Delays between actions and their outcomes severely hinder reinforcement learning systems, but little is known of the neural mechanism by which animals overcome this problem and bridge such delays. The nucleus accumbens core (AcbC), part of the ventral striatum, is required for normal preference for a large, delayed reward over a small, immediate reward (self-controlled choice) in rats, but the reason for this is unclear. We investigated the role of the AcbC in learning a free-operant instrumental response using delayed reinforcement, performance of a previously-learned response for delayed reinforcement, and assessment of the relative magnitudes of two different rewards.Groups of rats with excitotoxic or sham lesions of the AcbC acquired an instrumental response with different delays (0, 10, or 20 s) between the lever-press response and reinforcer delivery. A second (inactive) lever was also present, but responding on it was never reinforced. As expected, the delays retarded learning in normal rats. AcbC lesions did not hinder learning in the absence of delays, but AcbC-lesioned rats were impaired in learning when there was a delay, relative to sham-operated controls. All groups eventually acquired the response and discriminated the active lever from the inactive lever to some degree. Rats were subsequently trained to discriminate reinforcers of different magnitudes. AcbC-lesioned rats were more sensitive to differences in reinforcer magnitude than sham-operated controls, suggesting that the deficit in self-controlled choice previously observed in such rats was a consequence of reduced preference for delayed rewards relative to immediate rewards, not of reduced preference for large rewards relative to small rewards. AcbC lesions also impaired the performance of a previously-learned instrumental response in a delay-dependent fashion.These results demonstrate that the AcbC contributes to instrumental learning and performance by bridging delays between subjects' actions and the ensuing outcomes that reinforce behaviour. Animals learn to control their environment through instrumental (operant) conditioning. When an animal acts to obtain reward or reinforcement, there is often a delay between its action and the outcome; thus, animals must learn instrumental action-outcome contingencies using delayed reinforcement. Although such delays impair learning, animals can nevertheless bridge substantial delays to acquire instrumental responses . Little The nucleus accumbens (Acb) responds to anticipated rewards in humans, other primates, and rats -15, and To investigate whether the AcbC is a reinforcement learning system specialized for delayed reinforcement, we first determined the ability of AcbC-lesioned rats to detect instrumental contingencies across a delay. The ability of AcbC-lesioned rats to acquire instrumental responding with delayed reinforcement was compared to that of sham-operated controls; each subject was allowed to respond freely on two levers, one of which produced reinforcement after a delay of 0, 10, or 20 s  and subsequently a reinforcer magnitude discrimination task (Experiment 1B). In Experiment 2, naïve rats were trained on the free-operant task for delayed reinforcement; AcbC lesions were then made and the rats were retested.In Experiment 1, there were two postoperative deaths. Histological analysis revealed that the lesions were incomplete or encroached significantly on neighbouring structures in four subjects. These subjects were excluded; final group numbers were therefore 8 (sham, 0 s delay), 6 (AcbC, 0 s delay), 8 (sham, 10 s delay), 7 (AcbC, 10 s delay), 8 (sham, 20 s delay), and 7 (AcbC, 20 s delay). In Experiment 2, one rat spontaneously fell ill with a colonic volvulus during preoperative training and was killed, and there were three postoperative deaths. Lesions were incomplete or too extensive in seven subjects; final group numbers were therefore 7 (sham, 0 s delay), 5 (AcbC, 0 s delay), 8 (sham, 10 s delay), 4 (AcbC, 10 s delay), 8 (sham, 20 s delay), and 5 (AcbC, 20 s delay).Lesions of the AcbC encompassed most of the core subregion; neuronal loss and associated gliosis extended in an anteroposterior direction from approximately 2.7 mm to 0.5 mm anterior to bregma, and did not extend ventrally or caudally into the ventral pallidum or olfactory tubercle. Damage to the ventromedial caudate-putamen was occasionally seen; damage to AcbSh was restricted to the lateral edge of the dorsal shell. Schematics of the lesions are shown in Figure The imposition of response-reinforcer delays retarded the acquisition of free-operant lever pressing, in sham-operated rats and in AcbC-lesioned rats Figure . AcbC-le2 × delay3 × (session14 × lever2 × S) revealed multiple significant interactions, including lever × delay × lesion (F2,38 = 5.17, p = .01) and session × lever × delay (F6.0,229.1 = 5.47,  = .464, p < .001), justifying sub-analysis. All six groups learned to respond more on the active lever than the inactive lever .An overall ANOVA using the model lesionF2,21 = 11.7, p < .001;  = .276, p < .001; session × delay: F7.2,75.3 = 2.46,  = .276, p = .024). The presence of a delay also increased responding on the inactive lever slightly (delay: F2,21 = 4.06, p = .032), though not systematically (the 10 s group differed from the 0 s group, p = .036, but no other groups differed, p ≥ .153).For sham-operated rats, delays reduced the rate of acquisition of the active lever response and reduced the asymptotic level of responding attained . Responding on the inactive lever was not significantly affected by the delays (maximum F15.8,134.2 = 1.65,  = .607, p = .066). At 0 s delay, AcbC-lesioned subjects responded more than shams on the active lever  and the inactive lever (lesion: F1,12 = 9.12, p = .011). However, at 10 s delay, AcbC-lesioned rats responded significantly less than shams on the active lever ; there was no difference in responding on the inactive lever (F < 1, NS). At 20 s delay, again, AcbC-lesioned rats responded significantly less than shams on the active lever  and there was no difference in responding on the inactive lever (F < 1, NS).There was a further, delay-dependent impairment in AcbC-lesioned rats, who responded more than shams at 0 s delay but substantially less than shams at 10 s and 20 s delay. As in the case of sham-operated controls, delays reduced the rate of acquisition and the maximum level of responding attained in AcbC-lesioned rats  was calculated. This time can therefore be equal to or less than the programmed delay, and is only relevant for subjects experiencing non-zero programmed response-reinforcer delays. The response-to-reinforcer-collection ('response-collection') delays were also calculated: for every reinforcer delivered, the response most closely preceding it and the nosepoke most closely following it were identified, and the time between these two events calculated. This time can be shorter or longer than the programmed delay, and is relevant for all subjects.mean delay experienced by each rat were reflected in differences in the distribution of response-delivery and response-collection delays when the programmed delay was non-zero , which was analysed further. Increasing delays significantly reduced the rate of responding in this session for shams (F2,21 = 17.3, p < .001) and AcbC-lesioned rats (F2,17 = 54.4, p < .001). AcbC-lesioned rats responded more than shams at zero delay (F1,12 = 8.52, p = .013) but less than shams at 10 s delay (F1,13 = 4.71, p = .049) and at 20 s delay (F1,13 = 17.3, p = .001).There was a systematic relationship between the acquisition rate and the programmed delay of reinforcement, and this was altered in AcbC-lesioned rats. Figure 2 × experienced delaycov. Unlike a standard analysis of covariance, the factor × covariate interaction term was included in the model. This confirmed that the lesion retarded the acquisition of responding in AcbC-lesioned rats, compared to controls, in a delay-dependent manner, over and above the differences in experienced delay .Since the AcbC group experienced slightly longer response-delivery and response-collection delays than shams when the programmed delay was non-zero Figure , it was F1,38 = 1.69, NS). Experienced inactive-response-collection delays were 252 ± 19 s (sham, 0 s), 217 ± 29 s (AcbC, 0 s), 169 ± 23 s (sham, 10 s), 179 ± 33 s (AcbC, 10 s), 231 ± 65 s (sham, 20 s), and 136 ± 37 s (AcbC, 20 s). Again, ANOVA revealed no effects of lesion or programmed delay and no interaction (maximum F1,38 = 1.61, NS). When the square-root-transformed number of responses on the inactive lever in session 10 was analysed with the experienced delays up to that point as a predictor, using the model lesion2 × experienced inactive-response-collection delaycov just as for the active lever analysis, there was no lesion × experienced delay interaction (F < 1, NS).No such delay-dependent effects were observed for the inactive lever. Experienced inactive-response-delivery delays  were much longer and more variable than corresponding delays for the active lever, because subjects responded on the inactive lever so little. Means ± SEMs were 250 ± 19 s (sham, 0 s), 214 ± 29 s (AcbC, 0 s), 167 ± 23 s (sham, 10 s), 176 ± 33 s (AcbC, 10 s), 229 ± 65 s (sham, 20 s), and 131 ± 37 s (AcbC, 20 s). ANOVA of these data revealed no effects of lesion or programmed delay and no interaction  = rA/(rA+rB). Overmatching is said to occur if subjects prefer the schedule with the higher reinforcement rate more than predicted by the matching law; undermatching is the opposite. Both sham-operated and AcbC-lesioned rats were sensitive to the distribution of reinforcement that they received on two concurrent random interval (RI) schedules, altering their response allocation accordingly. Subjects preferred the lever on which they received a greater proportion of reinforcement. In general, subjects did not conform to the matching law, but exhibited substantial undermatching; this is common [Relative preference for two reinforcers may be inferred from the distribution of responses on concurrent variable interval schedules of reinforcement -33. Acco2 × (experienced reinforcer distributioncov × S); the factor × covariate term was included in the model. Analysis of sham and AcbC groups separately demonstrated that both groups altered their response allocation according to the distribution of reinforcement, i.e. that both groups discriminated the two reinforcers on the basis of their magnitude . There was also a significant lesion × reinforcer distribution interaction (F1,86 = 5.5, p = .021), indicating that the two groups' matching behaviour differed, with the AcbC-lesioned rats showing better sensitivity to the relative reinforcer magnitude than the shams , and the proportion of responses allocated to lever A, were calculated for each subject for the last session in each of the three programmed reinforcement distribution contingencies . Mean switch probabilities (± SEMs) were 0.41 ± 0.02 (AcbC) and 0.49 ± 0.01 (sham).Because switching behaviour has the potential to influence behaviour on concurrent schedules e.g. , we alsoDue to mechanical faults, data from four subjects in session 10 (preoperative) and data from one subject in session 22 (postoperative) were not collected. Both sessions were removed from analysis completely, and data points for those sessions are plotted using the mean and SEM of the remaining unaffected subjects .2 × delay3 × (session3 × lever2 × S), indicated that responding was affected by the delays to reinforcement , but there were no differences between the groups due to receive AcbC and sham lesions (terms involving lesion intent: maximum F was for session × lever × lesion intent, F2,62 = 1.844, NS). As expected, delays reduced the rate of responding on the active lever (F2,31 = 15.6, p < .001) and increased responding on the inactive lever (F2,31 = 8.12, p = .001) preoperatively.Preoperatively, the groups remained matched following later histological selection. Analysis of the last 3 preoperative sessions, using the model lesion intentAcbC lesions selectively impaired performance of instrumental responding only when there was a response-reinforcer delay. There was no effect of the lesion on responding under the 0 s delay condition, but in the presence of delays, AcbC lesions impaired performance on the active lever Figure . These cFs < 1, NS; for the inactive lever, lesion intent: F1,31 = 2.99, p = .094; lesion intent × delay: F < 1, NS). Analysis of the postoperative sessions, using the model lesion2 × delay3 × (session17 × lever2 × session-14-active-lever-responsescov × S), revealed a near-significant lesion × delay × session × lever interaction (F22.4,335.5 = 1.555,  = .699, p = .054). Furthermore, analysis of postoperative responding on the active lever, using the model lesion2 × delay3 × (session17 × session-14-active-lever-responsescov × S), revealed a session × delay × lesion interaction (F17.3,259.5 = 1.98,  = .541, p = .013) and a delay × lesion interaction (F2,30 = 3.739, p = .036), indicating that the lesion affected responding on the active lever in a delay-dependent manner. In an identical analysis of responding on the inactive lever (using inactive lever responding on session 14 as the covariate), no terms involving lesion were significant (maximum F: lesion, F1,30 = 1.96, p = .172), indicating that the lesion did not affect responding on the inactive lever.Subjects' responding on the relevant lever in the last preoperative session (session 14) was used as a covariate to increase the power of the analysis . As expeF2,20 = 11.78, p < .001; session × delay: F12.4,124.1 = 2.36,  = .388, p = .008) and in AcbC-lesioned rats . Shams continued to discriminate between the active and inactive lever at all delays . AcbC-lesioned rats continued to discriminate at 0 s and 10 s , but AcbC-lesioned subjects in the 20 s condition failed to discriminate between the active and inactive levers postoperatively .Postoperatively, response-reinforcer delays continued systematically to decrease responding on the active lever, both in shams . However, at 10 s, AcbC-lesioned rats responded significantly less than shams on the active lever . Similarly, at 20 s, AcbC-lesioned rats responded less than shams on the active lever (lesion: F1,10 = 6.282, p = .031). There were no differences on responding on the inactive lever at any delay (Fs ≤ 1.31, NS).Lesioned subjects responded as much as shams at 0 s delay, but substantially less than shams at 10 s and 20 s delay . In this session, there was no significant effect of delays on shams' performance (F2,20 = 2.15, p = .143), though there was for AcbC-lesioned rats (F2,11 = 9.01, p = .005). There were no significant differences in responding on this session between shams and AcbC-lesioned rats in the 0 s condition (F1,10 = 3.10, p = .109) or the 10 s condition (F < 1, NS), but AcbC-lesioned rats responded less at 20 s delay (F1,11 = 6.74, p = .025).There was a systematic relationship between the postoperative response rate and the programmed delay of reinforcement, and this was altered in AcbC-lesioned rats. Figure 2 × experienced delaycov, with the factor × covariate interaction term included in the model. This confirmed that the lesion affected responding in AcbC-lesioned rats, compared to controls, in a delay-dependent manner, over and above the postoperative differences in experienced delay .Since the AcbC group experienced slightly longer response-delivery and response-collection delays than shams when the programmed delay was non-zero Figure , as befoAcbC-lesioned animals were hyperactive compared to sham-operated controls, and gained less mass then shams across the experiments Figure , consistThese results establish that the AcbC contributes to learning of actions when the outcome is delayed. Lesions of the AcbC did not impair instrumental learning when the reinforcer was delivered immediately, but substantially impaired learning with delayed reinforcement, indicating that the AcbC 'bridges' action-outcome delays during learning. Lesions made after learning also impaired performance of the instrumental response in a delay-dependent fashion, indicating that the AcbC also contributes to the performance of actions for delayed reinforcement. Finally, the lesions did not impair the perception of relative reward magnitude as assessed by responding on identical concurrent interval schedules for reinforcers of different magnitude, suggesting that the impulsive choice previously exhibited by AcbC-lesioned rats  is attriDelays have long been known to retard instrumental learning ,37. DespN-methyl-D-aspartate (NMDA) glutamate receptors in the AcbC has been shown to retard instrumental learning for food under a variable-ratio-2 (VR-2) schedule [in which P(reinforcer | response) ≅ 0.5] [after instrumental training sessions impairs subsequent performance, implying that PKA activity and protein synthesis in the AcbC contribute to the consolidation of instrumental behaviour. Thus, manipulation of Acb neurotransmission can affect instrumental learning. However, it is also clear that excitotoxic destruction of the AcbC or even the entire Acb does not impair simple instrumental conditioning to any substantial degree. Rats with Acb or AcbC lesions acquire lever-press responses on sequences of random ratio schedules [in which P(reinforcer | response) typically declines from around 1 to 0.05 over training] at near-normal levels [P(reinforcer | response) = 1] in the absence of response-reinforcer delays, they acquire the response at normal rates.In the absence of response-reinforcer delays, AcbC-lesioned rats acquired an instrumental response normally, responding even more than sham-operated controls. In contrast, blockade of ) ≅ 0.5] , as has ) ≅ 0.5] . Concurr) ≅ 0.5] . Once th) ≅ 0.5] . Further) ≅ 0.5]  or a pro) ≅ 0.5]  into thel levels ,45. In sl levels ,45, consl levels ,44,45. Oper se, since they responded normally (in fact, more than shams) when the food was not delayed. Thus although the Acb and its dopaminergic innervation are clearly very important in motivating behaviour e.g. [In contrast, when a delay was imposed between responding and reinforcement, AcbC-lesioned rats were impaired relative to sham-operated controls, in a systematic and delay-dependent fashion. The observation that learning was not affected at zero delay rules out a number of explanations of this effect. For example, it cannot be that AcbC-lesioned rats are in some way less motivated for the food our e.g. ,46-48, tour e.g. ,48. Howeour e.g. ,29,49-52Our results also indicated that when there were programmed delays to reinforcement, AcbC-lesioned animals experienced longer response-reinforcer collection delays, partly due to their failure to collect the reinforcer as promptly as shams. These additional experienced delays probably retarded learning. However, in addition to this effect, there was a further deficit exhibited by AcbC-lesioned rats: even allowing for the longer response-collection delays that they experienced, their instrumental learning was impaired more by delays than that of sham-operated controls. Deficits in learning with delayed reinforcement may account for some of the variability in the effect of AcbC lesions or local pharmacological manipulations on instrumental learning across different schedules.P(outcome | no action) = 0. When they are then exposed to the instrumental contingency, such that P(outcome | action) > 0, this prior experience may enhance their ability to detect the instrumental contingency ΔP = P(outcome | action) - P(outcome | no action). In one aversive Pavlovian conditioning procedure in which a conditioned stimulus (CS) was paired with electric shock, AcbC lesions have been shown to impair conditioning to discrete CSs, but simultaneously to enhance conditioning to contextual (background) CSs [The fact that pre-exposure to the context improves instrumental learning in normal rats  suggestsund) CSs , though und) CSs ,55. It iThe instrumental task used requires animals either to associate their response with the delayed food outcome , or to strengthen a stimulus-response association (habit) when the reinforcer eventually arrives ,39. BothA delay-dependent impairment was also seen when AcbC lesions were made after training. This indicates that the AcbC does not only contribute to the learning of a response when there is an action-outcome delay: it also contributes to the performance of a previously-learned response. Again, AcbC-lesioned rats were only impaired when that previously-learned response was for delayed (and not immediate) reinforcement. Of course, learning of an instrumental response depends upon the animal being able to perform that response; preventing an animal from pressing a lever (a performance deficit) would clearly impair its ability to learn an instrumental response on that lever to obtain food. In the present set of experiments, it is clear that AcbC-lesioned rats were just as able to perform the response itself  as controls, as shown by their normal performance in the zero-delay condition, so it is not clear whether the delay-dependent impairments in learning and performance can be attributed to the same process. Again, since responding was unaffected in the zero-delay condition, many alternative interpretations (such as a lack of motivation to work for the food) are ruled out. It may be that AcbC-lesioned rats are impaired at representing a declarative instrumental action-outcome contingency when the outcome is delayed, or in forming or executing a procedural stimulus-response habit when the reinforcing event does not follow the response immediately. It may also be that they represent the action-outcome contingency normally but value the food less because it is delayed, and that this affects responding in a free-operant situation even though there is no alternative reinforcer available.Excitotoxic lesions of the whole Acb do not prevent rats from detecting changes in reward value  . Such leThe term 'reinforcement learning' simply means learning to act on the basis of reinforcement received; it is a term used in artificial intelligence research  that doeWe have demonstrated that excitotoxic lesions of the AcbC do not prevent rats from learning a simple instrumental response when the reinforcing outcome follows their action immediately. However, AcbC lesions impair rats' ability to learn the same instrumental response when the outcome is delayed. The lesions also impair performance of an instrumental response that was learned preoperatively, but again only when response-reinforcer delays were present. These results suggest that the AcbC makes a specific contribution to reinforcement learning and instrumental performance when reinforcing outcomes do not arrive immediately but are delayed. AcbC dysfunction, which is known to promote impulsive choice, appears to cause rats to be temporally short-sighted, learning preferentially about the proximal consequences of their actions and preferring immediate over delayed rewards.n = 26) or sham lesions (n = 24). Two died postoperatively. Subjects were next trained in a task in which they had continuous access to two identical levers; one lever delivered a single food pellet each time it was pressed, and the other lever had no effect. For some rats, the food pellet was delivered immediately after the lever press . For others, each pellet was delayed by either 10 s (8 AcbC, 8 sham) or 20 s (8 AcbC, 8 sham). Subjects were trained for 14 sessions.Fifty naïve rats received excitotoxic lesions of the AcbC , 10 s (n = 16), or 20 s (n = 16). One rat spontaneously fell ill with a colonic volvulus and was killed. Once the subjects had been trained for 14 sessions, they were allocated to receive either AcbC lesions or sham surgery . Sham and AcbC groups were matched for performance preoperatively: within each delay condition, rats were ranked by their rates of responding on the active lever at the end of training, and rats with equivalent levels of performance were randomized to receive sham or AcbC lesion surgery. They were then retested postoperatively on the same task for a further 18 sessions , with each rat experiencing the same delay as it had preoperatively. These rats then had their locomotor activity assessed, and were killed and perfused for histology.A further 48 naïve rats were trained to acquire an instrumental response as before, with delays to reinforcement of 0 s  housed in a temperature-controlled room (minimum 22°C) under a 12:12 h reversed light-dark cycle (lights off 07:30 to 19:30). Subjects were approximately 15 weeks old on arrival at the laboratory and were given a minimum of a week to acclimatize, with free access to food, before experiments began. Experiments took place between 09:00 and 21:00, with individual subjects being tested at a consistent time of day. Subjects had free access to water. During behavioural testing, they were maintained at 85–90% of their free-feeding mass using a restricted feeding regimen. Feeding occurred in the home cages at the end of the experimental day. All procedures were subject to UK Home Office approval (Project Licences PPL 80/1324 and 80/1767) under the Animals (Scientific Procedures) Act 1986.2HPO4, 0.028 M NaH2PO4 in double-distilled water, sterilized by filtration) and adjusted with NaOH to a final pH of 7.2–7.4. Toxin was injected over 3 min and the micropipette was left in place for 2 min following injections. Sham lesions were made in the same manner except that vehicle was infused. At the end of the operation, animals were given 15 ml/kg of sterile 5% w/v glucose, 0.9% w/v sodium chloride intraperitoneally. They were given a week to recover, with free access to food, and were handled regularly. Any instances of postoperative constipation were treated with liquid paraffin orally and rectally. At the end of this period, food restriction commenced or was resumed.Subjects were anaesthetized with Avertin  and placed in a Kopf or Stoelting stereotaxic frame  fitted with atraumatic ear bars. The skull was exposed and a dental drill was used to remove the bone directly above the injection and cannulation sites. The dura mater was broken with the tip of a hypodermic needle, avoiding damage to underlying venous sinuses. Excitotoxic lesions of the AcbC were made by injecting 0.5 μl of 0.09 M quinolinic acid (Sigma, UK) through a glass micropipette at coordinates 1.2 mm anterior to bregma, ± 1.8 mm from the midline, and 7.1 mm below the skull surface at bregma; the incisor bar was 3.3 mm below the interaural line . The toxBehavioural testing was conducted in one of two types of operant chamber of identical configuration (from Med Associates Inc, Georgia, Vermont, USA, or Paul Fray Ltd, Cambridge, UK). Each chamber was fitted with a 2.8 W overhead house light and two retractable levers on either side of an alcove fitted with an infrared photodiode to detect head entry. Sucrose pellets (45 mg, Rodent Diet Formula P, Noyes, Lancaster, New Hampshire, USA) could be delivered into the alcove. The chambers were enclosed within sound-attenuating boxes fitted with fans to provide air circulation. The apparatus was controlled by software written by RNC in C++  using thA variety of free-operant schedules may be used to assess instrumental acquisition with delayed reinforcement . We usedAlternative free-operant schedules for this purpose exist, such as one in which the first response sets up reinforcement, and a subsequent response made before the reinforcer is delivered postpones reinforcement, in order to keep the delay between the last response and the reinforcer constant . However, the tandem FR-1-DRO schedule constrains the maximum rate of reinforcement, which also decreases as the delay being used increases. Furthermore, it does not hold constant the probability of reinforcement given a response, and it introduces two opposing contingencies: some responses make reinforcement more likely, while others (those during the delay) make it less likely ,87 ratheImmediately after subjects were placed in the operant chamber, the sessions began. The houselight was illuminated, and remained on for each 30-min session. Two levers were extended into the chamber. All lever responses were first 'debounced' to 10 ms . Other than this, all lever presses and nosepokes into the food alcove were recorded. Responding on the left (active) lever caused a single pellet to be delivered following a delay, under a fixed-ratio-1 (FR-1) schedule  × 40 (D) × 18 (H) cm, equipped with two horizontal photocell beams situated 1 cm from the floor that enabled movements along the long axis of the cage to be registered. Subjects were placed in these cages, which were initially unfamiliar to them, and their activity was recorded for 2 h. All animals were tested in the food-deprived state. Locomotor hyperactivity and reduced weight gain have previously been part of the phenotype of AcbC-lesioned rats, though without alterations in the consumption of the reinforcer used in the present experiments ,29,36.x-second (RI-x) schedules were implemented by having a clock tick once a second; each tick set up reinforcement with a probability p = 1/x. Once reinforcement had been set up for a schedule, the next response caused reinforcement to be delivered. Multiple pellets were delivered 0.5 s apart. For concurrent RI schedules, a 2 s changeover delay (COD) was imposed to discourage frequent switching between schedules [Subjects were trained in 30-min sessions to respond on both levers separately under interval schedules of reinforcement. The two levers were designated A and B; these were counterbalanced left/right . As before, responses were debounced to 10 ms. Training and testing proceeded according to Table chedules -34,88. TRats were deeply anaesthetized with pentobarbitone sodium (200 mg/ml, minimum of 1.5 ml i.p.) and perfused transcardially with 0.01 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Their brains were removed and postfixed in paraformaldehyde before being dehydrated in 20% sucrose for cryoprotection. The brains were sectioned coronally at 60 μm thickness on a freezing microtome and every third section mounted on chromium potassium sulphate/gelatin-coated glass microscope slides and allowed to dry. Sections were passed through a series of ethanol solutions of descending concentration (3 minutes in each of 100%, 95%, and 70% v/v ethanol in water) and stained for ~5 min with cresyl violet. The stain comprises 0.05% w/v aqueous cresyl violet (Raymond A. Lamb Ltd, Eastbourne, UK), 2 mM acetic acid, and 5 mM formic acid in water. Following staining, sections were rinsed in water and 70% ethanol before being differentiated in 95% ethanol. Finally, they were dehydrated and delipidated in 100% ethanol and Histoclear  before being cover-slipped using DePeX mounting medium (BDH, UK) and allowed to dry. The sections were used to verify cannula and lesion placement and assess the extent of lesion-induced neuronal loss. Lesions were detectable as the absence of visible neurons (cell bodies of the order of 100 μm in diameter with a characteristic shape and appearance), often associated with a degree of tissue collapse (sometimes with consequent ventricular expansion when the lesion was adjacent to a ventricle) and gliosis .dependent variable = A2 × Bcov × (C5 × Dcov × S) where A is a between-subjects factor with two levels, B is a between-subjects covariate, C is a within-subjects factor with five levels, and D is a within-subjects covariate; S denotes subjects in designs involving within-subjects factors [ for any terms involving factors in which the sphericity assumption was violated [Data collected by the chamber control programs were imported into a relational database (Microsoft Access 97) for case selection and analysed with SPSS 11. Figures were created with SigmaPlot 2001/v7 and Adobe Illustrator 8. All graphs show group means and error bars are ± 1 standard error of the mean (SEM) unless otherwise stated. Count data (lever presses and locomotor activity counts), for which variance increases with the mean, were subjected to a square-root transformation prior to any analysis . Homogen factors . For rep factors  and the violated ., Huynh-Feldt epsilonAcb, nucleus accumbensAcbC, nucleus accumbens coreAcbSh, nucleus accumbens shellAMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolpropionateANCOVA, analysis of covarianceANOVA, analysis of varianceCOD, changeover delayDA, dopamineDRO, differential reinforcement of other behaviourFR, fixed ratioi.p., intraperitonealh, hourmin, minuteN-methyl-D-aspartateNMDA, P(A), probability of event A occurringP(A | B), probability of A occurring, given that B has occurredPBS, phosphate-buffered salinePKA, protein kinase A (cyclic-adenosine-monophosphate-dependent protein kinase)RI, random intervalSEM, standard error of the meanVR, variable ratiov/v, volume per unit volumew/v, weight per unit volumeRNC conceived and designed the studies, supervised THCC, wrote the software, and drafted the manuscript. THCC participated in the design of the studies and tested the animals. The work contributed to THCC's MPhil thesis. Both authors performed surgery, processed histological material, analysed the results, and read and approved the final manuscript.


It is now common for parents to measure tympanic temperatures in children. The objective of this study was to assess the diagnostic accuracy of these measurements.Parents and then nurses measured the temperature of 60 children with a tympanic thermometer designed for home use (home thermometer). The reference standard was a temperature measured by a nurse with a model of tympanic thermometer commonly used in hospitals . A difference of ≥ 0.5 °C was considered clinically significant. A fever was defined as a temperature ≥ 38.5 °C.The mean absolute difference between the readings done by the parent and the nurse with the home thermometer was 0.44 ± 0.61 °C, and 33% of the readings differed by ≥ 0.5 °C. The mean absolute difference between the readings done by the parent with the home thermometer and the nurse with the hospital thermometer was 0.51 ± 0.63 °C, and 72 % of the readings differed by ≥ 0.5 °C. Using the home thermometer, parents detected fever with a sensitivity of 76% (95% CI 50–93%), a specificity of 95% (95% CI 84–99%), a positive predictive value of 87% (95% CI 60–98%), and a negative predictive value of 91% (95% CI 79–98 %). In comparing the readings the nurse obtained from the two different tympanic thermometers, the mean absolute difference was 0.24 ± 0.22 °C. Nurses detected fever with a sensitivity of 94% (95 % CI 71–100 %), a specificity of 88% (95% CI 75–96 %), a positive predictive value of 76% (95% CI 53–92%), and a negative predictive value of 97% (95%CI 87–100 %) using the home thermometer. The intraclass correlation coefficient for the three sets of readings was 0.80, and the consistency of readings was not affected by the body temperature.The readings done by parents with a tympanic thermometer designed for home use differed a clinically significant amount from the reference standard  the majority of the time, and parents failed to detect fever about one-quarter of the time. Tympanic readings reported by parents should be interpreted with great caution. Parents and health care workers use temperatures measured by parents at home to determine if a child requires medical assessment. Health care workers sometimes make decisions about the need for investigations and hospital admission based on the temperature that parents report. This is especially true in the newborn or in the immunocompromised patient, where any temperature above the normal range can be indicative of bacterial infection.Health care workers have used commercial tympanic thermometers in hospitals for over 15 years. These thermometers are quick and easy to use and minimize the risk of nosocomial infection. Tympanic thermometers are now available for home use at a cost of about $50 US. There are no published studies comparing measurements obtained with tympanic thermometers designed for home use to measurements obtained with other types of thermometers, or comparing readings obtained by parents with these instruments to those obtained by health care workers. The primary objective of this study was to determine the diagnostic accuracy of readings obtained by parents with a tympanic thermometer designed for home use.The Health Research Ethics Board of the University of Alberta approved this study and parents and older children signed a consent form.Traditionally, rectal temperatures have been used as the reference standard in pediatrics. However, readings done by a nurse using a model of tympanic thermometer commonly used in hospitals were chosen as the reference standard for this study as they are easier to obtain than are rectal temperatures and tend to be closer to PA temperatures when the patient is febrile  or when Parents of patients from the General Pediatric Clinic, the Emergency Department, or on the inpatient units of the Stollery Children's Hospital were eligible for the study. Parents of febrile or afebrile children aged 6 months to 16 years were approached on the days when the study nurse was available, but preference was given to patients who were being assessed because of fever. Parents were not approached if it would be inconvenient for them to participate in the study, or if they did not communicate in English, but there were no other exclusion criteria.After informed consent was obtained from the parent , the parent was given the instruction sheet from the Braun Thermoscan (Thermoscan Inc., San Diego, CA) tympanic thermometer (home thermometer) to read. They were then asked to measure their child's temperature when they felt they understood the instructions. They then recorded the temperatures they measured on a sheet of paper. Without looking at this sheet of paper, one of three trained research nurses then measured the child's temperature using the same thermometer. The nurse then also measured the child's temperature with CORECHECK (ALARIS Inc., San Diego, CA) tympanic thermometer . All readings were done in the same ear with a new probe cover.The mean absolute difference between the parents' reading and the reading by the nurse using the same thermometer was calculated. Readings were then analyzed separately for the febrile children. A temperature of ≥ 38.5 °C on the reading taken by the nurse using the hospital thermometer was defined as a fever. The percentage of times that the readings differed by 0.5 °C or more was calculated as this was considered a clinically significant difference.Even if the parent and the nurse obtained similar readings from the home thermometer, it would still be possible that these readings are inaccurate, as there are no published studies demonstrating the accuracy of this thermometer. Therefore, the readings obtained by the nurse and the parent from the home thermometer were compared to those obtained by the nurse using the hospital thermometer (the reference standard for the study). If the readings obtained by the parent differed considerably from those obtained by the nurse, but the nurse then obtained very consistent readings with the two different thermometers, one could conclude that the parents were inaccurate because of human error. On the other hand, if the nurse obtained inconsistent readings with the two different thermometers, this would suggest that either instrument or human error could be the problem.The measures of central tendency were determined, and the intraclass correlation coefficient determined for the three sets of readings. The three sets of measurements were also compared using the Bland-Altman method  where thIt was not possible to calculate a sample size, as the expected standard deviation between the three sets of readings could not be predicted. We estimated that a sample size of 60 children would be adequate.Sixty parents were approached and all agreed to participate in the study. Their children were 35 males and 25 females aged 5 months to 15 years (median 3 years). Although the majority of patients were being assessed because of fever, only 17 children were febrile at the time of the study. All 3 measurements were obtained within 5 minutes for 55 of the 60 cases.Table The mean absolute difference between the temperatures measured by the nurse using the home thermometer versus the hospital thermometer was 0.24 ± 0.22 °C. The higher reading was from the hospital thermometer in 25 of the 60 cases. There was agreement between the readings taken by the nurse from the two thermometers with regard to the presence of fever, except in one case where one reading was 38.4 °C and the other was 38.6 °C . The absolute differences were similar for febrile and afebrile children. Using the reference standard of a tympanic temperature measured by a nurse with a model of thermometer commonly used in hospitals, the parents did not detect a fever in four of 13 cases . This suggests that the readings obtained by a parent with a tympanic thermometer are sometimes not reliable. In fact, previous studies showed that parents' subjective assessment of whether their child had a fever had a sensitivity of 81.8%  and 88.9In comparing the readings taken by a nurse with a home tympanic thermometer to a hospital tympanic thermometer, the mean absolute difference was 0.24 °C, with 13 % of the readings differing by a clinically significant amount. It is not clear if this degree of discrepancy is to be expected when a skilled operator takes two tympanic readings, or if it was a true difference between the two types of tympanic thermometer. The nurse would have identified all but one of the 17 children with fever as being febrile with either thermometer, and that child had a temperature of 38.4 °C on one thermometer and 38.6 °C on the other thermometer. Therefore, it appears that with a skilled operator, the tympanic thermometer designed for home use is reasonably reliable, and is likely to detect fever even if the actual reading is not always accurate.The chief limitation of this study is that readings obtained by a nurse from the hospital tympanic thermometer must be reliable for the results of this study to be accurate. Some studies have concluded that tympanic thermometers do not give an accurate measurement of body temperature ,9,10. HoIt is possible that parents would have performed better had they leisurely read the instructions in their own home rather than doing so in a hospital-based setting, or had they had time to practice. However, it is also possible that they performed better than they would have in the home, as they knew evaluation of their performance was occurring. It is also possible that parents who would choose to buy a tympanic thermometer will differ from our study population. It is not clear what advice to give to parents about purchasing a thermometer. The ability of parents from the inner city to correctly use and read a mercury glass thermometer was poor in previous studies ,13. An eWith the recent outbreak of Severe Acute Respiratory Syndrome, rapid measurement of body temperature  has become an important tool to indicate if people can board airplanes and if staff can work. It is therefore important to determine the accuracy of non-health care workers at measuring body temperature. This study showed that the temperatures measured by parents with a tympanic thermometer often differ by a clinically significant amount from those measured by a nurse with a tympanic thermometer. There is not a consistent gradient or direction of the gradient between the two temperatures, which makes it difficult to interpret the temperatures reported by parents. There was a smaller gradient between the readings taken by a nurse with a tympanic thermometer designed for home use compared with those taken with a model of tympanic thermometer commonly used in hospitals, but there were still potentially clinically significant discrepancies 14% of the time. This makes it difficult to conclude if the poor performance of the parents relates entirely to human error, or if there is also an element of instrument error. The fact that in comparison with our reference standard  the readings taken with the home thermometer by the nurse correlated much better than those taken by the parent suggests that the parents did not use ideal technique. In any case, one should interpret temperatures taken by parents with a tympanic thermometer with great caution. Although parents will detect the majority of fevers with these instruments, the absolute numbers obtained may not be accurate. Again, we find that in pediatrics, the clinical picture is a more useful piece of information than are "the numbers"!°C – degrees CelsiusPA: pulmonary arteryThe author(s) declare that they have no competing interests.JLR wrote the protocol and the manuscript. HJ helped with patient recruitment and reviewed the manuscript. DWS did the statistical analysis and reviewed the manuscript.The pre-publication history for this paper can be accessed here:


Recently, several members of the transforming growth factor-beta (TGF-beta) superfamily have been shown to be essential for regulating the growth and differentiation of ovarian follicles and thus fertility.Ovaries of neonatal and adult sheep were examined for expression of the TGF-betas 1–3 and their receptors (RI and RII) by in situ hybridization using ovine cDNAs. The effects of TGF-beta 1 and 2 on proliferation and differentiation of ovine granulosa cells in vitro were also studied.The expression patterns of TGF-beta 1 and 2 were similar in that both mRNAs were first observed in thecal cells of type 3  follicles. Expression of both mRNAs continued to be observed in the theca of larger follicles and was also present in cells within the stroma and associated with the vascular system of the ovary. There was no evidence for expression in granulosa cells or oocytes. Expression of TGF-beta 3 mRNA was limited to cells associated with the vascular system within the ovary. TGFbetaRI mRNA was observed in oocytes from the type 1  to type 5  stages of follicular growth and granulosa and thecal cells expressed this mRNA at the type 3  and subsequent stages of development. The TGFbetaRI signal was also observed in the ovarian stroma and vascular cells. In ovarian follicles, mRNA encoding TGFbetaRII was restricted to thecal cells of type 3  and larger follicles. In addition, expression was also observed in some cells of the surface epithelium and in some stromal cells. In granulosa cells cultured for 6 days, both TGF-beta 1 and 2 decreased, in a dose dependent manner, both the amount of DNA and concentration of progesterone.In summary, mRNA encoding both TGF-beta 1 and 2 were synthesized by ovarian theca, stroma and cells of the vascular system whereas TGF-beta 3 mRNA was synthesized by vascular cells. Luteinizing granulosa cells also responded to both TGF-beta 1 and beta 2 in vitro. These findings in sheep are consistent with TGF-beta potentially being an important autocrine regulator of thecal cell function and possibly a paracrine regulator of ovarian cell function at various development stages. ThreeExcept where indicated, laboratory chemicals were obtained from BDH Chemicals New Zealand Ltd , Invitrogen  or Roche Diagnostics N.Z. Ltd. .Total cellular RNA was isolated from ovine ovary using TRIzol according to manufacturer's instructions. First strand cDNA was produced from total cellular RNA using a poly t primer. Complementary DNAs encoding a portion of the coding sequence of the genes were isolated using standard PCR techniques. For individual cDNAs generated, primer sequences and annealing temperature are given in table ad lib access to pasture and water and lambs were kept with their mothers until just prior to tissue collection. Romney ewes and lambs were killed by administration of a barbiturate overdose . Recovered ovaries were fixed in 4% (w/v) phosphate-buffered paraformaldehyde and embedded in paraffin wax.All experiments were performed in accordance with the 1999 Animal Welfare Act Regulations of New Zealand. All animals had in situ hybridization protocol described previously with minor modifications [in situ hybridizations, 4–6 μm tissue sections were incubated overnight at 55°C with 45,000 cpm/μl (approximately 48,000 dpm/μl) of 33P-labelled antisense RNA. Non-specific hybridization of RNA was removed by RNase A digestion followed by stringent washes (2 × SSC, 50% formamide, 65°C and 0.2 × SSC at 37°C). Following washing, sections were dehydrated, air dried and coated with autoradiographic emulsion . Emulsion-coated slides were exposed at 4°C for 3 weeks, developed for 3 1/2 minutes in D19 developer (Eastman Kodak, Rochester, NY), development was stopped using a 1 minute incubation in 1% acetic acid and slides were fixed with a 10 minute incubation in Ilfofix II (Ilford Limited, Cheshire, England). Sections were stained with hematoxylin and then viewed and photographed using both light and dark field illumination on an Olympus BX-50 microscope . Non-specific hybridization was monitored by hybridizing at least two tissue sections from each age group (lamb and adult) with approximately equal concentrations of the sense RNA for each gene. Hybridization was considered to be specific when the intensity of silver grains, as measured by visual assessment, over a cellular type was greater than that observed in the area of the slide not containing tissue. For all genes, hybridization of the sense RNA over the tissue section was similar or lower in intensity to that observed on the areas of the slide not containing tissue of both the sense and antisense hybridized slides and thus was considered non-specific.Cellular localization of mRNAs was determined using the ications . Sense aClassification of follicles was based on the system outlined by Lundy et al. . BrieflyOvaries were collected from ewes following slaughter at the local abattoir, transported back to the laboratory at room temperature, washed in 3% bleach solution in PBS for 5 minutes, rinsed twice in PBS and stored in Leibovitz media containing 0.1% BSA, 100 U/ml penicillin and 100 μg/ml streptomycin. Follicles approximately 1–2 mm in diameter were dissected away from the ovaries and stored in Leibovitz media until collection of granulosa cells. The granulosa cells were collected by cutting follicles in half followed by manual scraping of cells from the follicular wall using a wire loop. Oocytes and follicular debris were removed from the cells using a micro-glass pipette. Remaining cells were collected by centrifugation at 300 g for 5 min at room temperature, washed once in 5 mls Leibovitz media, twice in 5 mls McCoys media  with 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM GlutaMAX-1 and resuspended using a syringe and needle. Cell viability was determined using trypan blue exclusion and 100,000 viable cells per well  were added in McCoys media containing 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM GlutaMAX-1, 5 ng/ml selenium (Sigma), 10 ng/ml insulin (Sigma), 5 μg/ml apo-transferrin (Sigma), 30 ng/ml androstenedione (Sigma), 3 ng/ml ovine FSH , 1 ng/ml IGF-1  with varying doses (0–10 ng/ml) of purified human TGF-β1 and recombinant human TGF-β2 (R & D Systems, Minneapolis, MN). Cells were cultured at 37°C in a 5% CO2 incubator. Every 48 hours, 200 μl of media was removed from each well and replaced with 200 μl of warmed media that had been prepared at the start of the culture and stored at 4°C. Media samples from the last 48 hours of treatment were collected on day 6 of treatment and frozen at -20°C for later determination of progesterone concentrations by RIA. Unattached cells were removed by 2 washes with McCoys media at 37°C. Attached cells were lysed by incubating cells at 37°C in 100 μl distilled water for 1–2 hours followed by freezing at -70°C. All treatments were performed at least in triplicate with three independent pools of granulosa cells. Within an assay, individual values outside of 20% of the mean value for the treatment were discarded. Points in which at least 2 of the replicates were not within 20% of each other were regarded as missing data. This occurred for the 10 ng TGF-β1 measure of DNA in a single pool of granulosa cells.The amount of DNA present in each well was determined by comparing binding of Hoechst 33258 dye  in samples to calf thymus DNA standard measured with a Wallac 1420 plate reader at 350 nm for excitation and 460 nm for emission. Sensitivity of the assay  was 33 ng per well and the intra- and inter-assay co-efficients of variation (CV), based on variability of the 100, 250, 1000 and 2500 ng standard curve points were 3.9% and 8.8%, respectively. No samples were below the sensitivity of the assay.Concentrations of progesterone in media were determined by RIA as described . The sen6 viable cells per well in 2 mls of control (i.e. no TGF-β) culture media described above for 48 hours. At this time, unattached cells were removed by washing the wells twice with PBS. RNA was collected using TRIzol according to the manufacturer's instructions. First strand cDNA was produced from total cellular RNA using the SuperScript™ preamplification system for first strand cDNA synthesis. An initial PCR was performed with 4 week old ovary RNA to obtain the ovine sequence of a region of the TGFβRII gene which spans introns 4 and 5 in the human sequence (AY675319) and a second set of primers was designed based on the ovine sequence (see table Granulosa cells were collected as described above and either frozen immediately after collection or plated in 6 well culture dishes at a density of 1.0–1.5 × 10Concentration of progesterone per μg DNA was calculated for individual wells before averaging for each treatment within each assay. Points in which at least 2 of the replicates were not within 30% of each other were regarded as missing data. Changes in the concentrations of progesterone in media and DNA content after culture were analysed with the general linear model procedure of SAS. Replicate was included in the model as baseline progesterone and DNA values varied among the granulosa cell pools. Differences between least square means were evaluated using least significant differences and were considered significant when p < 0.05. Data presented are least square means. The standard errors of least square means were 0.7 ng/well, 0.2 μg/well and 0.5 ng/μg for progesterone, DNA and p4 per DNA, respectively.The mRNA for TGF-β1 was not observed in granulosa cells or oocytes of any follicles Figure , table 2The pattern of expression of mRNA encoding TGF-β2 was similar to that observed for TGF-β1, with hybridization limited to the thecal cells of type 3 and larger follicles Figure , table 2Expression of TGF-β3 mRNA was exclusive to cells associated with the vascular system of the ovary. Expression was not observed in the granulosa, theca, or oocyte of any follicle examined Figure , table 2Expression of TGFβRI mRNA was observed in oocytes of all types of follicles Figure , table 2Expression of TGFβRII mRNA was not observed in types 1,1a or 2 follicles Figure , table 2in situ, freshly isolated or cultured granulosa cells expressed mRNA for the TGFβRII when assessed by RT-PCR (Figure Both TGF-β1 and TGF-β2 inhibited progesterone synthesis of cultured granulosa cells, whether expressed as a function of number of cells placed in culture (Figure R Figure .in vitro [In the ewe, expression of TGF-β1 and TGF-β2 mRNA in the follicle was limited to thecal cells during all stages of follicular growth examined. Furthermore, expression of TGF-β3 mRNA was not observed in any follicular cells. This is in contrast to the observed expression patterns for these proteins in other species where TGF-β1 and TGF-β2 have been localized to granulosa as well as thecal cells and sometimes also to the oocytes of many species ,13,31-33in vitro ,11. Thusin vitro .Similar to what has been observed in other species ,34,35, eExpression of mRNA encoding all three TGF-β isoforms and the TGF-β type I and II receptors were observed in cells associated with blood vessels and both receptor types and TGF-β1 and 2 mRNAs were observed in the stroma surrounding follicles indicating a potential role for TGF-β in regulating certain functions in the ovarian stroma and vascular network. TGF-β is known to be important in regulating angiogenesis ,39. MoreThe much more restricted pattern of expression of TGFβRII mRNA in sheep indicates that the TGFβRI may well be involved with other type II receptors in the signalling of other members of the transforming growth factor family. In agreement with this, TGFβRI has recently been shown to be involved in signalling of the oocyte-derived GDF-9 along with BMPRII ,42. In oThe suppression of progesterone production and DNA content in granulosa cell cultures by TGF-β1 or TGF-β2 is similar to inhibitory to mild stimulatory effects observed in bovine, ovine and porcine granulosa cell cultures and contrary to the strong stimulatory effects observed in rodents -18,20-26No differences in the efficacy of TGF-β1 and TGF-β2 were observed in ovine granulosa cells. Similarly, TGF-β1 and TGF-β2 were equally efficacious in stimulating inhibin production in luteinized human granulosa cells  and in min vitro, the lack of detectable expression of TGFβRII mRNA in these cells using in situ hybridization was very surprising. There are several potential explanations for these apparent conflicting results. It is possible that TGFβRII is expressed in ovine granulosa cells and the technique utilized simply failed to detect this message. The detection of mRNA encoding TGFβRII in isolated granulosa cells both before and after culture using RT-PCR would seem to support this assumption. However, it is possible that the isolation and culture of the granulosa cells potentially could be inducing expression of TGFβRII as most all cells in culture express TGFβRII [Given the potent effects of both TGF-β1 and TGF-β2 on granulosa cell function  TGFβRII . In addi TGFβRII  and cell TGFβRII .Expression of mRNAs encoding TGF-β1 and TGF-β2 as well as both type I and II TGF-β receptors were observed in the theca of small growing follicles indicating that TGF-βs may be regulating thecal cell function in an autocrine manner. Expression of mRNA encoding TGF-β type I and II receptors is also observed in luteal cells, stroma, the vascular system and surface epithelium suggesting that TGF-βs may also regulate other cell types in the sheep ovary. Since granulosa cells showed no evidence of expressing any of the TGF-β ligands and expression of the TGF-β type II receptor was equivocal, it seems likely that any TGF-β effects in granulosa cells in vivo are due to paracrine or endocrine actions and possibly regulated through an alternative type II receptor.in situ hybridizations and PCRs. SL and KLR performed the granulosa cell bioassays including progesterone and DNA measurements. JLJ and KPM designed and co-ordinated the experiments, performed statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.AHB, LDQ and LJH cloned the ovine TGF-βs and receptors, completed sequencing projects and alignments, and performed the


A survey for the year 2003 of significant developments in Australia's official international health relations, and their domestic ramifications, is presented. The discussion is set within the broader context of Australian foreign policy. Sources include official documents, media reports and consultations with officers of the Department of Health and Ageing responsible for international linkages. Australia's health relations with other nations in the field of health constitute an important sub-set of health policy not only because of the intrinsic significance of bi-lateral and multilateral linkages, but also because of their ramifications for health policy at the domestic level.In broad terms, these health relations encompass a range of interactions with consequences for health, including: membership of global and regional bodies; the negotiation of international agreements; action to counter particular external threats to health; assistance to developing countries; and international trade and investment in health-related goods and services. In 2003 there were continuing developments in all these areas within a wider foreign affairs context overshadowed by official policy concerns about global and regional security, the deployment of the Australian armed forces in various theatres of service, and renewed fears of the human and economic costs of infectious diseases. Balancing these concerns with national defence were renewed efforts to forge bi-lateral trade links in global trade environment characterized by the emergence of trade blocs centred in North America, Europe and Southeast Asia.Although consultation occurs with states and territories, it is the Australian Government that is constitutionally responsible for conducting Australia's international relations. These responsibilities include appointing representatives to international bodies and organizations, such as the United Nations and its various agencies, including the World Health Organization and assenting to agreements and regulations promulgated by international agencies. The formulation and implementation of policy with direct or indirect international health ramifications is not centralized, but is usually the result of consultations between various relevant government departments and statutory authorities.An important element of the Australian Government's foreign affairs powers relates to international treaties. While a degree of consultation with state and territory governments and with the public occurs, and the national parliament is able to scrutinize and comment upon international treaties, it is the executive that has the final decision on such agreements.In 2003 Australia continued to play a strategically important and respected role in international organizations concerned with health, especially the World Health Organization. At the World Health Assembly, the governing body of the WHO, the Australian delegation supported resolutions concerned with strengthening nursing and midwifery and child and adolescent health. In the wake of SARS, Australia also supported the review of the International Health Regulations and is likely to subscribe to them . The volThe Department of Health and Ageing was closely involved with international comparative health data projects including WHO's World Health Survey and the health systems performance survey of the Organization for Economic Cooperation and Development. Australia's participation in the health mandate of the Commonwealth of Nations was illustrated by the Therapeutic Goods Administration's provision of a secretariat for the Clearing House of Commonwealth Agencies for Chemical Safety. Australia also participated in the meeting of Commonwealth health ministers on the eve of the annual World Health Assembly of WHO in Geneva .In December Australia signed the Framework Convention on Tobacco Control (FCTC) the first multilateral treaty negotiated under the auspices of the World Health Organization. For the first time, nations were invited to implement control measures covering such issues as health warnings, advertising, packaging and labelling, sales, and smuggling. They were also called upon to embrace policy measures designed to counter the global tobacco epidemic . The FCTA reciprocal health care agreement with Norway was signed, further expanding the rights of Australian residents to immediate and necessary treatment in the national health systems of countries with which Australia has reciprocal treaties. These include New Zealand, UK, Italy, Malta, Holland, Sweden, Finland, and the Republic of Ireland. These arrangements are "cost neutral" and do not include costly accounting or administrative procedures. In terms of domestic policy, the continuing "internationalisation" of Medicare (pioneered by the Hawke Labor Party ministry at the time of Medicare's introduction) by the Liberal-National Party Coalition is paradoxical since local citizens are being encouraged to opt out of public hospital treatment through a rebate on private health insurance and penalties for higher income earners who do not insure privately. Whilst these agreements have cemented closer diplomatic ties, their potential benefits to international travellers, especially those subject to punitive insurance premiums or the refusal of insurance due to old age or infirmity, remain inadequately publicized. Treaties are also being negotiated with Denmark and Belgium.Following years of negotiations and planning, a treaty was signed with New Zealand establishing a single joint therapeutic goods agency. This body, due to commence operations in 2005, will regulate prescription and retail drugs, therapeutic devices and also complementary medicines. It will replace the Australian Therapeutic Goods Administration and its New Zealand counterpart. To a large extent, the two regulatory systems will have been integrated, although there are still areas of disagreement (e.g. policies on the advertising of PBS medicines) which will need to be negotiated. This joint agency creates a model in international health relations which other states could profitably emulate where they share common concerns and have similar health systems. In December 2002 the two countries finalized treaty arrangements establishing both a joint standards code and a joint statutory authority, Food Standards Australia New Zealand . These aThese developments have furthered Australian foreign policy concerned with establishing trans-Tasman free trade, commenced some two decades ago with the negotiation of the Closer Economic Relations agreement with New Zealand. The new regulatory arrangements have created a virtual trans-Tasman free market in food  and therapeutic drugs.While not having the legal status of a treaty, for some years the Department of Health and Ageing has had memoranda of understanding with its counterparts in China, Indonesia, Thailand and Japan. In 2003 further activities were undertaken under the auspices of these agreements. During the state visit of China's president Hu Jinta, a plan of action was signed between the two health ministries. The Indonesian relationship continued with the inclusion of a health delegation to the Sixth Australia Indonesia Ministerial Forum in Jakarta in March, preceded by two rounds of meetings between officials of the Indonesian and Australian health departments. The Australia-Japan Partnership in Health and Family Services formed the basis for negotiations for joint research on mental health and an international conference on suicide prevention . In a reIn the course of 2003 Australia finalized free trade agreements  with Singapore and Thailand and continued negotiations with the USA . From thThe free trade agreement with the USA raised controversies about attempts to include the Pharmaceutical Benefits Scheme (PBS) in concessions demanded by US negotiators. These issues have been outlined in the account of developments in the PBS elsewhere in this series of review articles.The emergence and rapid spread of Severe Acute Respiratory Syndrome (SARS) to several countries in East and Southeast Asia and to Canada revived popular atavistic fears of pandemics and damaged the tourism and travel industry, as well as some Australian suppliers of goods and services to Asia. WHO issued a global alert on the disease in March, and the last reported case of international occurred in July. So serious was the threat of SARS to the economies of some countries that a special meeting of health ministers, attended by Australia, was organized by Asia-Pacific Economic Cooperation (APEC) to discuss the situation. A task force was subsequently established by APEC to deal with SARS. An example of the economic costs of the disease was the decision by the Governments of Singapore and Australia to postpone negotiations on a greater share of the Sydney-Los Angeles air route (dominated by QANTAS) for the Singapore carrier, due to uncertainty about demand.In Australia SARS was declared a quarantinable disease under the Quarantine Act 1908 and policy guidelines for health professionals, airline and border control staff and the general public were developed by the Department of Health and Ageing, which also led an inter-departmental task force to monitor world developments. Until July 2003, when the WHO announced that no country was still considered SARS-affected, international aircraft arriving at Australian airports were required to obtain "SARS-free" clearance, nurses were posted at airports and restrictions on elective surgery were placed on travellers returning from affected countries . During Australia's official international development assistance programme is an important foreign policy tool, especially in the Asia-Pacific region. Some $225 m.  was allocated to health-related international development assistance in 2003–4 budget of the Australian Agency for International Development (AusAID). However, while Australia's contribution to HIV/AIDS control and its regional advisory role associated with SARS were acknowledged by the Foreign Affairs Minister in his report to Parliament, health assistance received little prominence. Security, good governance and counter-terrorism were emphasized as the focus for the official foreign aid programme. Support for essential services in Papua New Guinea continued as a major imperative [Medicare Plus policy initiatives [volte face from previous policies deliberately designed to discourage foreign doctors from immigrating in the belief that controlling the number of doctors would contribute to cost-containments of Medicare. It also continues to raise the ethical danger of Australia contributing to a "brain drain" of medical staff from countries that are themselves short of such expertise. In 2002 the Commonwealth of Nations had agreed to a code of practice for the international recruitment of health workers to help safeguard the interests of developing nations. Australia has endorsed the code.The fact that the domestic health workforce is now part of a global market for skilled workers was further demonstrated by continuing efforts to recruit nurses from overseas, the decision of the Australian Health Ministers Conference to sanction dentists from selected Commonwealth countries to work in public clinics. In addition, a scheme to recruit overseas-trained medical practitioners was included in the Australian Government's tiatives . It is iThe Australian Government will need to handle policies associated with the recruitment of overseas-trained health personnel with care due to professional sensitivities and the need for legislation at the state level to regularize the status of some professions.This brief review of Australia's international health relations in 2003 has demonstrated that health must be seen as an integral part of trade and security within the wider foreign policy context. The protection of health in free trade arrangements is important for their domestic legitimacy. It is vital that those involved in health policy are aware of its potential international dimensions, while those responsible for foreign affairs include health in their approach. Official health linkages have served to promote good will in some otherwise difficult relationships, as has been the case with Indonesia. They have also helped to promote a positive international image for Australia.The opinions expressed in this article are the sole responsibility of the author.


Acidithiobacillus ferrooxidans. There are a few studies about the benefit of metals of low value through bioleaching. From all of these, there are almost no studies dealing with complex minerals containing arsenopyrite (FeAsS). Reduction and/or elimination of arsenic in these ores increase their value and allows the exploitation of a vast variety of minerals that today are being underexploited.Bioleaching is a process that has been used in the past in mineral pretreatment of refractory sulfides, mainly in the gold, copper and uranium benefit. This technology has been proved to be cheaper, more efficient and environmentally friendly than roasting and high pressure moisture heating processes. So far the most studied microorganism in bioleaching is 4) formed is virtually insoluble and remains in the solids. The influence of seven factors in a batch process was studied. The maximum rate of arsenic dissolution in the concentrate was found using the following levels of factors: small surface area of particle exposure, low pulp density, injecting air and adding 9 K medium to the system. It was also found that ferric chloride and carbon dioxide decreased the arsenic dissolution rate. Bioleaching kinetic data of arsenic solubilization were used to estimate the dilution rate for a continuous culture. Calculated dilution rates were relatively small (0.088–0.103 day-1).Arsenopyrite was totally oxidized. The sum of arsenic remaining in solution and removed by sampling represents from 22 to 33% in weight (yield) of the original content in the mineral. The rest of the biooxidized arsenic form amorphous compounds that precipitate. Galena (PbS) was totally oxidized too, anglesite  of arsenic removal. Further studies are needed to determine other factors that influence specifically the solubilization of arsenic in the bioleaching system such as: pH, dissolved oxygen concentration, redox potentials, nature of concentrate and temperature among others.  When this happens, the metal is extracted into water [Acidithiobacillus ferrooxidans (At. ferrooxidans, previously Thiobacillus ferrooxidans), a gram-negative, acidophilic, chemolithoautotrophic, non-spore forming rod. Nutritionally, typical At. ferrooxidans isolates are considered obligate autotrophs. At. ferrooxidans is able to use either ferrous iron or a wide variety of reduced inorganic sulfur species as an electron donor compounds. It is also able to grow using ferric iron as an electron acceptor, provided that an electron donor, such as reduced inorganic sulfur compound is present. Energy is derived from the oxidation of reduced iron and sulfur compounds, including ferrous ion, sulfide, elemental sulfur and thiosulfate, with final oxidation products being ferric ion and sulfate [Acidithiobacillus thiooxidans, Acidithiobacillus caldus, Leptospirillum ferrooxidans, and Acidiphilium acidophilum [The term bioleaching refers to the bacterial conversion of an insoluble metal  into a soluble form , disseminated in a pyritic matrix. Besides of lead, zinc and copper as valuable metals, such deposits may contain significant quantities of silver, gold, arsenic, antimony, bismuth and mercury. Numerous economically important deposits of these ores exist in the world [A complex sulfide ore is an association of galena (PbS), sphaelerite (ZnS) and chalcopyrite  reported to inhibit bacteria to a greater degree than As(V) .The processing of complex arsenic bearing ores and concentrates requires a good understanding of the mechanisms involved previous to the design and development of an adapted sequence of process units. In particular, a complete chemical and mineralogical characterization of the ore is essential from both biological and metallurgical points of view in order to determine the possible inhibition problems and the requirement for effluent treatment processes .At. ferrooxidans in gold-arsenic concentrates. This study established the economic feasibility of this technology and its potential for alleviating some environment related problems [The interest in the study of bioleaching of arsenic-containing minerals, started two decades ago with a publication dealing with the degradation of arsenopyrite (FeAsS) through problems . Later, problems  studied There have been other studies aimed to determine the influence of several factors over the arsenic bioleaching in gold minerals and concentrates. These studies have been mainly focused to maximize gold extraction and considered bioleaching as a pretreatment in the cyanidation process, consequently leaving the importance of arsenic dissolution and extraction in a second term.et al [At. ferrooxidans and At. thiooxidans. They achieved an increase in gold extraction from 55.3% to 96.8% using the following conditions; pulp density 20%, pH 2, stirring conditions 200 rpm, temperature 30°C, time 7 days. Another research group used a domestic strain of At. ferrooxidans and a gold refractory mineral to study the influence of a magnetic field, surfactant addition and the presence of Ag+, Bi3+, Co2+ y Hg2+ ions over the bioleaching process [+ ion [Ubaldini et al  studied  process . They fos [+ ion .As indicated above, previous studies are focused in using bioleaching as a technique to eliminate arsenic in high-value minerals such as gold concentrates. However, there is no reported work on arsenic elimination or reduction in minerals of low value such as lead.This work is aimed to reduce the arsenic content in complex concentrates of galena and to generate preliminary data to allow us to conduct further studies to understand the complex behavior of the bioleaching process. This preliminary study deals with the influence of main factors in batch bioleaching over the arsenic solubilization from a complex lead concentrate.7–4), and they were: surface area, pulp density, carbon dioxide bubbling, air bubbling, 9 K medium addition, FeCl3 addition, and two different At. ferrooxidans strains.The main factors influencing the biooxidative treatment were tested using a two level fractional factorial plan of experiments  and AAS Hydride System for arsenic was: 3.85% As, 14.75% Fe, 23.53% S, 49.76% Pb, 2.48% Zn, 0.51% Sb, 0.29% Cu, 0.28% Bi. The remaining 4.56% being other elements such as: Cd, Ca, Ag, K, Mn, Na, Ni, Ba, Mo, Sn, Si, O.Table Photographs of prepared mounts Figures , 2 and 3At. ferrooxidans have provided great amount of basic knowledge about this process and have been useful in the understanding of the physicochemical and microbiological aspects of this phenomenon [Studies dealing with bioleaching through enomenon ,22.Biooxidative dissolution of arsenopyrite ,23-29 anThere are only a few of studies dealing with arsenic-bearing ores and concentrates of natural sources ,14,31,32The concentrated used in this study was highly complex as can be seen in Figures 4). Chemical analysis performed through AAS resulted in the presence of residual arsenic. This indicates that all crystalline species present in the concentrate were completely oxidized through the bioleaching process. This means that all arsenic present in arsenopyrite was either solubilized (22 to 33%) or precipitated in amorphous compounds (67 to 78%). Also, galena (PbS) was completely oxidized to anglesite. Only a very small portion of anglesite remained in solution 2(OH)6 and scorodite FeAsO4·2H2O [Formation of precipitates of arsenic and iron has been commonly observed with the use of ooxidans . SeveralsO4·2H2O ,25-27.+3 ions in the indirect oxidation, inhibiting the leaching of the mineral species [0 can be avoided by the use of bacterial consortia in which sulfur-oxidant bacteria such as At. caldus [At. thiooxidans are included [It has been reported that the elemental sulfur and ferric arsenate are accumulated in the surface of the grains forming a coating barrier that avoids the contact between the mineral and the Fe species ,27. The  species , while t. caldus  or At. tIn this study the precipitation of amorphous arsenic compounds was important during the bioleaching process. In samples taken at the end of the experiment there was a difference in the arsenic content in the digested mineral residue, which was treated by a hydrochloric acid digestion, to the non-digested residue :Table Arsenic = 52.0 - 66.3 Pulp Density - 32.6 Surface Area - 21.1 Ferric chloride - 12.5 Carbon dioxide + 29.3 Air + 29.3 9 K Medium + 3.00 Strain + 3.94 Days.     (1)2 = 0.834With a determination coefficient of R3 and CO2 addition, air and 9 K Medium addition and any strain present.The negative sign in some of the variables of the model indicates that in order to maximize bioleaching of arsenic, these factors must be kept in low levels. These were denoted with a zero value in the model. Thus, the levels of factors that should be considered in the model are the following: pulp density at 10%, low surface area, no FeClOne of the main disadvantages for the commercial use of bioleaching is the slow nature of this process, which is due mainly to the relative slow growth rate of the bacteria ,35, to tIn Figure The graphical pattern of the arsenic bioleaching for Run 1 Figure  shows soThe data for this run (Run 1) were fitted through a third order polynomial linear regression model and statistically analyzed over a 95% confidence interval Arsenic = 100.16 + 11.487 Days - 0.4705 Days2 = 0.6585With a determination coefficient of RBesides of all these convenient features in Run 1, the percentage of bioleached arsenic is still low. Proper conditions of solubilization of arsenic during bioleaching are key feature to improve the percentage (yield) of arsenic removal. Therefore, further studies are needed in order to determine the other factors (not considered in this work) that influence specifically the solubilization of arsenic in the bioleaching system such as: pH, dissolved oxygen concentration, redox potentials, nature of concentrate and temperature among others.When time (days) was used as the only independent variable, data collected in all runs were adjusted to a third order polynomial regression model and the analysis is shown in Figure 2 - 0.0131 Days3     (3)Arsenic = 23.888 - 1.0597 Days + 0.5161 Days2 = 0.3237, which represents a value relatively low due to the absence of the other significant factors (pulp density, CO2, 9 K medium, particle size, air injection and ferric chloride added).The determination coefficient of this model was RIt is worth to note that this model resembles the well known general three-phase bacterial growth model.The pulp density effect is shown in Figure In the case of arsenic, it is possible that a high pulp density may cause a greater interaction between the dissolved arsenic and other products of biooxidation. Thus, increasing the rate of formation of precipitates and consequently decreasing the concentration of arsenic in solution.In our study it was found a strong interaction between the pulp density and the air injection with respect to the arsenic removal. The ANOVA analysis of this interaction is shown in Table The effect of surface area (particle size) can be observed in Figure A possible explanation for the behavior observed in this study is based in the great complexity of the mineral concentrate that may enhance the specific interactions. These interactions are affected by the particles of smaller sizes where the greater surface area leaves each particle exposed to a greater amount of different mineral species. These species increase the complexity of the medium thus affecting directly the solubilization of arsenic during the bioleaching. The complexity of the medium due to small size particles can be further increased when a high pulp density is used as pointed out in the previous section. These two issues, complexity and pulp density, combined in the conditions above mentioned may produce a negative effect over the arsenic dissolution.At. ferrooxidans and /or At. thiooxidans [In the present study the effect of addition of ferric chloride during the arsenic bioleaching resulted in a decrease of the solubilized arsenic in the system as observed in Figure ooxidans -47.At. ferrooxidans require CO2 for its growth. This is due to the fact that these bacteria are able to use CO2 as its only source of carbon for their biosynthetic reactions. CO2 fixation is usually achieved through the so-called Calvin-Benson-Bassham cycle [2 is bubbled into the system. This behavior is in agreement with studies reported in literature that established that the consumption of carbon by the bacteria decreases at high CO2 concentrations implying the formation of intracellular bicarbonates [2 present in the air is enough to withstand the bacterial growth [It is well known that autotrophic organisms such as am cycle . In Figurbonates . Other al growth ,25.2 and air into the system. The ANOVA analysis of this interaction is also shown in Table 2 according to the results shown in Figure 2 on the dissolution of arsenic. Therefore, further studies oriented to establish the relationship between the dissolved oxygen and the concentration of CO2 in the system are needed in order to elucidate this effect.In our study, it was found a strong interaction between the two levels of COAcidithiobacillus ferrooxidans is an aerobic microorganism that uses oxygen as a final electron acceptor during the oxidation process. However, in absence of oxygen it is still able to grow in the presence of inorganic reduced sulfur compounds by using the ferric ion as an alternative electron acceptor [2 is admitted into the system. Again a more definite study targeted to measure dissolved oxygen in the system may bring light into the effect of air during the arsenic bioleaching.acceptor . In thesacceptor . In the The 9 K medium is the source of nitrogen and phosphorous  needed fAt. ferrooxidans strains used in this study are shown. Strain T1 presents the common bacterial model of growth in three stages, while strain T18 presents a straight model that requires less adaptation time. This can be due to the fact that T18 was previously grown in systematically increased arsenic concentrations as discussed in the material and methods section of this paper.In Figure Data generated from the batch arsenic bioleaching were used as a first approximation for the calculation of the dilution rate D for a future design of a continuous bioleaching system. The procedure used consisted in to derive the third order polynomial equations (2) and (3) with respect to time and plot each equation versus the arsenic concentration in the system. Then to draw a straight line from the origin to the maximum point in each curve Figure . The slowhereF = Rate of medium flow through the vessel (volume/time)V = Volume of the vessel (volume)D = F/V Dilution rate (time-1)Pn-1 = Inner product concentrationPn = Outer product concentrationdPn/dt = Total variation of the product concentration in the vesseldPn/dt)production= Product concentration variation due to the production in the vessel(Rearranging Eq. (4):dPn/dt = 0 and assuming that Pn-1 = 0 in a single vessel,In steady state, From whichEquation (7) represents a straight line through the origin having a slope of D (dilution rate) and corresponds to the straight line plotted in Figure -1, while for run 1 in equation (2) is of 0.103 days-1 (not shown in the plot). However, these dilution rates are still relatively small compared to the dilution rates used in industrial scale processes (0.05–0.6 h-1) [Using this method, the calculated dilution rate for the general model of equation (3) is 0.088 days0.6 h-1) .Since the bioleaching rate and the levels of leached arsenic are limited , proper conditions of solubilization of arsenic during bioleaching is the key feature to improve the percentage (yield) of arsenic removal. Therefore, further studies are needed in order to determine the other factors (not considered in this work) that influence specifically the solubilization of arsenic in the bioleached system such as: pH, dissolved oxygen concentration, redox potentials, nature of concentrate and temperature among others.At. ferrooxidans) was able to completely oxidize the minerals present during the arsenic bioleaching. Since, galena (PbS) was completely oxidized to anglesite (PbSO4) with only a very small portion of anglesite remaining in solution, while the main phase of this appeared as a solid precipitate. Other elements present originally in the concentrate such as Zn, Sb, and Cu were also solubilized.The performance of the bacteria used in this study .The increase in pulp density generates a decrease in the dissolved arsenic concentration. However, this decrease is greater in runs where air was not injected to the system.The maximum rate of arsenic dissolution in the concentrate was found using small surface area of particle exposure, low pulp density, injecting air and adding 9 K medium to the system.The effect of addition of ferric chloride during the arsenic bioleaching resulted in a decrease of the solubilized arsenic in the system.2 according to the results is associated to the decrease in arsenic dissolution. Further studies oriented to establish the relationship between the dissolved oxygen and the concentration of CO2 in the system are needed in order to elucidate this effect.The presence of CO2 is admitted into the system. A study using dissolved oxygen measurements is needed in the future to determine the effect of air during the arsenic bioleaching.Arsenic dissolution was increased with the aeration of the system. The availability of oxygen in the medium decreases when the pulp density increases and also when COThe flotation concentrate was obtained from La Soledad mine . Chemical analysis was carried out by atomic absorption spectrometry through AAS (GBC Avante Σ), arsenic was determined by AAS Hydride System. The major phases in the concentrate were determined by X-ray diffraction (Siemens D5000). Mineral samples were mounted in polyester resin blocks using approximately 0.2 g per mount and surface was polished. Mounts were examined using a microscope (Olympus AX70) and photographs were taken at various sites of each sample.k factorial series. In this series, there are k factors each at two levels. Hence there are a total of 2k treatments in the full factorial set. This series is particularly useful in the exploratory stages of an investigation because it permits the examination of a fairly large number of factors and their interactions in a trial of reasonable size [One of the most useful types of multifactor experiment is the 2ble size .In almost all experiments the investigator would like to reduce the number of observations required for a complete factorial. If certain assumptions can be met the use of fractional factorials is a most efficient technique to reduce the number of observations and still obtain the desired information. The usual fractional factorial is still orthogonal, which means that certain effects are estimated independently of one other .k design containing 2k-p runs is called a 1/2p fraction of the 2k or, more simply, a 2k-p fractional factorial design. It is possible to construct this type of designs for investigating up to k = N-1 factors in only N runs. If k = N-1 the fractional factorial design is said to be saturated. Of particular importance is a very useful saturated fractional factorial design for studying seven factors in eight runs; that is, the 27–4 design. This design is a one-sixteenth fraction of the 27 [A major use of fractional factorial designs is in screening experiments. These are experiments in which many factors are considered with the purpose to identifying only the important variables that affect the response and their interactions. The factors that are identified as important are then investigated more thoroughly in subsequent experiments. A fractional factorial of the 2f the 27 .This was the design used in this work to test the factors influencing the biooxidative treatment. The objective was to maximize the arsenic solubilization. The factors, levels and runs are presented in Table The bioleaching experiments were carried out using two solid concentrations. The low level for pulp density was settled as 10 % w/v; the high level as 20% w/v.μm sieve in order to obtain two fractions. Both of them were dried and analyzed for specific surface area by a laser scattering particle sizer . The specific surface area for fractions >75 μm and <75 μm were 0.42 and 1.65 m2/g and were established as low and high levels respectively.The lead concentrate was washed with distilled water; the mineral suspension was wet sieved using a 75 Addition of ferric chloride at concentration of 150 mg per liter of liquid medium was settled as high level, no ferric chloride addition was low level.-1.Low level: no carbon dioxide bubbling; high level: carbon dioxide flow from a compressed cylinder, injected into the mixture at a rate of 0.2 vol/vol min-1 was high level.No air bubbling was considered as low level; no sterile air bubbles injected into the liquid-concentrate mixture at a rate of 0.3 vol/vol min4)2SO4, 0.5 g of MgSO4·7H2O, 0.1 g of KCl, 0.5 g of K2PO4, 0.01 g of Ca(NO3)2 was settled as high level. The pH was adjusted to 5.7 with sulfuric acid.Pure distilled water as culture medium was established as low level; the use of 9 K medium , which cAcidithiobacillus ferrooxidans wild type strain termed T1 [-1 [4, 3.0 g (NH4)2SO4, 0.5 g KH2PO4, 0.5 g MgSO4· 7H2O, 0.1 g KCl, 0.01 g Ca(NO3)2 per liter, adjusted to pH 2.0 with sulfuric acid. After bacterial growth for ten days, cultures were filtered and the clear liquid was used as inoculum (20% v/v).A native ermed T1 , isolated T1 [-1 . The strThe eight biooxidation runs Table  were conAt the end of the experiment the resulting pulp in each run was filtered and the bioleached mineral was washed with distilled water and dried in a stove at 40°C. Two grams of this material were taken and were submitted to digestion using 10 ml of HCl 0.6 N during 3 hours at room temperature. The digested mineral was filtered, washed with distilled water and dried at 40°C. In both cases the arsenic content was determined through chemical analysis.Table Arsenic = 0 β+ 1 βPulp Density + 2 βSurface Area + 3 βFerric chloride + 4 βCarbon dioxide + 5 βAir + 6 β9 K Medium + 7 βStrain + 8 βDays.     (8)WhereArsenic Expected value of arsenic concentration in mg l-1 in leachate0β, 1β,... 8 βRegression coefficientsAs the experiment was monitored each 48 hours, and arsenic concentration was determined in leachate, arsenic dissolution data gathered during the bioleaching experiment were analyzed as time series (time as independent variable) by multiple regression to fit the third order polynomial model:Arsenic = β0 + β1t + β2t2 + β3t3     (9)WhereArsenic Expected value of arsenic concentration in mg l-1 in leachatet Time in days of bioleaching0β, 1β, 2β, 3 βRegression coefficientsAnalytical procedures and graphing was performed using MS Excel or Minitab 13.0MM designed and performed the experimental runs and prepared the manuscript. ME performed the statistical analysis. BP provided the bacterial strains and helped in the culture production. AL performed the technical revision of the manuscript. EO got the funds and provided the tutoring during the development of this work. All authors read and approved the final manuscript.