Timo Wille

Fredrik Ekström

Jong-Cheol Lee

Yuan-Ping Pang

Horst Thiermann

Franz Worek
email: franzworek@bundeswehr.org

Kinetic analysis of interactions between alkylene-linked bispyridiniumaldoximes and human acetylcholinesterases inhibited by various organophosphorus compounds

Keywords: Organophosphorus compounds, Acetylcholinesterase, Oxime, Reactivation, Kinetics, Structure-Activity Relationship

A c c e p t e d M a n u s c r i p t 2

Abstract

The therapeutic approach of organophosphorus compound (OP) intoxications is to reactivate the inhibited enzyme acetylcholinesterase (AChE). Numerous studies demonstrated a limited efficacy of standard oxime-based reactivators against different nerve agents such as tabun and cyclosarin. This emphasizes research for more effective oximes. In the present study, reactivation kinetics of tabun-, sarin-, cyclosarin-, VX-or paraoxon-ethyl-inhibited human AChE (hAChE) with a homologous series of bis-ortho-pyridiniumaldoximes, Ortho-4 -Ortho-9, was investigated with a robot-assisted setting, allowing determination of second-order reactivation rate constants as well as model calculations. The reactivation constants of Ortho-4 -Ortho-9 resulted in marked differences of affinity and reactivity depending on the OP structure and the linker length of the oximes. In general, the K D values decreased with increasing linker length.

Reactivity increased from Ortho-4 to Ortho-6 for PXE-and VX-inhibited hAChE and from Ortho-4 to Ortho-7 for GA-inhibited hAChE and decreased again with Ortho-8 and Ortho-9. In contrast, k r decreased with increasing linker length for sarin-and cyclosarin-inhibited hAChE. In view of the pronounced decrease of K D from Ortho-4 to Ortho-9, the k r2 values increased with all tested OP. Hence, the ratios of K I / K D and of K I / k r2 showed that in almost all cases the affinity of Ortho-N to the native hAChE was higher than to OP-inhibited enzyme. Model calculations indicated that Ortho-6 -Ortho-9 could be superior to obidoxime in reactivating tabun-inhibited hAChE. Finally, these data emphasize the need to develop oximes with a higher selective affinity towards OP-inhibited hAChE in order to minimize possible side effects. Organophosphorus compounds (OP) have been used as insecticides for pest control [START_REF] Eddleston | Pesticide poisoning in the developing worlda minimum pesticides list[END_REF] and as chemical weapons (tabun, sarin, VX) in military conflicts and in terrorist attacks [2;3]. The main mechanism of action of OP is a progressive inhibition of acetylcholinesterase (AChE) by phosphylation (denotes phosphorylation and phosphonylation) of its catalytic serine hydroxyl group leading to an inactive enzyme species [START_REF] Marrs | Organophosphate poisoning[END_REF]. The inability of inhibited AChE to hydrolyze the neurotransmitter acetylcholine results in an endogenous acetylcholine overflow followed by an over-stimulation of cholinergic receptors, a massive disturbance of numerous body functions, and finally in death by respiratory failure [START_REF] Grob | The manifestations and treatment of poisoning due to nerve gas and other organic phosphate anticholinesterase compounds[END_REF]. The causal therapeutic approach of OP intoxications is to use oximes as nucleophiles to remove the phosphyl moiety and to reactivate the enzyme. Numerous in vitro and in vivo studies demonstrated a limited efficacy of standard reactivators against different OP [START_REF] Eyer | Testing of antidotes for organophosphorus compounds: Experimental procedures and clinical reality[END_REF]. One particular challenge is the reactivation of tabun-inhibited AChE, which typically has a secondorder reactivation constant that is several orders of magnitude lower than those of other OP-inhibited AChE, e.g., sarin-inhibited AChE. This fact emphasizes the necessity to search for more effective oximes.

Recently, a homologous series of bis-ortho-pyridiniumaldoximes tethered with 4-9 methylene groups, designated as Ortho-4 -Ortho-9, has been reported as AChE reactivators with the objective of increasing the affinity to phosphorylated human AChE (hAChE), accelerating enzyme dephosphorylation, reducing the therapeutic dosage in order to minimize adverse effects and gaining the ability to counteract a broader range of OP [START_REF] Pang | Rational design of alkylene-linked bis-pyridiniumaldoximes as improved acetylcholinesterase reactivators[END_REF]. It was shown, that with an increasing A c c e p t e d M a n u s c r i p t 6

MATERIAL AND METHODS

Chemicals

Acetylthiocholine iodide (ATCh) and 5,5´-dithio-bis-2-nitrobenzoic acid (DTNB) were obtained from Sigma (Taufkirchen, Germany) and paraoxon-ethyl (PXE) was from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Sarin (GB), tabun (GA), cyclosarin (GF) and VX (>98% by GC-MS, 1H NMR and 31P NMR) were made available by the German Ministry of Defence. HI 6 (98%) was a gift from Dr. Clement (Defence Research Establishment Suffield, Ralston, Alberta, Canada). Ortho-4 -Ortho-9 were synthesized as described previously [START_REF] Pang | Rational design of alkylene-linked bis-pyridiniumaldoximes as improved acetylcholinesterase reactivators[END_REF]. All other chemicals were from Merck (Darmstadt, Germany).

Preparation of hemoglobin-free erythrocyte ghosts

Hemoglobin-free erythrocyte ghosts as a source of human erythrocyte AChE were prepared according to Dodge et al. [START_REF] Dodge | The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes[END_REF] with minor modifications [START_REF] Worek | Reactivation kinetics of acetylcholinesterase from different species inhibited by highly toxic organophosphates[END_REF]. The AChE activity was adjusted to a minimum of 9000 U/l by appropriate dilution with phosphate buffer (0.1 M, pH 7.4). Aliquots of the erythrocyte ghosts were stored at a temperature of -80°C. Prior to use, ghosts were homogenized on ice with a Sonoplus HD 2070 ultrasonic homogenator (Bandelin electronic, Berlin, Germany), three times for 5 s with 30 s intervals, to achieve a homogeneous matrix for the kinetic studies.

A c c e p t e d M a n u s c r i p t 7

Preparation of OP-inhibited hAChE

Ghosts were incubated with a small volume (1%, v/v) of appropriate OP concentrations (Fig. 1) for 30 min at 37°C to achieve an AChE inhibition by >95%. Thereafter, the treated samples were dialyzed (phosphate buffer, 0.1 M, pH 7.4) overnight at 4°C to remove residual inhibitor. Then, the absence of inhibitory activity was tested by incubation of treated and control ghosts (30 min, 37 °C).

Reactivation kinetics

Reactivation kinetics was conducted according to Worek et al. [START_REF] Worek | Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes[END_REF]. The liquid handling steps of the reactivation tests were done with a Tecan Freedom EVO, for details see [START_REF] Wille | Development of a high-throughput screening for nerve agent detoxifying materials using a fully-automated robot-assisted biological assay[END_REF]. Tests with this robot-assisted setting were done to allow high reproducibility and ensure minimal error probability. In brief, the time-and concentration-dependent reactivation of OP-inhibited hAChE by oximes was tested with Ortho-4 -Ortho-9 (1 -100 µM, 37°C; Fig. 1) in a 96-well using a discontinuous reactivation method. At specified time intervals (0, 1, 10, 20, 30 min) 10 µl aliquots were taken and transferred to a tempered measuring plate (24-well) pre-filled with 2400 µl TRIS-DTNB buffer (37°C) and 50 µl ATCh (0.65 mM final concentration). This resulted in a 160-fold dilution of AChE and oxime (final concentration of 0.00385 µM, 0.0385 µM and 0.385 µM for 1 µM, 10 µM and 100 µM oxime during incubation, respectively). Subsequently, the activity of the enzyme was recorded by a Safire 2 spectrophotometer (TECAN, Männedorf, Switzerland). The enzyme activities were referred to control hAChE activities in the presence of the respective oxime concentration resulting in identical oxime

t k v v v v ln obs t 0 i 0      (1)
with v 0 control activity, v i activity of inhibited hAChE in the absence of oxime, v t activity of reactivated hAChE at time t k r and K D were obtained by the nonlinear fit of the relationship of k obs versus oxime concentration (1, 10, 100 µM).

The second order rate constant k r2 describing the overall reactivating efficiency

was calculated by D r 2 r K k k  (2)

Secondary calculations

In addition, the ratio between the inhibition constant towards native hAChE, K I (data taken from [START_REF] Pang | Rational design of alkylene-linked bis-pyridiniumaldoximes as improved acetylcholinesterase reactivators[END_REF]), and the calculated K D and k r2 was formed. Hereby, the respective constants of Ortho-5 -Ortho-9 were referred to Ortho-4 constants (defined as 1) and the relative ratios between K I and K D or k r2 were calculated.

A c c e p t e d M a n u s c r i p t 9

Model calculations

The reactivation rate constants of Ortho-4 -Ortho-9 and obidoxime were used to calculate the time-dependent reactivations of GA-, GB-, VX-, and PXEinhibited hAChE (Eq. 3):

  t k 0 t obs e 1 A A      (3)
(with A t hAChE activity at time t, A 0 control hAChE activity, k obs first-order rate constant of reactivation).

The oxime concentration for the calculation was 50% of K i for Ortho-4 -Ortho-9

and 10 µM for obidoxime [12;13].

Data export and statistics

The raw data were transferred automatically via macros into Excel files and the

Reversible inhibition of hAChE by Ortho-N

It has been shown that reversible inhibition of native hAChE by Ortho-4 -Ortho-9 is related to the length of the linker, i.e., K I decreases from 32 μM (Ortho-4) to 0.3 μM (Ortho-9) [START_REF] Pang | Rational design of alkylene-linked bis-pyridiniumaldoximes as improved acetylcholinesterase reactivators[END_REF]. In this study, the reactivation of OP-inhibited hAChE by Ortho-N was determined with an oxime concentration of 1 -100 μM followed by extensive dilution for assaying hAChE activity. Accordingly, the final oxime concentrations during activity measurements were between 0.00385 and 0.385 μM. Under these conditions, Ortho-4 -Ortho-6 did not affect the control hAChE activity. On the other hand, Ortho-7 (at 0.385 μM) reduced the activity to 76%, and Ortho-8 reduced the activity to 89% and 44% at concentrations of 0.0385 μM and 0.385 μM, respectively. With Ortho-9 the hAChE activity was inhibited to 64% and 15% at assay concentrations of 0.0385 μM and 0.385 μM, respectively.

Reactivation kinetics of Ortho-N

The determination of reactivation rate constants of Ortho-4 -Ortho-9 with hAChE inhibited by a number of structurally different OP (Fig. 1) resulted in marked differences of affinity (Fig. 2) and reactivity (Fig. 3). The dissociation constant progressively decreased with increasing linker length. For example, the K D for VX-hAChE showed a 335-fold decrease when the linker length was increased from n = 4 to n = 9 and reached a nanomolar level with Ortho-9 (Fig. 2). Noteworthy, the differences in K D between different OP conjugates at any The second-order reactivation rate constant k r2 is the ratio of reactivity and affinity and is reflecting the reactivating efficiency of oximes. In view of the pronounced decrease of K D from Ortho-4 to Ortho-9 the k r2 values increased with all tested OP (Fig. 4). The range of k r2 values was between 0.093 mM -1 min - 1 (GA) and 75.6 mM -1 min -1 (VX) and GA-inhibited hAChE showed the largest difference among the Ortho-N series (418-fold).

Interaction of Ortho-N with native and OP-inhibited hAChE

Ortho-N exhibited a positive relationship between affinity towards native or OPinhibited hAChE and linker length. Hence, it was tempting to form the ratio of K I / K D and Fig. 5 shows that in almost all cases the ratio was > 1, i.e., the affinity of Ortho-N to native hAChE was higher than the one to OP-inhibited enzyme.

A c c e p t e d M a n u s c r i p t

12
This fact is also reflected by the ratio of K I / k r2 (Fig. 6). With the exception of GA-inhibited hAChE, the ratio of K I / k r2 is always >1 and increasing with the linker length. Generally, increase of inhibitory potency from Ortho-4 to Ortho-9 outweighs the increase in reactivating efficiency.

Model calculations

In view of the relationship shown in Figs. 5 and6, it was compelling to calculate the time-dependent reactivation of Ortho-N at concentrations determined by the respective K I . Concentrations used in these calculations were half of the K i for the respective Ortho-N and 10 μM for obidoxime. The data demonstrate that, under these conditions, the Ortho oximes would result in a slower reactivation of GB-, PXE-and VX-inhibited hAChE compared to obidoxime (10 μM; Fig. 7).

Ortho-6 -Ortho-9 would be superior to obidoxime in reactivating GA-inhibited hAChE but still would achieve only partial reactivation within a reasonable time frame.

A c c e p t e d M a n u s c r i p t 13 4. DISCUSSION

Reactivation kinetics of OP-inhibited hAChE by Ortho-N

The determination of the reactivation rate constants of Ortho-N revealed a relationship between linker length and affinity to hAChE inhibited by tabun (GA), sarin (GB), cyclosarin (GF), VX and paraoxon-ethyl (PXE; Fig. 2). These results are in agreement with previous findings on a continuous decrease of K D from Ortho-3 to Ortho-9 with echothiophate-inhibited hAChE [START_REF] Pang | Rational design of alkylene-linked bis-pyridiniumaldoximes as improved acetylcholinesterase reactivators[END_REF].

The comparison of K D of individual Ortho-N determined with hAChE inhibited by structurally different OP shows a rather small variation. For example, Ortho-4 -Ortho-8 the difference between the highest and lowest K D values ranges from 2.3 to 6.0 μM; only with Ortho-9, a higher variation was found due to an outstanding low K D with VX-inhibited hAChE (0.07 μM). The remarkable uniformity of the affinity of Ortho-N towards hAChE inhibited by different OP is in contrast to K D values determined with clinically used mono-(2-PAM) and bispyridinium oximes (obidoxime, HLö-7, HI-6, methoxime) [7;14]. For these clinically used oximes, huge differences in affinity were observed, e.g., a more than 30-or 100-fold difference in K D of obidoxime or 2-PAM between cyclosarin-and sarin-inhibited hAChE, respectively. The high and uniform affinity of Ortho-oximes indicates that the binding site for this series of reactivators is compatible, on the structural level, with GA-, GB-, PXE-and VXinhibited hAChE. This conclusion is in agreement with crystallographic studies of Ortho-7, which suggested that the interaction with the peripheral anionic site The reactivity of Ortho-N with OP-inhibited hAChE was rather low compared to other oximes (Fig. 3). The only exception was the reactivation of PXE-inhibited hAChE by Ortho-6 whose k r was higher than the k r values determined with methoxime, HLö 7, HI 6 and 2-PAM, although it did not reach the value of obidoxime (0.81 min -1 ) [START_REF] Worek | Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes[END_REF]. In contrast to the dissociation constant, the reactivity of Ortho-N was not related to the linker length. With PXE-, VX-and GA-inhibited hAChE, a bell-shaped curve of k r values was observed; while, with GB-and GF-inhibited hAChE, a gradual decrease in reactivity with increasing linker length was found. The overall reactivating efficiency of oximes is determined by the ratio of k r / K D [START_REF] Worek | Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes[END_REF]. Predominantly due to the decrease of K D the second-order reactivation constant k r2 increased with the linker length (Fig. 4). The difference between Ortho-9 and Ortho-4 was moderate with GB-and GF-inhibited hAChE but with GA-inhibited hAChE a more than 400-fold difference was observed. In comparison to obidoxime, 2-PAM, HLö 7, HI 6 and methoxime [START_REF] Eyer | Chemical Warfare Agents: Toxicology and Treatment[END_REF] the Ortho-N exhibited a comparable or even superior reactivating efficiency. In fact, Ortho-6 had a 10-fold and Ortho-9 a 90-fold higher k r2 than obidoxime which is considered to be one of the most effective reactivators of GA-inhibited hAChE [START_REF] Eyer | Chemical Warfare Agents: Toxicology and Treatment[END_REF]. These findings are in agreement with studies of Ortho-7, which showed a significantly higher reactivation activity than both obidoxime and HLö 7 on GA-inhibited AChE [14;16], and contradict the long standing paradigm that a para-substituted pyridiniumoxime is required for efficient reactivation of GA [START_REF] De Jong | The bispyridinium-dioxime HLö-7: A potent reactivator for acetylcholinesterase inhibited by the stereoisomers of tabun and soman[END_REF]. The underlying mechanism of the high

Relation between intrinsic inhibitory potency and reactivation efficiency

The Reversible inhibition of AChE by oximes is dependent on the oxime structure and is a determinant of adverse effects and toxicity [15;18;19].

Previously, the inhibitory potency of Ortho oximes was determined with hAChE [START_REF] Pang | Rational design of alkylene-linked bis-pyridiniumaldoximes as improved acetylcholinesterase reactivators[END_REF] and it was shown that the affinity increased more than 100-fold from Ortho-4

to Ortho-9 and the inhibition constant K I was only 0.3 μM with Ortho-9. These results are in line with results reported by Patocka and Bielavsky, showing a relationship between the chain length of dimeric para-oximes and the reversible inhibition [START_REF] Patocka | Affinity of bis-quarternary pyridinedialdoximes for the active centre of intact and isopropylmethylphosphonylated acetylcholinesterase[END_REF]. Hence, the Ortho-N series is characterized by a positive relationship between affinity towards native or OP-inhibited hAChE and the linker length. It is therefore desirable to determine whether the increase in affinity towards OP-inhibited hAChE may be outweighed by an increase in intrinsic inhibitory potency towards the native enzyme. By forming the ratio K I / K D , it became evident that, with few exceptions, the increase of affinity towards native hAChE was higher than the increase of affinity towards OP-inhibited A c c e p t e d M a n u s c r i p t 16 hAChE, i.e., a ratio > 1 (Fig. 5). In addition, the ratio of K I / k r2 demonstrates that, with GB-, GF-, VX-and PXE-inhibited hAChE, the linker length related improvement of reactivating efficiency is outweighed by increased intrinsic inhibition (Fig. 6). These findings underscore the importance of improving reactivation efficiency by increasing mobility of the reactive oxime moiety as reported by Ekström et al. [START_REF] Ekström | Structure of HI-6*sarin-acetylcholinesterase determined by xray crystallography and molecular dynamics simulation: Reactivator mechanism and design[END_REF].

Therapeutic use of Ortho oximes

As a consequence of the K I / K D ratio > 1, the therapeutic concentration of the Ortho-N in vivo will be substantially lower compared to the clinically used oximes (2-PAM, obidoxime, HI 6, methoxime). It has been reported that 1 μmol/kg Ortho-7 was well tolerated in rats while 3 μmol/kg killed four of six animals [22]. Due to the high in vivo toxicity of Ortho-7 this oxime was administered at a dose of 1 μmol/kg to echothiophate-poisoned rats resulting in less protection compared to 2-PAM (100 μmol/kg).

The impact of a potentially limited concentration of Ortho-N on the timedependent reactivation was calculated by using the determined reactivation rate constants, therapeutic concentration of obidoxime (10 μM) and concentration of Ortho-N at half of its K I value [START_REF] Pang | Rational design of alkylene-linked bis-pyridiniumaldoximes as improved acetylcholinesterase reactivators[END_REF]. These model calculations (Fig. 7) indicate that, due to the low concentration and subsequent low reactivity of Ortho-N, the reactivation would be markedly slower compared to obidoxime with GB-, VXand PXE-inhibited hAChE. In contrast, the high reactivating efficiency of Ortho-6 -Ortho-9 for GA-inhibited hAChE would result in a superior reactivation relative to obidoxime (10 μM). On the basis of these results, it is worthy of pursuing

Conclusions

The reactivation kinetics of Ortho-N with hAChE inhibited by different OP is characterized by an increasing affinity proportional to the linker length, a low to moderate reactivity, and a continuous increase of reactivating efficiency from Ortho-4 to Ortho-9. At any given linker length, the difference in K D between this collection of OP is comparatively small. The concomitant increase of the affinity of Ortho-4 to Ortho-9 towards native hAChE resulted in a disadvantageous ratio between intrinsic inhibition and reactivating efficiency with GB-, GF-VX-and PXE-inhibited hAChE. However, Ortho-6 -Ortho-9 showed an outstanding reactivating efficiency with GA-inhibited hAChE being up to two orders of magnitude higher compared to obidoxime. Due to the inhibitory potency towards native hAChE, the therapeutic concentration of the most effective Ortho-N will be limited and future in vivo studies are needed to assess the therapeutic value of these oximes in treating GA poisoning. Finally, these kinetic data emphasize the need to develop oximes with a more selective affinity towards OP-inhibited AChE and higher mobility for better reactivation efficiency, to minimize the adverse side effects caused by the reversible inhibition of AChE by oximes.

A

  c c e p t e d M a n u s c r i p t linker length of the oximes the affinity to echothiophate-inhibited AChE increased leading to a remarkable rise of the second-order reactivation rate constants. Now, it was tempting to analyze the reactivation kinetics of Ortho-4-Ortho-9 with tabun (GA)-, sarin (GB)-, cyclosarin (GF)-, VX-and paraoxon-ethyl (PXE)-inhibited hAChE.

  reactivation and control assay and are given as %reactivation. The observed first-order rate constant of reactivation k obs was calculated for each oxime concentration by linear regression analysis applying the equation 1. All curves fitted a R 2 ≥ 0.98:

  constants and percentage of hAChE inhibition were determined with Prism 5.0 (GraphPad Software, San Diego, CA). All data are means of n = 4 experiments.

  A c c e p t e d M a n u s c r i p t 11 given linker length are relatively small.The ability of oximes to remove the phosphyl residue from the active site of the enzyme, reflected by the reactivity rate constant k r , showed marked differences among the tested OP (Fig.3). Reactivity increased from Ortho-4 to Ortho-6 for PXE-and VX-inhibited hAChE and from Ortho-4 to Ortho-7 for GA-inhibited hAChE and decreased again with Ortho-8 and Ortho-9. In contrast, k r decreased with increasing linker length for GB-and GF-inhibited hAChE. The highest k r , 0.515 min -1 , was determined with Ortho-6 and PXE-inhibited hAChE and the largest difference in reactivity among the Ortho-N series was recorded with VX-inhibited hAChE (38.6-fold). Plotting the k r of VX and PXE related to the linker length resulted in similar formed bell-shaped curves, whereas reactivation per se was higher for PXE.

A c c e p t e d M a n u s c r i p t 14 in

 14 combination with a significant flexibility of the central linker are key factors in the ability of Ortho-7 to reactivate tabun-inhibited AChE[7;14].

A c c e p t e d M a n u s c r i p t 15 reactivating

 15 efficiency of Ortho oximes with GA-inhibited AChE may be related to a differential binding in the peripheral binding site and a favourable orientation in the active-site gorge of the enzyme[START_REF] Ekström | Crystal structures of acetylcholinesterase in complex with HI-6, Ortho-7 and obidoxime: Structural basis for differences in the ability to reactivate tabun conjugates[END_REF].

  therapeutic value of oximes in OP poisoning is determined by (a) the reactivating efficiency (k r2 ) with hAChE inhibited by a specific OP, (b) the pharmacokinetics, predicting the course of therapeutic oxime concentrations and thus determining the interval of oxime administration, (c) the intrinsic inhibitory activity of oximes towards native hAChE and (d) the tolerability.

A c c e p t e d M a n u s c r i p t 17 pharmacokinetic

 17 studies and reactivation studies using GA-poisoned animals to properly assess the therapeutic value of Ortho-N in treating GA poisoning.

  PI, Kern C, Hong F, Kollmeyer TM, Pang Y-P, Brimijoin S. Cholinesterase reactivation in vivo with a novel bis-oxime optimized by computer-aided design. J Pharmacol Exp Ther 2003;307:190-6.

A c c e p t e d M a n u s c r i p t 23 LEGENDSFigure 1 :

 231 Figure 1: Structures of OP and Ortho-N (N = 4 -9) used in this study.

Figure 2 :

 2 Figure 2: The dissociation constant K D (μM) for the oxime-induced reactivation

Figure 3 :

 3 Figure 3: The reactivation rate constant k r (min -1 ) for the oxime-induced

Figure 4 :

 4 Figure 4: The calculated second-order rate constant k r2 (mM -1 min -1 ) for the

Figure 5 :

 5 Figure 5: The ratio of K I / K D in dependence of the linker length of Ortho-N. K I

Figure 6 :

 6 Figure 6: The ratio of K I / k r2 in dependence of the linker length of Ortho oximes.

A c c e p t e d M a n u s c r i p t

Figure 7 :

 7 Figure 7: Calculation of the time-dependent reactivation of GB-, GA-, PXE-, and

A c c e p t e d M a n u s c r i p tFiguresFigure 1 :

 1 Figures

FigureA c c e p t e d M a n u s c r i p t 2 Figure 2 :A c c e p t e d M a n u s c r i p t 3 Figure 3 :A c c e p t e d M a n u s c r i p t 4 Figure 4 :A c c e p t e d M a n u s c r i p t 5 Figure 5 :

 22334455 Figure

Acknowledgments

Jong-Cheol Lee and Yuan-Ping Pang were supported by the US Defense Advanced Research Projects Agency (DAAD19-01-1-0322).

A c c e p t e d M a n u s c r i p t

Conflict of Interest Statement

The authors declare that there are no conflicts of interest


NEE WILLIAMSON Lisa M Mcdaid
email: l.mcdaid@sphsu.mrc.ac.uk

Graham J Hart

Increased HIV testing and reduced undiagnosed infection among gay men in Scotland, 2005-2008: support for the opt-out testing policy?

Keywords: HIV testing, undiagnosed infection, men who have sex with men, opt-out testing 2

Objective: To examine changes in HIV testing and undiagnosed infection among MSM in Scotland between 2005 and 2008. Methods: Self-complete questionnaires and Orasure™ oral fluid collection kits were distributed to men visiting the commercial gay scene in Glasgow and Edinburgh. Results: Questionnaires and oral fluid specimens were provided by 1350 men (51.6% response rate) in 2005 and 1277 (59.7% response rate) in 2008. 2572 men were eligible for inclusion in the analyses. Recent HIV testing increased from 33.2% in 2005 to 48.3% in 2008 (p<0.001). HIV prevalence was comparable in 2005 and 2008 (4.4% and 4.6%

respectively). Among HIV-positive men, there was a reduction in undiagnosed infection between 2005 and 2008 from 41.7% to 26.3% (p=0.08). Undiagnosed HIV did not differ between men who were and were not tested in the past year. In 2008, only 4 (26.7%) HIV-positive men tested in the past 6 months were undiagnosed, compared with 11 (42.3%) HIV-positive men who had not (p=0.03).

Conclusion:

There was a substantial increase in recent HIV testing between 2005 and 2008. Although there was a concurrent (non-significant) reduction in undiagnosed HIV, there was no difference in undiagnosed infection between men who had and had not tested recently. However, lower proportions of undiagnosed infection among the most recent HIV-positive testers suggest frequent testing could have a role in reducing undiagnosed HIV and should remain central to HIV prevention efforts.

INTRODUCTION

A resurgence in diagnosed HIV has been noted among men who have sex with men (MSM) in North America, Western Europe and Australia. [START_REF] Sullivan | Reemergence of the HIV epidemic among men who have sex with men in North America, Western Europe, and Australia, 1996-2005[END_REF] Evidence suggests increases in diagnoses among UK MSM are the result of increasing HIV testing, [START_REF] Dougan | Does the recent increase in HIV diagnoses among men who have sex with men in the UK reflect a rise in HIV incidence or increased uptake of HIV testing?[END_REF] and, since the introduction of opt-out testing (whereby all patients should be offered a test regardless of symptoms or risk factors), uptake in genitourinary medicine (GUM) clinics has increased markedly. [START_REF]HIV in the United Kingdom: 2009 Report[END_REF] In Scotland, opt-out testing was implemented as part of the Scottish Sexual Health Strategy, published in 2005. A concurrent increase in HIV testing was observed in our community-based surveys of MSM in Scotland. [START_REF] Williamson | HIV testing trends among gay men in Scotland, UK (1996-2005): implications for HIV testing policies and prevention[END_REF] MSM remain the group most at risk of acquiring HIV in the UK, and over one quarter of HIV-positive MSM is estimated to be undiagnosed. [START_REF]HIV in the United Kingdom: 2009 Report[END_REF] Although the increase in HIV testing in clinics has been accompanied by a fall in the proportions who leave clinics undiagnosed, [START_REF]HIV in the United Kingdom: 2009 Report[END_REF] reducing undiagnosed HIV remains a key policy aim, [START_REF]HIV in the United Kingdom: 2009 Report[END_REF][START_REF]HIV Action Plan in Scotland December 2009[END_REF] and further expansion of HIV testing in clinical and community settings is recommended. [START_REF]HIV in the United Kingdom: 2009 Report[END_REF] In Scotland, undiagnosed infection (measured by unlinked anonymous testing of Glasgow GUM clinic attendees) among MSM fell from 49% in 2004 to 8% in 2007 and, though recent figures show an increase to 41% in 2008, the previous decrease was attributed to the introduction of opt-out HIV testing. [START_REF] Health Protection | Unlinked anonymous HIV testing of genitourinary medicine clinic attendees in Glasgow: 2008[END_REF] Since 2005, we have included the collection of oral mucosal specimens to be tested anonymously for HIV antibodies in our community-based surveys, which allows us to examine HIV testing and its association with undiagnosed infection in this population. Here, we examine changes in between 4 2005 and 2008, the association between the HIV testing and undiagnosed infection, and discuss the implications for future HIV testing policy in the UK. months' or 'between 6 months and 1 year ago' (a total of 12 months). Oral fluid specimens were analysed at the West of Scotland Specialist Virology Centre (screened for anti-HIV using an enzyme immunoassay; positives re-screened, and repeat reactives confirmed using Western Blot). Data were analysed with SPSS 15.0. Logistic regression was used to estimate odds ratios and 95% confidence intervals (CI), adjusted for survey venue, age, qualifications and frequency of gay scene use, which differed between the surveys. Ethical approval was granted by University of Glasgow, Faculty of Medicine Ethics Committee. 1), as was prevalence of undiagnosed HIV in the sample as a whole (1.9% and 1.2% respectively; AOR=0.65, 95% CI 0.33-1.29). Among HIV-positive men, there was a (non-significant) reduction in undiagnosed infection from 41.7% to 26.3% (Table 1).

METHODS

RESULTS

Questionnaires

In multivariate analysis, controlling for the demographic differences between the surveys, the 2008 increases in ever and recent HIV testing remained significant (Table 2). Age and frequency of gay scene use were also significantly associated with ever and recent testing, as was survey venue with recent testing. In multivariate analysis, there was no significant change in HIV prevalence, or undiagnosed infection (among HIV-positive men), over time (Table 2). The odds of testing HIV-positive were higher among men surveyed in saunas (compared with bars) and lower among men with degree or postgraduate (compared with secondary) education. Compared with men aged <26 years, the odds of being HIV-positive were higher among men aged 26+ years, while the odds of being undiagnosed were lower.

In 2005, only 4 of the 25 men with undiagnosed HIV had never had an HIV test, while 11 reported recent (negative) testing; 3 perceived themselves to be HIV-positive. In 2008, only 2 of the 15 undiagnosed men had never tested and 7 reported testing in the past year; none perceived themselves to be positive. Prevalence of undiagnosed HIV did not differ between men who were and were not recent testers (1.8% and 1.4% respectively, p=0.4); among HIV-positive men, 18 (31.6%) who reported recent testing were undiagnosed, compared with 20 (40.8%) who did not (p=0.3). In 2008, 408 men (34.4%) reported having tested in the past 6 months. Only 4 (26.7%) HIV-positive men tested in the past 6 months were undiagnosed, compared with 11 (42.3%) HIV-positive men who had not tested in the past 6 months (p=0.03); 1.0% and 1.4% of the total sample respectively (p=0.5).

DISCUSSION

Between 2005 and 2008, there was a substantial increase in the proportion of men reporting ever and recent HIV testing in community-based surveys in Scotland (in continuation of a trend noted previously [START_REF] Williamson | HIV testing trends among gay men in Scotland, UK (1996-2005): implications for HIV testing policies and prevention[END_REF]). Collection of oral fluid specimens in 2005 and 2008 provides the unique opportunity to assess the association of this increase with undiagnosed infection in this population for the first time. There are some limitations to note when considering our results. This was a venue-based sample so caution should be taken when generalising to the wider population. The oral fluid specimen response rate was relatively low, and may not be representative. However, the overall survey response rate was higher (66% in 2005 and 71% in 2008), and there were few differences between the men who did and did not provide oral fluid specimens; [START_REF] Williamson | HIV prevalence and undiagnosed infection among a community sample of gay men in Scotland[END_REF][START_REF] Mcdaid | Circumcision among men who have sex with men in Scotland: Limited potential for HIV prevention[END_REF] suggesting that the men who provided these were representative of the larger venue-based sample. It should also be noted that the question used to derive recent HIV testing, and therefore the period defined as recent testing, was different in 2005 and 2008 (16 months in 2005 and 12 months in 2008). However, if this had affected the results then reported recent testing would almost certainly have been higher in 2005 than in 2008, which was not the case.

Finally, caution should be exercised in interpreting our findings on recency of HIV testing and undiagnosed infection, given the small numbers involved. This could also have limited power to detect a difference between years.

Although testing rates have traditionally been lower in the Scottish surveys than those observed in other UK cities, [START_REF] Williamson | HIV testing trends among gay men in Scotland, UK (1996-2005): implications for HIV testing policies and prevention[END_REF][START_REF] Williamson | Increases in HIV-related sexual risk behaviour among community samples of gay men in London and Glasgow: how do they compare?[END_REF] the rates are now more similar to those reported in comparable venue-based surveys in London, Manchester and Brighton. [START_REF] Dodds | A tale of three cities: persisting high HIV prevalence, risk behaviour and undiagnosed infection in community samples of men who have sex with men[END_REF] Almost half of the men surveyed in Glasgow and Edinburgh reported having tested in the past year. It is encouraging that recent HIV testing has continued to increase among MSM in Scotland; strong evidence of an impact beyond the clinical setting of the opt-out testing policy, now recommended throughout the UK. [START_REF]UK National Guidelines for HIV Testing[END_REF] However, although there was a concurrent, albeit non-significant, reduction in undiagnosed HIV, our results cannot address whether the two are causally linked and do not suggest the two are directly correlated, with no difference in undiagnosed infection between men who had and had not tested recently. This raises the question of what more can be done to reduce undiagnosed HIV in this population?

Estimates suggest one in five UK MSM newly diagnosed with HIV are diagnosed within six months of infection. [START_REF]HIV in the United Kingdom: 2009 Report[END_REF] Here, just under half of undiagnosed HIV-positive men reported having had a (negative) HIV test in the past year. With high viral loads immediately after seroconversion, [START_REF] Mindel | ABC of AIDS Natural history and management of early HIV infection[END_REF] the risk for onward HIV transmission is apparent.

Lower proportions of undiagnosed infection among the most recent HIV-positive testers (in the past 6 months) are consistent with even more frequent (than annual) testing having a role in reducing undiagnosed HIV. However, the decline in recent testing with age (noted here and in our previous analysis of trends between 1996 and 2005 [START_REF] Williamson | HIV testing trends among gay men in Scotland, UK (1996-2005): implications for HIV testing policies and prevention[END_REF]) suggests that men may not test regularly. This implies that the challenge now is to increase the frequency of HIV testing, and essentially "normalize" regular testing among gay men at risk of HIV. Current policy recommends annual testing, [START_REF]UK National Guidelines for HIV Testing[END_REF] but there is a case for testing the acceptability of greater frequencies of six, three or even one monthly intervals. This is no easy task, and as much attention must be given to why men would want (and therefore be willing) to increase the frequency of testing, [START_REF] Imrie | HIV testing in men who have sex with men: are we ready to take the next HIV testing test?[END_REF] as to achieving this.

There is growing interest and debate around the use of treatment for HIV transmission prevention. [START_REF] Montaner | The case for expanding access to highly active antiretroviral therapy to curb the growth of the HIV epidemic[END_REF][START_REF] Garnett | Treating our way out of the HIV pandemic: could we, would we, should we?[END_REF][START_REF] Cock | Can antiretroviral therapy eliminate HIV transmission?[END_REF][START_REF] Wagner | Should we try to eliminate HIV epidemics by using a 'Test and Treat' strategy?[END_REF] The premise for this is that if new HIV infections are predominantly transmitted from HIV-infected individuals not on treatment, treating all HIV-infected individuals regardless of CD4-cell counts could ultimately reduce HIV transmission at the community-level. [START_REF] Montaner | The case for expanding access to highly active antiretroviral therapy to curb the growth of the HIV epidemic[END_REF] For new infections to be promptly identified and treated, frequent, regular HIV testing among at-risk groups is central to this approach. Although the evidence is mainly limited to modeling or ecological and observational studies, [START_REF] Montaner | The case for expanding access to highly active antiretroviral therapy to curb the growth of the HIV epidemic[END_REF][START_REF] Granich | Universal voluntary HIV testing with immediate antiretroviral therapy as a strategy for elimination of HIV transmission: a mathematical model[END_REF][START_REF] Charlebois | Effect of expanded ART strategies on the MSM HIV epidemic in San Francisco[END_REF][START_REF] Das-Douglas | Decreases in community viral load are associated with a reduction in new HIV diagnoses in San Francisco[END_REF][START_REF] Dodd | Examining the promise of HIV elimination by 'test and treat' in hyperendemic settings[END_REF] a 'test and (immediate) treat' approach has been adopted in San Francisco. [START_REF] Russell | City endorses new policy for treatment of HIV[END_REF] The Strategic Timing of Antiretroviral Treatment (START) study (clinicaltrials.gov NCT00867048) will inform us whether earlier treatment initiation is of individual benefit.

If demonstrated to be the case, increasing the frequency of HIV testing will be vital.

There is also still some question as to whether treatment does reduce sexual transmission of HIV. [START_REF] Cohen | Universal voluntary HIV testing and immediate antiretroviral treatment (correspondence)[END_REF] Although there is evidence to suggest this could be the case within heterosexual partnerships, [START_REF] Attia | Sexual transmission of HIV according to viral load and antiretroviral therapy: systematic review and meta-analysis[END_REF] there is not currently the same for MSM. There are further concerns about the development of drug resistance, the interaction of other sexually transmitted infection, and the possible impact of increasing risk behaviours. [START_REF] Montaner | The case for expanding access to highly active antiretroviral therapy to curb the growth of the HIV epidemic[END_REF][START_REF] Garnett | Treating our way out of the HIV pandemic: could we, would we, should we?[END_REF][START_REF] Wagner | Should we try to eliminate HIV epidemics by using a 'Test and Treat' strategy?[END_REF] Studies have shown that most HIV-positive MSM reduce sexual risk behaviour after diagnosis, [START_REF] Gorbach | Transmission behaviours of recently HIV-infected men who have sex with men[END_REF][START_REF] Colfax | Sexual risk behaviours and implications for secondary HIV transmission during and after HIV seroconversion[END_REF][START_REF] Fox | Reductions in HIV transmission risk behaviour following diagnosis of primary HIV infection: a cohort of high-risk men who have sex with men[END_REF] however, a recent review reported prevalence of unprotected anal intercourse with a partner of unknown or discordant HIV status in the last 12 months was 26% among diagnosed HIV-positive MSM in the US. [START_REF] Crepaz | Prevalence of unprotected anal intercourse among HIV-diagnosed MSM in the United States: a meta-analysis[END_REF] We previously found similarly high rates of sexual risk among this group in the UK, [START_REF] Williamson | Sexual risk behaviour and knowledge of HIV status among community samples of gay men in the UK[END_REF] not all of which could be accounted for by risk reduction strategies, and there was some suggestion that it was maintaining safer sex behaviour that men find difficult (with higher risk among men diagnosed over a year earlier). Hence, behaviour change, particularly during the period immediately after seroconversion when HIV transmission could be more likely, [START_REF] Mindel | ABC of AIDS Natural history and management of early HIV infection[END_REF] remains a necessary goal of any HIV prevention strategy.

The UK policy change to opt-out HIV testing appears to have had a significant impact on the testing rates of MSM. However, with only one in five UK MSM newly diagnosed with HIV diagnosed within six months of infection, [START_REF]HIV in the United Kingdom: 2009 Report[END_REF] and high viral loads immediately after seroconversion, [START_REF] Mindel | ABC of AIDS Natural history and management of early HIV infection[END_REF] increasing earlier diagnosis is imperative. Further expansion of HIV testing in clinical and community settings has been recommended to further reduce undiagnosed HIV, [START_REF]HIV in the United Kingdom: 2009 Report[END_REF] but, beyond providing additional testing opportunities, there is little to suggest this would have any great impact on the frequency with which MSM test for HIV. It is the latter that remains the challenge to be met and may have the greatest chance of reducing the undiagnosed fraction and having a real impact on the HIV epidemic among MSM in the UK.

KEY MESSAGES

o Between 2005 and 2008, there was a substantial increase in the proportion of MSM reporting ever and recent HIV testing in community-based surveys in Scotland.

o Although there was a concurrent reduction in undiagnosed HIV, there was no difference in undiagnosed infection between men who had and had not tested recently.

o To further reduce undiagnosed HIV, the challenge now is to increase the frequency of HIV testing among gay men at risk of HIV. or 'between 6 months and 1 year ago'. † Among men with HIV-positive oral fluid specimens.

  The 2005 and 2008 MRC Gay Men's Surveys collected anonymous, self-complete questionnaires and (Orasure™) oral fluid specimens. Time and location sampling was used to recruit representative samples in commercial gay venues (11 bars/2 saunas in 2005; 12 bars/2 saunas in 2008) in Glasgow and Edinburgh, Scotland's two largest cities.[7] Questionnaires included demographics and HIV testing history. Recent HIV testing was categorised in 2005, as those tested in the year of, or immediately prior to, the survey (i.e. 2004 or 2005 -the 2005 survey was conducted in late April, giving a total of 16 months defined as recent testing), and in 2008, those indicating their last test was 'in the past 6

Diagnosed 3 *

 3 Recent HIV testing: for the 2005 survey, recent testers includes men reporting testing in the year of or immediately prior to the survey, i.e. 2005 or 2004; for the 2008 survey, recent testers includes men who indicated that their last test was 'in the past 6 months' or 'between 6 months and 1 year ago'.

  The proportion of men who had ever had an HIV test increased significantly from 60.8% in 2005 to 74.6% in 2008; recent testing increased from 34.0% to 48.3% (Table1). HIV prevalence was comparable in 2005 and 2008 (4.4% and 4.6% respectively, Table

	and oral fluid specimens were provided by 1350 men (51.6% response
	rate) in 2005 and 1277 (59.7% response rate) in 2008. 55 men are excluded from these
	analyses (2005: 1 specimen with insufficient volume, 5 unconfirmed cases; 2008: 45
	heterosexual men who reported no sexual contact with men in the previous 12 months, 4
	laboratory specimens not returned).

Table 2 : Unadjusted and multivariate logistic regression for recent HIV testing, HIV prevalence and undiagnosed infection among gay men in Scotland Ever had HIV test

 2 Recent HIV testing: for the 2005 survey, recent testers includes men reporting testing in the year of or immediately prior to the survey, i.e. 2005 or 2004; for the 2008 survey, recent testers includes men who indicated that their last test was 'in the past 6 months'

			Recent HIV test*	HIV-positive oral fluid	Undiagnosed HIV-
		(N=2512)	(N=2440)	specimen result	positive †
				(N=2572)	(N=117)
		OR 95% CI P	OR 95% CI P	OR 95% CI P	OR 95% CI P
		value	value	value	value
	Unadjusted
	Year
	2005	1	1	1	1
	2008	1.89 1.59-2.24 <0.001 1.81 1.54-2.14 <0.001 1.04 0.72-1.51 0.83	0.50 0.23-1.09 0.08

*

ACKNOWLEDGEMENTS

We thank the survey staff and fieldworkers in each city, the venue managers, their staff, and the men who agreed to participate in the survey. The UK Medical Research Council funds Lisa McDaid and the Gay Men's Survey as part of the Sexual and Reproductive Health Programme (WBS U.1300.00.005) at the Social and Public Health Sciences Unit.

COMPETING INTERESTS

None.

AUTHOR CONTRIBUTIONS


Multiple aims in the development of a major reform of the national curriculum for science in England

Keywords: curriculum, policy development, scientific literacy, secondary school, science education

In the context of a major reform of the school science curriculum for 14-16 year olds in England we examine the aims ascribed to the reform, the stakeholders involved and the roles of differing values and authority in its development. This reform includes an emphasis on socioscientific issues and the nature of science; curriculum trends of international relevance. Our analysis identifies largely 'instrumental' aims, with little emphasis on 'intrinsic' aims and associated values. We identify five broad categories of stakeholders focusing on different aims with, for example, a social, individual, political or economic emphasis. We suggest that curriculum development projects reflecting largely social and individual aims were appropriated by other stakeholders to serve political and economic aims. We argue that a curriculum reform body representing all stakeholder interests is needed to ensure that multiple aims are considered throughout the curriculum reform process. Within such a body the differentiated character of the science teaching community would need to be represented.

Introduction

Previous studies have identified the school science curriculum as 'contested terrain' [START_REF] Fensham | The link between policy and practice in science education: The role of research[END_REF] with many distinct aims ascribed to it [START_REF] Aikenhead | Science Education for Everyday Life[END_REF]Black & Atkin, 1996;[START_REF] Reiss | What should be the aim(s) of school science education?[END_REF]. For example, supporting the development of 'scientific literacy' for all students is an increasingly prominent aim for school science education [START_REF] Deboer | Scientific literacy: another look at its historical and contemporary meanings and its relationship to science[END_REF][START_REF] Laugksch | Scientific literacy: a conceptual overview[END_REF][START_REF] Roberts | Scientific literacy/science literacy[END_REF]. However, school science education is also the starting point for further science study and potentially a career in science, technology or engineering. In contrast to scientific literacy this is an aim relevant to a minority of students in schools. Such multiple aims can create tensions [START_REF] Roberts | What counts as science education?[END_REF]. For example, should the school science curriculum emphasise the traditional academic content of the separate physical, chemical and biological sciences (providing a solid foundation for further science study) or focus more on how science features alongside ethical, social and political issues within contexts such as air pollution or global warming (principally serving the aims of scientific literacy)? This paper considers how such multiple aims, and associated tensions, featured in the development of a major reform of the school science curriculum for 14-16 year olds in England.

Despite concerns over the failures of many previous science curriculum reforms [START_REF] Blades | Procedures of power and curriculum change: Foucault and the quest for possibilities in science education[END_REF][START_REF] Eijkelhof | ANW: A new course on public understanding of science for senior general secondary education in the Netherlands[END_REF] there has been relatively little research into the development of curriculum policy and its interaction with practice [START_REF] Aikenhead | Science Education for Everyday Life[END_REF][START_REF] Fensham | The link between policy and practice in science education: The role of research[END_REF]. Drawing upon Kogan's view of policy as the authoritative allocation of values [START_REF] Kogan | Educational policy-making: A study of interest groups and parliament[END_REF] [START_REF] Fensham | The link between policy and practice in science education: The role of research[END_REF] identifies two sets of important, but neglected, research questions focusing on the role of values and authority respectively in education policy (see also Corrigan, Dillon, & Gunstone, 2007). This paper contributes to this research agenda through an analysis of the development of a major reform of the school science curriculum in England. We consider the role of values by examining the aims ascribed to this curriculum reform and the stakeholders promoting these aims. We address the role of authority by identifying those stakeholders who were successful in shaping the reform, and also those stakeholders who were more peripheral. Significantly, we identify a key moment in the development of the reforms when the authority of one group of stakeholders was particularly decisive. Finally, we consider the implications of this analysis of curriculum reform within England for curriculum development initiatives internationally.

Several features of our chosen curriculum reform make it an appropriate context in which to explore these issues. The reform provides a range of science courses aimed at enabling teachers to match the perceived needs of their students (QCA, 2005b, p. 9). This includes an enhanced presence for courses focusing on science within employment settings ('applied science' courses). The reform also emphasises the teaching of socioscientific issues and the nature of science. This is a curriculum emphasis reflected in current science curriculum reform initiatives in many countries (Black & Atkin, 1996;[START_REF] Van Den Akker | The science curriculum: between ideals and outcomes[END_REF], for example those emphasising science inquiry [START_REF] Rudolph | Inquiry, instrumentalism, and the public understanding of science[END_REF] and science-society-technology (STS) teaching [START_REF] Solomon | Teaching Science, Technology and Society (STS)[END_REF]. Thus, multiple aims feature strongly across these different courses.

Furthermore, it is a statutory reform: all publically funded schools in England are required to respond to it. Thus, the reform impacts on virtually all stakeholders in the

A conceptual framework for examining curriculum policy development

The focus of this paper can be expressed in terms of a policy process 'cycle' [START_REF] Bowe | Reforming Education and Changing Schools[END_REF]. The representation shown in Figure 1 moves away from a linear view of policy generation followed by policy implementation, reflecting trends in the analysis of the policy-practice relationship in a range of policy contexts [START_REF] Elmore | Curriculum Policy[END_REF][START_REF] Hill | Implementing Public Policy. An Introduction to the Study of Operational Governance[END_REF].

[Insert Figure 1 about here] Figure 1 emphasises the complexity of the policy process, the different contexts in which policy is developed, and the ways in which these contexts interact over time.

Crucially, in this view policy can only achieve meaning through practice. The use of the term 'policy cycle' reflects this dynamic character of the policy process. Three interacting policy contexts are identified. Policy is initiated and constructed within the 'context of influence'. This includes both private arenas of influence (e.g. social networks in and around government) and public arenas of influence (e.g. curriculum committees). Secondly, in the 'context of policy text production' policy is 'represented' through policy texts: statutory policy statements, official commentaries and speeches. The term 'represented' is important here: the language of these policy texts tends to be 'articulated in the language of general public good' (Bowe et al., 1992, p. 20) whilst representing hidden values and interests generated within contexts of influence. Reflecting the interactive, cyclical nature of the policy process such and debate within the 'contexts of influence'. The third policy context is the 'context of practice'. It is here, in those places to which the policy is addressed, that a policy is interpreted and then 'recreated'. This process involves continuous interactions with policy texts, and perhaps more likely, official and unofficial 'commentaries', i.e. interpretative texts and media reports which attempt to 'make sense of' these policy texts.

Our analysis focuses largely on the 'context of policy text production'. We examine official government curriculum documents and published reactions and commentaries from a range of non-governmental stakeholders. Such texts provide the 'public face' of curriculum policy through which policy aims are communicated to stakeholders.

Whilst we do not claim to have examined all relevant documents, we did review all documents in the public domain that, in our judgement, had a significant impact on the formation of these reforms. In addition we provide some insights into the 'context of influence' through consideration of the activities of government-initiated curriculum projects and curriculum development projects funded by charitable organisations. For example, we are interested in who was involved in such activities. However, we did not attempt systematically to examine sources such as minutes of government meetings or, through formal interview, the reflections of those involved.

Our experience has been that such sources are difficult to access and interpret.

Finally, the context of practice features in our analysis through consideration of the outcomes and influence of several evaluation studies of piloted curriculum initiatives. We examine three sequences of activities that played a major role in shaping the reform: the Beyond 2000 seminar series; a government-funded curriculum development project; and the design and evaluation of the Twenty First Century Science courses. We also refer to broader policy initiatives in England with implications for the school science curriculum. Table 1 gives an overview of the activities referred to.

The development of the 2006 reform

[Insert Table 1 about here] Beyond 2000 seminar series From January 1997 to April 1998 a series of six seminars led by university-based science education researchers was held in the UK. The aim of the Beyond 2000 seminar series was to 'consider and review the form of science education required to prepare young people for life in our society in the next century' (Millar & Osborne, 1998, p. 1). Three distinct aims for school science education appeared in the early part of the Beyond 2000 report: enhancing student interest by promoting a sense of wonder and curiosity about the achievements of science; supporting the development of scientific literacy; and preparation for more advanced science study. In the report scientific literacy was characterised as follows:

School science education should aim to produce a populace who are comfortable, competent and confident with scientific and technical matters and artefacts. The science curriculum should provide sufficient scientific knowledge and understanding to enable students to read simple newspaper articles about science, and to follow TV programmes on new to express an opinion on important social and ethical issues with which they will increasingly be confronted' (Millar & Osborne, 1998, p. 9).

Of the three aims identified above that of achieving scientific literacy was the most prominent in the main body of the report. The report recommended the development of a core science course for all students focusing on scientific literacy with a flexible suite of additional courses to match the needs of particular students. The report also outlined a set of 'ideas-about-science' to be taught alongside more traditional science content. These 'ideas-about-science' included aspects of the nature of science and socioscientific issues; these themes would reappear in the 2006 reform.

The Beyond 2000 project was funded by the Nuffield Foundation a charitable organisation with a long history of supporting influential curriculum development in the sciences (The Nuffield Foundation). The majority of those involved in the seminars were university academics with a professional interest in school science education (Millar & Osborne, 1998, p. 32). The principal motivation for the project, as expressed by the authors in the opening paragraph of the report, was to address their growing concern about the lack of relevance of the current science curriculum to the needs and interests of all young people. Whilst professional scientists had some representation at the seminars their role appears not be have been a prominent one. an example of a non-governmental, non-legitimised interest group [START_REF] Kogan | Educational policy-making: A study of interest groups and parliament[END_REF] exerting authority in the development of national curriculum reform, illustrating the multiple stakeholders working within the 'context of influence' represented in Figure 1.

Qualifications and Curriculum Authority curriculum project

In 2000 the Qualifications and Curriculum Authority (QCA) initiated a curriculum development project 'Keeping School Science in Step with the Changing World of the 21 st Century'. The QCA was the government body responsible for the National Curriculum in England at that time. The project was the government's response to the Beyond 2000 report [START_REF] Qca | Written evidence from QCA to the House of Lords Science and Technology Select Committee 'Science Teaching in Schools Report[END_REF]. Three separate studies were commissioned. Study 1 developed a definition of scientific literacy that might underpin the school science curriculum for 14-16 year olds. School science teachers and a broad range of additional stakeholders were then asked for their responses to this definition [START_REF] Leach | Teachers' views on the future of the secondary science curriculum[END_REF]. Study 2 evaluated methods for assessing student understanding of the nature of science and socioscientific issues [START_REF] Osborne | Developing effective methods of assessing ideas and evidence[END_REF]. Study 3 developed curriculum models for science that might address the aims of scientific literacy [START_REF] Millar | Twenty First Century Science: Insights from the design and implementation of a scientific literacy approach in school science[END_REF].

Stakeholders within the QCA stated that this curriculum development project aimed to: address poor student motivation for science education; support students' future engagement with science issues outside of school; and provide students with a foundation for further study in the sciences (Hollins, 2001, p. 22). The inclusion of Study 2, which examined the assessment of the nature of science and socioscientific issues, reflects recognition within the QCA that assessment in schools is a significant There was a striking level of continuity of stakeholder involvement in the Beyond 2000 and QCA curriculum development projects. The key authors of the QCA reports were university-based science education researchers many of whom had also contributed to the Beyond 2000 seminar series. However, studies also involved additional stakeholders, particularly school science teachers. In engaging with teacher stakeholders Study 1 in particular identified many 'critical voices' in relation to the meaning and feasibility of the goals of scientific literacy. For example, there was little agreement amongst those consulted about the content of any future curriculum that might support students as 'consumers and users' of science. Study 1 also found that 'there was some doubt as to whether pupils could be prepared to engage with expert science through the science curriculum' and no consensus on the impact of such teaching on student motivation (Leach, 2002, p. 49). However, as shown below these concerns appear to have been given limited attention within subsequent developments. Science Project Team, 2003). The Twenty First Century Science (21CS) project set out to address two aims for science education: to develop the scientific literacy of all students to support their engagement with science-related issues in later life, and to provide the foundations for more advanced courses in science [START_REF] Millar | Twenty First Century Science: Insights from the design and implementation of a scientific literacy approach in school science[END_REF]. The project aimed to achieve this through a flexible suite of courses for 14-16 year olds to be taken in the last two years of compulsory schooling (OCR, 2009; University of York/Nuffield Foundation, 2009). The 21CS suite would be available as an option for schools. All students following 21CS would complete a 'core' 21CS course. This course provided 'a broad, qualitative grasp of the major science explanations' [START_REF] Millar | Twenty First Century Science: Insights from the design and implementation of a scientific literacy approach in school science[END_REF](Millar, , p. 1507) ) and also included insights into the nature of science and its relation to [START_REF] Burden | Evaluation of the Twenty First Century Science Pilot[END_REF]. However, final reports from the second set of evaluations were completed in 2006; too late to influence the formation of the 2006 reform.

Twenty First Century Science

Introduction of the science curriculum reform at national level

In February 2004 the government published the revised 'programme of study' for science: the statutory science curriculum content to be followed within all publically funded schools in England from September 2006 (DfES/QCA, 2004). Featuring The purpose of the changes is to increase the flexibility of qualifications [for 14-16 year olds] to provide for the wide range of student interests and aptitudes so that more students will be encouraged to study more science (DfES/QCA, 2004).

The focus of this statement of aims was on encouraging greater post-compulsory participation in science education; there is no explicit reference here to achieving the aims of scientific literacy. This was in contrast to the focus on scientific literacy within the Beyond 2000 seminar series, the reports of the QCA curriculum projects and the 21CS project. The programme of study included a later section entitled 'the importance of science'. Here, additional aims for school science education were suggested: '[science] does provide us with the most robust information about the way the universe works that has so far become available to us'; '[science] trains the mind in a way that industry prizes'; 'science stimulates and excites pupils' curiosity'; '[pupils] learn to question and discuss science-based issues that may affect their own lives' (DfES/QCA, 2004, pp. 14-15). However, whilst the programme of study explicitly recognised a range of aims for science education, the aim of enhancing postcompulsory participation in science education appeared most prominently. Skills, 2004). The SIIF identified low numbers of scientists and engineers as a major cause for concern. Promoting increased student enjoyment of school science was identified as important in order to enhance continued participation in science education. This reflects an increasing articulation of education policy as economic policy within the 'knowledge economy' [START_REF] Ball | The Education Debate[END_REF].

Another policy strand in England at this time was the development of a variety of progression routes through 14-19 education broadly, including vocational routes, in order to provide a curriculum that is motivating for all students [START_REF] Tomlinson | 14-19 Curriculum and Qualifications Reform: Final Report of the Working Group on 14-19 Reform[END_REF].

Enhancing educational opportunity was identified in a government policy paper as 'vital for social justice -giving us the chance to break forever the historic link between social background, educational achievement and life chances that have dogged us as a nation' (DfES, 2005, p. 3). However, in the context of the science curriculum for 14-16 year olds, the emphasis returned to the need to improve science attainment and increase post-compulsory participation in science courses in order to sustain the supply of scientists and engineers (DfES, 2005, p. 39).

The revised statutory curriculum framework, whilst different from that of the 21CS pilot, shared several characteristics with it (e.g. a 'core' science course for all students focusing on scientific literacy with the flexibility of additional options; an emphasis on aspects of the nature of science and socioscientific issues). For many stakeholders the decision to adopt this curriculum framework at a national level ahead of the & Technology Select Committee, 2006, p. 22). For example, the final chapter of the Beyond 2000 report recommended that curriculum innovations should be piloted and evaluated in a representative range of schools and the outcomes used to inform subsequent changes at the national level (Millar & Osborne, 1998, p. 30). The motivations of the government officials involved in the decision to implement the revised statutory curriculum framework were not recorded, at least within the publically accessible documents examined here. It is possible that they felt that a science course (21CS) whose initial evaluation indicated enhanced interest amongst teachers and students might result in greater participation in post-compulsory science courses. The emphasis on scientific literacy also corresponded with the development by QCA of a whole school curriculum policy that emphasised citizenship and public engagement across the school curriculum [START_REF] Qca | The new secondary curriculum: What has changed and why[END_REF]. Irrespective of their motivations stakeholders within government made this critical decision from a position of authority over stakeholders such as those involved in the Beyond 2000 and 21CS development activities and science teachers working in schools.

Discussion

Table 2 summarises the multiple aims identified in the case study presented above.

[Insert Table 2 about here]

The first column summarises the key changes associated with the 2006 reforms. The next two columns distinguish between 'immediate aims' within compulsory 14-16 science education and 'longer term aims' related to post-compulsory education and 2 to identify the links different stakeholders make between specific changes to the curriculum and immediate and/or future aims. Any such links can be considered as 'routes' through Table 2. These 'routes' are also indicative of the values of different stakeholders [START_REF] Kogan | Educational policy-making: A study of interest groups and parliament[END_REF]. For example, the Beyond 2000 report advocated teaching about the nature of science and socioscientific issues in order to increase student motivation and interest in their science education with the longer term goal of supporting them in engaging effectively with science-related issues as citizens. Such links form a 'route' across the top row of Table 2 and emphasise values associated with self-determination and equity. There was little emphasis within Beyond 2000 on supporting post-compulsory participation in science education. By contrast, many government documents (e.g. the Science and Innovation Investment Framework) emphasised the need to improve student interest and attainment in science in order to increase the pool of students participating in post-compulsory science education thereby ensuring an adequate supply of future scientists and engineers. The emphasis here is on values of progress and institutional maintenance.

Other government documents refered to increased flexibility of provision leading to improvements in student attainment and post-compulsory participation (DfES, 2005). These developments were seen as serving the aims of enhancing general employability within a highly developed science/technology workforce and promoting values of social mobility and inclusion. Such links form 'routes' through the middle and lower sections of Table 2.

Overall, the activities examined in this paper tended to take an instrumental view of the aims of science education. For example, they focused on specific future functions that science education might serve (e.g. increasing post-compulsory participation in engaging with science issues in the popular news media). The activities tended not to reflect a more liberal or intrinsic view of education: introducing students to the power and wonder of the science worldview with educational aims 'grounded in intellectual and personal outcomes for pupils' (Donnelly, 2005, p. 294). These educational aims reflect the nature of the subject itself; they are intrinsic to the subject and independent of the uses to which any learning might be put. These purely educational values did not have a strong presence within the documents examined here.

The range of distinctive aims associated with the 2006 reform gives the potential for significant tensions. For example, the 2006 reform provides schools with a wider choice of science courses than was available previously. Stakeholders within the QCA have suggested that this flexibility should enhance student motivation for science education by enabling them to follow a science course that matches their needs and interests [START_REF] Qca | Science. Changes to the Curriculum from 2006 for Key Stage 4[END_REF]. However, in practice these course 'choices' follow largely from student attainment. For example, science courses focusing on science within employment settings ('applied science' courses) tend to be the preserve of mid-tolower attaining students, at least within England [START_REF] Bell | Positioning applied science in schools: uncertainty, opportunity and risk in curriculum reform[END_REF]. Such stratification of students by attainment within compulsory science education is likely to work against the achievement of broader social mobility and inclusion in later life [START_REF] Ball | The Education Debate[END_REF][START_REF] Gorard | The impact of socio-economic status on participation and attainment in science[END_REF]; an explicit aim within other government documents associated with the 2006 reform (DfES, 2005). Our analysis provides no evidence that such a critical tension was recognised or considered by the stakeholders involved. [START_REF] Fensham | Familiar but different: Some dilemmas and new directions in science education[END_REF][START_REF] Fensham | The link between policy and practice in science education: The role of research[END_REF]. These demands reflect many of the values identified earlier.

Curriculum demands and associated stakeholders

[Insert Figure 2 about here]

Here we use the representation in Figure 2 to consider the different roles of five distinct categories of stakeholder who feature in our analysis: university-based science education researchers; professional scientists; representatives of charitable organisations (e.g. the Nuffield Foundation); school teachers; and government policy makers. We also examine different positions of authority across these stakeholders, and how such authority was manifested. In Fensham's view political, economic and subject maintenance demands usually carry the most weight in determining science curriculum and assessment, with individual, social and cultural factors 'often given prominence in the preambles to a curriculum as some sort of consolation prize' (Fensham, 2009, p. 5). Certainly the economic demand to increase the supply of scientists had a strong presence in many of the documents examined here. Political demands were also prominent, e.g. enhancing student attainment, as measured by national or international assessments, and improving social inclusion and gender equity. In the context of the reforms in England the principal stakeholders associated with these demands were government policy makers.

Other demands also had a strong presence. For example, publications associated with the Beyond 2000 and 21CS projects addressed both social demands (e.g. enhancing In the context of the 2006 curriculum reform in England, the demands of 'subject maintenance' are less visible. Traditionally the stakeholders highlighting the need to maintain the profile and identity of the separate science subjects within the school curriculum have been professional scientists, acting as 'guardians of the disciplines' (Gaskell, 2003, p. 140). However, professional scientists did not feature as central players in the activities described above. This reflects what has been identified as a shift of ownership of the science curriculum since the latter half of the 20th century away from professional scientists (Black & Atkin, 1996, p. 60). Our analysis shows that the most prominent subject-related stakeholders within the curriculum projects influencing the 2006 reform were university-based science education researchers. These academics are likely to have stronger links with university education departments than with university science departments. A similar outcome has been identified in relation to the redesign of the chemistry curriculum towards the aims of scientific literacy in China [START_REF] Wei | Rationale and Approaches for Embedding Scientific Literacy into the New Junior Secondary School Chemistry Curriculum in the People's Republic of China[END_REF]. The voices of science teachers (and through them those of their students) were not prominent in the development of the 2006 reforms in England. Referring to the cyclical model of the policy process introduced earlier, recreating curriculum policy within schools involves a continuous interaction with curriculum policy texts within contexts of practice; science teachers are 'curriculum creators' rather than 'curriculum deliverers' [START_REF] Pring | Education for All: The Future of Education and Training for 14-19 year olds[END_REF]. This process takes many years; a point highlighted by Kahle in her review of 60 years of science education reform in the US [START_REF] Kahle | Systemic reform: Research, vision, and politics[END_REF].

Teacher stakeholders were involved in the piloting of 21CS. However, the 2006 reforms were finalised ahead of the publication of the more extended evaluation of this pilot. Furthermore, teacher responses critical of the meaning and feasibility of scientific literacy within the science classroom identified within Study 1 of the QCA curriculum project appear not to have been engaged with in subsequent developments.

There was also insufficient attention given to the differentiated character of the science teaching community [START_REF] Donnelly | Science education: policy, professionalism and change[END_REF]. Science teachers differ in their skills, aspirations and identities, the places in which they work and the students that they work with [START_REF] Banner | The Enactment and Impact of Science Curriculum Reform[END_REF][START_REF] Witz | Science as an ideal: teachers' orientations to science and science education reform[END_REF]. The teachers involved in the piloting of 21CS (or at least their heads of department) had volunteered to be involved in this curriculum innovation. However, the voices of other, perhaps more traditional, science teachers did not feature strongly. Ultimately, the authority of government stakeholders resulted in the premature installation of a national statutory curriculum framework sharing many features of the 21CS courses being piloted at that time. The implications of this policy decision within schools is the focus of ongoing work [START_REF] Banner | The Enactment and Impact of Science Curriculum Reform[END_REF]. Finally, we have shown that professional scientists, school science teachers and their students had little authority within the development of this reform.

Implications

Here we consider messages that might be drawn from our analysis for the development of science curriculum reform policies internationally. We first discuss the need for a significant school-based pilot phase ahead of any national or regional rollout of curriculum reform. From the theoretical perspective represented by the policy process cycle in Figure 1 curriculum reform necessarily involves engaging with the context of practice. The purpose of a pilot phase is therefore to engage with practice in a planned and controlled fashion, with evaluation tools built in, such that any lessons learnt can be used to further develop the reform ahead of any broader roll-out. There are also pragmatic reasons for a significant pilot of curriculum reform. Highly motivated 'early adopter' teachers who volunteer to be involved in a curriculum pilot will develop pedagogic resources associated with the innovative elements of any course, e.g. teaching activities and assessment instruments in the context of socioscientific issues. One outcome of a pilot would be that this essential practitioner expertise will be available to support 'later adopters' and even 'reluctant adopters'

following any broader roll-out. Finally, any pilot phase would also need to represent the differentiated character of the teaching community; to involve both those teachers looking to change their practice, but also (in the case of a statutory reform) those teachers who are broadly comfortable with existing curricula.

In addition to teachers and their students other stakeholders also need to have a voice in the development of curriculum reform. Curriculum reform is a necessary compromise between competing stakeholder demands [START_REF] Roberts | What counts as science education?[END_REF]. Not paying sufficient attention to such multiple demands lies behind the failure of many previous reforms of science education with a focus on scientific literacy [START_REF] Blades | Procedures of power and curriculum change: Foucault and the quest for possibilities in science education[END_REF][START_REF] Eijkelhof | ANW: A new course on public understanding of science for senior general secondary education in the Netherlands[END_REF]. For example, earlier we identified a shift of ownership of the science curriculum away from professional scientists. However, as reform is enacted their role is likely to grow. For example, professional scientists working in universities are involved in selecting those students who are chosen to follow sciencerelated courses within universities. Furthermore, returning to the role of teachers in curriculum reform, these key stakeholders need to be involved centrally in all of the policy contexts represented in Figure 1, and not simply at the 'pilot' or 'implementation' phase [START_REF] Kirk | Teacher voice and ownership of curriculum change[END_REF].

Our analysis lends support to calls for a body representing all stakeholders to have responsibility for national or regional curriculum reform (House of Lords Science & Technology Select Committee, 2007, p. 17;The Royal Society, 2008). Within such a body all five categories of stakeholder identified in our analysis would have an active and ongoing role within each of the policy contexts represented in Figure 1. Additional stakeholders would also need to be involved such as representatives of professional organisations for scientists, teachers, parents, students, school inspectors and examining bodies. Such a body would draw upon an understanding of the successes and failures of previous curriculum reform initiatives. It would also be responsible for ensuring that any reform is preceded by a significant pilot phase within a representative sample of schools. An allocation of funds to support such piloting and associated evaluation would be needed. It is unlikely that all demands on school science curricula could be catered for, all stakeholders satisfied. However, previous analyses suggest that without an explicit identification and consideration of multiple aims and associated tensions successful curriculum reform is unlikely. Demands on the school science curriculum [START_REF] Fensham | Familiar but different: Some dilemmas and new directions in science education[END_REF][START_REF] Fensham | The link between policy and practice in science education: The role of research[END_REF] ENVIRONMENTAL

  , providing a rich context in which tensions between distinct aims for school science are likely to surface.

  developed over time, and are likely to involve extended contestation

  with interest. Such an education should enable them

  Despite being neither initiated or funded by the government the Beyond 2000 report has played a significant role in the development of the reform of the National Curriculum for Science in England; an influence acknowledged explicitly by curriculum officials in the government (QCA, 2006). Thus the Beyond 2000 project is

  ways in which teachers 'recreate' curriculum reforms in their classrooms. There was a strong emphasis within Study 1 and Study 3 on the need to address the dual goals of science literacy and preparation for post-compulsory science study. For example, the definition of scientific literacy presented to respondents in Study 1 included a focus on preparing students to engage with science and technology issues as 'future consumers and users of science'. This focus was contrasted with the preparation of students for a career in science, i.e. as future 'producers' of scientific knowledge.

  In 2002 the development of a new suite of science courses began (21st Century

  social and ethical issues. Within the 21CS curriculum model the majority of students would also opt for one of two additional science courses offering either traditional science content or a focus on the applications of science within everyday and workrelated contexts. The 21CS curriculum framework had much in common with the outcomes of Study 3 (curriculum models) of the QCA curriculum development project referred to earlier. Indeed, Robin Millar, a Professor of Science Education based at the University of York and a lead member of the 21CS development team, had been involved centrally in Beyond 2000 and Study 3 of the QCA curriculum project. Taken together these activities comprised a long term, coherent sequence of projects focused on the development of a curriculum emphasising scientific literacy, alongside other goals, and involving a common core of university-based science education researchers.

  The development of 21CS teaching materials and training resources was funded by three charitable trusts: the Nuffield Foundation, the Salters' Institute and the Wellcome Trust. The latter two organisations in particular have interests in the appreciation of science (chemistry and the biomedical sciences respectively) amongst young people. Indeed, the Wellcome Trust is the largest non-governmental funder of biomedical research in the UK. Their interest in the promotion of scientific literacy may be a response to concerns about adverse public responses to issues such as genomics and the use of animals in research[START_REF] Levinson | Valuable lessons: Engaging with the social context of science in schools: Wellcome Trust[END_REF]. In addition to this development work the QCA commissioned a pilot of 21CS in 78 volunteer schools and colleges across England from September 2003. Two early evaluation studies of this pilot were conducted by the QCA (QCA, 2005a). The broadly positive indications from these studies were used by the government to justify the introduction of related reforms on a national scale (House of Lords Science and Technology Select Committee, 2007, p. 5). A more substantial evaluation, involving three linked studies, was commissioned by the charitable organisations funding the 21CS project in 2004

  inside front cover of the programme of study was a statement of aims for the new national curriculum:

  This emphasis on enhancing post-compulsory participation within the official government statement of curriculum was reflected in similar priorities within other government policy initiatives at that time. Two examples are given here. In 2004 the Multiple aims in the development of curriculum reform 13 UK government published a ten-year Science and Innovation Investment Framework (SIIF) aimed at increasing the contribution made by science to the national economy (H.M. Treasury, Department of Trade, & Industry & Department for Education and

  of the 21CS pilot was premature (House of Lords Science

  ) or specific capacities that students might develop (e.g.

Figure 2

 2 Figure 2 characterises the range of demands made of school science curricula

  science-related social and ethical issues such as genetic engineering) and individual demands (e.g. enabling individuals to deploy science understandings in making personal decisions about diet, nutrition and exercise). The associated environmental demand was also present; issues such as climate change and sustainability feature strongly within 21CS resources. Far from being a 'consolation prize' individual, social and environmental demands were the principal focus of at least some of the curriculum development activities examined here. In terms of the stakeholders involved, university-based science education researchers and representatives of charitable organisations were the main stakeholders advocating these demands.

  Our case study has shown the differential authority positions held by each of these groups of stakeholders. The Beyond 2000 project exerted authority by initiating a sequence of influential curriculum development activities. The principal stakeholders in Beyond 2000 were university-based science education researchers and organisations. However, the locus of authority shifted towards government stakeholders as these development activities progressed.

  Figure1A conceptual framework for examining policy reform[START_REF] Bowe | Reforming Education and Changing Schools[END_REF]

uk International Journal of Science Education

	Page 29 of 29

URL: http://mc.manuscriptcentral.com/tsed Email: ijse_editor@hotmail.co.

Acknowledgements

The authors thank Jim Donnelly for helpful comments on an earlier draft of this paper. The work reported here is one outcome from a larger study funded by the Economic and Social Research Council and the Gatsby Charitable Foundation, grant number RES-179-25-0004.