permissive for both signalling events associated with toxicity and for enzymatic H 2 O 2 metabolism (i.e., detoxification). These experiments measured the number of viable cells after increasing time intervals, a parameter potentially affected by growth inhibition and by cell death. H 2 O 2 induced apoptosis in both EM223/07 and Cr 48 cells (not shown) thereby confirming the notion that functional aprataxin is dispensable for survival under these conditions. Importantly, the same assay was successfully employed to demonstrate that AOA2 cells are hypersensitive to H 2 O 2 (Fig. 2L and [START_REF] Airoldi | Characterization of two novel SETX mutations in AOA2 patients reveals aspects of the pathophysiological role of senataxin[END_REF]. As a final note, identical results were also obtained with the alkylating agent MMS (Fig. 3A).

Our findings therefore provide unambiguous evidence for lack of hypersensitivity of AOA1 cells to apoptosis induced by H 2 O 2 . Additional studies provided results in keeping with the notion that the same is true for necrosis. We indeed found that high concentrations of H 2 O 2 rapidly promote an identical accumulation of trypan blue positive EM223/07 and Cr 48 cells, whereas hypersensitivity was once again detected in AOA2 cells (Fig. 2S). Lack of hypersensitivity is therefore further established and even appears somewhat surprising, as catalase activity in AOA1 cells is about 25% lower than that detected in the normal counterpart. Future studies should investigate whether reduced catalase activity is a general feature of the AOA1 phenotype; previous studies providing evidence for hypersensitivity to H 2 O 2 may have employed AOA1 cells with remarkably low catalase activity. The notion that AOA1 lymphoblast cells display reduced rates of SSB removal, but are nevertheless as sensitive as normal cells to H 2 O 2 or MMS may be explained by the lack of contribution of SSBs in the molecular events mediating toxicity. Indeed SSBs are normally associated with poor toxicity in non-neuronal cells, in particular those caused by H 2 O 2 , continuously produced in oxygenated tissues. In this perspective, it should be reminded that the AOA1 neurological phenotype is not associated with gross damage in non-neuronal tissues. However, inefficient repair of SSBs continuously produced by reactive oxygen species promotes the accumulation of these lesions in non-replicating post-mitotic neurons, thereby interfering with transcription (Wilson and Mattson, 2007) and causing neurodegeneration [START_REF] Caldecott | XRCC1 and DNA strand break repair[END_REF].

In conclusion, although the exact contribution of delayed repair of SSBs to the AOA1 pathology is still to be demonstrated, we herein provide experimental evidence that aprataxin is required for the normal repair rate of SSBs induced by H 2 O 2 or MMS. Cells were exposed for 15 min to increasing concentrations of H 2 O 2 at 4°C (A) or 37°C (F) and immediately analysed for DNA strand scission with either the alkaline halo (circles) or the comet (squares) assays, as detailed in the Methods section. (B) Cells were pre-exposed (15 min) to 50 µM H 2 O 2 (4°C), and analysed with the alkaline halo (circles) or the comet (squares) assays either immediately or after increasing time intervals of post-incubation in complete culture medium at 37°C. (G) The cells were pre-exposed (15 min) to 10 (Cr 48) or 5 µM (EM223/07) H 2 O 2 at 37°C and analyzed for SSB repair with the alkaline halo or the comet assays as in B. Results represent the means ± SD calculated from three to five separate experiments. *P < 0.01; **P < 0.001 compared to Cr 48 cells (two-way ANOVA followed by Bonferroni's test).

(C, D) Counts of Cr 48 and EM223/07 cells exposed to increasing concentrations of H 2 O 2 for either 15 min at 4°C (C-E) or for 30 min at 37°C (H-L) and post-incubated for increasing time intervals in fresh culture medium. (E, L) The results obtained at the 72 h time point are normalized vs the respective untreated control. The results obtained with EM518 cells, derived from an AOA2 patient [START_REF] Airoldi | Characterization of two novel SETX mutations in AOA2 patients reveals aspects of the pathophysiological role of senataxin[END_REF], are also shown to provide a positive control. Results represent the means ± SD calculated from three to five separate experiments. **P < 0.001 compared to Cr 48 cells (two-way ANOVA followed by Bonferroni's test). The family pedigree in figure 1 shows 2 affected siblings: the proband (II-2) and her brother (II-3).

The proband is a 53 years old female born to non-consanguineous parents, at term following an uncomplicated pregnancy. From the age of 6 she presented slurred speech, frequent falls, progressively more severe walking difficulties, leading in 2 years to the need of assistance in walking and to complete loss of ambulation by the age 18.

Neurological examination at the age of 14 showed ataxic gait possible only with bilateral help, gross dysmetria of voluntary movements, diffuse distal leg and hand hypotrophy, absence of deep tendon reflexes (DTR), pes cavus, with intact strength and sensation. There was marked dysarthria and difficulties in visual pursuit with ocular esotropia. Cognition was intact. The disease progressed slowly and at the last examination (at 52 years of age) the patient presented flaccid tetraplegia, severe hypotrophy, hypotonia and areflexia. The difficulties in visual pursuit and the dysarthria were unchanged.

Blood exams were remarkable for hypoalbuminemia (1.6 g/l, n.v. 3.4-4.8), hypercholesterolemia (344mg/dL, n.v. <200), high α1-fetoprotein (12.1ng/ml, n.v. <10) Neurophysiology showed severe axonal neuropathy more severe at the lower limbs. Needle EMG was compatible with severe chronic denervation. Acoustic and visual evoked potential were within the normal range. MRI imaging showed moderate to severe cerebellar atrophy. II-3. The clinical history of the second born of this family was quite similar to the elderly sister, presenting his first motor and coordination problems at school age, and progressing to flaccid tetraplegia by the age of 25. When first examined at age 46 he was tetraplegic, with severe hypotrophy and hypotonia, absent DTRs. Cognition and sensation were preserved. His oropharingeal and laringeal function was severely impaired, resulting in dysarthria, dysphonia and dysphagia causing recurrent respiratory infections. Ocular movements were mildly impaired, with slow saccades and minimal nystagmus. MRI imaging at age 36 showed moderate cerebellar hypotropy. Blood tests showed hypoalbunimemia (2.4 g/l, n.v. 3.4-4.8) and elevated α1-fetoprotein (15.5, n.v. <10). The patient died for respiratory infection at age 49. For analysis of mutant aprataxin deadenylation activity (removal of an adenylated residues at 5'-end of a single strand break, SSB) we used a 5'-AMP and 3'-FAM labeled 23-mer oligonucleotide (AMP-23-FAM; 5'-App-TCTAGCACTTGAGGCTATCCATG-FAM-3'), a 3'-hydroxyl 21-mer oligonucleotide (21-OH; 5'-CTACGTCAGATCTGCGGATGT-3') and a complementary 45-mer oligo (45-mer; 5'-CATGGATAGCCTCAAGTGCTAGAGACATCCGCAGATCTGACGTAG-3') (Eurogentec, Seraing, Belgium) as described (Takahashi et al., 2007, Reynolds et al., 2009b).

Supplementary Methods

DNA deadenylation reaction

Oligonucleotides were annealed to produce a duplex substrate with an adenylated single strand break and a 1-bp gap with a 3'-hydroxyl terminus (OH).

Cell extracts were prepared from Cr 48 and EM223/07 lymphoblast cell lines. Cells lysis was performed in 20 mM Tris-HCl pH 7.5, 10 mM EDTA, 1 mM EGTA, 100 mM NaCl, 1% Triton X-100 and protease inhibitor cocktail (Sigma-Aldrich, San Louis, MO, USA), by incubation on ice for 10 minutes and then clarified by centrifugation. For DNA adenylate hydrolysis assay 0,1 µg, 1 µg or 5 µg of lymphoblast cell extracts were incubated with 100 nM of annealed oligonucleotides for 1 h at 30°C in 25 mM Hepes pH 8.0, 130 mM KCl, 1 mM DTT, 10 mM MgCl 2 , 100 µM dNTPs and 1 mM ATP. Reactions were stopped by addiction of an equal volume of formamide gel loading buffer and incubation at 95°C for 5 min. Products were fractionated on a 15% denaturing polyacrylamide/urea gel and images were acquired with a GelDoc-It-UVP (EuroClone, Milano, Italy) using an UV transilluminator and an emission filter (513-557 nm).

  mutation in the APTX gene associated with delayed DNA single-strand break removal fails to enhance sensitivity to different genotoxic agents

  Tada M, Igarashi S, Koyama A, Date H, Yokoseki A, Shiga A, Yoshida Y, Tsuji S, Nishizawa M and others. 2007. Aprataxin, causative gene product for EAOH/AOA1, repairs DNA single-strand breaks with damaged 3'-phosphate and 3'-phosphoglycolate ends. Nucleic Acids Res 35(11):3797-3809. Takashima H, Boerkoel CF, John J, Saifi GM, Salih MA, Armstrong D, Mao Y, Quiocho FA, Roa BB, Nakagawa M and others. 2002. Mutation of TDP1, encoding a topoisomerase I-dependent DNA damage repair enzyme, in spinocerebellar ataxia with axonal neuropathy. Nat Genet 32(2):267-272. Tranchant C, Fleury M, Moreira MC, Koenig M, Warter JM. 2003. Phenotypic variability of aprataxin gene mutations. Neurology 60(5):868-870. Wilson DMr, Mattson MP. 2007. Neurodegeneration: nicked to death. Curr Biol 17(2):R55-R58.

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Formation/repair of SSBs, and toxicity, induced by H2O2 exposure at ice-bath or physiologic temperature.

Figure 3 .

 3 Figure 3. Formation/repair of SSBs, and toxicity, induced by MMS exposure at physiologic temperature.(A) Cells were exposed for 1h to increasing concentrations of MMS at 37°C and immediately analysed for DNA strand scission with either the alkaline halo (circles) or the comet (squares) assays, as detailed in the Methods section. (B) Cells were pre-exposed (1h) to 50 µg/ml MMS and analysed with the alkaline halo (circles)

  Figure 1S. 5'-AMP single strand break (SSB) repair in control and AOA1 lymphoblastoid cell lines. (Top) Cartoon of the duplex substrate used in the experiment with a 5'-AMP terminus, a 1-bp gap and a 3'-hydroxyl terminus. (Bottom) 0,1 µg, 1 µg or 5 µg of Cr 48 or EM223/07 lymphoblast cell extracts were incubated with a FAM labeled 5'-AMP SSB substrate for 1h at 30°C. Reaction products were fractionated on a 15% denaturing polyacrilamide/urea gel and image was acquired with a GelDoc-It-UVP. The AMP-23-FAM oligonucleotide was fractionated in parallel as size marker (Mk). The position of the repaired 45-FAM oligonucleotide is indicated. The position 23-FAM indicates both AMP-23-FAM and 23-FAM generated from AMP adenylation in Cr 48 extracts, as shown in the lower part of the gel as separated bands with a longer gel run.

Figure 2S .

 2S Figure 2S. Toxicity induced by high concentrations of H 2 O 2 in Cr 48, AOA1-EM223/07 and AOA2-EM518 cells.Cells were exposed to increasing concentration of H 2 O 2 for 30 min in saline A at 37°C and postincubated for 30 min in fresh culture medium. Toxicity was then assessed by using the trypan blue exclusion assay. Results represent the means ± SD calculated from three to five separate experiments. *P < 0.001 as compared to Cr 48 cells (one-way ANOVA followed by Dunnett's test).

  Figure 1S

Table 1S

 1S Non-protein thiol content and catalase, glutathione peroxidase or superoxide dismutase activities in Cr 48 and EM 223/07 cells

		Cr48	EM223/07
	Non-protein thiols	21.9 + 2.2	19.8 + 1.0
	(nmol/mg protein)
	Catalase	4.08 + 0.2	3.07 + 0.4*
	(U/mg protein)
	Glutathione peroxidase	41.8 + 3.9	43.2 + 4.7
	(mU/mg protein)
	Superoxide dismutase	12.2 + 1.3	12.5 + 1.4
	(U/mg protein)

Results are means ± S.E.M. from five separate experiments. * P < 0.01 as compared to Cr 48 cells (one-way ANOVA followed by Dunnett's test).

John Wiley & Sons, Inc.Human Mutation

Acknowledgements

The work was supported by funds from the Italian ministry of Health RC2008-2009, RF2006-70, RF2007-75 (MTB, AM, OC, NB) and PS-NEURO ex art.56/05/7 (MTB, NB, AM), from the CARIPLO foundation grant # 2007.5156 (MTB) and from the Istituto Superiore di Sanita' grant # 526D/10 (MTB, NB). The APTX mutation herein described will appear in the DDBJ/EMBL/ GenBank nucleotide sequence databases with the accession number AB603741.

Contract grant sponsor: Italian ministry of Health; the CARIPLO foundation; the Istituto Superiore di Sanita'; Contract grant number: RC2008-2009, RF2006-70, RF2007-75 and PS-NEURO ex art.56/05/7; grant # 2007.5156; grant # 526D/10.


Ivonne A Herrera
email: ivonne.a.herrera@sintef.no

Erik Hollnagel

Solfrid Håbrekke

Proposing safety performance indicators for helicopter offshore on the Norwegian Continental Shelf

Keywords: Resilience Engineering, Risk Analysis, Safety Management, Leading and Lagging Safety Indicators

Over last 10-years period there has been just one helicopter accident (with no fatalities) in the Norwegian sector of helicopter offshore operations. In this case, safety monitoring cannot be based on the absence of accidents. The main objective of this paper is to suggest a combination of leading and lagging indicators to monitor safety performance for helicopter offshore operations. An approach is described to identify indicators using different perspectives: a Risk Influence Model, the Functional Resonance Analysis Method (FRAM), and lessons learned from previous studies. The approach uses accident and incident data, as well as normal operations (when nothing goes wrong). The suggested indicators were evaluated through observations and interviews/workshop with helicopter operators, air traffic controllers, helicopter deck operators and regulators. The paper discusses the approach and proposes a set of domain specific safety performance indicators.

INTRODUCTION 1.Background

Measurements of safety performance in aviation traditionally rely on lagging indicators such as accident rates, which may be further decomposed to identify particular safety issues. This categorization of accidents has enabled several improvements on specific issues. However, there is a growing concern that this information does not provide the required basis for the prevention of future accidents. The International Civil Aviation Organization (ICAO) recommended the establishment of an effective Safety Management System (SMS) [1]. Indicators are therefore needed to provide an adequate understanding of the current state of the system, and to predict possible future events or consequences of changes; i.e., leading rather than lagging indicators. Yet despite the benefit of a proactive SMS, the aviation industry generally still focuses on the reactive part of safety management.

Helicopter transport is essential for petroleum activities in the North Sea, since there is no other effective way to transport personnel. Over the last 10-years period there has been just one helicopter accident (with no fatalities) in the Norwegian sector of helicopter offshore operations. In this case, monitoring of safety cannot be based on the absence of accidents. This paper presents the results of work carried out under the Helicopter Safety Study -3 (HSS-3) [START_REF] Herrera | Helicopter Safety Study 3[END_REF], which had the overall objectives to contribute to improve safety and to set a reference standard for methodologies to analyse risk of offshore helicopter transportation. The HSS-3 project was a follow-up of previous studies: HSS-1 (period 1966-1990) [START_REF] Ingstad | Helicopter Safety Study[END_REF] and HSS-2 (period 1990-1998) [START_REF] Hokstad | Helicopter Safety Study 2[END_REF]. For the development of indicators, an important mandate for HSS-3 is to use experience from previous helicopter studies [START_REF] Ingstad | Helicopter Safety Study[END_REF][START_REF] Hokstad | Helicopter Safety Study 2[END_REF][START_REF]NOU 2002: 17 Helikoptersikkerheten på norsk kontinentalsokkel. Delutredning nr. 2: Utviklingstrekk, målsettinger, risikoinfluerende faktorer og prioriterte tiltak, Ministry of Transport[END_REF]. To complement this approach, HSS-3 incorporates development within safety thinking using a resilience engineering perspective to identify safety indicators.

Purpose of the paper

The main objective of this paper is to suggest a combination of leading and lagging indicators to monitor safety performance for helicopter offshore operations. An approach is described that identifies indicators, using different perspectives: (1) the Functional Resonance Analysis Method (FRAM), (2) a Risk Influence Model, and (3) lessons learned from previous studies. Data from normal operations (when nothing goes wrong), were used together with accident and incident data. The paper discusses the approach and proposes a set of domain specific safety performance indicators.

Delimitations

The main focus is the indicators within aviation safety in relation to major accidents, hence excludes occupational accidents. The FRAM method was used for the identification of indicators in relation to a specific scenario landing on helicopter deck. Several publications have described the use of risk influence models [START_REF] Herrera | Helicopter Safety Study 3[END_REF][START_REF] Ingstad | Helicopter Safety Study[END_REF][START_REF] Hokstad | Helicopter Safety Study 2[END_REF]. The paper emphasized indicators identified through monitoring normal operations.

APPROACH

Combining perspectives to identify indicators

Different perspectives were used to identify safety indicators as illustrated in Figure 1. The literature survey enabled a theoretical understanding of safety indicators, identification of relevant criteria and indicators from other studies. Resilience Engineering represents an alternative perspective on safety that takes into account successes and failures [START_REF] Hollnagel | Resilience Engineering: Concepts and precepts[END_REF]. Resilience is defined as the intrinsic ability of a system to adjust its functioning prior to, during, or following changes and disturbances, so that it can sustain required operations under both expected and unexpected conditions. Resilience Engineering aims to develop theories, tools and methods to support resilient organizations. The FRAM method is based on resilience engineering principles and was used to identify leading indicators. The HSS-3 RIF model is an update of previous HSS-2 model. The RIF model was explored to identify lagging indicators. In combination with a literature survey, this provided candidates for indicators that were assessed in close consultation with the industry using indicators criteria, leading to a final set of leading and lagging indicators.

Data gathering

The identification of indicators was based on an iterative process:

 An initial set of indicators was identified based on literature review, application of RIF and FRAM method.  A workshop assessed indicators against indicator criteria.  Interviews with operational staff (pilots, engineers, training, helicopter deck, air traffic controllers, petroleum representatives and regulator) assessed indicators against indicator criteria.  Observations of helicopter landing on helicopter deck during simulator session helped to improve modeling and improved analyst understanding of the context of operations.

Important lessons from literature survey

Baseline for HSS-3 was the recommendations in the public report "Helicopter Safety on the on the Norwegian Continental Shelf, Part 2: Trends, objectives, risk influencing factors and recommended measures" [START_REF]NOU 2002: 17 Helikoptersikkerheten på norsk kontinentalsokkel. Delutredning nr. 2: Utviklingstrekk, målsettinger, risikoinfluerende faktorer og prioriterte tiltak, Ministry of Transport[END_REF]. The indicators that can be used to monitor risk were: Over the last 10-years period there has been just one helicopter accident (with no fatalities) in the Norwegian sector. In addition, changes in regulations contribute to a reclassification of incidents and an increased of number of reports. An increased number of reports do not necessarily provide an indication of poor safety performance. Fatality rate and increased number of reports are therefore not suitable as sole indicators for safety performance. To complement this view, it is necessary to look for accident precursors to assess safety performance. In general, leading indicators are defined as conditions, events or measures that can be used to predict the future occurrence of an event, e.g., as accident precursors. The literature shows that there is no consistency between the definition of indicators and their application [START_REF] Hale | Special Issue on Process Safety Indicators[END_REF]. Special attention should therefore be given to the definition of indicators each time they are addressed.


Based on literature review, discussion in international forums and author's experience the following definitions are used:

 Lagging indicators measure results after unwanted events.  Leading indicators refer to current system status and their interpretation may be used to say something about future performance

The literature presents an extensive list of characteristics for indicators. There is a need for a realistic approach for the selection. The following characteristics are adopted:

 Meaningful: the value can be correlated to accident frequency or consequence, a RIF for the risk model on accidents, or with FRAM functions for the risk model for normal operations.  Available or affordable: it is possible to gather data with a reasonable cost.  Reliable: The data should as far as possible be either objective or intersubjectively verifiable.  Operational: It is possible to use the indicator to identify specific improvement measures in an operational context.  Ownership: The indicators are "owned" by the personnel which performance is measured.

The Accident Investigation Board/Norway (AIBN) presents a study regarding the relation between concurrent organizational changes and safety [START_REF]Safety in Nowegian Aviation during the Process of Change[END_REF]. In this study 5 outcome-based and 38 activitybased performance indicators for flight safety were proposed [START_REF]Safety in Nowegian Aviation during the Process of Change[END_REF][START_REF] Tinmannsvik | Ytelsesindikatorer for flysikkerhet -noen resultater fra svensk luftfart[END_REF]. The development of indicators and determination of importance for flight safety are based on safety audit checklists and discussions with experienced people from the Norwegian and Swedish civil aviation authorities. These indicators are considered as candidates for HSS-3 recommended indicators. Another significant finding in the study showed is that there is a strong focus on learning from rare accidents and failures. There is no tradition to analyze successes (normal operations with no delays) [START_REF] Herrera | Aviation safety and maintenance under major organizational changes, investigating non-existing accidents[END_REF]. This trend has changed for flight operations and air traffic management with the introduction of Line Operations Safety Audit (LOSA) and Normal Operations Safety Survey (NOSS) respectively. These safety management tools are mainly based on managing errors and threats.

Functional Resonance Analysis Method (FRAM)

Resilience Engineering provides a practical basis for the development of systemic models in order to describe the characteristic performance of a system as a whole. It can therefore also be used as the starting point for developing a systemic or functional risk model (FRM). The purpose of a systemic model is to describe the dynamic and non-linear nature of what happens within a system. This should be seen as a complement to the traditional view where accidents are described either as sequences or as concatenation of latent conditions. Hollnagel presents a new method to perform accident investigation and safety assessment, called the Functional Resonance Analysis Method (FRAM) [START_REF] Hollnagel | Barriers and accident prevention[END_REF].

Resilience engineering sees success is as a consequence of the ability of groups, individuals, and organizations to anticipate the changing shape of risk before damage occurs; failure is simply the temporary or permanent absence of that. Adopting this view means that there is a need for models that can represent the variability of normal performance and methods that can use this both to provide more comprehensive explanations of accidents and to identify the possible risks. The helicopter safety study adopts this view to identify leading indicators.

In its present form, FRAM comprises the following five steps [START_REF]A White paper on Resilience Engineering for ATM[END_REF]:

Define the purpose of the analysis, since FRAM can be used for both accident investigation and safety assessment. Identify and describe system functions. The result of the second step is the model. Every function can be characterized by six basic aspects: Input (I, that which the function uses or transforms), Output (O, that which the function produces), Preconditions (P, conditions that must be fulfilled to perform a function), resources (R, that which the function needs or consumes), Time (T, that which affects time availability), and Control (C, that which supervises or adjusts the function). A FRAM function is shown in Figure 2. Identify functional resonance by means of instantiations. An instantiation illustrates aspects and the potential links among the functions in a defined context [START_REF] Herrera | Comparing a multi-linear (STEP) and systemic (FRAM) method for accident analysis. Safety, Reliability and Risk Analysis: Theory, Methods and Applications[END_REF]. Figure 3 shows an instantiation for approach planning. The aim of this step is to determine the possible ways in which the variability from one function could spread in the system and how it may combine with the variability of other functions. This may result in situations where the system loses its capability safely to manage variability. The propagation may be both indirect via the effects that the variability may have on the general conditions or direct via the output from a function.

Identify effective countermeasures or barriers that can be introduced in the system. In FRAM, prospective countermeasures aim at dampening performance variability in order to maintain the system in a safe state. But it is consistent with the principle of Resilience Engineering to consider also measures that can sustain or amplify functional resonance that leads to desired or improved outcomes. Besides recommendations for countermeasures or barriers, FRAM can also be used to specify recommendations for the monitoring of performance and variability, in order to be able to detect undesired variability at an early stage. Performance indicators may thus be developed for individual functions and for the couplings among functions.

Risk Influence Modeling (RIF)

The risk influence model in the HSS-3 project is an update of the model developed in the previous helicopter safety study [START_REF] Hokstad | Helicopter Safety Study 2[END_REF]. This approach assumes that accidents and incidents can be described as the result of cause-effect relations, sometimes as a single cause-effect chain but more often as a combination of multiple cause-effect chains.

The risk influence model is based on a number of Risk Influencing Factors (RIFs) arranged in influence diagrams. A RIF is defined as a set of conditions that influence the risk, either positively or negatively. The RIFs are likely to have a varying degree of importance for the different categories of accidents. Eight different accident categories are defined in the model. These categories are: accident by take-off or landing on heliport, accident by take-off or landing on helicopter deck, accident following critical aircraft system failure during flight, near miss or mid-air collision with other aircraft, collision with terrain, sea or building structure, accident exposing passengers inside the helicopter, accident exposing passengers outside the helicopter and other/unknown (i.e. lightening). The status of a RIF may be improved by specific actions or become worse due to changes and threats.

The RIFs are split into two categories; risk frequency influencing factors (as shown in Figure 4) and risk consequence influencing factors, and are organized in three levels. Operational RIFs (Level 1) are risk influencing factors related to activities directly influencing the risk and that are necessary to provide safe helicopter operations on a day-to-day basis. These activities include conditions related to technical dependability, operational dependability, provision of necessary external services and surroundings. Organisational RIFs (Level 2) are defined as risk influencing factors related to the organizational basis, support and control of running activities in the helicopter transport. These factors are related to helicopter manufacturers or design organizations, helicopter operators, maintenance organizations, air traffic and navigation services, heliport and helicopter deck operators. Regulatory and customer related RIFs (Level 3) are defined as risk influencing factors related to requirements and controlling activities from international organizations, authorities and customers.

MAIN RESULTS

Leading indicators identified applying FRAM

The scenario described in this paper is an approach to and landing on a floating platform during night with good visibility and no unusual events. Results from the application of FRAM referred to the five steps described in section 2.2. In the first step FRAM was used as safety assessment looking into normal operations to identify relevant indicators. In the second step a corpus of 21 functions was identified as relevant for the scenario landing on helicopter deck, e.g., Table 1. Verify position Land Support helicopter landing Each function was characterized in terms of six aspects (an example is shown on table 2). The granularity of the description of functions was based on iterative assessment of the scenario and the functions between the analyst and pilot. The third step was the assessment of the potential variability for each singular function. Landing on helicopter deck was analyzed in relation to landing on fixed and floating oil and gas installation during day and night. In this way, it was possible to determine variability related to normal operations. The fourth step is the determination of the ways in which variability is spread through the system.

Instantiations were used to illustrate the combinations of variability. Then indicators were determined based on significant combinations of variability (bold letters illustrated on Figure 5). 3). This was achieved through discussions with operational and management personnel.

DISCUSSION AND CONCLUDING REMARKS

Helicopter safety is a result of something that the system does and not a passive property of the system. Safety is a dynamic characteristic and the result of interaction between several organizations. This view guides the indicators that are identified in the study. Each method represents different ways of understanding, it is necessary to be aware of their advantages and limitations. The paper demonstrates how a combination of several approaches provides a set of lagging and leading indicators. Since we are addressing a dynamic characteristic, it is recommended periodically to review the indicators to see if they are still relevant or whether new indicators should be considered.

The literature review show that the majority of indicators are selected from check lists or because they are easy to collect. This approach does not necessarily support indicators relevance towards safety.

The FRAM modeling provides a more dynamic approach to helicopter operations. The use of instantiations enables to illustrate how variability spreads and which variability is significant to a successful landing. The main advantage of FRAM is that this approach considers the influence of the context on actual performance. Indicators identified using FRAM are leading, these indicators show a correlation to a successful operation. Indicators identified using RIF model are mainly lagging, these indicators are based on incidents and accidents information. The RIF model provides an static picture of the overall helicopter offshore operations. The recommended set of indicators represents a combination of quantitative and qualitative data. Experience from previous studied show that quantitative information does not provide enough information towards the quality aspect. This shortcoming compensated emphasizing the importance of observations and use qualitative data.

This study represents a step forward from mainly learning from failures to consider also normal operations without failures. This approach has helped to identify alternative indicators. The identification of indicators using FRAM and the modeling enhanced understanding of the system. The indicator discussions with the industry helped to identify recommended measures to improve safety relevant to actual performance. The RIF approach allows a perspective of helicopter performance during the last 30 years. The FRAM approach represents a step forward in using new methods to improve aviation safety. While RIF method is widely recognized for risk assessment. The FRAM approach will require more applications to demonstrate its capability and have wider acceptance within the safety community.

Figure 1 :

 1 Figure 1: HSS-3 overall approach to propose lagging and leading indicators

Figure 2 :

 2 Figure 2: The six aspects of a FRAM function

Figure 3 :

 3 Figure 3: Instantiation landing on helicopter deck -flight planning (1 hour before departure)

Figure 4 :

 4 Figure 4: Risk Influence Model HSS-3 for the frequency of accidents

Figure 5 :

 5 Figure 5: FRAM instantiation for landing on helicopter deck during night

Table 1 : Example of FRAM functions for scenario landing on helicopter deck

 1

	Landing on helicopter deck FRAM functions
	Manage contracts	Manage competence
	Perform weight & balance calculations	Manage procedures
	Approach planning	Fix approach on GPS
	Do pre-landing preparations	Arrive to minimum descend
	Approach near by obstruction	Establish visual
	Decide approach type (see

Table 2 )

 2

Table 2 : FRAM function characterization FRAM Function Decide Approach type

 2

	Input	Helicopter deck personnel on helicopter deck
	Input	Helicopter deck obstacles report
	Input	Cockpit display information: wind and position
	Output	Decision
	Precondition	Helicopter airworthy
	Precondition	Company message " Over deck & over circle"
	Time	Visual
	Control	Approach procedures

Table 3 : Example of indicators identified for normal operations

 3 There are individual differences in relation to installation type i.e. fixed or floating. Use of observations to provide a qualitative evaluation.Procedures quality and complianceUse of audits and/or observations to provide an assessment of procedures revision and compliance Manage contracts -use of penalties Number of free days that have been negotiated, use of overtime Visual references Helicopter deck status in relation to regulation and recognized guidelines

	Indicator	Operationalization
	Helicopter airworthy	Indicators related to maintenance performance, Vibration Health
		Monitoring and Minimum Equipment List
	Quality of communication helicopter
	crew and helicopter deck personnel
	3.2. Lagging indicators identified using RIF approach
	The indicators are based on number of incidents and are mainly related to the operational level.
	Examples of identified indicators are:
	 Technical RIF: Windshield cracking, chip warning, oil leakage detected by walk-around  Operational RIF: Overload of cargo, incorrect marking or improper handling of dangerous
	goods, fuelling event, wrong charts in flight folder
	 Helicopter deck RIF: Crane or other obstacles on rig near to helicopter deck, incorrect
	helicopter deck position, incorrect information of pitch/roll/heave from moving helicopter
	decks
	 Weather and other RIF: Incorrect weather information, bird strikes

Paper presented at PSAM 10, June 7-11 2010, Seattle, USA

Acknowledgements

The Helicopter Safety Study 3 is sponsored by the A/S Norske Shell, BP Norway, Civil Aviation Authority Norway, ConocoPhillips Norge, Eni Norge, GDF SUEZ E&P Norge AS, Marathon, Nexen Exploration Norge AS, Statoil and Total E&P Norge AS. We thank Norwegian technical, operative and administrative personnel from helicopter operators, air navigation providers, helicopter deck personnel and emergency preparedness organizations for their participation, as well as petroleum and civil aviation (Norway and UK) authorities for their constructive comments and openness in the best interest to improve helicopter safety. The authors acknowledge Eduardo Runte for his contribution in the initial phase of FRAM functional modelling. We thank the other members of the project team Lars Bodsberg, Per Hokstad, Erik Jersin and Tony Kråkenes for their valuable comments. The work presented in this paper has also benefit from discussions on indicators during the 3 rd symposium on Resilience Engineering in 2008 and information from the European Helicopter Safety Team. We are grateful to John Wreathall and Teemu Reiman for sharing their knowledge and publications regarding the identification of leading safety indicators for the Nuclear Industry. Finally, we thank Andrew Hopkins for his views on safety indicators for the process industry presented during a visit to Norway (2009).

The work was carried out under the Norwegian Helicopter Safety


Dominique Besagni
email: dominique.besagni@inist.fr

Claire François
email: claire.francois@inist.fr

Juan Gorraiz
email: juan.gorraiz@univie.ac.at

Christian Gumpenberger
email: christian.gumpenberger@univie.ac.at

Ivana Roche
email: ivana.roche@inist.fr

Edgar Schiebel
email: edgar.schiebel@ait.ac.at

Martin Wieland
email: martin.wieland@univie.ac.at

Exploring the bibliometric and semantic nature of negative results

Keywords: bibliometrics, scientometrics, negative results, negative result publication, S&T information

Background

Scientific progress in some disciplines is hampered by researchers' tendencies to bin negative results. However, their publication is beneficial in order to prevent duplication of effort, save public money and to facilitate and promote scientific communication and progress.

A bibliometric analysis of negative results literature was performed in order to identify their most important attributes, to study their outcome and to explore their hidden relationships, focussing on the distribution of publications openly declared as containing negative results and published in journals completely devoted to this kind of publications. The Journal of Negative Results in BioMedicine (JNRBM) was used as a role model, since all the other relevant journals are not indexed in the largest citation databases. In order to evaluate the prestige of this journal, the impact factor as well as the new alternative indicators SJR and SNIP were looked up for all assigned categories. The complete article records of JNRBM, the number of citations and the citation percentiles & averages were retrieved from Scopus and Web of Knowledge to generate an impact profile.

Data analysis and visualization of relationships were performed using the software tool BibTechMon. A co-author and a co-affiliation map were produced in order to examine the plurality of communities based on the existing relationships between the authors publishing in JNRBM.

Furthermore two samples of "positive results" based on related articles containing the same number of items, either sharing the same descriptors or the same references (bibliographical coupling) were compiled in Scopus.

The first sample, obtained from Scopus by retrieving articles sharing the maximal number of references, was used to test if bibliographic coupling revealed other publications of negative results. The second sample of "positive results", sharing the maximal number of descriptors, was used to enable a comparison with the sample of "negative results". Thereafter, their abstracts were subjected to assisted data extraction, in order to identify vocabulary characterising negative results publications.

Results

The unofficial impact factor of JNRBM is 1.64 based on the information gained from the official journal website. The median impact factor (IF) in the corresponding WoS category "Medicine, research & experimental" (to which biomedical science journals are assigned) is 2.023, and the aggregate IF is 3.474. Thus the IF of JNRBM is below these values and corresponds to quartile 3 (Q3). According to Scopus data, in SCImago Journal Rank JNRBM has a SJR of 0.124 and is assigned to the categories "Medicine" (Q1) and "Pharmacology, Toxicology and Pharmaceutics" (Q2), whereas in CWTS Journal Indicators a SNIP of 0.43 is reached in the category "Biochemistry, Genetics and Molecular Biology (all)" (Q2).

JNRBM publications (93% of them articles) are cited by a broad spectrum of journals rather than by specific titles. Journals exclusively devoted to their official publication like JNRBM have a rather low impact. Only 11% of their items are above citation average. However, only one third of the publications remain uncited.

Figure 1. Average citations of publications in JNRBM (2002-2009)

Apart from single publications there is no specific community for the publication of negative results in devoted literature like JNRBM. Authorship is widely spread, with 95% of the authors contributing with a unique paper and the most active author publishing 6 papers.

Our first analysis shows that bibliographic coupling can be useful for the identification of other negative results, however, can only be regarded as a supplementary method.

A text mining approach was applied to the JNRBM publications and resulted in a list of around 50 terms expressing negative assertions. These were employed to query the database PASCAL. An indicator measuring the degree of negativeness of a publication is defined. This indicator can be then applied to both the PASCAL corpus and the JNRBM publications and the obtained results compared.

At this stage it is impossible to distinguish between the various possible negation meanings. This exploratory work opened a challenge in the refinement of mining techniques -not only to detect negative assertions, but especially to allow categorization by their real semantic meaning.

Acknowledgments

We express our gratitude to Mr. Abel L. Packer, Director of BIREME/OPS/OMS for his inspiring suggestions and helpful feedback.

Neither the co-author nor the co-affiliation analyses indicate a strong interconnectivity of authors or affiliations. There are only few clusters, whereas the majority of the publications have affiliations originating from a single country.

Figure 2. Co-authorships and co affiliations of publications in JNRBM (2002-2009) and similar publications (high concordance in descriptors) in Scopus