Johan T Den Dunnen

Dr Peter E M Taschner
email: p.taschner@lumc.nl

John Wiley

Inc Sons

Describing structural variants by extending HGVS sequence variation nomenclature

Keywords: sequence variation, complex mutation, mutation detection, mutation database, nomenclature sequence variation, complex mutation, mutation detection, mutation database, nomenclature, MDI sequence variation, complex mutation, mutation detection, mutation database, nomenclature, MDI ¶

Introduction

The standard human sequence variation nomenclature (http://www.hgvs.org/mutnomen/) has gradually evolved as the result of continuous additions and changes [START_REF] Antonarakis | Recommendations for a nomenclature system for human gene mutations[END_REF][START_REF] Den Dunnen | Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion[END_REF].

The standard nomenclature was predominantly designed and used to describe sequence variants in gene sequence variant databases or in tabular format in publications. The updated standard nomenclature supports the need of the (clinical) genetics community in describing simple variants at different levels, including the non-coding DNA level. Large-scale rearrangements detectable by cytogenetic techniques traditionally belong to the genetic changes covered by the International System for Human Cytogenetics Nomenclature [START_REF] Iscn | An International System for Human Cytogenetics Nomenclature[END_REF]].

The application of array technology by the cytogenetics community and next generation sequencing technology in DNA diagnostics result in the increased detection of complex changes at relatively high resolution. These complex changes include exon duplications with additional variants in the duplicated exon, but also structural variants (SVs) such as large scale rearrangements (segmental duplications, inversions, translocations and transpositions) and copy number variations. The molecular nature of the data generated by the new techniques demands extensions of the standard ISCN and HGVS nomenclature systems. We believe that both systems will gradually approach each other and ultimately merge. As a first step, an extension of the HGVS standard nomenclature is proposed to allow accurate and unambiguous description of more complex changes including SVs. The aims of this paper are: 1) to investigate the problems encountered when describing more complex variants, 2) to suggest a solution by extending the guidelines to provide sufficient flexibility and consistency for the description of complex sequence variants, 3) to provide a description format, which can be generated and interpreted by dedicated software tools, such as the Mutalyzer sequence variant nomenclature checker [START_REF] Wildeman | Improving sequence variation descriptions in locus-specific mutation databases and the literature using the MUTation AnaLYZER (MUTALYZER) mutation nomenclature checker[END_REF].

Current standard human sequence variation nomenclature

The standard nomenclature for simple sequence variants, its basic rules and variant type preference have been summarized in Tables 1 and2. The standard nomenclature regarding complex sequence variants is recapitulated here to allow assessment of its limitations:

• Complex changes are sequence changes involving two or more changes occurring at the same location. The combination of changes may include substitutions, deletions, duplications, insertions and inversions, which affect either a single nucleotide or a range of nucleotides, but also larger scale events (translocation, gene conversion and transposition). When descriptions become too complex, the recommendation is to submit the sequence that has been determined to GenBank and to use the accession and version number in the description. For example, c.123+54_123+55insAB012345.2:g.76_420 denotes an insertion of nucleotides 76 to 420 from GenBank file AB012345 version 2 between nucleotides c.123+54 and 123+55 of the intron.

• Two or more changes in one individual (allele, haplotype and genotype descriptions) are described by combining the changes, per allele (chromosome) between square brackets ("[]"). When changes occur at flanking positions, it is recommended to merge the separate descriptions. For example, the combination of c.76A>C and c.77T>G should not be described as c.[76A>C; 77T>G], but as c.76_77delinsCG.

Limitations of the current nomenclature

The description of complex variants using "delins" and a Genbank accession number is convenient in case of large changes where the inserted sequence is a perfect copy. However, duplications, gene conversions, and inversions often have additional small changes and can be regarded as "imperfect". According to the standard HGVS nomenclature, these "imperfect" rearrangements have to be described as deletioninsertions. This makes the descriptions long and complex. In addition, the similarity between the imperfect copies is not easily recognized (Fig. 1).

Furthermore, the standard HGVS nomenclature defines duplication as "a sequence change where a copy of one or more nucleotides is inserted directly 3'flanking of the original copy". This means that the original sequence (5' copy) is in the same orientation (i.e. head-to-tail) as the inserted copy (3' copy). Therefore, the HGVS definition applies only to tandem duplications. The ISCN nomenclature describes these as direct duplications to distinguish them from inverted duplications, in which the order of the bands with respect to the centromere has changed. In cytogenetic terminology, either one of the copies involved may have been inserted in an orientation opposite to the original sequence.

EXTENSIONS AND ADDITIONAL SUGGESTIONS

To remove the limitations mentioned above, we suggest the following extensions (See Table 1 for a summary of the extended rules): i) Nested changes We propose the description format using nesting to describe complex variants. This extension could be included in the "Symbols" section of the standard HGVS nomenclature as:

• Curly braces { and } are used to indicate "sub-alleles", containing one or several changes within insertions, gene conversions, inversions and duplications. The suballele is used as a suffix to the original change (e.g., format: dup{change}). The description g. 200_400dup{280A>C}, for example, indicates that the 3' copy of the sequence from nucleotide position 200 to nucleotide position 400 also contains an A to C substitution at position 280. The nested changes follow the numbering scheme of the main variant, unless g. or other position number prefixes are included between braces.

To demonstrate the use of the nested change description format, we will apply it to describe several theoretical changes in the copies of a region with a duplication (g. 200_400dup) and a substitution (g.280A>C) (Fig. 1, alleles A-D, description underlined). The substitution in the 5' copy can be described as described by adding the change within inserted sequence as a "sub-alleles" to the accession numbers of the inserted sequence. In the description g.400_401ins{AB567890.1:g.200_201insTA} for allele F, the accession number of the inserted sequence is contained within the curly braces. As a consequence, the suballele description of inserted sequences can be evaluated according to the standard nomenclature. Other complex variants, such as changes within sequences involved in inversions, gene conversions or other exchanges, can be described by adding these changes as "sub-alleles" to the accession numbers of the exchanged sequences.

Application of the nested change format to theoretical "imperfect" inversions in a region with a substitution (g.158A>C) and an inversion (g. 100_200inv) is also easy (Fig. 2). Consider the deletion-insertion allele g.100_200delinsAB987654.1 (Fig. 2, allele D, description not underlined), which becomes g.100_200inv{158A>C} in nested change format. The position of the nested change refers to the original position within the reference sequence, i.e. before inversion. In addition, the nucleotide specified is also the original one, i.e. not its reverse complement in case of inversions.

If the inverted region contains an additional substitution (g.176T>C), the standard allele description rules can be used to create "sub-alleles" containing the variants separated by a semi-colon, for instance g.100_200inv{158A>C;176T>C} (Fig. 2, allele E, description underlined).

The nested change format is also applicable to "imperfect" duplications with changes in the orientation of one or both copies (Fig. 3). For clarity, in this text (but not in the nomenclature) the orientation of the copies relative to the reference sequence specified by the start and end positions. Perfect duplications have copies in the same head-to-tail orientation (→→) without any change (g.200_400dup, Fig. 3, allele A). A small insertion following the 5' copy can be described as an allele: g.[200_400dup;400_401insATAC] (Fig. 3, allele B). The same insertion between the copies can be described as duplication with nested insertion: g.200_400dup{insATAC} (Fig. 3, allele C). In this case, the nested insertion has no position number to indicate its insertion before the 3' copy. A small insertion within the 3' copy also can be described as duplication with nested insertion: g.200_400dup{280_281insAC} (Fig. 3, allele D). Standard HGVS nomenclature can describe an inversion where the 5' copy is inverted (tail-to-tail orientation: ←→) in an allele format combining inversion and duplication: [inv;dup]. The other copy orientations, head-to-head (→←) and tail-to-head (←←) can be regarded as duplications with nested inversions, which are easily described as dup{inv} and inv;dup{inv}, respectively. Thus, inversion of the 3' copy of allele D could be described in the format dup{inv} with two levels of nesting: g.200_400dup{inv{280_281insAC}} (Fig. 3, allele E). The inversion of the 5' copy of allele D uses the format inv;dup with one level of nesting: Consider a region in which an insertion of the sequence AB567890.1 was found (Fig. 4, alleles A and B). Occasionally, similar insertions are observed, but more nucleotides are inserted than those contained within the AB567890.1 record (Fig. 4, allele C). Allele C is described as g.200_201ins{ATAC;AB567890.1;AT} in composite change format. For optimal flexibility, the composite change format can be combined with nesting to describe the result of mutation events involving sequences of known and unknown origin. Thus, the insertion of a sequence, which differs from AB789012.1 by the additional insertion of nucleotides GC between positions 24 and 25 in AB567890.1, can be described as g.100_200ins{ATAC;

{AB567890.1:g.24_25insGC};AT} (Fig. 4, allele D).

iii) Variant type preference and the order of descriptions

The standard nomenclature uses different variant types to achieve the shortest description of a specific change. The standard nomenclature contains a hierarchy to indicate which variant type is preferred for a given change (Table 2). For inserted sequences resulting from a duplication of the immediately preceding sequence (tandem duplication), the variant type duplication is preferred over insertion.

Similarly, three other variant types, substitution, inversion and gene conversion, are preferred above the insertion/deletion (delins) type. New in this extension is that The variant type preference should be applied within the "sub-alleles" in the nested or composite change formats introduced here. For the unambiguous reconstruction of the variant sequence, it is important to evaluate the descriptions in the correct order (See Table 1). In general, variants in "sub-alleles" should be ordered from 5'to 3' similar to allele descriptions. In case of descriptions with different levels of nesting, variant type preferences and order have to be applied first on the deepest sub-allele level (within the most inner braces). For example, working upwards in g.200_400dup{280_281insAC}, allows unambiguous recognition of the 3' copy as the target location of the AC insertion. In addition, effects on the size of the copy are more easily calculated. In composite change formats, descriptions separated by semicolons should be treated independently in the order from left to right. For example, g.100_200ins{ATAC;{AB567890.1:g.24_25insGC};AT} is not equal to g.100_200ins{AT;{AB567890.1:g.24_25insGC};ATAC}, although the different parts of the inserted sequence are the same. The extended description by itself should not be interpreted as an evolutionary sequence of events, i.e. the molecular mechanism leading to the observed complex variant. It should be regarded simply as a description to support automatic conversion of any reference sequence into the sequence observed by the submitter. Similarly, the new formats could be used to describe somatic changes during tumor progression and to follow the evolution of cancer cell genomes. The same reference sequence file would also suffice for other genomic rearrangements, such as gene conversions and translocations leading to gene fusions in leukemia. Furthermore, the use of the same reference file may help to identify variants resulting from retrotransposition of reverse transcribed transcripts in LSDBs or whole genome resequencing data. The new formats can be expected to remove the limitations of the current version of the standard nomenclature mentioned above. The increased flexibility will, however, add a new layer of complexity to the standard nomenclature. Evaluation of a combination of nested and composite changes will be relatively straightforward and should allow unambiguous reconstruction of the complete complex variant sequence.

We have implemented the standard nomenclature in a software tool, the Mutalyzer nomenclature checker [START_REF] Wildeman | Improving sequence variation descriptions in locus-specific mutation databases and the literature using the MUTation AnaLYZER (MUTALYZER) mutation nomenclature checker[END_REF]]. The nomenclature extensions suggested here are planned to be included in a future version of Mutalyzer and should not be difficult to incorporate in other software with similar functionality.

Formatted

Formatted:

  Indent: First line: 36 pt Deleted: regarding complex sequence variation descriptions (http://www.hgvs.org/mutnomen/)

Formatted:

  Indent: First line: 36 pt Formatted: Indent: First line: 36 pt Deleted: the standard HGVS nomenclature can only be used to describe Deleted: (i.e. head-to-tail) without changes in orientation of the duplicated units (or duplicons) Deleted: duplicons Deleted: one Deleted: The difference between the two possible inversions is not clear in the popular short ISCN nomenclature version, but obvious in the less-used detailed version (See ISCN2009, p. 69).Deleted:The following extensions are suggested t

  g.[200_400dup;280A>C]. According to the standard nomenclature, the same substitution in the 3' copy should be described after submission of the sequence involved to Genbank. Then, the corresponding accession and version number (AB456789.1) are used to describe the change as g.400_401insAB456789.1 (Fig.1, allele D, description not underlined). For this allele, the nested change format uses the main change type dup followed by the substitution between braces, e.g., g.200_400dup{280A>C}. The position of the nested change refers to the original position within the reference sequence, i.e. before duplication. Due to the nested change format, "imperfect" 3' copies are easily distinguished from insertions of nonhomologous sequences in Fig.1. The similarity between alleles E and F can also be Formatted: Indent: First line: 36 pt

  sequence is represented by arrows. The → arrow indicates a copy in the original orientation with the sequence specified by the start and end positions; the ← arrow indicates a copy in the opposite orientation with the reverse complement of the Deleted: , descriptions underlined

  g.[200_400inv;200_400dup{280_281insAC}] (Fig.3, allele F). The inversion of both copies of allele D (or the 5' copy of allele E) is described in the format inv;dup{inv}with two levels of nesting: g.[200_400inv;200_400dup{inv{280_281insAC}}] (Fig.3, allele G).ii) Composite changesWe propose the composite change description format to describe different insertions of similar sequences. This format uses the "sub-allele" braces of the nested Formatted: Indent: First line: 36 pt enclose a description of the inserted sequences, which are separated by a semi-colon (;) in their order of appearance from 5' to 3' (e.g., format: ins{sequence; accession number.version number; sequence}, g.200_201ins{ATAC; AB567890.1;AT}. The composite change description format allows a combination of accession numbers and IUB nucleotide codes.

Formatted

  should be used to indicate a duplication in tail-to-head (←←) orientation.

  proposed here have been available for comments since June 2010 on the HGVS sequence variation nomenclature website (http://www.hgvs.org/mutnomen), but no suggestions for modifications have been received. The increased flexibility of the nomenclature extensions will allow simple and unambiguous descriptions of closely related complex variants. The new formats support descriptions at the nucleotide level for duplications with nested inversions, which correspond to the large scale rearrangements known by cytogeneticists as inverted duplications [ISCN2009, p. 69]. For whole genome resequencing projects, the extensions might minimize the submission of sequences involved in SVs as separate entities to Genbank or other primary sequence repositories. The extensions will allow comparable descriptions of similar repetitive elements at different locations.

Formatted

Fig. 1 .

 1 Fig. 1. Nested change format reflects similarity between duplications containing different SNP alleles. A genomic region (dashed arrow)(Allele A) has undergone duplication (Allele B) in combination with substitution (Alleles C and D). The imperfect duplication (allele D) is described using the nested change format (underlined) and according to current HGVS nomenclature (not underlined). The presence of the same substitution in different copies is easily recognized (Alleles C and D). The nested change format helps to recognize the difference between the variants with 3' copy changes and those with insertion of an unrelated sequence (Alleles E and F). Current HGVS nomenclature requires submission of the sequences involved and the use of the corresponding accession numbers to describe the complex variants (Alleles D-F).

Fig. 2 .Fig. 3 .

 23 Fig. 2. Nested change format reflects similarity between inversions containing different SNP alleles. In a genomic region (dashed arrow)(Allele A) containing a SNP (Allele B) and an inversion (Allele C), two complex variants (imperfect inversion alleles D and E) are described using the nested change format (underlined) and according to current HGVS nomenclature (not underlined). The presence of the same substitution in B, D, and E is easily recognized. Current HGVS nomenclature requires submission of the sequences involved and the use of the accession numbers AB987654.1 and AB876543.1 to describe the complex variants. Please note that the substitutions in alleles D and E are represented relative to the sequence in its original orientation, whereas the inversion will result in the insertion of its reverse complement.

Fig. 4 .Fig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .

 41234 Fig. 4. Composite change format reveals the similarity between different insertions at the same location. In genomic region A, insertions of similar sequences (dashed arrow), which only differ by a few flanking nucleotides are observed (Alleles B-D).The composite change format (underlined) captures both the similarity and the difference between the inserted sequences by concatenation of individual nucleotide sequences and accession number AB567890.1. The GC insertion within the AB567890.1 sequence is described using nesting (Allele D). Current HGVS nomenclature requires submission of the sequences involved and use of the corresponding accession numbers (not underlined).

John Wiley & Sons, Inc.Human Mutation

Acknowledgements

The authors wish to thank Jeroen Laros for stimulating discussions, Emmelien Aten for critically reading the manuscript, Ivo Fokkema for help with the figures, and the participants of the Human Variome Project Paris 2010 meeting for suggestions.

Part of the research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement nº 200754 -the GEN2PHEN project.

Formatted: Indent: First line: 36 pt


Sabrina Pottinger

Vaios Lappas

Christine Charles

Rod Boswell

Performance Characterisation of a Helicon Double Layer Thruster using Direct Thrust Measurements

The performance of a Helicon Double Layer Thruster (HDLT) has been characterised using a pendulum type thrust stand and retarding field energy analyser. Data recorded for a fixed propellant flow rate of 16 sccm of krypton and fixed magnetic field topology show that the thrust generated increases linearly with increasing radio frequency input power over a range of 250 W to 650 W. Over the power range investigated thrust levels of approximately 1 to 2.8 mN were achieved. A maximum effective specific impulse of 280 s was determined using the thrust data. Ion energy distribution functions indicate that increasing power corresponds to improved plasma generation processes as general trends show increasing plasma and beam currents with increasing power.

Introduction

The helicon double layer thruster (HDLT) is a relatively new electric propulsion concept that generates thrust by accelerating an RF generated plasma across a double layer that forms in the presence of a diverging magnetic field [START_REF] Charles | Plasmas for spacecraft propulsion[END_REF]. This technology offers advantages compared to conventional gridded ion engines and Hall effect thrusters in terms of the simplicity of the thruster design [START_REF] Martinez-Sanchez | Spacecraft electric propulsion-an overview[END_REF]. The lifetime of the HDLT is not limited by grid erosion mechanisms and the thruster produces a quasi neutral plume, therefore a hollow cathode or alternative beam neuralising device is made redundant. Proof of concept and thruster functionality has been demonstrated for a variety of propellants, magnetic field topologies and source tube dimensions [START_REF] Charles | An experimental investigation of alternative propellants for the helicon double layer thruster[END_REF][START_REF] Charles | Effect of exhaust magnetic field in a helicon double-layer thruster operating in xenon[END_REF][START_REF] West | Testing a helicon double layer thruster immersed in a space-simulation chamber[END_REF][START_REF] Pottinger | Helicon double layer thruster development for low power operation[END_REF].

A comprehensive evaluation of the HDLTs capabilities in terms of electric propulsion performance indicators such as thrust to power ratios and specific impulse (I sp ) has yet to be performed. This information is necessary for HDLT development and optimisation for mission applications as well as providing data to aid in the understanding of the underlying physics of thruster operation. To date, direct thrust measurement data has not been published. Momentum flux measurements have been used to determine the thrust generated by a HDLT [START_REF] West | A high sensitivity momentum flux measuring instrument for plasma thruster exhausts and diffusive plasmas[END_REF]. An instrument with a measurement range of 0.02 -0.5 mN and resolution of 15 µN measured a maximum thrust of 0.2 mN for a thruster operating at a power of 100 W, a mass flow rate of 25 sccm of argon and a maximum magnetic field strength of 138 Gauss, the source tube dimensions in this case were 150 mm diameter and 290 mm length. Indirect thrust measurements may provide an estimate of expected thrust levels but there are several issues associated with this measurement technique that can only be overcome with the use of direct measurement methods. The dimensions and placement of the target must be such that the entire cross section of the plasma plume impinges on the target. The insertion of a deflecting plate in the thruster plume results in plasma perturbations, the target will undergo charging and heating that will impact the properties of the plume environment. The impact and rebounding of ions and neutrals may also lead to erroneous results. These factors considered indirect thrust measurements often require verification by means of conventional direct thrust measurement, and discrepancies between the results determined using the two techniques have been demonstrated [START_REF] Longmier | Validating a plasma momentum flux sensor to an inverted pendulum thrust stand[END_REF]. The current investigation aims to evaluate the performance of a HDLT for set operating parameters as a function of power and reports the first direct thrust measurements recorded for a HDLT. The evaluation of HDLT performance has been undertaken within a space simulation chamber capable of achieving background pressures of the order of 10 -7 mbar without load, the chamber has dimensions of 1.2 m length and 1.2 m diameter. The HDLT shown in Fig. 1(a) consists of a Pyrex source tube with an outer diameter of 80 mm and length 172 mm. The source tube is surrounded by a source solenoid and an exhaust solenoid that terminates flush with the source tube exit. For all results presented the HDLT was operated using the exhaust solenoid alone to generate the necessary diverging magnetic field. An applied current of 3.5 A was supplied to the exhaust solenoid producing a peak magnetic field of approximately 100 Gauss. A helicon discharge is generated by the deposition of radio frequency (RF) power into a copper antenna at a driving frequency of 13.56 MHz. The antenna type used in the current investigation is a double saddle antenna also known as a Boswell type antenna. The RF power is delivered via an impedance matching network incorporating two variable vacuum capacitors. The ratio of forward to reflected power is monitored using two independent methods: 1. A conventional standing wave meter and 2. The RF generator's in built current -voltage sensor which accurately measures and displays load conditions. For all results presented the HDLT has been operated with krypton propellant at a flow rate of 16 sccm (1 mgs -1 ), which corresponds to a background pressure of 6.3 × 10 -4 mbar. Thrust measurements were recorded over an input power range of 250 to 650 W in 50 W increments and RFEA measurements were taken for powers of 250 to 500 W.

Experimental Apparatus

Thrust Stand Design

An in house developed pendulum type thrust stand is used to characterise thruster performance, see Fig. 1(b). The thrust stand operates by comparing the deflection caused by the HDLT's applied thrust to the deflection caused by a calibrating force. The deflection of the thrust stand is measured with a laser optical displacement sensor with a resolution of 3 µm at a sampling rate of 312.5 Hz to 2.5 kHz. The light from the semiconductor laser is deflected from a central support beam on the thrust stand and displacements are determined by the sensor unit via triangulation. A measuring rate of 312.5 Hz and 3 point averaging has been adopted for all testing.

A stepper motor and pulley apparatus has been incorporated into the thrust stand structure in order to allow in situ calibrations to take place during experimental trials. This apparatus functions by suspending a weight of a precisely known mass from a pulley that transfers this force directly to the thrust stand. The stepper motor can also be used to position the weight in a configuration where it applies zero force to the thrust stand. The response of the thrust stand to a static applied force over a range of 1 to 10 mN has been evaluated and shown that the response of the system is linear over the force range of interest. The standard uncertainty of the response of the thrust stand is approximately ±3 % of the measured value.

The calibration process also accounts for the impact of electrical harnessing. The system has been designed in order to minimise the number of mechanical connections to the thruster that could result in damping of the system. The only possible source of damping is the electrical connection to the HDLT solenoids, flexible wiring has been installed in order to mitigate this issue. The RF antenna and propellant feed line are mechanically isolated from the thruster. The fact that the propellant injector system is decoupled from the source means that the contribution of the expansion of neutral gas to the thrust is omitted from the measurement. This fact combined with the use

Ion Energy Distribution

A retarding field energy analyser (REFA) has been used to record the ion energy distribution function of the HDLT plume. The probe consists of 4 electrically isolated grids (an earthed grid, repeller, discriminator, suppressor) and a collector plate, which are sequentially stacked to form the probe head [START_REF] Rudakov | Overview of probe diagnostics on the H-1 heliac[END_REF]. The probe orifice was placed on the thruster's central axis, all data was recorded at a single axial position approximately 5 cm downstream of the source tube exit. The positioning of the probe ensures that plasma perturbations caused by the presence of the RFEA do not interfere with the critical region in the vicinity of the source tube exit that is thought to be key in the formation of stable double layers. The influence of neutral -ion charge exchange collisions were also considered when selecting the probe position. Ion beams decay exponentially due to charge exchange collisions with neutrals [START_REF] Charles | Plasmas for spacecraft propulsion[END_REF]. As the mean free path for such collisions is of the order of 10 cm for the plasma conditions under investigation the probe was placed at half the mean free path to minimise these effects [START_REF] West | A high sensitivity momentum flux measuring instrument for plasma thruster exhausts and diffusive plasmas[END_REF]. Ion energy distributions have been determined for input powers of 250 W to 500 W and have been used to estimate the ion velocity of the HDLT plasma.

Experimental Results and Discussion

The thrust generated by a non optimised laboratory prototype HDLT is presented in Fig. 2. For the test range investigated the data shows that thrust is a linear function of input RF power, each data point is the average result of 10 test runs in identical conditions. The main thrust generating mechanisms for the HDLT are the reaction forces produced as the ions accelerate across the double layer and then detach from the diverging magnetic field. The strength of the double layer is not highly dependent on RF power. The RFEA measurements presented in Fig. 3 correspond to a change in beam energy over the RF powers investigated of ∆E beam ≤ 1 eV. Theoretical studies verified by experiment have shown that the key parameter for double layer strength is background operating pressure which influences the ratio of ions to neutral species as well as the electron temperature [START_REF] Charles | A review of recent laboratory double layer experiments[END_REF]. The observed increase in thrust with increasing power may be attributed to increasing ion density at higher power and improved ion detachment. The beam current has been shown to increase linearly with power (see Fig. 4), as a consequence the number of ions contributing to the high ion energy population has increased. Many studies have been performed on the process of ion detachment, which is a crucial factor for the performance of electromagnetic thrusters. Gesto et. al. modelled ion trajectories in the plume of a HDLT and demonstrated that detachment occurs as the curvature of ion motion approaches zero. In this study electron dynamics were omitted, hence the electric field produced by ambipolar diffusion was neglected [START_REF] Gesto | Ion detachment in the helicon double-layer thruster exhaust beam[END_REF]. A two fluid plasma model was developed to investigate the optimisation of ion detachment mechanisms, this study showed that the initial azimuthal velocity profile at the boundary of the exhaust aperture i.e. the r -z plane located at the source tube exit dictates the properties of an ions gyro motion and its capability to detach from the magnetic field [START_REF] Schmit | Magnetic detachment and plume control in escaping magnetized plasma[END_REF]. Calculations of the gyroradius of electrons and ions in the plume of a HDLT using an experimentally determined electron temperature of T e = 5.2 ± 0.5 eV show that it is of the order of the radius of the experiment [START_REF] West | Testing a helicon double layer thruster immersed in a space-simulation chamber[END_REF]. Therefore, the plasma located downstream of the source tube exit which also corresponds to a region of weaker magnetic field strength, is non magnetised. These studies agree that the critical factor dictating ion detachment is the ion velocity on entering the plume region of the plasma which in turn dictates the rotational profile of ion motion. The application of RF power results in resonant high frequency fields being established that heat the plasma and increase the ionisation rate, resulting in higher ion densities. Further work is required to relate the alignment of the RF electric and magnetic fields with respect to the velocity components of the ions, in this way the influence of applied RF power on ion detachment may be evaluated. It should also be noted that increasing the magnitude of RF input power may initiate plasma mode change. Mode change corresponds to jumps in plasma density as a consequence of improved power coupling, this phenomena may be manipulated to enhance thruster performance [START_REF] Franck | Mode transitions in helicon discharges[END_REF].

Figure 3 shows the ion energy distribution functions for the HDLT plasma for various input power levels. The data is normalised to the peak current produced for an RF input power of 500 W. All distributions show two peaks which is characteristic of the presence of a high energy ion population (the ion beam) superimposed on the bulk plasma population. It can be seen that increasing power from 350 to 500 W results in a shift in plasma and beam potential to lower potentials. Increases in the collected ion current and beam current are observed with increasing power. The ratio of the number densities of the high energy plasma population to bulk plasma population increases with increasing power, which is confirmed by the data presented in Fig. 4. The ion current shown in Fig. 4 corresponds to the peak current collected by the RFEA at the plasma potential and the beam current is the current corresponding to the beam potential. The collected ion and beam currents do not display a strong dependence on the input power. The ion current plateaus for powers greater than 350 W and the beam current displays a linear relationship with power. This indicates that at higher powers a greater proportion of the plasma contributes to the higher energy population than observed at lower power levels. Assuming the HDLT plasma consists of a monoenergetic beam with zero beam divergence the ion velocity v ion may be determined applying the conservation of energy shown by Eq. 1, 1 2

mv 2 ion = e(V b -V p ) (1)
Where m is the ion mass, V p is the plasma potential, V b the beam potential and the elementary charge, e = 1.602×10 -19 C. The effective exhaust velocity v exh is determined for a measured thrust T at a mass flow rate ṁ where I sp is the specific impulse and g is the acceleration due to gravity at the Earth's surface.

I sp = T g ṁ = v exh g (2)
The values of ion velocity presented in Fig. 5 determined using measured beam energies in conjunction with Eq. 1 show good agreement with the velocity determined in previous investigations where v ion ∼ 6 kms -1 was found for a HDLT operating with xenon propellant [START_REF] Charles | Xenon ion beam characterization in a helicon double layer thruster[END_REF]. Using Eq. 2 to determine the expected thrust for ion velocities shown in Fig. 5 results in T ∼ 5 mN. A maximum thrust of 2.8 mN was measured using the apparatus described in section 2.1, this corresponds to an effective specific impulse of 280 s which is determined using Eq. 2. The difference between the ion velocity determined using the RFEA and the effective exhaust velocity calculated via direct measurement may be attributed to the assumptions made in the calculation of v ion with the use of Eq. 1. By assuming the beam is fully ionised the impact of neutral species which leave the source channel with a thermal velocity is neglected, the beam is also assumed to have zero divergence. These factors result in an over estimate of the exhaust velocity and calculated thrust. From Eq. 2 we may define the thrust as T = ṁv exh , taking the ratios of the exhaust velocities determined using the RFEA and direct measurement provides the ratio of the ion flow rate and the mass flow rate which describes the mass utilisation of the HDLT. For the test conditions under investigation a minimum mass utilisation of 20% is achieved for a RF input power of 250 W and 35% is achieved at 500 W. This is a key result which confirms that in order to improve the performance of the HDLT the ion fraction must be increased. The thrust and specific impulse of conventional electric thrusters with proven flight heritage is significantly greater than the levels demonstrated by the non optimised laboratory model HDLT. Electron bombardment ion thrusters such as the T6 by Qinetic, NSTAR and XIPS -25 thrusters have demonstrated a specific impulse of 4120 s, 3100 s and 3800 s respectively [START_REF] Snyder | Performance evaluation of the T6 ion engine[END_REF][START_REF] Wilbur | Ion thruster development trends and status in the United States[END_REF]. These thrusters operate at powers in excess of 4 kW and achieve thrust to power ratios of approximately 0.03 mNW -1 compared to 0.004 mNW -1 produced by the HDLT in this investigation. Hall effect thrusters have shown a throttling capability of 75 -250 mN corresponding to a nominal specific impulse of 1500 s over a power range of 1.5 to 6 kW [START_REF] Walker | Performance characteristics of a cluster of 5-kW laboratory Hall thrusters[END_REF]. Gridded radio frequency ion thrusters such as the RIT series produce thrust ranging from 10 mN to 250 mN with increasing discharge chamber diameter [START_REF] Groh | State of the art of radio-frequency ion sources for space propulsion[END_REF]. The RIT 10 which operates at input powers comparable to the HDLT is a 10 mN thruster with a specific impulse of 3300 s. It should be noted that one of the key advantages of the HDLT is the capability to accelerate ions without the use of a system of grids. This process elimates a wear mechamism but also limits the energy of the accelerated ions to the potential of the double layer. Although this self imposed limit prevents the HDLT from achieving specific impulse comparible to that achieved by established technologies this limit prevent damage to the spacecraft due to sputtering by high energy ions which is advantageous from a systems engineering perspective. The HDLT remains at an early stage of development and this initial characterisation highlights areas for future work and performance enhancement.

Conclusions

This investigation has demonstrated the linear relationship between thrust and applied RF power for a HDLT using direct thrust measurements. The observed increase in thrust is supported by probe measurements which indicate the number of beam ions comprising the high energy population of the plasma increases with increasing power. These results provide confirmation that ion detachment occurs and is consistent with the results reported in literature. Double layer strength has not varied greatly as a function of power, therefore maximising ionisation efficiency and ion detachment are critical factors for enhancing thruster performance. For the test conditions investigated a propulsive thrust efficiency (η T = T 2 2 ṁP ) of η T ≤ 1% is attained. The low thrust efficiency reiterates the fact that ion fraction must be increased and the thruster requires further optimistation in order to provide a viable alternative to conventional electric propulsion technologies. Based on the indication that HDLTs possess the capability to operate over extended lifetimes future mission applications for this technology may be interplanetary scientific missions or in the long term cargo transfer missions where rapid transfer times are not imposed. On orbit applications such as orbit maintenance and reorbit at end of life are also feasible for missions requiring on orbit operations in excess of 15 years. In order to provide a competitive propulsion option thruster performance optimisation must take place accompanied by an increased technology readiness level.

Further work is required to confirm the physics of thrust generating mechanisms, in particular combining the processes associated with double layer acceleration and ion detachment. The relative contribution of each thrust generation mechanism to the net thrust should also be evaluated. Experimental characterisation of HDLTs for various operating parameters is necessary to identify high performance regimes and provide reliable data for the validation of theoretical studies. This data is crucial for the optimisation of HDLT designs and developing the fundamental physics of thruster operation.

  (a) The HDLT within the propulsion test facility (b) CAD model of the HDLT mounted on the thrust stand. Courtesy of T. Harle

Figure 1 .

 1 Figure 1. Experimental apparatus and the pendulum thrust stand

Figure 2 .

 2 Figure 2. Thrust characterisation for a krypton discharge with ṁ = 16 sccm (1 mgs -1 ) and applied solenoid current 3.5 A

Figure 3 .Figure 4 .

 34 Figure 3. Normalised ion energy distribution functions for ṁ = 16 sccm Kr (1 mgs -1 ) and applied solenoid current 3.5 A

Figure 5 .

 5 Figure 5. The exhaust velocity determined from the beam energy (circles) and the effective velocity (squares)

Acknowledgements

This research has been funded through the University of Surrey's strategic partnership agreement with EADS Astrium.


E Punzón Quijorna

V Torres Costa

F Agulló-Rueda

Herrero Fernández

A Climent

F Rossi

Manso Silván

P Herrero Fernández

M Manso

TiN x O y /TiN dielectric contrasts obtained by ion implantation of O 2 + ; structural, optical and electrical properties

Keywords: TiN, oxygen implantation, electric/dielectric contrasts, HR-TEM

come   L'archive ouverte pluridisciplinaire

Introduction

TiN is a reputed material exploited in the fields of electronics [1], optics [2,3] and hard-anticorrosive thin films [4,5]. With regard to the former, the good electrical conductivity of TiN oriented to its application as diffusion barrier for Cu contacts in very large scale integration for microelectronics [1]. Optical studies on TiN have been mainly an accurate resource to determine the dielectric function of TiN preparations [2] or describing its plasmonic properties [3]. In fact, the fabrication of micro-nano structured TiN materials in the appropriate matrix would allow the exploitation of plasmonic devices [6].

The search for adapted TiN properties for specific applications opened the path for the proposal of derived ternary compounds among which, TiCN [7] and TiBN [8] for tribomechanics, TiAlN for corrosion resistance [9], TiCrN for magnetic properties [10] or TiNO [11,12] for modified optical properties. The exploitation of TiN in plasmonic devices passes through structural requirements such as patterning in dielectric micro and nano environments [6]. In this frame, an accurate localized transformation of TiN into TiNO could be an attractive way to produce functional electric-dielectric electromagnetic bandgap structures [13]. However, processing challenges arise in the preparation of such structures.

The controlled growth of TiNO is extremely difficult in view of the drastic integration of oxygen in the structure when mixed plasmas are used [14,15] or unexpected O sources are present in the chamber [11] (e.g. very low O 2 partial pressures lead to O rich TiNO).

Modification approaches have also been proposed; anodic oxidation [16] or O plasma immersion methods [12] were described as potential processes to avoid thermal modification, which produces extended diffusion profiles of oxygen that limit the possibility of creating structured domains [17]. Bearing in mind the stability against ion mixing of TiN films bombarded in the 100 KeV range [18], in this work we evaluate the possibility of producing modification of electronic and optical characteristics of TiN by O 2 + ion implantation to give rise to sharp TiNO/TiN interfaces.

Materials and Methods

2.1 Materials processing and modification.

TiN films used for ion implantation in this work were obtained by reactive magnetron sputtering in an Ion Tech sputtering system. The deposition pressure was 0.2 Pa and a power of 300 W DC was applied to the magnetron, which contained a high purity Ti (99.995%) target within a mixed argon-nitrogen discharge (Ar/N=9/1). A steady atmosphere was reached by 30 min pre-sputtering with the reactive gas mixture attaining growth rates of 200 nm/h. respectively) and fluences of 5. 10 13 , 10 15 or 5.10 16 cm -2 (samples labeled F1, F2 and F3, respectively) was carried out by cooling at room temperature and a chamber operation pressure of less than 10 -5 Pa.

Substrates

Materials Characterization.

Rutherford backscattering spectrometry (RBS) was performed at the Centro de Micro-Análisis de Materiales-Universidad Autónoma de Madrid (Spain) using the 5 MeV tandetron accelerator [20]. Measurements were performed with a 3.035 MeV He + beam to exploit the resonance of the 16 O( , ) 16 O elastic scattering cross section. The incidence angle with respect to the surface normal was 74º, and the scattered particles where detected at an angle of 170º by using a silicon surface barrier detector. The experimental spectra have been fitted for quantitative purposes by using the SIMNRA code [START_REF] Mayer | Application of Accelerators in Research and Industry[END_REF] to determine the film structure.

X-Ray diffraction (XRD) was performed in a Siemens diffractometer using /2 configuration with 0.02º scan step and 6 s integration time. The presence of strain in the modified samples was obtained from the analysis of the diffractograms using the Stokes

Wilson formula [22]. Raman spectra were measured with a Renishaw Ramascope 2000 spectrometer and an argon ion laser (operating at a wavelength of 514.5 nm). Laser power on the sample was around 3 mW. An optical microscope (100× objective) was used to focus the laser light on the sample and to collect the scattered light in a backscattering geometry. The system was configured in a quasi-confocal mode. No polarization analyzer was used before the spectrometer.

Cross-sectional samples (TiN reference and TiNE1F3 samples) for transmission electron microscopy (TEM) were prepared by dimple-grinding with a Gatan 656 dimpler and ion-milling with a Fischione 1010 as previously described [23]. Transmission electron microscopy studies were done in a Philips Tecnai 20F FEG analytical microscope operating at 200 kV and equipped with a dark field high angle annular detector (HAAD) for Z-contrast analysis. Electron diffraction patterns were obtained by the selected area method (SAED).

Iimage processing of the high-resolution images was carried out using Gatan Digital Micrograph and ImageJ software packages.

Four probe measurements were performed in a home made system located at the microelectronics laboratory (UAM) by scanning currents from -100 to 100 mA. In this range all the samples showed an ohmic linear regime. Sheet resistance (R s ) values were obtained considering a correction factor of CF=4.2357 [START_REF] Sze | VLSI technology[END_REF]. Spectroscopic Ellipsometry (SE)

Measurements were performed in a FLS-300 (JA Woollam Co., Inc) using a low power illumination at fixed angle (72,5º) and covering the spectral range of 250-1800 nm with simultaneous detection by using a CCD. indicates that the original TiN films contained already O (up to 3 at. %). However, previous studies suggest that O adsorbs along TiN crystal boundaries upon atmospheric exposure [26] leading to a base O concentration of circa 3% as established by RBS [27]. Spectra corresponding to modified TiNO films were simulated according to a simplified three layer model (see continuous line spectra in figures 2.a and 2.b). Total thickness after implantation was conserved so that sputtering effects could be considered as negligible at the energy and fluence ranges considered in this work. This is in agreement with what is observed by RBS characterization of anodically oxidized TiN [16] and results in a clear advantage with respect to the oxidation induced by plasma immersion under bias potential [12], which induces considerable layer sputtering.

Results and discussion

From the analysis of data corresponding to the sweep in O 2 + implantation energy, it can be observed that different energies gave rise to oxygen containing layers with thicknesses of circa 35, 50 and 75 nm for E1, E2 and E3, respectively. For a given fluence (figure 2 films grown by magnetron sputtering [28], which can be optimized in controlled conditions [29]. The diffraction diagrams for implanted samples presented no new peaks but notable distortions of peak intensity/width ratios and peak position shifts with respect to the TiN reference. In fact, damage induced by implantation is known to induce such shifts related to a slight reduction of lattice parameter. From Figure 3.a it can be observed that the higher the implantation energy, the closer the diffraction pattern to the one of reference TiN. This feature is a clear indication that the damage is produced by accumulation of oxygen rather than by direct impact damage during irradiation. This is in agreement with the fact that simulated ionization energy is similar for the three energies studied. Clearly, for a given implantation energy (see i.e. for E= 10 KeV in figure 3.b) the higher the dose, the higher the deviation of the diffraction pattern from that of the reference. These distortions can be interpreted in terms of damage accumulation in the form of stress in the films.

In order to estimate such modifications, the Stokes Wilson formula has been used to determine the strain for the most relevant modification at E1 (figure 4). It can be derived from these data that, especially at the 111 direction, that the higher the implantation fluence, the higher the strain introduced in the modified layer. In fact, the widening of diffraction peaks can be attributed to the modification of lattice parameters with respect to the original cell. It is worth noting that, though the modified layer reaches a composition of TiN 0.51 O 1.26 , the structure remains face centered cubic with a lattice parameter close to that of TiN. This reveals the relevant tightening effect of the unmodified TiN film below since relevant structural changes have been described for TiN 0.74 O 0.36 and TiN 0.6 O 0.4 (JCPDS files 00-049-1325 and 00-044-0951, respectively), which contain a considerably lower structural O. These data point out to the presence of considerable stress fields in the implanted regions. Further insight into this phenomenon can be extracted from the estimations of lattice parameters. The results indicate that the cell tends to stretch, which is compatible with a reduction of N in the crystals [30].

These new data reveal that 111 planes are preferentially affected and become closer after modification. This lattice distortion in the modified layer correlates with the creation of a strain field at the interface with the native TiN.

In order to get an insight into the local structure of the irradiated areas a TEM study was performed on samples treated at the lower energy and higher dose (E1F3) to compare with TiN/SiO 2 /Si(100). The general view of the reference and irradiated structures can be seen in figure 5.a and 5.b, respectively. The TiN films of 90 nm thickness present the typical columnar growth on the SiO 2 substrate, as previously observed for Si substrates [28]. The To deepen our understanding of the microstructure of the irradiated films we have also measured the Raman spectra for various irradiation energies and fluencies on TiN/Si(100)

samples. The largest variation is found for the lowest energy E1 because the Raman spectrum is more sensitive to the upper layers due to the light absorption in the material (for higher energies the material is modified at deeper layers). Therefore only the spectra for E1 are shown in Figure 6. The crystal structure of TiN is cubic, with all the atoms at a center of inversion symmetry. Therefore the first order Raman spectrum is forbidden for a perfect crystal. However, the usual presence of nitrogen vacancies and other defects induces some broad band first order Raman spectrum [31]. The non-irradiated TiN film shows main bands at 198 (transverse acoustic phonon, TA), 289 (longitudinal acoustic, LA), 456 (second order, 2A), and 548 cm -1 (transverse optical, TO) [32]. The sharp peak at 520 cm -1 arises from the silicon substrate.

On irradiation with oxygen ions the TiN Raman bands become more intense with fluence (Figure 6) because the number of defects increases and so does the intensity of the defect-induced first-order Raman spectrum. In particular the acoustic phonon bands become stronger relative to the optical phonon band. Since the latter is at a higher frequency it involves mostly the vibration of the lighter nitrogen atoms, whereas acoustic modes involve mostly vibrations of the heavier Ti atoms. Therefore the decrease of the TO intensity indicates that the nitrogen content decreases with fluence. The frequencies of the TiN bands increase significantly with fluence (up to 100 cm -1 for the TO phonon), pointing to a shortening of the Ti-N bond distance with irradiation, that correlates to the decrease in the lattice parameter observed by x-ray diffraction. No evidence of new Raman peaks due to transformed phases like TiO 2 anatase or rutile has been found. It is interesting to note that the silicon peak intensity increases strongly with irradiation. Since the substrate is buried under the TiNO film, whose thickness does not change with irradiation, we attribute the intensity enhancement to a decrease of the absorption coefficient for the irradiated film, which allows more laser light to reach the substrate and also more scattered light from the substrate to reach the detector. The Raman intensity from the substrate buried under a TiNO film of thickness d is

I = I 0 exp(-2 d)
, where I 0 is the laser intensity with no film and the linear absorption coefficient of the film. From the increase of the silicon Raman intensity and taking d = 180 nm, we estimate a decrease on = 43000 cm -1 at the laser wavelength (514.5 nm) for the E1F3 film with respect to the TiN film.

Electrical and optical properties

The modification of the film properties was initially probed by sheet resistance measurements on films deposited on high dielectric constant substrates (TiN/SiO2/Si(100)).

The results of these measurements are presented in figure 7. These resistivity measurements indicate that the process of O 2 + implantation is especially adapted for gradual modifications.

Both, variation in energy and fluence produce a scaled increase in the sheet resistance. The results further indicate that the lower energy is better adapted towards production of a deeper electrical modification. For this energy, the highest fluence induces in fact a resistivity value four times higher than the corresponding to the reference TiN (R s = 7.7 Ω/ , in agreement with the sheet resistance measured for TiN films of thicknesses higher than 50 nm) [33].

Films with increased contrasting properties (E1 energy implantation) were further characterized by spectroscopic ellipsometry (figure 8). The Psi and Delta curves show that a controlled and scalable modification is indeed obtained by the implantation process. The evolution of the Psi and Delta plots reveal an increasing depolarization with implantation fluence. This depolarizing effect is usually attributed either to an increasing surface roughness or a stronger substrate influence. Since no significant roughness change has been observed on implanted samples, it may be concluded that the increasing depolarization is due to a stronger substrate effect. This result further supports the idea of a decrease in light absorption (i.e. an increase in the transparency of the implanted film) as suggested by the Raman measurements.

In an analogue but reversed process, such light absorption decrease is in agreement with the increased absorption in the visible range observed in highly N doped TiN 2 films [34]. In fact, a clear interference effect can be detected in the low wavelength range for the films implanted at the higher fluence (see especially in this case the Delta curve).

Conclusions

We have described an oxygen implantation process that allows the shallow modification of TiN into TiN x O y . The depth and degree of modification can be tailored by accurately selecting the implantation energy and fluence of implantation. For a determined fluence, implanted oxygen is restricted to a narrower region in depth, giving rise to higher oxygen concentrations as demonstrated by RBS. However, the modification is not only an issue of formation of a ternary compound but also the product of intense structural damage in the implanted TiN.

Such modification induced partial amorphization of an outer layer and transferred strain to the interface with the underlying TiN. In particular, high resistivity and optical contrasts have

Acknowledgements.

We acknowledge funding provided by grants MAT2008-06858-C02-01, MAT2008-06858-C02-01 and CONSOLIDER-Ingenio FUNCOAT CSD2008-00023 from Ministerio de Ciencia e Innovación (Spain), Madrid Regional Grant "Microseres" and Fundación Domingo Martínez. The technical support from L. García Pelayo and the electrical measurement facilities from the Microelectronics Laboratory (UAM) are greatly appreciated.

  were Si(100) or thermal SiO 2 /Si(100) depending on the characterization technique envisaged. Final TiN film thicknesses was circa 180 and 90 nm for Si(100) and SiO 2 substrates, respectively. The implantation of O ions into the TiN films was simulated by using the "Stopping and Range of Ions in Matter" (SRIM) software code [19]. Simulation of atomic O implantations in spite of the molecular O 2 + helps in determining a maximum in the penetration depth. The process does not take into account the broad spectrum of distribution energies for the dissociated O atoms induced upon interaction of the molecular O 2 + beam with the TiN sample during the experiment. The results are presented considering a stoichiometric 1:1 TiN. After selection of the energy range (1-40 KeV), simulations were performed using the "Surface Sputtering" calculation for 10000 ions. Simulations were performed to estimate the implantation depth and to illustrate the distribution of atoms at the first stages of implantations. The TiN coatings were implanted with O 2 + in a DanFysik high current ion implanter. Irradiation at energies of 10, 20 and 40 KeV (samples labeled E1, E2 and E3

  selected on the basis of simulation of O ion implantations that allow predicting a maximum in the penetration depth of O. It is considered that molecular oxygen will dissociate upon interaction with the sample distributing the energy between the two dissociated atoms (not in identical parts). Distributions are only considered to illustrate the damage induced at the first stages of implantation (i.e. disregarding accumulated damage). The corresponding O ion and recoil ionization profiles generated by SRIM (mainly N recoils) (Figure1.a) illustrate that, at energies between 10 and 40 KeV, the incident ions easily generate recoils, which become active species in producing considerable damage by nuclear energy loss. In fact, at the range of depths in this study, the energy released by recoil ionization corresponds to a notable fraction of that released by incident O ions (circa 2/3, 1/2 and 1/3 for 10, 20 and 40 KeV implantation respectively). Note also that at 20 KeV a maximum is reached in the energy released per O ion, which justifies the selection of the energy ranges in this study. Furthermore, these ionization events can be induced up to depths of 100 nm, 60 nm or 30 nm for implantation energies of 40, 20 or 10 KeV, respectively. By looking at the final ion and recoil in depth distribution (figure1.b), one observes that a very intense relocation of recoiled atoms is induced (see two order of magnitude scale correction between ion and recoil axis) with a maximum and attenuation of distribution at depths related directly to those mentioned previously for ionization energy. On the other hand, O ions accumulate considerably deeper than recoils with concentration maxima at depths much deeper than those where ionization damage occurs (i.e. for 40 KeV, incident O ionization damage maximum takes place at circa 10 nm while final O distribution maximum appears at circa 50 nm). It can be also observed that the higher the implantation energy, the broader the final O ion distribution. These considerations are important in order to compare with experimental data obtained by the subsequent multi-technique analysis but should be improved by alternative simulations to describe with more precision the dynamic changes in the composition due to accumulated damage[START_REF] Moller | [END_REF]. 3.2 Oxygen integration. The in-depth composition and degree of O incorporation in the TiN/Si(100) films was probed by non Rutherford RBS (O resonance with 3.035 KeV He + beam) to enhance O sensitivity. Data were obtained for the reference TiN sample and the TiNO samples produced at different energies for a fixed intermediate fluence (Figure 2.a) and different fluencies for the lower energy (Figure 2.b). The fitting of the spectra corresponding to the control sample

  .a), results comply with the rule foreseen by simulation results: the lower the implantation energy, the lower the implantation depth but the higher the O concentration (note inset with magnified O peak intensity). An increase of fluence produces the expected increase in the O concentration. However, it should be mentioned that the distribution of O does not exactly reproduce the simulated distributions (not even in the low implantation regime). This is related to the fact that simulations are performed with single atom ions, while experimental implantations are performed with O 2 + , which drastically enlarges the distribution energy of O when reaching the surface giving rise to considerable O concentration on the surface. With regard to the aims of this work, increased surface contrasting properties are obtained for samples with lower energy implantation and higher fluence. It is important to remark that the surface composition of this particular sample presents the stoichiometry of a material closer to TiO 2 than to TiN (TiN 0.51 O 1.26 ). This stoichiometry indicates the presence of extra N and O with respect to what would be expected for derived Ti compounds. 3.3 Structural Modification. The XRD diagrams corresponding to TiN/Si(100) and O 2 + implanted TiN/Si(100) are presented in Figures 3.a and 3.b for implantations at increasing energy and increasing fluence, respectively. All the films present the characteristic peaks of TiN (JCPDS 38-1420) with a diffraction peak intensity distribution that was slightly modified as a function of the O 2 + implantation treatment. In the reference TiN sample, the peaks corresponding to (111) planes presented a remarkably higher intensity/width ratio than those corresponding to (200) planes (diffraction maximum reference). This indicates a texturization previously observed in TiN

  figure 5.b. A transition line to the gluing material of the preparation has been drawn to guide

  been determined by implantations at 10 keV and fluence of 5.10 16 cm -2 , which gave rise to TiN 0.51 O 1.26 compositions. The outer layer shows increased transparency with respect to the original TiN as illustrated by Raman spectroscopy and ellipsometric measurements. This suggests a neat increase of the dielectric function of the film, which is in agreement with the increase in sheet resistance up to 4 times the value of reference TiN. These results point out to the possibility of growing surface TiNO structures in TiN with potential optical and biomedical applications.

Figure 1 :

 1 Figure 1: Simulations of oxygen ion and recoil ionization profiles (a) and, oxygen ion and

Figure 2 :

 2 Figure 2: RBS at oxygen resonance (3.035 MeV He + beam) of unmodified sample (TiN/Si),

18 Figure 3 :

 183 Figure 3: XRD diagrams corresponding to TiN/Si reference and oxygen implanted TiN/Si

Figure 4 : 20 Figure 5 .

 4205 Figure 4: Effect of varying fluence (for the lower E1 energy) in TiN/Si lattice strain calculated

21 Figure 6 :

 216 Figure 6: Room temperature Raman spectra of the TiN/Si films before and after irradiation

Figure 7 :

 7 Figure 7: Four point probe measurements of film sheet resistance for TiN/SiO 2 /Si samples

Figure 8 :

 8 Figure 8: Spectroscopic Ellipsometry measurements of TiN/Si sample and samples irradiated


Kornelis ; Van Hateren
email: k.j.j.van.hateren@isala.nl

Kornelis J J Van Hateren

Gijs W D Landman

Nanne Kleefstra

Iefke Drion

Klaas H Groenier

Sebastiaan T Houweling

Henk J G Bilo

MD. Kornelis J J Van Hateren

Glycemic control and the risk of mortality in elderly type 2 diabetic patients (ZODIAC-20) Running title: Glycemic control and mortality in old age

Keywords: Diabetes mellitus type 2, Aged, Mortality, Hemoglobin A, Glycosylated, Longitudinal studies

Aims Studies on macrovascular consequences of glucose control in elderly patients (>75 years) with type 2 diabetes mellitus (T2DM) are lacking. The present study aimed to investigate the relationship between HbA 1c and mortality in this specific population.

Methods

Between 1998 and 1999, 374 primary care patients with T2DM aged older than 75 years participated in the ZODIAC study, a prospective observational study. Early 2009, data on mortality were collected. Updated means for annually measured HbA 1c values were calculated after a follow-up time of 10 years. Updated mean HbA 1c was used as a timedependent covariate in a Cox proportional hazard model. Main outcome measures were allcause and cardiovascular disease (CVD) mortality. Analyses were performed in strata according to diabetes duration (<5, 5-11 and ≥11 years).

Results

In the group with a diabetes duration <5 years, an increase of 1% in the updated mean HbA 1c level was associated with an increase in all-cause and CVD mortality risk of 51% (95%CI 17%-95%) and 72% (95%CI 19%-148%), respectively. Glycemic control was not related to mortality for patients with a diabetes duration ≥5 years.

Conclusions

Poor glycemic control is related to increased all-cause and CVD mortality in patients >75 years with T2DM of short duration (<5 years).

Discussion

Because of the observational study design, our results should be interpreted with caution. Nevertheless, they are suggestive that improving glycemic control may be beneficial in elderly patients with T2DM, especially in those with recently diagnosed T2DM.

Randomised controlled trials are necessary to investigate whether this holds true.

The beneficial effects of improved glycemic control decrease with longer diabetes duration and with increasing age. However, there are no clinical data on the macrovascular and microvascular consequences of (intensive) glucose control in the very old (patients >75 years).

What's new?

Higher levels of HbA 1c are related to increased all-cause and CVD mortality in diabetic patients aged older than 75 years, but only in those with diabetes of short duration (<5 years). Randomised controlled trials are necessary to investigate whether improving glycemic control in specific elderly diabetic populations, for example patients with newly diagnosed T2DM, may be beneficial.

Introduction

In a recent response to a meta-analysis of the Collaborators on Trials of Lowering Glucose (CONTROL) Group, the differences between patients with type 2 diabetes mellitus (T2DM) of short and long duration were emphasized [START_REF] Emanuele | Duration of diabetes, glucose control and cardiovascular risk[END_REF][START_REF] Turnbull | Intensive glucose control and macrovascular outcomes in type 2 diabetes[END_REF]. Based on the heterogeneous results of four large randomised controlled trials, it seems that intensive glucose control is only beneficial in those with diabetes of short duration [START_REF] Holman | 10-year follow-up of intensive glucose control in type 2 diabetes[END_REF][START_REF]The Action to Control Cardiovascular Risk in Diabetes Study Group. Effects of intensive glucose lowering in type 2 diabetes[END_REF][START_REF] The | Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes[END_REF][START_REF] Duckworth | Glucose control and vascular complications in veterans with type 2 diabetes[END_REF]. A meta-analysis, published in 2006, already showed that the beneficial effects of improved glycemic control decreased with longer diabetes duration and with increasing age [START_REF] Stettler | Glycemic control and macrovascular disease in types 1 and 2 diabetes mellitus: Meta-analysis of randomized trials[END_REF]. Unfortunately, there are no clinical data on the macrovascular and microvascular consequences of (intensive) glucose control in adults older than 75 years. Although guidelines recommend to apply less stringent targets to frail older adults and those with limited life expectancy, the level of evidence of this advice is low and mainly based on expert opinion [START_REF] Diabetes | Standard of medical care in diabetes -2010[END_REF]. We aimed to explore the relationship between HbA 1c and (cardiovascular) mortality, and the role of diabetes duration in this relationship, in a prospectively designed cohort of elderly patients (>75 years) with T2DM.

Patients and methods

Study population

This study is part of the ZODIAC (Zwolle Outpatient Diabetes project Integrating Available Care) study; the design and details of which have been presented elsewhere [START_REF] Ubink-Veltmaat | Shared care with task delegation to nurses for type 2 diabetes, a prospective observational study[END_REF]. In this study, general practitioners are assisted by hospital-based diabetes specialist nurses in their care of patients with T2DM. At baseline, patients with a very short life expectancy (including patients with active cancer) or insufficient cognitive abilities were excluded (~5%). Four patients were excluded because of insufficient baseline data. Nearly 90% (n=1357) of the remaining patients agreed to participate. For the present study, we selected all patients aged older than 75 years (n=374).

Data collection

Baseline data were collected in 1998 and 1999, and consisted of a full medical history including macrovascular complications, medication use, and tobacco consumption. Patients were considered to have macrovascular complications when they had a history of angina pectoris, myocardial infarction, percutaneous transluminal coronary angioplasty, coronary artery bypass grafting, stroke or transient ischaemic attack. Laboratory and physical assessment data, such as HbA 1c , lipid profile, serum creatinine, the urinary albumin-tocreatinine ratio, blood pressure, weight, and height were collected annually. An updated mean of annually measured HbA 1c was calculated for each individual from baseline to the end of the follow-up period by averaging the baseline values with the mean annual values.

For example, at one year the updated mean HbA 1c is the average of the baseline and one year values, and at three years it is the average of baseline, one year, two year and three year values. This technique is similar to the one used in the United Kingdom Prospective Diabetes Study (UKPDS) [START_REF] Adler | Association of systolic blood pressure with macrovascular and microvascular complications of type 2 diabetes (UKPDS 36): prospective observational study[END_REF]. We examined two clinical endpoints in this study: all-cause and cardiovascular disease (CVD) mortality. Early 2009, the vital status and cause of death were retrieved from records maintained by the hospital and the general practitioners. The causes of death were coded according to The International Classification of Diseases, 9 th revision (ICD-9).

Statistical analyses

Continuous variables are presented as mean (± standard deviation) for normally distributed values and as median (interquartile range) for non-normally distributed values. Normality was evaluated using Q-Q plots. Nominal variables are presented as the total number of patients (percentage). A Cox proportional hazard model was used to investigate the relationship between the updated mean HbA 1c , as a time dependent covariate, and mortality with and without adjustment for selected confounders. We used two different models. In model 1, only age and gender were taken into account as possible confounders. In model 2, we adjusted for the following variables: age, gender, smoking (yes or no), BMI, duration of diabetes, serum creatinine, macrovascular complications (yes or no), albuminuria (yes or no), systolic blood pressure, total cholesterol-HDL ratio, and use of insulin (yes or no). Analyses were repeated in strata according to diabetes duration. The diabetes duration variable at baseline was categorised into tertiles: <5 (n=111), 5-11 (n=139) and ≥ 11 years (n=124). In order to estimate the possible implications of higher HbA 1c levels on mortality, we calculated the population attributable risk percent (PAR%) of HbA 1c levels ≥7% for all-cause and cardiovascular mortality [START_REF] Natarajan | A simple method of determining confidence intervals for population attributable risk from complex surveys[END_REF]. In our analyses PAR% can be interpreted as the percentage by which mortality rates could be reduced if all patients would have had HbA 1c levels <7%. The

Results

Baseline characteristics of our study population are presented in table 1. Approximately one third of our study population was male. Median age (interquartile range) was 80 (78-83) years and median diabetes duration was 8 (4-13) years. Patients with a diabetes duration ≥11 years (tertile 3) had lower mean BMI and were more often smokers compared to patients with shorter diabetes duration. The number of patients treated with only a diet was the highest in the group with a diabetes duration <5 years (tertile 1); also, use of insulin was the lowest in this group compared to patients with longer diabetes duration. After a follow-up time of 10 years, 304 out of 374 patients (81%) had died, of whom 127 deaths (42%) were attributable to cardiovascular causes.

Analyses overall group

In multivariate analyses (model 2), an increase of 1% in HbA 1c led to an increase in CVD mortality risk by 26% (95% confidence interval (CI) 6-49%). The unadjusted hazard ratio, and the age-and gender adjusted one, were not relevantly different. The relationship with allcause mortality was not significant in both models.

Analyses stratified according to diabetes duration (table 2)

In the group with a diabetes duration <5 years (tertile 1), the level of HbA 1c as a continuous variable was positively related to both all-cause and CVD mortality. In multivariate analyses, an increase of 1% in HbA 1c was associated with an increase in all-cause and CVD mortality risk of 51% (95%CI 17%-95%) and 72% (95%CI 19%-148%), respectively. All results for patients with a diabetes duration ≥5 years were not significant. The PAR% of HbA 1c levels ≥7% for all-cause mortality in patients with diabetes of short duration was 23% (95%CI 2%-36%). For CVD mortality the PAR% was 39% (95%CI 17%-48%). Again, all results for patients with a diabetes duration ≥5 years were not significant.

Population attributable risk percent

All analyses were repeated with only the baseline HbA 1c value as variable of interest (data not shown). Results did not relevantly change. The proportional hazards assumptions were met for all analyses.

Discussion

Poor glycemic control is related to increased all-cause and CVD mortality in patients with T2DM aged over 75 years, but only in those with diabetes of short duration. In the lowest tertile (duration <5 years), the all-cause mortality risk was 51% higher for every 1% increase in HbA 1c . For CVD mortality, the increase in mortality risk was even 72%.

To our knowledge, the relationship between HbA 1c and mortality in elderly patients with T2DM has not been described before. In previous observational and intervention studies elderly patients were either not included or subanalyses were not performed for this specific population. More recently, a large retrospective observational study showed that there seems to be a U-shaped association between HbA 1c and mortality [12]. Although an estimated 16% of the study population was aged over 75 years, no subanalyses were performed.

It is important to emphasize that the associations found between HbA 1c and mortality in this study do not imply causality. Because of the observational nature of our study, we can only speculate about the underlying mechanisms. Firstly, poor glycemic control itself may indeed affect mortality risk in elderly patients with recently diagnosed diabetes. The heterogeneous results of four large randomised controlled trials in younger patients already suggested that intensive glucose control may only be beneficial with regard to mortality in those with diabetes of short duration [START_REF] Emanuele | Duration of diabetes, glucose control and cardiovascular risk[END_REF][START_REF]The Action to Control Cardiovascular Risk in Diabetes Study Group. Effects of intensive glucose lowering in type 2 diabetes[END_REF][START_REF] The | Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes[END_REF][START_REF] Duckworth | Glucose control and vascular complications in veterans with type 2 diabetes[END_REF]13]. Secondly, it may be possible that our results are influenced by confounders we did not adjust for. For example, the results for all-cause mortality may be confounded by co-morbidities such as cancer or infectious diseases. In order to reduce the impact of reverse causality we performed additional analyses for the overall group, in which we excluded the deaths in the first year of follow-up. This did not relevantly change the results.

Besides its observational design, there are other reasons why our results should be interpreted with caution. Firstly, our study cohort is rather small and only comprises 374 elderly patients with T2DM. Since we also stratified our cohort into tertiles, the number in these tertiles are even smaller. Secondly, the heterogeneous health status of elderly patients Deleted: Since it is not possible to draw conclusions about causality, results from observational data should always be interpreted with caution. Bearing this limitation in mind, our results do seem to correspond with the results of large randomised controlled trials, in which benefits of (intensive) glucose control were only observed in patients with diabetes of short duration [START_REF] Holman | 10-year follow-up of intensive glucose control in type 2 diabetes[END_REF][START_REF] The | Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes[END_REF]. In a recent retrospective observational study, low levels of HbA1c were related to increased all-cause mortality [12]. Unfortunately, no subanalyses for elderly patients or patients with diabetes of short duration were performed. Based on our results, one could hypothesize that the so-called legacy effect of intensive control as observed in the United Kingdom Prospective Diabetes Study (UKDPDS) may also be present in old age [START_REF] Turnbull | Intensive glucose control and macrovascular outcomes in type 2 diabetes[END_REF]. However, it is also Deleted: it may be possible that 11 makes it more difficult to identify the implications of our results for clinical practice. However, additional analyses revealed that for patients with a diabetes duration less than 5 years, the all-cause mortality rate could theoretically have been lowered by 23% if all patients had had HbA1c levels <7%. An important strength of our study is its prospective design. Other strengths of our study are the high number of deaths after 10 years follow-up, the use of the updated mean method and the number of variables we adjusted for in our model.

Although our study is the first study linking higher levels of HbA 1c to increased mortality in elderly patients with recently diagnosed diabetes, we do not recommend aiming for intensive glycemic control for all subjects in this specific patient category. Intensive control may also lead to an increased risk of hypoglycemia causing possible adverse events such as fall accidents and fractures. Physicians caring for older patients should take comorbidity, frailty and estimated life expectancy into account when setting treatment goals for individual patients. Confirmation of our results in other cohorts would be interesting, because if confirmed, randomised controlled trials are necessary to investigate whether improving glycemic control in specific elderly diabetic populations, for example patients with newly diagnosed T2DM, may be beneficial.

Tables

7 The

 7 assumption of proportional hazards was checked by inspecting the Schoenfeld residual plots for the baseline predictor variables. All analyses were performed with SPSS version 15.0.1 (SPSS inc., Chicago, Illinois, USA). ZODIAC study and the informed consent procedure were approved by the local medical ethics committee. Informed consent was obtained from all patients.

Formatted

  CJ, Peters JR, Tynan A et al. Survival as a function of HbA1c in people with type 2 diabetes: a retrospective study. Lancet 2010; 375: 481-9. 13. UK Prospective Diabetes Study (UKPDS) Group. Effect of intensive blood-glucose control with metformine on complications in overweight patients with type 2 diabetes (UKPDS 34). Lancet 1998; 352: 854-65.

Table 1

 1 Baseline characteristics. SD), medians (interquartile range) or n (%). One-way ANOVA, Chi square, or Kruskal-Wallis test was used where appropriate to test for differences between groups.

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						Formatted: English (U.S.)
		Overall		Diabetes duration		p-value
						Formatted: English (U.S.)
			Tertile 1	Tertile 2	Tertile 3
			(<5 years)	(5-11 years)	(≥11 years)
	Characteristic	n=374	n=111	n=139	n=124
	Age (years)	80 [78-83]	80 [78-83]	80 [77-82]	80 [78-84]	0.887
	Male sex	130 (34.8)	34 (30.6)	57 (41.0)	39 (31.5)	0.148
	Body mass index (kg/m 2 )	27.8 (4.4)	28.6 (4.4)	28.0 (4.3)	26.9 (4.2)	0.012
	Duration of T2DM (years)	8 [4-13]	2 [1-3]	7 [6-9]	16 [13-20]	-
	Systolic blood pressure (mm Hg)	155.7 (24.7)	153.1 (24.3)	156.8 (24.9)	156.7 (24.9)	0.416
	Current smoking	33 (8.8)	4 (3.7)	11 (8.0)	18 (14.8)	0.011
	HbA1c (%)	7.4 (1.2)	7.3 (1.3)	7.5 (1.1)	7.4 (1.2)	0.292
	Albuminuria present	206 (55.1)	53 (47.7)	84 (60.4)	69 (55.6)	0.133
	Cholesterol-HDL ratio	4.9 (1.6)	5.2 (1.7)	4.8 (1.6)	4.7 (1.5)	0.099
	Serum creatinine (µmol/L)	98 [86-115]	95 [82-111]	99 [87-123]	98 [87-111]	0.165
	Macrovascular complications present	162 (43.3)	45 (40.5)	62 (44.6)	55 (44.4)	0.780
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Table 2

 2 Analyses stratified according to diabetes duration. Hazard ratios and the 95% confidence intervals of HbA 1c , for all-cause and cardiovascular disease (CVD) mortality.

	Mortality	Model		Diabetes duration
			Tertile 1	Tertile 2	Tertile 3
			(<5 years)	(5-11 years)	(≥11 years)
			n=111	n=139	n=124
	All-cause	Unadjusted	1.24 (1.01-1.52) 1.01 (0.83-1.24) 0.99 (0.82-1.20)
		Model 1 a	1.27 (1.03-1.55) 1.04 (0.85-1.26) 1.03 (0.84-1.26)
		Model 2 b	1.51 (1.17-1.95) 1.04 (0.84-1.28) 1.05 (0.85-1.30)
	CVD	Unadjusted	1.35 (1.00-1.81) 1.17 (0.87-1.57) 1.19 (0.91-1.55)
		Model 1 a	1.37 (1.02-1.84) 1.19 (0.99-1.15) 1.28 (0.98-1.68)
		Model 2 b	1.72 (1.19-2.48) 1.18 (0.87-1.60) 1.16 (0.86-1.58)

a Adjusted for age and gender; b Adjusted for age, gender, smoking (yes or no), BMI, duration of diabetes, serum creatinine level, macrovascular complications (yes or no), albuminuria (yes or no), systolic blood pressure, total cholesterol-HDL ratio, and use of insulin (yes or no).

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