A Gutierrez
email: a.gutierrez@brgm.fr

; Barrat
email: barratjm@gmail.com

Groundwater resources of Burundi. New elements and decision making tools

Burundi, which stands just below the equator line, is a small (28 000 km 2 ) mountainous country with contrasted elevation (between 772 m in the rift area up to2 670 m.a.m.s.l.), and rainfall ranging from 700 to 2 200 mm/y. On geological point of view, Precambrian (basement) rocks are prevailing (up to 90% of granite, schist and quartzite), covered from time to time with alluvial formations in the inland valleys and the collapse zones related to the East-African rift (Imbo area in the eastern and northern edge of the Lake Tanganyika and in the Moso area in the south-eastern part of the country, bordering Tanzania).

The way of tapping water resources is mainly through springs : 22 000 springs are tapped for water supply compared to no more than 30 boreholes in 2010. Recent studies [START_REF] Barrat | Programme Sectoriel Eau -Burundi -Cartes des Potentialités en Eaux Souterraine BRGM/RC-59751-FR[END_REF], [START_REF] Barrat | Forages de Karusi (Burundi) -Interprétation des pompages[END_REF], [START_REF] Barrat | Supervision des opérations de construction de piézomètres dans les communes de Kirundo[END_REF], however, prove that groundwater resource should not be neglected. A groundwater potential map for the country (figure 1) was carried out in 2011 at the scale of a quarter million, which was validated by a series of boreholes in 2013 & 2014. Due to the tectonic activity in the region (rift), the basement is well fractured and proper siting of boreholes along fault zones result in high transmissivity (100-1000 m 2 /d), in addition to the high development of the weathered zone in granites & schists (up to 100m thickness) which provides high storativity. However, faulting system also results in aquifer compartmenting leading to variable sustainability. The area of Gitega is located in such a structure where water abstraction needs to be monitored carefully to avoid overexploitation.

Water obtained from boreholes is generally of good quality, except for iron and manganese in some places.

Inventory of springs in the Ngozi region [START_REF] Gutierrez | Sources de la Province de Ngozi (Burundi). Critères pour un réseau de suivi[END_REF] gives insights of how rainfall water infiltrates and incidentally becomes mineralized. [START_REF] Barrat | Programme Sectoriel Eau -Burundi -Cartes des Potentialités en Eaux Souterraine BRGM/RC-59751-FR[END_REF] References:

Figure 1 :

 1 Figure 1: Map of groundwater potential of Burundi[START_REF] Barrat | Programme Sectoriel Eau -Burundi -Cartes des Potentialités en Eaux Souterraine BRGM/RC-59751-FR[END_REF]


M.D. Nadia Bouabdallaoui
email: nadia.bouabdallaoui@gmail.com

MD Hassan Debbagh

Thibaut Schoell

Guillaume Lebreton

Surgical Management of undiagnosed laceration of superior vena cava due to blunt trauma

Keywords: blunt thoracic trauma, great vessels injury, superior vena cava injury, isthmic aortic rupture, cardiac tamponade

Intrapericardial rupture of the superior vena cava resulting from blunt thoracic trauma is a rare and life-threatening condition that has to be ruled out in the presence of signs of cardiac tamponade and history of blunt thoracic trauma. We report the case of undiagnosed superior vena cava laceration caused by a high speed road traffic accident in a 25-years old patient revealed by cardiac tamponade. We highlight the need of urgent surgical exploration in all unstable patients in the setting of blunt thoracic trauma regardless of imaging conclusions.

Introduction

Great vessels injuries are frequently associated with severe blunt trauma to chest, mostly in high-speed deceleration. Traumatic laceration of the superior vena cava (SVC) is rare and often fatal. It is a challenging diagnosis that has to be ruled out in deceleration trauma.

Surgical exploration is urgent in unstable patients.

Case report

A 25-year-old female patient was admitted to our emergency room complaining from severe chest pain after a head on collision at 80 Km/h responsible for blunt thoracic trauma. At admission, Glasgow Coma Score was 15, systolic blood pressure 110 mm Hg and pulse rate 100 bpm. Initial CT-scan evaluation displayed multiple trauma including contained isthmic aortic rupture, left clavicle fracture, nasal pyramid fracture and pericardial effusion first described as insignificant (Figure 1A). As the patient was hemodynamically stable, endovascular treatment was first proposed for the ruptured aortic isthmus. Unfortunately, she rapidly deteriorated. Bedside echocardiography showed massive pericardial effusion. Rescue median sternotomy was performed. Upon opening the tensed pericardium, 500 cc of venous blood was quickly evacuated with prompt hemodynamic stabilization. Surgical inspection revealed the source of hemorrhage as a misdiagnosed 2-cm laceration of the superior vena cava (SVC) at the atriocaval junction (Figure 1B). Hemorrhage was immediately controlled by digital pressure and the injury rapidly repaired (Figure 1C). No cardiopulmonary bypass was required. Adequate hemostasis was achieved and the sternum was closed. Uncomplicated endovascular treatment of traumatic thoracic aortic injury was secondly performed.

Postoperative course was uneventful. The patient was discharged on day 10. She is doing well at one-year follow-up.

Comment

We present here in the case of a patient with two major intrathoracic vascular injuries complicating blunt chest trauma: partial rupture of the intrapericardial SVC and isthmic aortic rupture. Deceleration and traction are the usual mechanisms proposed to explain the occurrence of these injuries at the level of vulnerable sites i.e. the aortic arch -descending aorta junction (explaining the aortic isthmus rupture) and the superior vena cava -right atrial junction (explaining the SVC rupture) [START_REF] Prêtre | Blunt trauma to the heart and great vessels[END_REF][START_REF] O'connor | Vascular injuries after blunt chest trauma: diagnosis and management[END_REF] . Aortic isthmus rupture has catastrophic outcome as it rapidly leads to death in the great majority of patients. Without surgery, almost one third of survivors will die within few hours and more than one half will die within one week [START_REF] Nan | Blunt traumatic cardiac rupture: therapeutic options and outcomes[END_REF] . Even if unusual, multiple life-threatening vascular lesions after blunt chest trauma may occur and focus on the ascending aorta is unwise.

Chest CT-scan is key in the diagnosis of traumatic great vessels injury. Nevertheless, surgical exploration must always be the rule in unstable patients admitted for chest trauma with severe deceleration and no further diagnostic testing is needed 2 .

Great vessels injuries are usually repaired without circulatory support because of frequent favourable anterior location of the lesions [START_REF] Nan | Blunt traumatic cardiac rupture: therapeutic options and outcomes[END_REF] .

Conclusion

Traumatic laceration of the superior vena cava is rare and often fatal. It is a challenging diagnosis that has to be ruled out in the setting of cardiac tamponade complicating chest trauma with severe deceleration. Preoperative work up is often limited by hemodynamic compromise. Surgical exploration is urgent in unstable patients.

Conflict of interest: none

Legends


Mathieu Acher

François Esnault

Large-scale Analysis of Chess Games with Chess Engines: A Preliminary Report

Keywords: chess game, data analysis, artificial intelligence

The strength of chess engines together with the availability of numerous chess games have attracted the attention of chess players, data scientists, and researchers during the last decades. State-of-the-art engines now provide an authoritative judgement that can be used in many applications like cheating detection, intrinsic ratings computation, skill assessment, or the study of human decision-making. A key issue for the research community is to gather a large dataset of chess games together with the judgement of chess engines. Unfortunately the analysis of each move takes lots of times. In this paper, we report our effort to analyse almost 5 millions chess games with a computing grid. During summer 2015, we processed 270 millions unique played positions using the Stockfish engine with a quite high depth (20). We populated a database of 1+ tera-octets of chess evaluations, representing an estimated time of 50 years of computation on a single machine. Our effort is a first step towards the replication of research results, the supply of open data and procedures for exploring new directions, and the investigation of software engineering/scalability issues when computing billions of moves.

Analyse large échelle de parties d'échecs avec des moteurs: un rapport préliminaire Résumé : La force des moteurs d'échecs et l'existence de nombreuses parties d'échecs ont attiré l'attention des joueurs d'échecs, des scientifiques de la donnée, et des chercheurs au cours des dernières années. Les meilleurs moteurs fournissent un jugement autoritaire qui peut être utilisé dans de nombreuses applications comme la détection de triches, le calcul d'une force intrinsèque, l'évaluation d'aptitudes, ou l'étude de prises de décision par des humains. Un problème important pour la communauté de chercheurs est de collecter un large ensemble de parties d'échecs avec les jugements des moteurs. Malheureusement l'analyse de chaque coup peut prendre énormément de temps. Dans ce rapport, nous décrivons notre effort pour analyser près de 5 millions de parties d'échecs avec une grille de calcul. Durant l'été 2015, nous avons analysé 270 millions de positions uniques issues de parties réelles en utilisant le moteur Stockfish avec une assez grande profondeur (20). Nous avons construit une base de données d'évaluation d'échecs de plus de 1 tera-octet, représentant un temps estimé de 50 années sur une machine seule. Notre travail est une première étape vers la réplication de résultats de recherche, la mise à disposition de données ouvertes et de procédures pour explorer de nouvelles directions, et l'étude de problèmes de génie logiciel pour calculer des milliards de coups.

Mots-clés : jeu d'échecs, analyse de données, intelligence artificielle

Introduction

Millions of chess games have been recorded from the very beginning of chess history to the last tournaments of top chess players. Meanwhile chess engines have continuously improved up to the point they cannot only beat world chess champions but also provide an authoritative assessment [START_REF] Hsu | Behind Deep Blue: Building the computer that defeated the world chess champion[END_REF][START_REF] Lai | Giraffe: Using deep reinforcement learning to play chess[END_REF][START_REF][END_REF][START_REF] Stockfish | [END_REF]. The strengths of chess engines together with the availability of numerous chess games have attracted the attention of chess players, data scientists, and researchers during the last three decades. For instance professional players use chess engines on a daily basis to seek strong novelties; chess players in general confront the moves they played to the evaluation of a chess engine for determining if they do not miss an opportunity or blunder at some points. From a scientific point of view, numerous aspects of the chess game have been considered, being for quantifying the complexity of a position, assessing the skills, ratings, or styles of (famous) chess players [START_REF] Biswas | A comparative review of skill assessment: Performance, prediction and profiling[END_REF][START_REF] Tamal | Quantifying depth and complexity of thinking and knowledge[END_REF][START_REF] Guid | How trustworthy is crafty's analysis of world chess champions[END_REF][START_REF] Regan | Depth of satisficing[END_REF][START_REF] Regan | Human and computer preferences at chess[END_REF][START_REF] Han | A psychometric analysis of chess expertise[END_REF], or studying the chess engines themselves [START_REF] Diogo | The impact of search depth on chess playing strength[END_REF][START_REF] Mccrossan | Gentlemen, stop your engines![END_REF]. Questions like "Who are the best chess players in history?" can potentially have a precise answer with the objective (and hopefully optimal) judgement of a chess engine. So far numerous applications have been considered, such as methods for detecting cheaters [START_REF] Barnes | On the limits of engine analysis for cheating detection in chess[END_REF][START_REF] Regan | A computer program to detect possible cheating in chess[END_REF], the computation of an intrinsic rating or the identification of key moments chess players blunder [START_REF] Wingate | Intrinsic chess ratings[END_REF].

A key issue for the research community is to gather a large dataset of chess games together with the judgement of chess engines [START_REF] Biswas | A comparative review of skill assessment: Performance, prediction and profiling[END_REF]. For doing so, scientists typically need to analyze millions of games, moves, and combinations with chess engines. Unfortunately it still requires lots of computations since (1) there are numerous games and moves to consider while (2) chess engines typically need seconds for fully exploring the space of combinations and thus providing a precise evaluation for a given position. As a result and due to the limitation of computing storage or power, chess engines have been executed on a limited number of games or with specific parameters to reduce the amount of computation.

Our objective is to propose an open infrastructure for the large-scale analysis of chess games. We hope to consider more players, games, moves, chess engines, parameters (e.g., the depth used by a chess engine), and methods for processing the overall data. With the gathering of a rich and large collection of chess engines' evaluations, we aim to (1) replicate state-of-the-art research results [START_REF] Biswas | A comparative review of skill assessment: Performance, prediction and profiling[END_REF] (e.g., on cheat detection or intrinsic ratings); [START_REF] Biswas | A comparative review of skill assessment: Performance, prediction and profiling[END_REF] provide open data and procedures for exploring new directions; (3) investigate software engineering/scalability issues when computing millions of moves; (4) organize a community of potential contributors for fun and profit.

In this paper, we report our recent effort to analyse almost 5 millions chess games with a computing grid. During summer 2015, we processed 270 millions of unique played positions using the Stockfish [START_REF] Stockfish | [END_REF] chess engine with a quite high depth (20). Overall we populated a database of 1+ tera-octets of chess evaluations, representing an estimated time of 50 years of computation on a single machine.

Data analysts or scientists can use the dataset as well as the procedures to gather novel insights, revisit existing works, or address novel issues. The lessons and numbers of our experience report can also be of interest for launching other large-scale analysis of chess games with other chess engines, games, and settings.

Dataset and Chess Games

We gathered 4.78 million unique games publicly available on some Web repositories. In this section we report on some properties of the dataset.

From a technical point of view, we parsed Portable Game Notation (PGN) files with a Java parser1 and pgn-extract2 . As a preprocess we notably eliminated a significant number of duplicated games with pgn-extract. We used the R programming language to produce plots and results.

Interestingly we can confront our results to a previous attempt by Randal Olson that explored a dataset of over 650,000 chess tournament games (see a series of blog posts3 ). We consider similar properties (such as distribution of Elo ratings) as well as additional ones (such as most represented events). In our case we have much more games and it is worth comparing our numbers to Olson's results. Our objective was to have a better understanding of the quality of the dataset, since some factors can have a negative impact on their exploitations by chess engines.

Elo Ratings

The Elo rating system is intensively used in chess for calculating the relative skill levels of players. Figure 1a gives the distribution of Elo ratings thanks to PGN headers. It should be noted that the Elo rating was adopted by the World Chess Federation (FIDE) in 1970. As such oldest games do not appear in Figure 1.

Players with > 2000 Elo rating are considered as experts in Chess; between 2200 and 2400, players are national masters; above 2400, international masters and above 2500 international grand masters. Figure 1a shows that most records come from games with players > 2200 Elo, with a large proportion played by international masters, grand masters, and top players. In general, the difference of Elos between two opponents is quite close (< 100), see Figure 1b. It is in line with what observed in chess tournaments (e.g., round-robin or swiss systems).

Ply per Games

Our 4.78 million unique games have a mean of 80 ply per game (40 moves). Figure 2 shows that the number of moves follows two trends: With <200 Elo difference, the number of ply 4 per game varies between 78 and 85. It is not clear why there is a slight increase. We can formulate the assumption that some games end early with a draw in case the Elo difference is closed. With a larger Elo difference, the number of Ply starts decreasing until 60 ply per game. It is quite intuitive since strongest players gain the upper hand quickly over their opponents. The number of ply in some games can be suspicious or presents limited interests:

• Klip,H (2305) -Bottema,T (2205) 1. e4 f6 2. d4 g5 3. Qh5# 1-0 (1990)

• Landa,K (2678) -Grall,G (

Elo Difference and Winning Chance

The difference in Elo rating strongly predicts the winner of the game. With a difference of 100, the chance for the higher player to win is 70% -not counting draws. The graph of Figure 3 is in line with the probabilities and expected outcomes of the Elo rating system. In general our dataset provides PGN headers information (Elo, winner) we can trustfully exploit in the future. We have also some exceptional records/anecdots:

• Vera Gonzalez,J (1551) -Hernandez Carmenates,Hold (2573): 1-0 (Elo difference: 1022)

• Freise,E (2018) -Anand,V (2794): 1-0 (Elo difference: 776)

• Sikora,J (1833) -Movsesian,S (2710): 1-0 (Elo difference: 877)

Colors and Percentage Win

Figure 4a shows that the percentage games won by white players depends of Elo Rating. Below 2200 Elo, chances to lose are higher (even with white colors) because such players face to better opponents and simply have less chance to win. On the other side, the percentage to win for a 2500 white player is 70% -not counting draws. Specifically, we have 120K games with white player having a Elo rating >2600: They win more than 55K games, draw 50K and lose only 15K games. Figure 4b shows the importance of having white pieces. For low Elo ratings, the draw rarely happens whereas it happens half the time for stronger players. All these results are coherent with what is usually observed in chess.

First moves and Openings

Figure 5a shows that the period 1900 -1960 was more prone to fluctuations/experiments for the choice of the first moves since the openings theory was not developed. d4, e4, Nf3, and c4 are the most popular first moves to start a game (see Figure 5b). It should be noted that for the period 1960 -2015 the top first moves slightly change over the time and tend to stabilize. It is consistent with existing databases and current practices. Figure 7a and Figure 7b show that the percentage win by white players tends to decrease steadily even if the first-move still provides a small advantage.

Inria

Comparison with Olson's dataset

Compared to Olson's results, we observe similar properties like:

• the distribution of Elo ratings and difference of Elo ratings between two opponents

• the importance of white and first-move advantage

• the increase of draws when chess experts are involved

• the proportion of first moves and openings

• the fact Elo ratings tend to predict game outcome We also observe some differences; we comment two of them here. First, the number of moves depending on difference Elo Rating (see Figure 2, page 5): Olson's dataset does not exhibit an increase curve between 0 and 200 difference Elo rating. Moreover the number of ply is slightly higher all along the graph. Second, the proportion of games win by white players depending on the date (see Figure 7a, page 9). We concur with the conclusion that having white pieces is an advantage for all periods. However we also observe a linear decrease that is not apparent in Olson's dataset, especially for old periods. Our conclusion is that there is no fundamental difference, i.e., the number of games considered in the two datasets can explain such differences.

Summary

Appendix B provides further results related to kinds of moves (promoted rates, queen side castling rates, etc.). Several other information can be extracted from the dataset as well. Our results so far suggest that (1) the database contains numerous interesting games with rather strong players; (2) headers information such as Elo rating or results of the games are coherent. Though some games can certainly be removed or corrected, we consider the dataset is representative of existing chess databases and consistent with chess practices and trends. We also obtain similar properties than in other datasets or databases. Finally, the number of games (almost 5 millions) is significant.

Large-scale Analysis of Chess Games with Chess Engines

We have now a better understanding of our database of chess games. We consider that the properties of the dataset are reassuring and justify the analysis of all games by chess engines -our original motivation. In a sense our next objective is to produce a dynamic analysis of chess games (by opposition to a static analysis as we made in previous section). The exploitations of the chess engines analysis (for blunder detection, players' ratings, etc.) are left as future work. In this section and preliminary report, we describe how we performed a large-scale analysis.

Analysis process

270 millions FEN positions. We encoded each game position using the Forsyth-Edwards Notation (FEN) notation. Some positions are equals (including the context leading to the position): a FEN encoding allows us to detect such equality. The underlying idea is that a chess engine is executed only one time per equal position. We also observe that some positions are theoretical openings and quite well-known, presenting limited interests for an analysis: we used Encyclopaedia of Chess Openings (ECO) code to detect such positions. Our dataset originally exhibits 380 millions positions. By exploiting FEN encoding and ECO classification, we only had to consider 270 millions positions. With these simple heuristics, we drastically reduced the number of games to analyze with chess engines.

Stockfish chess engine depth=20, multipv=1. We used Stockfish [START_REF] Stockfish | [END_REF] (version 6) to analyze all FEN positions. Stockfish is an open source project and a very strong chess engineone of the best at the time of writing [START_REF][END_REF]. Other researchers have also considered Stockfish in prior works. We used the UCI protocol to collect analysis. Importantly we set the depth to 20. It is a quite high depth (prior works usually set lower depths because of the computation cost). We also set multipv5 to 1.

Structuring data. We used a simple database schema (see Figure ??, page 13). PGN headers informations are stored and structured for retrieving games, positions, players, etc. For each position (FEN), we associate the score and log (multipv=1) computed by Stockfish. We developed several proof-of-concepts to validate the schema: https://github.com/ChessAnalysis/ chess-analysis-database. For instance, we can gather all positions of a game and depict the scores' evolution.

Distributing the computation. Our experiments suggested that it takes about 6 seconds to analyze a FEN on a basic machine. The use of a single machine was simply not an option since we have to analyze 270 millions position. It would require 270 * 106 * 6 = 1620 * 10 6 seconds, 450K hours, 18K days, and around 50 years of computation. The third step of our process was thus to distribute the computation on a cluster of machines. We used IGRIDA 6 , a computing grid available to research teams at IRISA / INRIA, in Rennes. The computing infrastructure has 125 computing nodes and 1500 cores.

Computational and storage cost. We split the FEN positions for distributing the computation on different nodes. We processed in batch (without user intervention) and the analysis was incremental. We used in average 200+ cores during night and day during 2 months. We gathered around 1,5 tera-octets of data (FEN logs).

Conclusion and Future Work

Our experience showed that it is practically feasible to analyze around 5 millions chess games with state-of-the-art chess engines like Stockfish.

Our next step is naturally to process and exploit data for either replicating existing works or for investigating new directions [START_REF] Biswas | A comparative review of skill assessment: Performance, prediction and profiling[END_REF]. The amount of analysis we have collected is superior to what have been considered so far in existing works: We expect to gain further confidence in existing results or methods (e.g., for players' ratings, blunders detection, influence of depth engines, etc.). Another direction is to collect more data. We used Stockfish with depth=20 and multipv=1 (single-pv mode). It has some limitations; for example, some methods/applications require multipv mode. We can rely on other chess engines. We can also use different settings for Stockfish (e.g., a higher depth and multipv). A possible threat is that the computation and storage cost can be very important.

We believe more data can help to better assess methods based on chess engines. From this perspective, we are happy to share our results with scientists, chess experts or simply data hobbyists. We hope to confront methods or interpretations and have different perspectives on data. More information can be found online: https://github.com/ChessAnalysis/chess-analysis Our long-term goal is to better understand the underlying beauty and complexity of chess thanks to the incredible skills of contemporary chess engines. The rise of computing power, software, and chess data can also be seen as an opportunity to address very old questions in the fields of cognitive and computer science (e.g., artificial intelligence, computational complexity, data and software engineering).

A Database schema

RT n°479

  (a) Distribution of Elo ratings (b) Differences of Elo

Figure 1 :

 1 Figure 1: Distribution and differences of Elo

Figure 2 :

 2 Figure 2: Ply per Game w.r.t. difference in Elo rating 4 A ply refers to one turn taken by one of the chess players

  1812) 1. e4 e5 2. Bc4 Bc5 3. Qh5 Nf6 4. Qxf7# 1-0 (2007) • Strekelj,V (1843) -Kristovic,M (2328) 1. f3 e5 2. Kf2 d5 3. Kg3 Bc5 4. Nc3 Qg5# 0-1 (2011) On the other hand, we have very long games, for example: • Sapin -Hyxiom (2003-01-01) 600 plies • Felber,J (2150) -Lapshun,Y (2355) (1998-09-05) 475 plies • Sanal,V (2286) -Can,E (2476) (2012-03-29) 456 plies

Figure 3 :

 3 Figure 3: Elo difference and games win (similar probabilities graph/outcomes of Elo rating system)

Figure 4 :Figure 5 :

 45 Figure 4: Colors and percentage win

Inria

  (a) Years of games (b) Ply per game depending on the date

Figure 6 :

 6 Figure 6: Games and years/dates

( a )

 a Percentages games win by white player depending on the date (b) Percentages games win by color depending on the date

Figure 7 :

 7 Figure 7: Percentages games win by color w.r.t. dates

Figure 9 :

 9 Figure 9: The ratio of captured pieces during a game has slowly decreased

Figure 10

 10 Figure 10: Pieces' rates

https://github.com/jvarsoke/ictk

https://www.cs.kent.ac.uk/people/staff/djb/pgn-extract/

http://www.randalolson.com/2014/05/24/chess-tournament-matches-and-elo-ratings/ Inria

UCI chess engines like Stockfish support multi best line or k-best mode. It means they return the k best moves/lines. When k=1, Stockfish returns the best line and evaluation. The increase of multipv is possible and has practical interests (see, e.g.,[START_REF] Biswas | A comparative review of skill assessment: Performance, prediction and profiling[END_REF]), but it has also a computational and storage cost.Inria

http://igrida.gforge.inria.fr/ RT n°479

Acknowledgement. We would like to thank our colleagues at DiverSE (http://diverse. irisa.fr) for their discussions and feedbacks.

B Misc: checkmates, captured pieces, promoted rates, and kinds of moves Inria


Clément Dégut

Pierre Barraud
email: pierre.barraud@cnrs.fr

Valéry Larue

Carine Tisné
email: carine.tisne@cnrs.fr

Backbone resonance assignments of the m1A22 tRNA methyltransferase TrmK from Bacillus subtilis

Keywords: backbone resonance assignment, heteronuclear NMR, unlabeling, methyltransferase, TrmK, tRNA, m1A

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Biological context

The biosynthesis of transfer RNAs (tRNAs) is a complex process composed of several steps leading to the formation of mature tRNAs with correct structures and functionalities. Apart from the processing of their extremities, the most salient property of the maturation process consists in the posttranscriptional incorporation of a large number of chemical modifications by the so-called modifica-tion enzymes. Among the modification enzymes, the methyltransferases (MTases) are the most frequent and diverse [START_REF] Hori | Methylated nucleosides in tRNA and tRNA methyltransferases[END_REF]. They catalyze the transfer of a methyl group from a methyl donor, mostly the S-adenosyl-L-methionine (SAM), towards different positions of the nucleotides. The m 1 A modification, which consists in the incorporation of a methyl group at position 1 of adenines, occurs on nucleotides 9, 14, 22 and 58 of tRNAs. This modification brings a positive charge on the adenine aromatic ring. We focused our work on the methyltransferase TrmK from B. subtilis that catalyzes the methylation of adenine 22 of tRNA Ser and tRNA Tyr [START_REF] Roovers | The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK)[END_REF]. B. subtilis TrmK belongs to the COG2384 (Cluster of orthologous groups). The members of this family are found in Gram-negative and Gram-positive bacteria. Their sequences are well-conserved in many bacterial pathogens (L. Monocytogenes, V. Cholerae, S. Pneumoniae. . . ). Since TrmK is essential for cell viability in S. Pneumoniae and since no homologues are found in humans, it was proposed to be a good target for the discovery of novel antibiotics [START_REF] Thanassi | Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae[END_REF]. We solved the X-ray structure of B. subtilis TrmK (Dégut et al., manuscript in preparation) and used NMR chemical shift mapping to get insight on the protein-RNA recognition mode. We report here the backbone chemical shift assignments of TrmK, assignments that were necessary to interpret the NMR chemical shift mapping with its tRNA substrate.

Methods and experiments

Protein expression and purification

Recombinant B. subtilis TrmK was expressed and purified with a protocol adapted from Roovers et al. [START_REF] Roovers | The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK)[END_REF]. Since wild-type TrmK is prone to aggregation through cysteine oxidation, we produced a protein variant with cysteine to serine mutations (i.e. C35S and C152S), which abolished protein aggregation and retained full enzymatic activity (Dégut et al., manuscript in preparation). Mutagenesis was performed by use of the Quickchange site-directed mutagenesis kit (Stratagen). The presence of the desired mutations in trmK was checked by sequencing. This variant was overexpressed in BL21(DE3) E. coli cells, in rich labeled media or minimum media (see below) supplemented with kanamycin at 30 µg.mL -1 . The cells were grown at 37 °C to OD600 ∼0.6, cooled down at 18 °C and induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Cells were harvested 24 h after induction by centrifugation and frozen at -80 °C until further use. The frozen cells were suspended in 1/50 of the culture volume of a 50 mM Tris-HEPES buffer pH 8.2 containing 500 mM NaCl, 5% glycerol and 1 mM of phenylmethanesulfonylfluoride (PMSF). The suspension was sonicated and the lysate was centrifuged for 30 min at 45'000 g. The resulting supernatant was loaded to a 5 mL Nickel Sepharose column (HisTrap, GE Healthcare) previously equilibrated with a 50 mM Tris-HCl buffer pH 8.0 containing 500 mM NaCl and 5% glycerol (equi-libration buffer). The resin was then washed with 30 mL of buffer and the protein was eluted with a gradient of the equilibration buffer supplemented with 500 mM imidazole pH 8.0. The N-terminal His 6 -tag of TrmK was removed by thrombin cleavage (25 U thrombin/mg of protein) performed overnight at 4 °C. PMSF at 250 µM, and EDTA at 1 mM were then added to the protein sample. The sample was concentrated with Amicon 10'000 MWCO (Millipore) and injected on a size exclusion chromatography column (Superdex-75 26/60, GE Healthcare) equilibrated with a 50 mM sodium phosphate buffer pH 7.0 containing 500 mM NaCl and 2% glycerol. Doubly labeled ( 15 N/ 13 C) and triply labeled ( 2 H/ 15 N/ 13 C) TrmK samples were obtained by growing the cells in Spectra-9CN and Spectra-9DCN media, respectively (Spectra Stable Isotopes, Inc.). Singly labeled ( 15 N) TrmK samples were obtained by growing the cells in M9 minimum media supplemented with 15 NH 4 Cl. To achieve high yield of protein in the fully deuterated medium, a double selection protocol of the strain was performed as previously described [START_REF] Sivashanmugam | Practical protocols for production of very high yields of recombinant proteins using Escherichia coli[END_REF]. Besides, specifically unlabeled samples were produced in M9 minimum media supplemented with 15 NH 4 Cl and with non-labeled histidine, arginine, or lysine at a final concentration of 1 mM, 1 hour before induction [START_REF] Rasia | Selective isotopic unlabeling of proteins using metabolic precursors: application to NMR assignment of intrinsically disordered proteins[END_REF]. NMR samples of TrmK at ∼0.7 mM were prepared in a 50 mM sodium phosphate buffer pH 7.0, 500 mM NaCl, 2% glycerol, 1 mM EDTA, 250 µM PMSF and 10% D2O. The sample was put in a 3 mm-diameter NMR tube.

NMR experiments

All NMR spectra were recorded at 15 °C on Bruker 600, 800, or 950 MHz spectrometers equipped with cryogenic probes. Backbone assignment was performed using the following standard 3D NMR experiments [START_REF] Salzmann | TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins[END_REF]): TROSY-HNCA, TROSY-HNCACB, TROSY-HN(CO)CACB and [ 1 H-15 N] NOESY-HSQC. A 3D (H)N(COCA)NH experiment was also measured [START_REF] Bracken | H)N(COCA)NH and HN(COCA)NH experiments for 1H-15N backbone assignments in 13C/15N-labeled proteins[END_REF]. Internal DSS standard was used for direct referencing of the 1 H chemical shifts, and indirect referencing of 15 N and 13 C shifts [START_REF] Wishart | 1H, 13C and 15N chemical shift referencing in biomolecular[END_REF]. Data processing was carried out with Topspin 3.2 for standard acquisition, and with MddNMR (Orek-hov and Jaravine 2011) for non-uniform sampling acquisition. Analysis of spectrum and backbone assignment was performed with the Sparky suite (Goddard and Kneller). Table 1 summarizes the NMR experiments used for the assignment.

Resonance assignments and data deposition

Before tackling the assignment of TrmK NMR signals, we needed to cope with problems of precipitation and proteolysis of TrmK. Indeed, TrmK precipitated during concentrations steps, needed high concentrations of salt (500 mM NaCl) and was subjected to intensive proteolysis within a few days after purification. Numerous conditions were tested using dialysis buttons. They allowed us to test the stability of TrmK in various conditions of buffer and temperature while working at high concentrations of TrmK (15 mg/mL) and low volumes (50 µL). As a result, TrmK does not precipitate at high concentration (> 15 mg/mL) in a phosphate buffer whereas we observed precipitation more rapidly in a Tris-HCl buffer. A high concentration of salt (400 to 500 mM NaCl) limited the precipitation of TrmK and its proteolysis. Anti-proteases (PMSF, Pepstatine A, Leupeptine, EDTA) were tested. PMSF and EDTA revealed to be efficient to stop proteolysis. PMSF was added in the lysis buffer and at different stages of purification and in the NMR tube. Lastly, glycerol was necessary to prevent precipitation when concentrating TrmK. 5% of glycerol was an optimal concentration during purification steps and for storage of the protein.

For the NMR studies, 2% of glycerol was used as the best compromise between the stability of the protein and the linewidth of NMR signals.

The protein sample in this study consists in 238 residues after the cleavage of the N-terminal His-tag and contains 5 prolines. NMR assignments were based on 3D heteronuclear NMR experiments performed on 2 H/ 15 N/ 13 C-labeled TrmK. Interestingly, the use of the (H)N(COCA)NH experiment [START_REF] Bracken | H)N(COCA)NH and HN(COCA)NH experiments for 1H-15N backbone assignments in 13C/15N-labeled proteins[END_REF], which correlates each NH group to the nitrogen of the amide group of the following residue in the sequence of the protein, revealed to be very efficient for the achievement and the validation of the assignments. This experiment is not sensitive and is commonly used for unfolded protein. However, with a fully deuterated protein, this experiment turned out to be very helpful in the assignment procedure. Besides, we used several specifically unlabeled samples to confirm the assignment or to provide starting point for the assignment (Fig. 1). For instance, Fig. 1 shows the unlabeling of all the lysines of TrmK. In this sample, all the amide groups of TrmK are 15 N-labeled except those of lysines. Consequently, the lysines are not observable in the red spectrum of Fig. 1. The superimposition with a reference TROSY spectrum (in black, Fig. 1) allows one to easily localize the peaks originating from lysine amide groups.

Backbone amide 1 H-15 N resonance assignment of TrmK was achieved for 222 of 233 non-proline residues, corresponding to 96% of completeness (Fig. 2). Amide groups from residues M1, S28, H30, A31, L39, N40, H41, K42, T113, E116 and R117 could not be assigned the majority of which are found in loop regions. In addition, 98% of Cα 98% of Cβ and 92% of CO were assigned. The backbone chemical shift assignments of TrmK were deposited in the BioMagResBank (http: //www.bmrb.wisc.edu) under accession number 26744.

An analysis of the chemical shifts of HN, N, CO, Cα and Cβ atoms was conducted with the TALOS+ webserver [START_REF] Shen | TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts[END_REF]. Fig. 3 compares the prediction of TrmK secondary structure obtained from TALOS+ with the secondary structure observed in the crystal structure of TrmK (Dégut et al., manuscript in preparation). The only light dif-ferences lie in the prediction from the NMR data of short α-helical turns (2 or three residues) around residue 68 and 165 that are not observed in the crystal structure. Therefore, both data are in very good agreement. The TrmK fold in solution and in the crystal is thus identical. Subsequently, the NMR footprint of binding of the tRNA on TrmK will be mapped on the X-ray structure of TrmK.

Figure 1 .Figure 2 .

 12 Figure 1. Superimposition of two 1 H-15 N TROSY spectra of TrmK in black for TrmK uniformly 15 N-labeled and in red for TrmK 15 N-labeled except on lysines. Lysines are thus not observable on the red spectrum confirming immediately the assignment of amide group for each lysine of the protein.

Figure 3 .

 3 Figure3. Prediction of TrmK secondary structure based on its backbone NMR chemical shifts using TALOS+[START_REF] Shen | TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts[END_REF]. The secondary structure prediction obtained with TALOS+ is shown as red bars for α-helices and yellow ones for β-strands, the height of the bars represent the probability of the secondary structure assigned by the software. Secondary structure of the TrmK X-ray structure is reported under the graph with yellow arrows for β-strands and red cylinders for α-helices.

Preprint submitted to Biomolecular NMR Assignments

Acknowledgments

Financial support from the TGIR-RMN-THC FR3050 CNRS for conducting the research is gratefully acknowledged. We thank Nelly Morellet for helpful discussions and technical advice.


MD Alexandre Fabre

PhD Sylvie Caspar-Bauguil

MD Jean Gaudart

MD Julien Mancini

Jésus Garcia

MD Marie Mittaine

MD Jean-Luc Rittié

MD François Brémont

MD Emmanuel Mas

Pre

Keywords: oxidative stress, fatty acids, nutritional status, respiratory system, children

Objectives: Oxidative stress is involved in cystic fibrosis (CF), but no guidelines for its assessment or its treatment exist. Our aim was to evaluate a test that measures the whole antioxidant capability in CF children.

Methods:

The whole blood antioxidant capability was assessed by the KRL test in 44 CF children (24 boys). We recorded several data on the same day: anthropometric measures, pulmonary function, CF severity scores, and plasma nutritional and inflammatory parameters (proteins, vitamins, erythrocyte fatty acids, micronutrients). We performed univariate and multivariate analyses with linear regression models.

Results:

The mean age at the first KRL assessment was 12.2 ± 3.8 years. The factors correlated to a diminution of the antioxidant capacity were mostly related to the severity of pulmonary disease (FEV1, acute exacerbation, and congestion). In multivariate analysis, the correlation between Brasfield score and erythrocyte antioxidant potential remained significant (β=-0.611, p<0.001).

Among nutritional factors, 6/3 ratio was significantly correlated to erythrocyte antioxidant potential (β=-1.213, p=0.01).

Conclusion:

The whole antioxidant capability, measured by the KRL test, should be a useful biomarker to assess oxidative stress in CF patients. This study suggests that 6/3 ratio should be relevant nutritional end-point in the management of antioxidant potential in CF children.

Introduction

It was already demonstrated that oxidative stress was involved in the pathophysiology of cystic fibrosis (CF), while recurrent pulmonary infections, chronic pulmonary inflammation and abnormal lung epithelium homeostasis increase the release of oxidative molecules, and antioxidants like micronutrients and fat-soluble vitamins are decreased [1][2][3][4]. However, no guidelines are established for the follow-up of oxidative stress or antioxidant potential in clinical practice in CF.

Oxidative stress is defined as an abnormal oxidant/antioxidant balance, secondary to either an increase of oxidants or a decrease of antioxidants [1]. In CF, large amounts of reactive oxygen species (ROS) are released by neutrophils that are present in bronchial lumen [1]. Other sources of ROS come from bronchial cilated and alveolar type II epithelial cells, and Pseudomonas aeruginosa [1]. These ROS alter bacteria phagocytosis. ROS are second messengers that can activate, through NF-B pathway, the transcription of inflammatory cytokines, mainly IL-6 and IL-8 [1]. Oxidants can also increase lipid peroxidation, producing several pro-inflammatory lipid mediators.

Oxysterols, which are produced by oxidation of cholesterol, are increased in CF [7]. The oxidation of arachidonic acid (AA) produces acid 8-isoprostane prostaglandin F2 [8]. Plasma level of this eicosanoid is increased after pulmonary exacerbations [8]. The imbalance is also due to a decreased level of glutathione (GSH), an impaired NO metabolism, and an abnormal intracellular H2O2 production [1]. Antioxidants present at respiratory surface are ascorbate, urate, -tocopherol, GSH, and mucins [6]. GSH is the only antioxidant present in larger amount in epithelial lining fluid than in plasma; however GSH levels are decreased to 5-10% in epithelial lining fluid and to 50% in plasma of CF patients [6].

Nutritional status is important in CF management. It is associated with both pulmonary status and survival [9]. In the setting of oxidative stress, protein malnutrition contributes to decreased albumin level and secondarily to low GSH levels [6]. Albumin can not provide all cysteine required for GSH production [6]. Pancreatic insufficiency causes malabsorption of fat-soluble antioxidants such as carotenoids, vitamin E, and coenzyme Q-10 [1,6]. There is also an abnormal fatty acid pattern in CF that is characterized by a decrease in the plasma levels of linoleic acid and docosahexaenoic acid (DHA) and an increase in the levels of AA and eicosapentaenoic acid [10,11]. These abnormalities are present even in well-nourished children, suggesting an involvement of the protein CFTR in fatty acid metabolism rather than fat malabsorption [11]. AA promotes the production of inflammatory mediators while 3 polyunsaturated fatty acids (PUFA), DHA and eicosapentaenoic acid (EPA), are involved in the resolution of inflammation [1,12]. Around 90% of CF patients require pancreatic enzyme replacement therapy (PERT) and fatsoluble vitamin supplementation. However, the amount of antioxidant vitamin supplementation could be different to obtain normal plasma vitamin levels or to correct oxidant/antioxidant balance [5,6]. For example, CF Foundation recommends 40-400 IU/d of vitamin E to normalize plasma levels [13]. To reach an antioxidant effect, 1600 IU/d of vitamin E were required in hypercholesterolemic subjects [1]. However, some studies reported that highdosage vitamin E supplementation increased risk of heart failure, coagulopathy, and death [6,14]. Carotenoid supplementation had also positive results on oxidative stress in CF trials. Vitamin E and carotenoid induced a decrease of markers of lipid peroxidation, like 8-isoprostane prostaglandin F2 and malondialdehyde respectively [6]. However, the clinical relevance of these improvements is unknown. Of note, large cancer prevention trials that used tocopherol and -carotene (ATBC trial) or -carotene and retinyl palmitate (CARET trial) lead to an excess lung cancer incidence and an excess mortality in the active vitamin treatment arm [15]. The cardiovascular mortality was also increased. Finally, micronutrient deficiencies, mainly selenium and zinc, which are frequent in CF, can alter the function of enzymatic antioxidants [1,6]. Zinc is a cofactor of superoxide dismutase and selenium is en essential component of GSH peroxidase and thioredoxin reductases [6].

Oxidative stress can be assessed by several biomarkers [1,5,6]. The aim of this study was to use a test that measured the whole antioxidant capability in CF children, using a rapid and standardized test (KRL test). We also looked for a correlation with appropriate clinical, biochemical, and radiological parameters.

Subjects and methods

Subjects

Between 09/11/2006 and 09/03/2008, oxidative stress was measured on blood samples in our pediatric CF unit. We retrospectively analysed blood samples of 44 CF children collected during this period. Children were assessed once (n=19), twice (n=22) or three times (n=3), resulting in a total of 72 samples. During this out-clinic visit, anthropometric parameters (weight, height, body mass index (BMI) z-score, height to weight (H/W) ratio) and respiratory status (congestion, exacerbation, forced expiratory volume in 1 second (FEV1) (%) and maximum midexpiratory flow (MMEF) [START_REF] Zapletal | Maximum expiratory flow-volume curves and airway conductance in children and adolescents[END_REF]) were noted in a computerized database. Their treatments, like fat-soluble vitamins, PERT, selenium supplementation, and ursodeoxycholic acid, were registered. A single physician (FB) also retrospectively evaluated CF severity scores (Shwachman and Brasfield scores). Shwachman score is composed of 4 items (general activity, physical examination, nutrition status, and radiological findings). The note of each item is 5, 10, 15, 20, or 25, ranging from severe to excellent. Thus, Shwachman score can be excellent (86-100), good (71-85), average (56-70), poor (41-55), or severe (≤40). Brasfield radiological score describes several items (air trapping, linear markings, nodular cystic lesions, large lesions, and general severity. This score varies from 0 to 25 (normal to severe).

We recorded the anthropometric parameters and FEV1 values obtained 6 months before to look for patient evolution prior to oxidative stress analysis.

We calculated the evolution of BMI z-score and FEV1 during this period, respectively (z-BMIz-BMI M-6 )/ z-BMI M-6 and (FEV1 -FEV1 M-6 )/ FEV1 M-6 , respectively defined as BMIev and FEV1ev.

Biological parameters

A blood sample was usually drawn for biological analyses. For this study, we collected the values of liver function tests (aspartate aminotransferase (AST), alanine amino-transferase (ALT), gamma-glutamyl transpeptidase (GGT)), blood cell count, creatinin level, nutritional and inflammatory proteins (prealbumin, albumin, retinol binding protein, ceruloplasmin, transferin, orosomucoid, and C reactive protein), micronutrients (zinc, cupper, and selenium), fat-soluble vitamins (retinol, and -tocopherol), and lipids levels (total cholesterol, triglycerides, and erythrocyte fatty acids).

Erythrocyte fatty acid composition was determined by capillary gas chromatography. Red blood cells were separated from plasma by centrifugation at 1730 g for 10 minutes at 4 •C and washed three times with an equal volume of cold physiological (9 g/L NaCl) solution. Lipid extraction was performed with a 3-mL chloroform/methanol mixture (V/V). Phospholipids were isolated by thin layer chromatography on silica gel 60 plates (E Merck, Darmsmat, Germany) using petrol ether/diethylether/acetic acid (80 V/20 V/1 V) as the mobile phase. Fatty acid methyl esters were generated by transmethylation of the phospholipid fraction in 5% acetyl chloride/95% methanol and separated by gas chromatography. The fatty acid analysis was carried out with a Dani GC 1000 gas chromatograph equipped with a CP-Wax 58 capillary column, 50 m in length, 0.25 mm external diameter, 0.2 µm thickness of the stationary phase (Varian Inc., Les Ulis, France), with helium 1 mL/min as carrier gas and a programmed temperature (60 to 230°C). Programmed temperature vaporization injector and flame ionization detector were used. 1-2 dinonadecanoylsn glycerol-3 phosphatidyl choline (Interchim, Montluc¸on, France) was used as internal standard.

Oxidative stress measurement

KRL test ("SPIRAL" laboratories, Couternon, France) is currently used to test the capability of erythrocytes to resist a standardized production of free radicals (http://www.nutriteck.com/sunyatakrl.html). The extracellular and intracellular antioxidant defenses contribute to maintain red blood cell membrane integrity and function until cell lysis. Blood samples were taken into evacuated tubes containing EDTA and kept at 4°C. Total blood or red blood cells separated from plasmas by centrifugation at 1730 g for 10 min at 4°C were diluted to 1/50 in isotonic saline solution and submitted to organic free radicals produced at 37°C under air atmosphere from the thermal decomposition of a 27 mmol/L solution of 2,2´-azobis (2-amidinopropane) dihydrochloride during 3 hours. Cell lysis was recorded using a 96-well microplate reader by measuring the optical density decay at 620 nm. Half-lysis time (T1/2 in min), the time at which 50% of cells have been lysed, is determined by calculating the time corresponding to OD1/2 according to the curve equation OD=f(time) and the highest value of the curve (Vmax). Results are expressed as mean of T1/2 of erythrocytes (T1/2 erythro) and T1/2 of whole blood cells (T1/2 whole blood).

Data analysis

Numerical data were expressed as mean  SD (minimum-maximum). A descriptive analysis of the main patient's clinical and biological characteristics (displayed in Table 1) was performed using a principal component analysis with varimax rotation. Univariate analysis was performed with non-parametric tests, Mann-Whitney test and Spearman correlation test. Then factors independently associated with T1/2 erythro/T1/2 whole blood were identified using stepwise multiple linear regression models (entry threshold: F test pvalue <0.1). All these analyses were performed on 44 cases, i.e. one per patient. When more than one analysis was performed for one patient, we chose the first measure. Then, a second multivariate analysis was performed on all available measures using linear models fitted with the Generalized Estimating Equation method. We therefore analysed 72 cases, accounting for the nonindependence of measures among patients who had more than one oxidative stress evaluation. We used the SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). All tests were two-sided and considered statistically significant at p<0.05.

Results

Description of the population

In this study, 44 children (24 boys and 20 girls) were included. The mean age at the first KRL assessment was 12.2  3.8 (5.9-19.1). Clinical characteristics of these children were reported on Table 1. Among these children, 5 (11.4%) had a z-BMI lower than -2; the H/W ratio was lower than 85% in 7 children (15.9%), between 85-90% in 3 children (7%). None of these malnourished children was on enteral feeding at that time. Ninety-one percent of these patients were pancreatic insufficient. They were supplemented with vitamin A (43%), vitamin E (91%) and selenium (64%) according to their respective blood values. Forty-three percent of patients were on ursodeoxycholic acid treatment.

Their respiratory status was correct. The mean value of FEV1 was 94% (Table 1). Only 2 patients had a FEV1 ≤ 50 %. Congestion and exacerbation were reported respectively in 57% and 14% of the visits. Pseudomonas aeruginosa was found in 16% of the cultures performed in all of the visits.

Only 2 patients did not have an antibiotic prophylaxis. At the time of evaluation, 15 patients (34%), i.e 25 visits (35%), were treated with itraconazole. In 38% of the visits, Brasfield score was ≥ 10. Finally, in 86% of the visits, the patients had a good or excellent score of Shwachman (≥ 70); none of them had a severe score.

The C reactive protein was available in 42 patients. Results were < 3 mg/L, between 3-10 mg/L, and > 10 mg/L, respectively in 69%, 21%, and 10% of the patients. Other biochemical results were reported in Table 2.

Oxidative status

The mean of T1/2 whole blood (n=72) was 90.3  11.4 min (68.5-121.7) (normal range: 85.15-102.60). The mean of T1/2 erythro (n=60) was 72.0  14.8 min (24.9-125.0) (normal range: 65.35-81.78). As expected, the correlation between T1/2 whole blood and T1/2 erythro was statistically significant (=0.525, p=0.001).

Principal component analysis

The plane identified by the first two factorial axes, was found to explain 38% of the total inertia in the data (Figure 1). The variables that are close to one of the axes are correlated together. For example, the first factorial axis (factor 1) was mainly positively correlated to FEV1 and retinol/RBP, and mainly negatively correlated to orosomucoid, ceruloplasmin, triglycerides, and Brasfield score. The second factorial axis (factor 2) was mainly positively correlated to retinol, pre-albumin, and RBP. Inversely, the principal component analysis should suggest that age and GGT were not correlated to the other variables.

Univariate analysis (Table 3)

There is an association between sex and antioxidant potential. T1/2 whole blood and T1/2 erythro were significantly shorter in females than males, 92.2 ± 12.4 vs. 88.4 ± 10.2 min (p=0.011), and 75.0 ± 11.5 vs. 68.9 ± 17.2 min (p=0.052) respectively. There was no correlation of T1/2 whole blood or T1/2 erythro with age, and anthropometric parameters. We did not find any difference between CF patients who were treated or not with antioxidants (vitamin A, vitamin E, ursodeoxycholic acid (UDCA), or selenium). The amount of PERT was negatively correlated to T1/2 erythro (ρ=-0.36, p=0.039).

As it could be expected, the levels of selenium in the serum were positively correlated to T1/2 erythro (ρ=0.35, p=0.047) and T1/2 whole blood (ρ=0.44, p=0.004). Concerning PUFA of membrane phospholipids of erythrocytes, the 6/3 ratio was negatively correlated to T1/2 erythro (ρ=-0.37, p=0.030).

However, there was no correlation with levels of PUFA (DHA, EPA, and AA), retinol, and -tocopherol.

Pulmonary functional tests revealed that only FEV1 was positively correlated to T1/2 erythro (ρ=0.39, p=0.018). Concerning the pulmonary infectious diseases of these patients, Pseudomonas aeruginosa colonisation was not correlated to antioxidant potential. However, patients that were on itraconazole treatment had lower antioxidant potential than patients that were not, statistically significant for T1/2 erythro, 65.7 ± 12.6 min vs. 75.8 ± 14.9 min (p=0.025). To note, these patients were not treated with itraconazole for acute broncho-pulmonory aspergillosis but for chronic colonisation. A shorter time of T1/2 erythro was found for acute exacerbation and congestion, 73.3 ± 15.7 min vs. 65.2 ± 5.3 min (p=0.022) and 76.1 ± 18.6 min vs. 68.7 ± 10.1 min (p=0.009). Finally, Brasfield score was negatively correlated to T1/2 eryhtro (ρ=-0.48, p=0.03) and T1/2 whole blood (ρ=-0.32, p=0.036).

We expected a negative correlation of antioxidant potential with inflammatory proteins. If there was a trend with C-reactive protein (CRP) and T1/2 erythro (p=0.063), we found a negative correlation of inflammatory proteins with T1/2 erythro: orosomucoid (ρ=-0.62, p<0.001) and ceruloplasmin (ρ=-0.57, p<0.001). The number of blood leucocytes was negatively associated with T1/2 whole blood (ρ=-0.36, p=0.018) and T1/2 erythro (ρ=-0.39, p=0.025).

Multivariate analysis

For T1/2 erythro, 3 parameters were independently associated: Brasfield score, 6/3 ratio, and levels of selenium in the serum. However, this latter parameter could be excluded when we analysed all the results of KRL test performed for the same patient. Finally, Brasfield score and 6/3 ratio remained independently negatively correlated to T1/2 erythro, β=-0.611 (p<0.001) and β=-1.213 (p=0.013) respectively. For T1/2 whole blood, 2 parameters were independently associated: sex and MMEF. After accounting for all the available measures, only the sex remained significantly correlated to T1/2 whole blood (p=0.015).

Discussion:

The KRL test represents the capability of a person to react to an oxidative stress. In our study, T1/2 erythro was significantly associated with a decrease of plasma level of selenium. The decrease of this antioxidant could alter the selenium-dependant GSH peroxidase, as it was reported [1]. T1/2 erythro was also associated with pulmonary congestion and exacerbation, i.e. may be with an increase of ROS in the lungs. To our knowledge, the correlation of oxidative stress and Brasfield score was not previously described.

In our study, this score was the only respiratory parameter that remained statistically significant in multivariate analysis. More than air trapping, this association could be due to bronchiectasis. In the literature, oxidative stress was present during acute respiratory exacerbations and it improved after treatment of exacerbation [START_REF] Range | Treatment of pulmonary exacerbations of cystic fibrosis leads to improved antioxidant status[END_REF]. The oxidation of proteins that were present in bronchoalveolar lavage fluids was negatively correlated to FEV1 values [START_REF] Starosta | Oxidative changes of bronchoalveolar proteins in cystic fibrosis[END_REF].

However, oxidative stress remains present in stable CF patients and it could also contribute to chronic pulmonary inflammation [5]. Similarly, a recent study assessed plasma and cellular oxidative stress biomarkers in stable adults with bronchiectasis (36 CF, and 54 without CF), and in 50 healthy controls [START_REF] Olveira | Cellular and plasma oxidative stress biomarkers are raised in adults with bronchiectasis[END_REF]. Oxidative stress biomarkers were increased in patients with bronchiectasis compared with controls, without difference between CF and not CF patients.

The relation of good nutritional status and pulmonary function is well established [START_REF] Munck | Nutritional considerations in patients with cystic fibrosis[END_REF]. We did not find any association between antioxidant capability, assessed by the KRL test, and anthropometric parameters. However, the fact that the amount of PERT was associated in univariate analysis with T1/2 erythro should suggest that pancreatic insufficiency by itself rather than clinical nutritional status is involved in oxidative stress in CF. Lezo et al. recently reported that CF patients with pancreatic insufficiency had higher levels of lipid peroxidation biomarkers than patients with pancreatic sufficiency [5]. We did not find any significant differences between oxidative stress and fat-soluble vitamins. The antioxidant capabilities of patients that were not treated with antioxidant vitamins were similar to those of supplemented patients. Abnormalities of fatty acids that occurs in CF seems to be of importance [7,10]. In multivariate analysis, we found a significant negative correlation of 6/3 ratio and T1/2 erythro. The increase of 6/3 ratio is associated with a decrease of antioxidant potential of CF patients, with synthesis of pro-inflammatory lipid mediators and reduction of resolvins, involved in the resolution of inflammation [12]. A limitation of our study is that we did not test these lipid mediators, but they were not routinely available.

In further studies, we should assess plasma levels of protaglandins (PGE2), leucotriens (LTB4), and eicosanoids (5-HETE) that are the products of the oxidation of AA by cyclooxygenase and 5-lipoxygenase. They could be of better interest than AA, EPA, and DHA levels that were not associated with oxidative stress in our study. However, ratio of 6/3 or AA/DHA could be of greater importance than the quantity of fatty acids [7,10].

In order to effectively manage oxidative stress in CF, we could hypothesize that: (i) plasma antioxidant normal values are too low in the setting of CF; (ii) antioxidant levels within the airway are more important than the plasma levels; (iii) trials are needed to determine appropriate antioxidant regimen [5,6]. New formulations of fat-soluble vitamins and antioxidants have been tested since few years [START_REF] Papas | A pilot study on the safety and efficacy of a novel antioxidant rich formulation in patients with cystic fibrosis[END_REF][START_REF] Sagel | Effect of an antioxidant-rich multivitamin supplement in cystic fibrosis[END_REF]. The formulation increased plasma levels of -carotene, -tocopherol and CoQ-10. Improvement of their antioxidant score was correlated to a decreased airway inflammation assessed by levels of IL-8 and total neutrophils in sputum. This score combined the values of these levels of several antioxidants pre and post-treatment. However, this score was complex and only determined in 10 patients. Moreover, pre and post-levels of IL-8 and especially total neutrophils only decreased in few patients [START_REF] Papas | A pilot study on the safety and efficacy of a novel antioxidant rich formulation in patients with cystic fibrosis[END_REF]. The formulation is now commercialised as AquADEKs®, a formulation containing several fat-soluble vitamins, water-soluble vitamins, micronutrients, and

antioxidants. An open-label non-randomised study enrolled 14 CF patients [START_REF] Sagel | Effect of an antioxidant-rich multivitamin supplement in cystic fibrosis[END_REF].

The authors only found an improvement of plasma levels of some antioxidants (-carotene, CoQ-10, and -tocopherol). No correlation was found with urine 8-isoprostane levels. In recent reviews, details on antioxidant trials in CF were summarized [1,6]. Our data suggest that the KRL test is an interesting tool to assess antioxidant potential in CF. Moreover, its correlation with pulmonary status could dispense of the measure of BAL oxidative biomarkers that are more invasive. This test could allow to follow the antioxidant potential of CF children and to monitor the effectiveness of antioxidant intervention.

In conclusion, the present study demonstrated that the whole antioxidant capability, measured by the KRL test, should be a useful biomarker to assess oxidative stress in CF patients. It was associated with pulmonary status. The better respiratory parameter correlated to antioxidant potential was Brasfield score. The efficiency of the KRL test was greater in erythrocytes than in whole blood cells. This study should suggest that 6/3 ratio could be more relevant than fat-soluble antioxidant vitamin levels to assess oxidative stress in CF. Finally, an interventional trial is required to evaluate the usefulness of this test to detect post-treatment improvements.

Figure 1 .

 1 Figure 1. Principal Component Analysis, plane of the first two components (Factor 1 and Factor 2). To note, this two factorial axes explained 38% of the total inertia in the data. The variables that are close to one of the axes are correlated together.

Table 1 : Clinical characteristics of CF children

 1

		N	mean	SD	Min	Max
	Age (year)	44	12.2	3.8	5.9	19.1
	z-BMI	44	-0.5	1.2	-3.8	2.7
	H/W ratio	44	96	11	68	122
	FEV1 (%)	44	94	22	33	142
	Brasfield score	44	7.5	3.3	1	14
	Shwachman score	44	82	9	60	100
	PERT (U lipase/kg/d)	40	4501	1478	2455	8471
	Sputum		positive		negative
	Staphylococcus aureus		54		18
	Pseudomonas aeruginosa	13		59
	Genotypes	N	Mutation 1		Mutation 2
		4	c.1521_1523delCTT (p.Phe508del)	c.1624G>T (p.Gly542X)
		2	c.1521_1523delCTT (p.Phe508del)	c.3605delA (P.Asp120AlafsX9)
		2	c.1521_1523delCTT (p.Phe508del)	c.617T>G (p.Leu206Trp)
		8	c.1521_1523delCTT (p.Phe508del)	other mutation
		4	other mutation	other mutation
	FEV1: forced expiratory volume in 1 second, H/W ratio: height to weight ratio,
	PERT: pancreatic enzyme replacement therapy, z-BMI: z-score of body mass
	index.

Table 2 : Biochemical characteristics of CF children

 2

		N	mean	SD	Min	Max
	AST (ULN)	40	0.7	0.2	0.3	1.6
	ALT (ULN)	40	0.6	0.4	0.2	2.0
	GGT (ULN)	40	0.5	0.3	0.2	2.0
	Albumin (g/L)	42	39.4	4.1	31.0	49.9
	Pre-albumin (g/L)	42	0.20	0.05	0.11	0.31
	Orosomucoid (g/L)	42	0.92	0.37	0.40	2.50
	Ceruloplasmin (g/L)	42	0.39	0.15	0.20	0.90
	Transferrin (g/L)	42	2.9	0.4	2.1	3.9
	Retinol binding protein (g/L)	42	0.033	0.010	0.013	0.062
	Whole blood selenium	38
	(µmol/L)		0.97	0.20	0.63	1.45
	Serum selenium (µmol/L)	41	0.86	0.19	0.53	1.39
	Serum zinc (µmol/L)	42	12.9	2.0	8.5	17.0
	Erythrocyte zinc (µmol/L)	42	165	31	95	220
	Retinol (mg/L)	42	0.38	0.12	0.15	0.68
	Retinol/RBP	42	0.85	0.08	0.67	1.06
	-tocopherol (mg/L)	42	8.5	2.2	4.8	13.6
	Total cholesterol (mmol/L)	41	3.14	0.64	1.70	4.90
	Triglycerides (mmol/L)	41	0.83	0.37	0.38	1.90
	EPA (mg/L)	42	10.5	4.2	3.5	22.8
	DHA (mg/L)	42	67.2	18.4	33.8	115.6
	AA (mg/L)	42	257.9	28.9	199.0	328.0
	6/3	42	4.7	1.1	2.8	8.6

Table 3 : Univariate analysis of continuous variables (Spearman

 3 Cu: cupper, DHA: docosahexaenoic acid, EPA: eicosapentaenoic acid, FEV1: forced expiratory volume in 1 second, H/W: height to weight ratio, MMEF: maximum midexpiratory flow, PERT: pancreatic enzyme replacement therapy, Se: selenium, T1/2 erythro : half-lysis time of erythrocytes, T1/2 whole blood : half-lysis time of whole blood cells,

	correlation test)
		T1/2 whole blood	T1/2 erythro
			p		p
	Anthropometric parameters
	Age	-0.067	0.665	-0.206	0.229
	Weight	0.012	0.940	-0.096	0.579
	Height	-0.040	0.797	-0.112	0.517
	H/W	0.193	0.209	0.107	0.533
	Pulmonary functional tests and severity scores
	FEV1	0.122	0.428	0.392	0.018
	MMEF	0.279	0.066	0.329	0.050
	Brasfield score	-0.317	0.036	-0.475	0.003
	Shwachman score	0.168	0.276	0.321	0.056
	Treatment
	UDCA (mg/kg/d)	0.134	0.387	0.234	0.169
	PERT (U lipase/kg/d)	-0.125	0.443	-0.361	0.039
	Biological parameters
	Leucocytes	-0.363	0.018	-0.385	0.025
	Serum Se	0.435	0.004	0.349	0.047
	Total Se	0.278	0.091	0.058	0.759
	Serum Cu	-0.153	0.340	-0.354	0.043
	Retinol	0.050	0.752	0.241	0.170
	-tocopherol	0.116	0.465	-0.041	0.819
	Orosomucoid	-0.236	0.132	-0.617	<0.001

UDCA: ursodeoxycholic acid.

Acknowledgements:

Emmanuel Mas was the recipient of a grant from Lactalis under the aegis of the Groupe Francophone d'Hépatologie, Gastroentérologie et Nutrition Pédiatrique.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. AA: arachidonic acid, ALT: alanine amino-transferase, AST: aspartate aminotransferase, DHA: docosahexaenoic acid, EPA: eicosapentaenoic acid, GGT: gamma-glutamyl transpeptidase, ULN: upper limit of normal.


Franck Mady
email: franck.mady@unice.fr

Jean-Bernard Duchez
email: jean-bernard.duchez@unice.fr

Yasmine Mebrouk
email: yasmine.mebrouk@unice.fr

Mourad Benabdesselam

Equilibrium Degradation Levels in Irradiated Erbium-Doped Fiber Amplifiers

35 words) -We show that local EDFA degradation can be tuned reversibly across equilibrium levels determined by local pump power and dose rate, while the dose is cumulated. This remarkable property inspires a physical, validated degradation model.

I. INTRODUCTION

Erbium-doped fiber amplifiers (EDFA) could be used in high-output power fiber laser sources operating in space, as those required in optical inter-satellite links (OISL) or remote sensing (embedded LIDAR). They have been flying around the Earth already, in satellite fiber optic gyroscopes (FOG). Mechanisms of the radiation-induced degradation of EDFA remain nevertheless unclear despite the significant amount of studies that have been published for more than 20 years [1]. Degradation consists in "darkening", an excess optical loss resulting from the formation of color centers upon ionization effects. It is characterized by the socalled radiation-induced attenuation (RIA), most often at pump or signal wavelengths. Unfortunately, guided radiations do not act as probes only. It is well known, notably, that light at pump wavelength (typically 980 nm) can mitigates the RIA. This effect being most probably due to photo-bleaching (PB), its efficiency depends on pump power. Pump-induced PB and its interplay with darkening have therefore to be properly characterized and understood to elucidate basic mechanisms and design physical explanatory/predictive models. To date, published works have generally not paid in the pump effect the attention it deserves: the pump power is not always specified (see, e.g. [2,3]), not taken into account in the interpretation [3], and EDFA lengths have been taken in the 1-3 m range [2]. These lengths are chosen to comply with operation conditions, but they result in an inhomogeneous distribution of pump power along the fiber (Fig. 1) and do not offer convenient conditions to elucidate basic mechanisms. In what follows, we use short fiber pieces to demonstrate new remarkable features revealing mechanisms that are included in a physical degradation model.

II. EXPERIMENTAL

The measurement set-up is similar to that used in [4], except that the probe at 633 nm is not used. The fiber under test (FUT) is in-core pumped at 976 nm (temperature and fiber-Bragg grating stabilized laser diode) while being homogeneously irradiated in the beam of an X-ray generator (Inel XRG 3500, copper anode, 45 kV accelerating voltage). Dose rates given to the FUT are monitored by an ionization chamber (PTW 23342, UnidosE), placed right below the FUT. Two InGaAs photodiodes served as power-meters to estimate launched and transmitted pump powers as well as RIA:

! RIA 980 nm (t) = " 10 L # ln(10) ln P out (t) P in (t) # P in (0) P out (0) $ % & ' ( ) in dB/m.
L = 2 cm is the FUT length, P in (t) and P out (t) are input and output powers at time t, respectively. P in is constant (P in (t) = P in (0)) within a 1% fluctuation.

Samples consisted of various COTS and tailor-made erbium-doped fibers fabricated and drawn in our laboratory by the standard MCVD and solution doping techniques. This summary particularly reports on the Er80-4/125 fiber commercialized by Liekki (n-light) as a very highly doped erbium fiber designed for fiber lasers and amplifiers. We used very short fiber pieces to avoid amplification of the spontaneous emission (ASE) and ensure a uniform distribution of both pump power and excitation ratio of Er 3+ ions before irradiation. The pump power was always virtually constant across our 2 cmlong samples (varying by less than 0.8%). Fig. 1 presents the calculated pre-irradiation pump and excitation ratio profiles that would exist in a 2 m-long Er80 fiber pumped at P in = 100 mW. In long fibers, ASE powers are not negligible and their profiles, together with those of pump power, excitation ratio, and PB efficiency, strongly depend on L and P in . The subsequent introduction of a signal in the fiber will also change these distributions. By taking short fiber pieces, we free the problem from extrinsic spatial variations to retain a well-defined pump power as the only parameter. Profile variation effects, that additionally accompany changes in P in or L and are necessarily integrated when the RIA is averaged over several meters, are thus ruled out.

III. RESULTS

A typical experiment is reported in Fig. 2 for the Er80 fiber. A unique fiber sample has been irradiated and pumped continuously for almost 3 days. The dose rate D' and the pump power P in have been switched between various constant values, as indicated in graphs. The upper part of the figure displays the evolution of the RIA measured at 980 nm whereas the lower part gives the cumulated ionizing dose. The experiment can be divided into 3 parts: A, B and C. In A, the pump power was constant (P in = 132 mW). The dose rate has been first set at 60 krad/min and then decreased by steps down to 40, 20, 11, 5, 2.6, 1.28 and 0 krad/min before being raised again to 5 and 11 krad/min. In the B region, the dose rate was constant (D' = 11 krad/min) but P in was switched from 132 to 167.8 mW, 48.2 mW, and raised back to 132 mW. The pump power was again dropped to 48 mW to enter the C region where we started with D' = 0 before switching to 20, 40, and 60 krad/min. Each couple (P in ,D') was maintained a sufficiently long period of time to observe the RIA can reach a steadystate level (hereafter termed equilibrium degradation level, EDL). EDL are found to increase with the dose rate and when pump power is decreased. This well reveals the competition between darkening and PB kinetics. After its initial steep increase, the RIA decreases in zone A despite the continuous increase of the dose! This uncommon fact would not be observed if the dose rate was switched before entering EDLs. EDLs are entirely determined by the couple (P in ,D'). They neither depend on the cumulated dose, i.e. the exposure history of the sample, nor on the starting darkening level. For instance, the couple mW, 11 krad/min) is formed twice in A and once in B, at various total doses and different initial RIA levels, but it always results in the same EDL around 30 dB/m (dashed horizontal lines highlight EDLs obtained for identical pump/dose rate couples). In the absence of ionizing radiation (D'= 0), the RIA does not decrease down to zero: the pump does not permit full recovery of the transmittance, suggesting that part of color centers is not bleachable. EDLs(P in ,0) appearing in Fig. 2 (in regions A and C) are lower at higher pump powers (about 13.1 and 18.5 dB/m at P in = 132 and 48.2 mW, respectively). The zero-dose rate case is actually a particular one because the EDL then depends on the initial darkening level. We examined for instance the case of pure photo-darkening, when the pump is turned on at 132 mW, without x rays, starting from a new pristine Er80 sample (no initial RIA). As expected for an Er-doped fiber, the photodarkening equilibrium level was very low, at 0.87 dB/m, far below the 13.1 dB/m value obtained when the same pump power is applied at D' = 0 starting from a 20 dB/m darkened state. EDLs extracted from Fig. 2 are plotted in Fig. 3 as a function of dose rate for P in = 132 mW (EDLs of zone A), and 48.2 mW (EDLs in zone B and C). Data are well fitted by a power law with almost the same exponent close to 0.17. EDLs extracted from Fig. 2, zone B, are plotted in Fig. 4 as a function of pump power for D' = 11 krad/min. Their pump power dependence again obeys a power law, with a -0.48 exponent.

Further experiments are needed to determine whether this value is dose rate sensitive (final paper).

A power law dependence of the RIA on the dose rate is not -a priori -an original finding, since the RIA was empirically found to vary as D' (1 -f) D f , where D is the dose and f an exponent (close to 0.8 in [5]). What is new here is the fact that we deal with EDLs that do not depend on the dose (RIA is frozen at the EDL, even under continuous irradiation at constant P in and D'). We have therefore EDL !(D' 0.17 /P in 0.48 ), but EDL !D 0 ! To highlight how the RIA probe light affects the RIA level, tests were made on new fiber pieces at various dose rates, "without pump". To measure the RIA, the pump was just turned on periodically for a few seconds. Results are shown in Fig. 5 (one data point per second). The black plot has been obtained at indicated dose rates by lighting the pump at 132 mW for 5 s every 15 minutes (pump ignition time ratio 0.56%), or every hour (0.14%). RIA levels are higher than those of Fig. 2, but the pump effect is still strong, preventing the RIA from a continuous increase and leading to its stabilization around 100 dB/m. To get the blue plot, the pump impact was reduced by dropping P in to 7.7 mW and the pump ignition time ratio to 0.06 and 0.03% (2 s ignition every 1 or 2 hours). Despite this reduction, PB is still sufficient to make the RIA stabilize, now around 260 dB/m. Even very short, low-frequency, low-power pump actions yield efficient mitigation. This reveals that measuring the 'intrinsic' RIA is a hard task! We finally estimated the room temperature annealing (RTA) by stopping xrays and waiting for 9 h before the last RIA record. RTA is more significant when it starts from the higher RIA at 270 dB/m (blue data points, almost -40 % in 9 h). The relative anneling is 4 times smaller when starting from 90 dB/m (black data points). In Fig. 2, RIA levels are such that RTA is rather weak. In presence of significant RTA, we could not reach almost perfectly the same EDL at 132 mW and 11 krad/min, as we did 3 times in 21 h.

IV. DISCUSSION AND MODELING

Our investigation of the interplay between radiation induced darkening and pump-induced PB evidences remarkable properties. Under spatially homogeneous pumping, the RIA can be tuned reversibly by varying P in and/or D' while the dose is being accumulated. This could not be demonstrated using standard amplifier lengths. At fixed launched power and dose rate, regions of long fibers experiencing higher pump power (see e.g. Fig. 1) would reach their EDL rather rapidly, whereas RIA in low pump power regions would not stabilize readily. This kinetics difference would be reinforced with the passage of time, since lower pump power also means faster RIA build-up and higher pump losses… Therefore it will take much more time for the lengthintegrated RIA to reach a clear stationary level. This considerable delay makes very hard to observe "rapid" EDL switches on long fibers. Basically, EDLs provide a most relevant reference to assess the radiation-resistance of EDFA. They indeed set the maximum, i.e. the worst degradation level achievable at given (P in ,D'). Optimizing EDLs, rather than lower dose-dependent levels, focuses the discussion on 'rates' (pump energy rate and dose rate), while offering the best safety margin. For actual EDFA lengths, local EDLs increase as the fiber is exposed to irradiation, due to the development of pump absorption and power loss (P in decreases). Regions with initially higher (resp. lower) pump power will experience more (resp. less) efficient PB so their RIA, together with their local EDL, will increase slowly (resp. rapidly). In other words, lower EDLs are less rapidly damaged (raised). As regards the PB efficiency, the notion of high and low pump power is obviously related to the dose rate. A high power, i.e. yielding very efficient PB, refers to a power which is capable of photo-ionizing color centers at a frequency which is comparable to that of their formation (set by D'). For space-based applications, concerned with very low dose rates, "high pump powers" can be actually low. Not only the small D' will make initial EDLs low (2-3 dB/m at worst, at a point initially at 50 -100 mW and 2"10 -6 krad/min for Er80. Value extrapolated from fits of Fig. 3) but it will also ensure a very slow increase of this EDL and of the RIA towards it. As a result, the 980 nm RIA of the Er80 fiber should not be significantly damaged in space. This conclusion is opposite to that of enhanced low dose rate sensitivity (ELDRS) found in [3], resulting in the prediction of higher degradation levels in space than in ground accelerated tests. Of course, our qualitative analysis must be refined and confirmed quantitatively by an accurate modeling. We have been showing for a few years that physical models based on simple energy level scheme considering trapping, detrapping and recombination transitions, together with related set of coupled rate equations, represent a powerful tool to explain a variety of experimental behaviors in systems damaged by carrier ionization and trapping. We gave a global and validated modeling of dose rate effects inherent in such systems [6], and recently explained successfully on this basis [START_REF] Mady | AIP Conf. Proc[END_REF] the interplay between pump and ionizing radiation in ytterbium-doped fibers previously demonstrated by our team [4]. For erbium-doped fibers, the physical model was built by inserting the minimal ingredients required to explain what we observed: electron-hole pair ionization, trapping (to form color centers) on 2 types of traps with different activation energies, photo-ionization of the shallowest trap (the second is optically disconnected to account for the non-bleachable part of the RIA), and recombination. The model cannot be presented in detail in this summary; it is of the same type as those introduced in [6,[START_REF] Mady | AIP Conf. Proc[END_REF]. In its present form, it is rigorously valid for fiber lengths tending to 0 (local model); longer fibers will be simulated after the implementation of this first module in a spatiallyresolved self-consistent simulation code, eventually coupled with an EDFA simulator. Fig. 6 and 7 present a typical example of simulations aiming at reproducing the experimental protocole of Fig. 2. We show the simulation of the first 6 steps of zone A (Fig. 6) and of the sequence of zone B (plus beginning of C, Fig. 7). EDLs are correctly generated and well depend on the couples (P in , D') only. The power law dependence of the EDL on D' and P in are qualitatively reproduced. The computed dose rate exponent (0.31) is almost twice the measured one, but this discrepancy most probably arises from the fact that simulation depicts too short fibers. The overall qualitative agreement with data, which is very good, validates our minimal physical model. The final paper will present additional results, notably on other erbium-doped fibers, other L, and EDLs at zerodose rate. Physical differences with ytterbium-doped fibers will be explained. The model will be introduced in detail (physical bases, equations, determining parameters, limits…) and thoroughly confronted to data. Beyond the basic investigations performed here at high doses and high dose rates, practical predictions for low dose rates and low doses applications, including possible ELDRS, will be discussed.

Fig. 1 .

 1 Fig. 1. Pump power (blue), co-and counter-propagative ASE powers (black) and the resulting excitation ratio (green) typical profiles in a 2 m-long Er80-4/125 fiber pumped at 980 nm with P in = 100 mW (simulated with the FiberLaserAmplifierToolbox freeware).

Fig. 2 .

 2 Fig. 2. Temporal evolution of the RIA under various pump power/dose rate couples (upper part), and the corresponding evolution of the cumulated dose in silica (lower part), applied continuously on the same 2 cm-long piece of the COTS Er80-4/125 fiber.

Fig. 3 .

 3 Fig. 3. EDLs as a function of dose rate for P in = 132 and 48.2 mW and power law fits. Inset: the same in log-log plots.

Fig. 4 .

 4 Fig. 4. EDLs as a function pump power for D' = 11 krad/min and power law fit to data.

Fig. 5 .

 5 Fig. 5. RIA at 980 nm measured by short periodic pump ignition. Black: P in = 132 mW during 5 s each 15 min (and sometimes 1 h). Blue: P in = 7.7 mW during 2 s, pump ignition period = 1 or 2 h.

Fig. 6 .

 6 Fig.6. Simulation of the first irradiation sequences of Fig.2 (zone A) (red curve) and a variant (blue curve) showing that EDLs given by the model are well reproducible for identical (P in , D') couples. Inset: EDL as a function of the dose rate and its power law fit.

Fig. 7 .

 7 Fig. 7. Simulation of irradiation sequences of Fig. 2, zone B and beginning of zone C. Inset: EDL as a function of pump power and its power law fit.


Franck Mady

Jean-Bernard Duchez

Yasmine Mebrouk

Mourad Benabdesselam

A sufficient model of the photo-, radio-, and simultaneous photo-radio-induced degradation of ytterbium-doped silica optical fibres

Keywords: radiation effects (on silica fibres, harsh environment) PACS Fiber optics, Radiation effects on optical elements, devices and systems

We propose a physical model of photo-, radio-, and simultaneous photo-radio-darkening of Yb-doped silica optical fibers. This work is complementary to the submission by J-B. Duchez et al., also in this conference, which reports a detailed characterization of the interplay between the pump (photo-darkening) and an external ionizing radiation (radio-darkening). The model predictions are in full agreement with data on photo-darkening and with all of the experiments reported in the Duchez's work.

INTRODUCTION

A detailed characterization of simultaneous photo-radiodarkening (PRD) is proposed in this conference by J-B. Duchez et al.. The present complementary work presents a model based on the minimal physical mechanisms (free of any semi-empirical law or parameter) that are shown to be sufficient to reproduce all of the experimental features reported in literature for photo-darkening (PD), and in the Duchez's abstract for radio-darknening (RD) and PRD. Refinements could be proposed to improve, e.g., the quantitative kinetics. Such a successful agreement between a wide variety of experimental features and the model has important consequences in the understanding and assessment of the YDF resistance. Even if PD and RD mitigation routes have been found (as phosphorus codoping for PD or cerium co-doping for PD and RD), our work sets a novel and proper understanding framework, beyond empirical recipes.

MODEL AND RESULTS

The physical processes of PD and RD build-ups are still rather controversial. The experimental validation of the present model provides a strong support of the retained mechanisms and should put an end to the main controversies. In [1], we concluded that the RD of X-ray irradiated YDF originated from the reduction of Yb 3+ ions, forming Yb 2+ ions and trapped holes. We also highlighted that trapped-hole-centres resulting from PD were identical to those produced by RD. At last, we showed that the thermal PD and RD bleaching was due to the release of trapped holes and their subsequent recombination to Yb 2+ ions. These findings are consistent with the proposal by the Engholm's team that PD is due to an Yb 3+ to Yb 2+ conversion, again with hole trapping, upon absorption of a UV photon (resulting from the cooperative de-excitation of several excited Yb 3+ ions) into the charge transfer (CT) bands of Yb 3+ ions [2][3][4]. Darkening can be bleached at 355 nm [5], 543 nm [6], and 915 nm [7]. If photobleaching (PB) results from the photoionisation (PI) of trapped holes, this variety of energies suggests that PI takes place between a localized trapping state and the valence band (VB), and that trapping states involved in darkening may start around ∼1.26 eV (980 nm) above the VB (consistent with [8,9]). Based on these hypotheses, we propose the model of Fig. 1, cast into the form of an energy level scheme. In this minimal model, we deal with a single trap, but a distribution of trapped-hole centres is required to reproduce accurate darkening kinetics. Thermal hole detrapping is also neglected here, but it could be added as a refinement. The pump is responsible for the direct CT of holes from Yb 3+ ions to traps, as shown by the σ PD transition, and the trapped-hole state is shared with RD [1]. Following [3], we do not consider hole transfer to the valence band because this CT requires a higher energy and is much less probable in the case of PD since this energy is gathered from several excited Yb 3+ ions. CT also increases the densities of Yb 2+ ions (n) and colour centres (h). Both species contribute to the excess loss in their respective absorption bands. PI of trapped-hole centres corresponds to the σ PB transition.

Released holes can recombine to Yb 2+ ions (PB). Transient carrier populations in the bands are always small so n and h are virtually identical, proportional to the excess absorption coefficient. We consider that 3 cooperative de-excitations of Yb 3+ ions are necessary to reach the first CT absorption band [3]. If the ionization energy of trapped-hole states is around 1.3 eV [8,9], it also takes the energy of k = 2 or 3 excited Yb 3+ ions to reach a significant PI cross-section.

Transitions of Fig. 1

€ h H = r n N = N r x inv α σ PD σ PB N V τ , for k = 2. (1)
If PI involves 3 excited ions, the PDEL increases linearly with x inv if α < 1 and α/r < h/H < max(1,1/r). Then:

€ h H = r n N = N r x inv σ PD σ PB N V , for k = 3. (2)
In ( 1) and ( 2), τ is the non-radiative lifetime of Yb 3+ ions and N v is the equivalent density of states in the VB. The dimension of the σ PB coefficient depends on k. To decide between these 2 cases, it would be interesting to examine the experimental dose rate dependence of RD in YDF. We indeed showed recently [10] that trap systems with small α ratios should present much higher dose rate effects than those with α ≥ 1 (study currently in progress). Whatever the case, the general behaviour of the calculated PRD is always the same. In Fig. 2 to 4, we simulated the same protocols as in the Duchez's experiments, here for k = 2, α = 1 (other parameters are not discussed in this short article). Fig. 2 presents the transmission decay in a PRD sequence, at various doses, followed by PD or PB under the sole action of the pump. It illustrates the crucial role of the PDEL by showing that the primary action of the pump (PD or PB) depends on the ionizing dose! The pump is mainly responsible for PD as long as the dose has not degraded the system below the PDEL. Then it essentially leads to PB (the reduced dose D is the ratio of the number of electronhole pairs created by the ionizing dose to N).

Fig. 2. Simulated transmission decays at various reduced doses D and the role of the pump after X-ray stops

In Fig. 3 (similar to Fig. 3 in the Duchez's work), 2 ionizing doses have been imparted with or without pump (PRD or RD) before leaving the pump acting alone. At the end of the dose absorption, the pump has slowed-down darkening in PRD, compared with RD, at doses yielding a PRD level above PDEL. It has conversely enhanced darkening at doses where the PRD level is below the PDEL. At high doses (longer irradiation times, dotted line in Fig. 3), the PRD level exceeds PDEL but reaches a plateau at a given dose due to the balancing PB pump action. Higher doses above this threshold will not be more harmful to the YDF. Increasing the inversion rate will increase the PDEL, but also the "restoring force" of the pump above the PDEL: the PRD equilibrium level will get closer to the PDEL and the PRD saturation level may decrease. Such a radio-hardening effect by pumping will be enhanced in PD-resistant YDF (low PDEL even at high x inv ). The effect of a decreasing dose rate is illustrated in Fig. 4, where simulations of the fractional irradiation experiments displayed in Fig. 4 of the Duchez's abstract are presented. Calculations are in very good agreement with data, except maybe for the decay kinetics after X-ray shuts-down (to be improved by a model refinement). We added, in dashed lines, the PRD obtained for permanent irradiations, the constant dose rate being equal to the mean dose rate achieved in the 3-and 6fraction irradiations. We also display the PRD obtained for the same dose, but with a constant dose rate of one tenth and one thousandth of the 6-fraction mean dose rate. By exploring, through a validated simulation, such low dose rates that are not achievable in practise (too long irradiations), we demonstrate that the PRD level decreases with the dose rate (at high dose) and that it saturates to its minimal value, equal to the PDEL, at very low dose rates. This is an important conclusion, notably as regards the use of YDF in space-based applications (PD-resistant YDF will be PRD resistant!) Fig. 4. Simulation of PRD for a dose given in 1, 3 and 6 fractions and comparison with permanent irradiations at similar mean dose rates. Inset: the same in log-log scales.

Fig. 1 .

 1 Fig. 1. Energy level scheme of the model. RD transitions in black, PD and PB ones in orange. Thick arrows show the relevant energy transitions. Dashed arrows just illustrate routes of valence changes. The ionizing radiation, responsible for RD, generates electron-hole pairs at a rate g 0 , representative of the dose rate. Electrons injected in the conduction band (CB) can be trapped by Yb 3+ ions (initial density N) to form Yb 2+ ions (density n), whereas holes get trapped on gap states to form trapped-hole colour centres (H states occupied by h holes).In this minimal model, we deal with a single trap, but a distribution of trapped-hole centres is required to reproduce accurate darkening kinetics. Thermal hole detrapping is also neglected here, but it could be added as a refinement. The pump is responsible for the direct CT of holes from

Fig. 3 .

 3 Fig. 3. Simulated degradation under X rays for RD and PRD and post-x-rays evolution due to the pump

  can be rationalized by a set of coupled non-linear differential equations and solved numerically to provide time-resolved PD, RD or PRD. Because of PB, simulated PD and PRD always reach a stationary level at long irradiation times, in agreement with data ([7] and Duchez's submission). The RD evolution is determined by a few ratios: r = N/H, α = β/γ, θ = δ/γ. PD and PRD are also controlled by the Yb 3+ ions inversion rate x inv and, notably,

σ PD /σ PB . The model was first checked against literature data for pure PD (g 0 = 0). It was found to reproduce all the PD features reported by Jetschke et al.

[7]

, in particular the linear dependence of the PD equilibrium level (PDEL) on the inversion rate x inv . We established that, below trap saturation, 2 physical cases lead to this linearity. If PI involves 2 excited Yb 3+ ions, the PDEL is proportional to x inv if α ≥ 1. Stationary hole trap filling (h/H) and conversion rate of Yb 3+ ions (n/N) are then: