Scutellospora calospora (Nicol. & Gerd.) Walker & Sanders; Glomus sp.; and Acaulospora laevis Gerdemann & Trappe associated with Trifolium subterraneum L. The model and findings are expected to contribute to the quantification of the role of AM fungi in plant mineral nutrition and the interpretation of different foraging strategies among fungal species.In order to quantify the contribution of arbuscular mycorrhizal (AM) fungi to plant phosphorus nutrition, the development and extent of the external fungal mycelium and its nutrient uptake capacity are of particular importance. We develop and analyse a model of the growth of AM fungi associated with plant roots, suitable for describing mechanistically the effects of the fungi on solute uptake by plants. The model describes the development and distribution of the fungal mycelium in soil in terms of the creation and death of hyphae, tip–tip and tip–hypha anastomosis, and the nature of the root–fungus interface. It is calibrated and corroborated using published experimental data for hyphal length densities at different distances away from root surfaces. A good agreement between measured and simulated values was found for three fungal species with different morphologies:  Arbuscular mycorrhizae are mutualistic symbiotic associations between plant roots and specific soil fungi and are formed by more than 90% of all terrestrial plants . They maIn spite of the worldwide occurrence of the AM symbiosis, mathematical models on mycorrhizae are scarce. In order to quantify the increased surface area provided by the fungal mycelium and its effect on nutrient uptake, it is necessary to quantify the dynamics of the external fungal mycelium. A first model to quantify the contribution of the external fungal mycelium to plant phosphorus uptake was developed by et al. .We now consider the general functional form of r scheme . The numδ=d/b. This dimensionless parameter represents the ratio of hyphal death rate to tip branching rate. If δ≪1, hyphal death rate is negligible compared with tip branching and biomass is accumulated in the centre of the colony (a). On the other hand, if δ≫1, tip branching is negligible compared with hyphal death and biomass is significantly reduced in the centre of the colony and the peak of colony biomass moves away from the root surface. This means that, when δ is large, the front of the colony is the most active region even if we assume that hyphae take up nutrients along their entire length. The value of the parameter δ is different for different fungal species as we show in In this paragraph we discuss the effect of the dimensionless parameters derived in appendices A and B on the model behaviour. We show in e colony a. On theρT is given byb shows that the total hyphal length of the colony increases exponentially with time, damped by a factor dependent on the value of the dimensionless parameter δ. The smaller the parameter δ, the larger the total colony biomass.Integrating the solution for the hyphal length density over the colony length, we obtain an equation for the total colony biomass. At any given time, the overall hyphal length α1 on the hyphal length density. If α1 is small, the result is close to the linear branching model, as expected. The parameter δ was set equal to 1, so that the curve in α1=0, is the same as the curve for δ=1 in figure 2a. If α1 is large, the production of new tips is reduced when the tip density is large. α2. When it is small, tip–hypha anastomosis is negligible, and the result is again similar to the linear model. As α2 increases, the production of new tips and hyphae is increasingly inhibited when both tip and hyphal length densities are large. A typical feature of tip–hypha anastomosis when compared with tip–tip anastomosis is a local peak near the front of the colony.We now consider the general model that includes tip–tip and tip–hypha anastomosis. In Scutellospora calospora (Nicol. & Gerd.) Walker & Sanders; Glomus sp.; and Acaulospora laevis Gerdemann & Trappe associated with clover (Trifolium subterraneum L.); these data appear suitable for comparison with modelled hyphal length densities. Hyphal length densities were measured at different times and distances from a cylindrical root compartment in a soil that initially had a small mycelium density. Plants with a previously established mycorrhiza were transferred to a two-compartment system, where root growth into a hyphal-only compartment was restricted by a fine nylon mesh. The root compartment was a cylinder with a diameter of 6 cm. The spread of external hyphae from the root compartment into the soil was measured at different distances after 0, 7, 14, 28 and 47 days. The measured values represent the total hyphal length density of mycorrhizal and other soil fungi. A control experiment conducted without mycorrhizae allowed for the estimation of the length densities of mycorrhizal fungi. At 3 cm and 7 days and 2 cm and 14 days, no control values were available. We estimated them by linearly interpolating the neighbouring values to be 1.06 and 2.00 m g−1, respectively. The data are presented in units of m g−1 dry soil. In order to convert them to units of cm cm−3 soil volume as required by the model, we need the soil bulk density (SBD). From the detailed information about the size of the hyphal compartment and amount of soil used, we estimated the SBD to be 1.02 g cm−3. AM fungi are obligate symbionts. Therefore, measurement of fungal growth can only be made in the presence of plants and is difficult. Experimental data for calibrating the spatial and temporal explicit models are scarce. v come from the adaptation of −1 for S. calospora, 0.07 cm d−1 for Glomus sp. and 0.31 cm d−1 for A. laevis. The guess values for the initial length densities at the root surface were taken to be the measured initial length densities of the three fungal species. Our initial estimate for the death rate d is motivated by v is the tip elongation rate and Δ is the distance between branches. According to Glomus mosseae. Assuming that they are equally spaced, distances between branches range from 0.115 to 0.132 cm. Using these values and our initial guesses for v, our estimates for bn range from 0.53 to 2.70 d−1. To determine an initial estimate for the hyphal tip density initially present at the boundary, we take the measurements of the hyphal length density inside the root compartment to represent the value at the interface between root and fungus compartment. We compute the hyphal tip density from a known value of the hyphal length density as follows. The data show that the hyphal length density at the root–fungus interface increases with time. Following the boundary condition ρb0, is the hyphal length density initially present at the root–fungus interface and a, the reciprocal of maximum tip density 1/nmax and the two anastomosis rates α1 and α2 to zero because these parameters represent processes not encountered in all fungal species.For model calibration, we take starting estimates for the model parameters from published data and try to fit both the linear and nonlinear models to measured hyphal length densities. Our initial estimates of v was fitted first since it is the only parameter that determines the extent of the colony at any given time. Then, the parameters b, d, nmax, α1 and α2 were varied in order to get the correct colony shape. Afterwards, these values were used as initial guesses for the Matlab function ‘fminsearchbnd’ in order to find the parameter set that minimizes the root mean squared error (RMSE) between measured and calculated hyphal length densities. This optimization algorithm uses the Nelder–Mead simplex method , while no significant tip–hypha anastomosis occurs. The net branching rate b is large while fresh supply from the root surface is relatively small. The dimensionless parameter δ=0.16 is small, which means that hyphal death is negligibly small. The values for v and d that produced a good fit to the measured data are approximately two times larger than the initial guess values while the branching rate is within the initially estimated range. Simulated values slightly underestimate measured values in the first few days of the experiment, which indicates that there might be additional relevant processes not yet included in the model.The linear branching model was not satisfactory for the other two fungal species,  outlier . When thAcaulospora laevis. According to the parameter values in α2 describing tip–hypha anastomosis in relation to tip branching to be 2.50. The dimensionless parameter α1 is small, hence nonlinear branching/tip–tip anastomosis is negligible. The dimensionless parameter δ is small, hence hyphal death is negligible, and the large value for the parameter a indicates that there is a substantial supply of new tips at the root surface. The value for v that produced a good fit to the measured data is approximately the same as the initial guess value based on S. calospora, indicating that nonlinear branching and both kinds of anastomosis are negligible for this fungal species. We also found that it was necessary to adapt the original model equations for the two other fungal species considered. The nonlinear branching/tip–tip anastomosis model could be satisfactorily fitted to the data for Glomus sp. and the one including tip–hypha anastomosis performed well for A. laevis.We have provided analytical and numerical solutions to a growth model of the external mycelium of AM fungi. We adapted the model of S. calospora imply that the fungus is mainly growing and allocating resources for getting a wider catchment area rather than local scavenging, for example, for mineral resources, via branching. Thus, for this fungus, the intraradical mycelium is very important for the function of its extraradical mycelium. Indeed, Glomus sp. imply that local scavenging for resources is important for this fungus as long as the hyphal tip density is small. Reaching near the maximum tip density, branching ceases and no more carbon resources are allocated for branching in the respective area. For saprophytic fungi, such local scavenging for resources is important (A. laevis imply that this fungal species develops a network of interconnected extraradical hyphae via tip–hypha anastomosis. This suggests that nutritional flow between hyphae is important for this fungal species. Note that we consider only anastomosis between hyphae from the same host plant. The formation of anastomosis between AM extraradical mycelium developing from different plants supports the creation of a large interconnected net between plants (Symbioses between a given host plant and different AM fungi have been shown to differ functionally . Such fumportant . The parv are up to three times larger than the rates of speed measured by v in the model is a hyphal-scale parameter. The rates of spread estimated by In Further development of the fungal growth model would consider a more sophisticated description of the extra- and intraradical mycelium.


Hyperplastic and neoplastic lesions of the liver occurring “spontaneously” in C3H × Y male mice were transplanted autologously and isologously. Hyperplastic lesions did not survive in the same animal or in other animals of the same strain. Carcinomas grew in the host or in isologous mice and killed the animals. Most of the lesions were hyperplastic, and few were carcinomas. It is concluded that the histologic pattern of hyperplastic and neoplastic lesions can be correlated with the results obtained on transplantation.


The question of which strategy is employed in human decision making has been studied extensively in the context of cognitive tasks; however, this question has not been investigated systematically in the context of perceptual tasks. The goal of this study was to gain insight into the decision-making strategy used by human observers in a low-level perceptual task. Data from more than 100 individuals who participated in an auditory-visual spatial localization task was evaluated to examine which of three plausible strategies could account for each observer's behavior the best. This task is very suitable for exploring this question because it involves an implicit inference about whether the auditory and visual stimuli were caused by the same object or independent objects, and provides different strategies of how using the inference about causes can lead to distinctly different spatial estimates and response patterns. For example, employing the commonly used cost function of minimizing the mean squared error of spatial estimates would result in a weighted averaging of estimates corresponding to different causal structures. A strategy that would minimize the error in the inferred causal structure would result in the selection of the most likely causal structure and sticking with it in the subsequent inference of location—“model selection.” A third strategy is one that selects a causal structure in proportion to its probability, thus attempting to match the probability of the inferred causal structure. This type of probability matching strategy has been reported to be used by participants predominantly in cognitive tasks. Comparing these three strategies, the behavior of the vast majority of observers in this perceptual task was most consistent with probability matching. While this appears to be a suboptimal strategy and hence a surprising choice for the perceptual system to adopt, we discuss potential advantages of such a strategy for perception. number of correct answers. An optimal decision strategy for a task is one that maximizes the utility. Because the utility functions and decision strategies used in perception have not been empirically investigated, it remains unclear what decision-making strategy is used, and whether the choice of strategy is uniform across individuals and tasks. In this study, we computationally characterize a decision-making strategy for each individual participant in an auditory-visual spatial localization task, where participants need to make implicit inferences about whether or not the auditory and visual stimuli were caused by the same or independent objects. Our results suggest that a) there is variability across individuals in decision strategy, and b) the majority of participants appear to adopt a probability matching strategy that chooses a value according to the inferred probability of that value. These results are surprising, because perception is believed to be highly optimized by evolution, and the probability matching strategy is considered “suboptimal” under the commonly assumed utility functions. However, we note that this strategy is preferred  under utility functions that value learning.For any task, the utility function specifies the goal to be achieved. For example, in taking a multiple-choice test, the utility is the total  Human performance in perceptual tasks is often benchmarked by optimal strategies. An optimal strategy is one that performs best with respect to its objectives or maximizes expected reward or equivalently, minimizes a cost function In this study we aim to computationally characterize human perceptual decision making strategies. As different strategies may be used across individuals, we characterize the strategy used by each individual observer instead of modeling the behavior of an “average observer”. We used a spatial localization task, as it is simple and fundamental to perceptual processing. While spatial localization has been studied extensively, it has not been investigated in the context of decision making strategies. In nature, at any given moment, we are typically exposed to both visual and auditory stimuli, and scene perception and analysis requires simultaneous inference about the location of auditory and visual stimuli . Therefore, multisensory spatial localization represents a task that the perceptual system is implicitly engaged in at all times. This task is particularly useful for probing decision-making strategies because it involves an automatic causal inference about the sources of stimuli, and distinct patterns of behavior corresponding to different strategies. For each observer we examined which of three plausible decision making strategies best accounts for their performance. We use a Bayesian causal inference model of multisensory perception model averaging, i.e., weighted averaging of the estimate derived from two different causal structures One strategy tested was the objective of minimizing the mean squared error. This is a commonly used loss function in normative models of human behavior model selectionAnother strategy we tested was to minimize the error in the inferred causal structure as well as the error in the spatial estimate. This strategy in the context of our task translates into probability matchingThe third strategy tested is This study was conducted according to the principles expressed in the Declaration of Helsinki. All participants in the experiment provided written informed consent in approval with the UCLA Institutional Review Board.One hundred and forty six subjects participated in the experiment. We used a large sample because we wanted to be able to detect even small subpopulations  who may adopt a different strategy. Participants sat at a desk in a dimly lit room with their chins positioned on a chin-rest 52 cm from a projection screen. The screen was a black acoustically transparent cloth subtending much of the visual field (134° width×60° height). Behind the screen were 5 free-field speakers , positioned along azimuth 6.5° apart, 7° below fixation. The middle speaker was positioned below the fixation point, and two speakers were position to the right and two to the left of the fixation. The visual stimuli were presented overhead from a ceiling mounted projector set to a resolution of 1280×1024 pixels with a refresh rate of 75 Hz.2) with a Gaussian envelope of 1.5° FWHM, presented 7° below the fixation point on a black background (.07 cd/m2), for 35 ms. The visual stimuli were always presented so that their location overlapped the center of one of the five speakers behind the screen positioned at −13°, −6.5°, 0°, 6.5° 13°. Auditory stimuli were ramped white noise bursts of 35 ms measuring 69 dB(A) sound pressure level at a distance of 52 cm. The speaker locations were unknown to the participants.The visual stimulus was a white-noise disk . On bisensory trials, subjects were asked to report Each trial started with fixation cross, followed after 750–1100 ms by the presentation of the stimuli. After 450 ms, fixation was removed and a cursor appeared on the screen vertically just above the horizontal line where the stimuli were presented and at a random horizontal location in order to minimize response bias. The cursor was controlled by a trackball mouse placed in front of the subject, and could only be moved in the horizontal direction. Participants were instructed to “move the cursor as quickly and accurately as possible to the exact location of the stimulus and click the mouse”. This enabled the capture of continuous responses with a resolution of 0.1 degree/pixel.i is indexed over sensory channels). Similar to A and σV respectively. We assume there is a prior bias for the central location, as modeled by a Gaussian distribution centered at 0°. The strength of this bias, σP, is a free parameter. The causal inference model infers the underlying causal structure, C, of the environment based on the available sensory evidence and prior knowledge using Bayes' rule shown in Equation 1.C = 1, C = 2, We used a Bayesian causal inference model of multisensory perception augmented with one of the three decision strategies described above to classify the decision making strategy used by each participant. Details of the model can be found elsewhere . 3) see . The fintructure .  is comA and σV, the standard deviation of the prior over space, σP, and the prior probability of a common cause, p(C = 1) = pcommon. We fit subject responses to the causal inference model for each of the three strategies and determine the best strategy based on the maximum likelihood fit for each subject , even when the probability of a common cause is high, there is a small probability of providing an auditory response consistent with the independent causal structure. Due to the high uncertainty in the auditory signals , this can even be observed when the stimulus locations are identical (left column).The three decision strategies produce distinct patterns of responses across trials and stimulus conditions. 2 = 0.83±0.11). The model fits for the probability-matching group data is also shown for all stimulus conditions in Supplementary For each participant, each of the decision strategy models was fitted to the data, and the observer was classified by the strategy that explained the data best. In order to be highly confident in the classifications, for an observer to be included in the sample we required that the log-likelihood difference between the best-fitting model and the second best-fitting model exceed a value of 3—which is considered substantial evidence for the support of one model vs. another The number of participants classified as utilizing the matching, selection, or averaging strategy is provided in We also tested whether the model selection strategy could explain the data better than the other two strategies if we allow a bias in choosing a model . Despite the additional free parameter for this model, we find similar proportions of categorization: N = 110, matching = 72%; selection = 13%; averaging = 15% – and after applying Bayesian Information Criteria regularization for the additional free parameter: matching = 72%; selection = 11%; averaging = 17%.We aimed to gain insight into the decision making strategy used in a perceptual task, by comparing three strategies and testing which one accounts best for the observers' data. Our computational modeling tools allow us to perform this type of analysis for each individual observer. Perceptual functions, in particular the basic ones that are shared across species  such as spatial localization, are often thought to be optimal. Perceptual functions are evolutionarily old and thus, it is argued that there has been sufficient amount of time for them to have been optimized by evolution We examined the decision strategies in an auditory-visual spatial localization task on a large sample of observers consisting of 110 individuals. First, we found that not all observers appear to utilize the same strategy. This variability across individuals suggests that this localization process is not predestined or hard-wired in the nervous system. More importantly, the vast majority of participants seem to use a probability matching strategy. This finding is surprising because this strategy is not statistically optimal in the conventional sense.Why should the majority of individuals use a “suboptimal” strategy in this basic task? To address this question, it is best to step back and re-examine the notion of optimality. While a probability matching strategy may not be optimal in a static environment, it may be optimal or close to optimal in a dynamic one The observed probability matching behavior suggests that the nervous system samples from a distribution over model hypotheses on each trial. Sampling-based representational coding has been proposed to account for neurophysiological phenomena such as spontaneous neural activity While probability matching was the modal response strategy found in the current study, we are not claiming that probability matching is used in all perceptual tasks, or even in all spatial tasks. Optimal performance in perceptual tasks has been reported by some previous studies. A recent study found observers' behavior to be consistent with the expected loss function in a visual discrimination task Probability matching has been shown to happen when people's response probability matches the relative frequency of the presented stimuli. Here we show that the nervous system can even match the probability of a more abstract construct such as the probability of causal structure of the stimuli which is one step removed from the stimuli themselves. This finding suggests that probability matching may be a general decision-making strategy operating at multiple levels of processing. The results of this study altogether suggest that the nervous system does not necessarily use the commonly assumed least squared error cost function in perceptual tasks, and underscore the importance of considering alterative objectives when evaluating perceptual performance.Text S1Model fitting and goodness of fit procedure.(0.03 MB DOC)Click here for additional data file.Figure S1Model fits to probability matching group. Shaded areas show the log-probability of response for the 82 subjects classified as using a probability matching strategy. Thick lines show the model fits averaged across individual subject fits. Vertical blue and magenta dotted lines show the location of the auditory and visual stimulus, respectively. The first row shows the five unimodal auditory conditions, ordered from leftmost to rightmost positions along the azimuth as shown by the blue vertical dotted line. The first column shows the five unimodal visual conditions, ordered from leftmost (top) to rightmost (bottom) as shown by the magenta vertical dotted line. The central 25 plots show data from the bisensory conditions with both the visual (magenta) and auditory (blue) response distributions.(2.70 MB TIF)Click here for additional data file.


Sequencing by hybridisation is an effective method for obtaining large amounts of DNA sequence information at low cost. The efficiency of SBH depends on the design of the probe library to provide the maximum information for minimum cost. Long probes provide a higher probability of non-repeated sequences but lead to an increase in the number of probes required whereas short probes may not provide unique sequence information due to repeated sequences. We have investigated the effect of probe length, use of reference sequences, and thermal filtering on the design of probe libraries for several highly variable target DNA sequences.We designed overlapping probe libraries for a range of highly variable drug target genes based on known sequence information and develop a formal terminology to describe probe library design. We find that for some targets these libraries can provide good coverage of a previously unseen target whereas for others the coverage is less than 30%. The optimal probe length varies from as short at 12 nt to as large as 19 nt and depends on the sequence, its variability, and the stringency of thermal filtering. It cannot be determined from inspection of an example gene sequence.Optimal probe length and the optimal number of reference sequences used to design a probe library are highly target specific for highly variable sequencing targets. The optimum design cannot be determined simply by inspection of input sequences or of alignments but only by detailed analysis of the each specific target. For highly variable sequences, shorter probes can in some cases provide better information than longer probes. Probe library design would benefit from a general purpose tool for analysing these issues. The formal terminology developed here and the analysis approaches it is used to describe will contribute to the development of such tools. There is currently an intense effort to improve the throughput as well as reducing the cost of DNA sequencing One application where cheap analysis has the potential to make a significant impact is in the sequence analysis of genes for drug targets in clinical samples A standard approach is to use a tiling array which covers the entire gene and has all four possible variants at the central position of each probe N) of probes for relatively short probe length. For practically accessible library sizes, probe lengths cannot be easily extended beyond 10 nt An alternative approach is to attempt to capture all possible variation. The logical extreme of this is ‘universal’ libraries where the probe library contains all possible sequences of a given length, initially these used very short probe length, circa 5 nt, but more recent implementations have extended this to 10 nt A third approach is to use known sequence variation to design a library. Even in genes that are known to be highly variable, such as drug targets in the HIV and influenza genome, the variation is often highly constrained. Specific variants occur at specific positions, generally with only two of the possible four bases observed at these positions, for example if the wildtype is C then only G mutants will be observed but not A or T. It is therefore possible to consider building a library that contains all possible sequence variants that can be formed by combinations of previously observed variants. The library would be constructed by comparing a set of previously determined reference sequences. A consensus sequence would be built making a note of all the observed variants at each position. Probes would then be designed by tiling along the consensus sequence with probes designed to hybridise to all possible combinations of variants that had been previously observed. Such a library would correctly identify more sequence variants than a traditional tiling library with variation at the single position where multiple mutations occur frequently within single probes and those mutations reflect a limited set of possible variants. However, the library would be limited to the detection of mutations that have been previously observed, i.e. any specific sequence variant not seen in any of the reference sequences will not be detected. Additionally the generation of all possible combinations may generate libraries with huge sizes. The effectiveness and practicality of using such libraries would therefore depend on a number of factors, the identity of specific variations that occur, their distribution along the gene sequence, the length of the gene, and the quantity and quality of reference sequences that are available for library design.Here we investigate the ability of probe libraries generated by recombination of reference sequences to re-sequence highly variable genes, with a focus on viral drug targets. We show that such an approach is applicable to some targets, but that it will be unsuccessful for others. The potential effectiveness of these libraries is highly dependent on target identity, variability, composition and probe length, thermal filtering and the number of reference sequences used. A critical factor in the effectiveness of the libraries for each target is the probe length chosen. Probe length has two important consequences. Firstly, it determines in a simple way how many probes uniquely identify a specific sequence region within a target gene. Secondly, it has a very strong effect on the number of probes required to build the library. In some of the cases investigated here libraries based on reference sequences would outperform traditional tiling libraries. This combined with the ability to reuse these libraries for multiple analyses makes this an approach for library design with significant potential. However, for several of the targets analysed this approach will not work, making a detailed analysis of a specific target gene, such as that reported here, important before making a decision on the design approach. We define a number of variables that are important in determining the feasibility of sequencing a putative target sequence by sequencing by hybridization. In particular, we show some cases where sequencing by hybridization using reference sequences will not be feasible due to the amount of variability in the target sequence. In those cases where it is feasible we analyse the optimal probe length for a number of scenarios and show that this also varies wildly from target to target. Thus when applying similar re-sequencing approaches to highly variable target genes it is necessary to examine their viability and optimal design on a case by case basis. The analysis reported here provides a basis on which to consider and optimize the design of such probe libraries.As case studies for this work we selected sequences from human immunodeficiency virus (HIV), hepatitis C (HCV) and influenza, all of which are highly variable organisms lL by sliding a window of length l over a consensus sequence that covers each base in a gene several times. Subsequently, we retain only those probes that are thermodynamically equivalent using a method that we recently developed lT of a selected target sequence that was not used as reference  which is calculated for a set of N sequences N,LS, each of length LTo quantify the variability of a gene, we define a variability ns4A is the most variable gene with a relative variability order of R = 0.526 and np5 is the least variable with R = 0.041  the probe length required to overcome repetitions is l = 16. As a result E1 can be analysed with a much smaller probe library, than HIV RT, by using a shorter probe length, as shown in The first problem which needs to be addressed for resequencing is that of sequence repetitions. We have previously shown that for a wide range of sequences the degree of repetition is strongly dependent on the probe length  library ; as moreThis shows that optimisation of probe length is an important consideration in the design of resequencing libraries. The length of the probes used can have a significant impact on the type and quality of information retrieved in a sequencing by hybridisation experiment. The propensity of mutations in a target sequence to form non-unique reads and therefore the probe length required to overcome this is target specific. Errors that result from ambiguous reads are currently dealt with in the information processing after the experiment. Investigating these issues during the process of probe design could aid the information processing, as well as identifying potential pitfalls in experimental design.TN) of the same lengths as the probes and determine how many of these match exactly to a probe in the library. Because repetitions have been avoided, as described previously, each matching probe relates unambiguously to a single position in the target sequence  of targets found ft with increasing RefN in RefN is increased. However, this does not lead to an increase in ft. This is in contrast to E1 and np5 which tend toward some limiting value in both 〈R〉 and ft. Resequencing using sequencing by hybridisation is feasible for those targets where ft reaches a limiting value at a useful level with reasonable numbers of probes. For np5, 90% of targets are found with RefN = 5. The average library size for RefN = 5 is 4600. Therefore, in these cases using a small number of RefN will minimise the size and therefore the cost of the library whilst maintaining the percentage of targets correctly identified by the library. By comparison, for ns4a with RefN = 5 only 13% of targets are found. And even if the RefN is increased to ten, doubling the average size of the library, only 20% of targets are found. This is clearly not high enough to make this approach viable for this target. Increasing RefN does increase the library size; however, in the examples shown , it does not lead to an adequate increase in the number of targets found. From ns4a has the highest relative variability and this may explain the difficulty in capturing all possible variants. Therefore, a more conventional approach to resequencing will be required in this case.The apparent saturation for R〉 or do indicate saturation but not at an acceptable level of Those targets in The examples in The difference in the number of reference sequences required to cover effectively a target shows that different types of variability may require different approaches. Sequencing by hybridisation may not be applicable to all types of resequencing applications. Our work provides a starting point for analysing the feasibility of short read resequencing to tackle some of these problems. This type of analysis is missing from current resequencing array design and it is clear from our results that there is a potential benefit to be gained from considering the number of reference sequences required. A reduction in library size, while retaining high coverage, can reduce costs. Our analysis provides an approach for measuring the likely effectiveness of sequencing by hybridisation and the optimal number of reference sequences for resequencing highly variable targets.GC content higher that 80%, since such probes are usually difficult to synthesise and offer lower quality information ft and the probe length, as well as the effect of thermal filtering, for Influenza np5. For unfiltered libraries, the number of targets found ft decreases slowly and monotonically with longer probes. However, for thermal filtering we notice oscillations in ft mainly due to the discrete nature of the equivalence parameter ω as well as due to an uneven distribution of this parameter along the gene. The number of targets found when only the probes with high GC content are removed is very similar to the number of targets found with no filtering and therefore is not drawn. For hybridisation experiments it is important to ensure that all probes hybridise selectively under the same conditions to ensure a minimum of false positives and negatives. Therefore, probes with melting temperatures that fall outside a specified temperature range need to be removed from the library since these probes are the most likely to give misleading results. The actual temperature range could be either a predefined experimental requirement or adjusted such that only the minimum number of probes are removed. For the temperature filtering we employ a new technique which we developed for evaluating the thermal equivalence of short DNA sequences np5) and which probes would be removed by the different thermal filtering criteria  such libraries can provide useful levels of sequence information (>90% coverage). However, in several cases the nature of the variation make this approach not viable (coverages less than 30% for ns4a and core). Where it is viable there is a complex interplay between the requirement to both reduce the number of probes in the library, and reduce the number of variable positions in each probe, which tends to lead to shorter probes, and the requirement to ensure that the majority of probes are unique within the target sequence, which tends to lead to longer probes.We have examined a number of highly variable genes that are desirable targets for a cheap and effective re-sequencing technology. Sequencing by hybridization is an appealing approach for such targets but the variability of these genes precludes the use of traditional tiling libraries. In this work we have examined the potential of probe libraries based on information on known variation to solve this problem. In specific cases  and (3), the multiset V = 1 if all sequences are identical and V = 4L if all four nucleotides occur at all positions. This assumes independence between sites or linkage equilibrium. Therefore, since the variability may take very large numbers for large L, we introduce, for practical purposes the logarithm to base 4 of the relative variability order (R),V can take is 4L, the maximal for R will be 1.The limit values for the variability are To complete the characterisation or the variability of sequence sets we also define the number of variable positionsN = 3 sequences of length L = 7,W = 2, which areV = 4, relative variablity order R = 1/7 and geometric mean of the variation is u = 2.For example consider a set of The characteristics of the sequences used for building the libraries in this work are summarised in N,LS, the consensus sequence cons.s can be obtained fromExtending the procedure to calculate the variations of a set of sequences S3,7 of the previous section, the consensus sequence would beFor the example set l,mP of length l starting at nucleotide position ml,We can now define a set of probes lL, for probes of length l, for a set N,LS is then formed out the set of sequences lQ and their complementary set asP, the reduced library L′ may be smaller than L. This reduction in size is due to the intrinsic repetition of the sequence as well as combinations of mutations resulting in non-unique probes and is discussed in The library S3,7 of the previous sections, we could form a library L with probes of length l = 4 nt,Using the example set t,Ls. This target sequence is not included in any of the RefN reference sequences used for building the libraries. We obtain a set T of subsequences of size l for this target sequence by using eqn. (13), i.e. lT = lL a ‘library’ formed of only one sequence. Since we are dealing only with one sequence, the number of test subsequences can be obtained immediately as |lT| = 2(L−l). The fraction of test subsequences found is the cardinality, i.e. the number of elements of the intersection of lT and the library For each gene we chose one particular sequence at random to serve as the test target sequence N sequences at our disposal, we can buildRefN sequences. In this work we take the averages by calculating a given quantity for all possible libraries and represent these averages by angle brackets. For instance the average of the relative variability order ft〉 would be all ft calculated for each of the c possible libraries that can be formed with RefN reference sequences.If we have a,bN are the number of nearest-neighbours of type . Base-pair types are grouped according to the number of hydrogen bonds, i.e., with strong s and weak w representing CG and AT base-pairs respectively. The sum of the number of all nearest-neighbours is simply the length of the probe, i.e., skw, = 0.4795 and w,wk = 1.205 as described in s,wN = w,sNf in the library. For a sufficiently symmetric distribution the most frequent equivalence parameter ωf will usually be a value very close to the mean, i.e. ωf≈〈ω〉 in most cases.To evaluate if probes in a library will hybridise at the same temperature range we use the approximate form of the thermal equivalence parameter ω l, the filtering interval Δω can be used independently of probe length. Consider anb, to be the changes to the number of nearest-neighbours aNb,, such thatsnw, = wns,. Note that eqn. (17) does not depend on the probe length l. In other words, any pair of probes of the same length and which differ in configuration by the same set of changes {anb,} will have the same difference in equivalence parameter ω′−ω independently of the probe length itself. This is an important result since it means that the same Δω can be used to filter probes of any length, e.g. using Δω = 0.5 is not more or less restrictive if the probe length is 10 or 20 nt. As a result, the thermal filtering for a given set of probes depends only on the statistical distribution of the values of ω.While the thermal equivalence ω depends on the length of the probe GC content have been shown to be difficult to synthesise and have a low discriminatory power (increasing the likelihood of cross-hybridisation), in this work we remove probes with GC content above 80% Since probes with a high Text S1Target sequences used. The additional file Sequences.fa.tar contains the sequences used to carry out these experiments. Each file in the archive has the sequence data that we used to create the library for each target sequence. Each file in the archive corresponds to one target, for example, protease.fasta corresponds to the data we used to analyse HIV protease.(0.01 MB TAR)Click here for additional data file.Text S2Example probe library generated by this analysis. This additional file Probes.fa.tar contains an example of a probe library set, for the gene np5 at read length 17, generated in this analysis. The files are labeled in a format to indicate the stringency of filtering (GC content followed by Δω). These are also available from the author and on the 4g.soton.ac.uk website.(0.17 MB TAR)Click here for additional data file.


Bacteriophage Φ12 is a member of the Cystoviridae and is distinct from Φ6, the first member of that family. We have recently isolated a number of related phages and five showed high similarity to Φ12 in the amino acid sequences of several proteins. Bacteriophage Φ2954 is a member of this group.Pseudomonas syringae HB10Y rather than for the rough LPS to which Φ12 attaches. Reverse genetics techniques enabled the production of infectious phage from cDNA copies of the genome. Phage were constructed with one, two or three genomic segments. Phage were also produced with altered transcriptional regulation. Although the pac sequences of Φ2954 show no similarity to those of Φ12, segment M of Φ2954 could be acquired by Φ12 resulting in a change of host specificity.Φ2954 was isolated from radish leaves and was found to have a genome of three segments of double-stranded RNA (dsRNA), placing it in the Cystoviridae. The base sequences for many of the genes and for the segment termini were similar but not identical to those of bacteriophage Φ12. However, the host specificity was for the type IV pili of We have isolated a new member of the bacteriophage family Cystoviridae and find that although it shows similarity to other members of the family, it has unique properties that help to elucidate viral strategies for genomic packaging and gene expression. Bacteriophage Φ6 was the first member of the Cystoviridae to be isolated . In 1999Pseudomonas syringae LM2489 which is a rough LPS derivative of the original host strain for the cystoviruses .pLM1454 was cut with The cDNA pieces were assembled to form complete copies of the three genomic segments. In many cases, the connections could be made by using unique restriction sites made evident by the sequencing project. The ends of segments were prepared by using oligonucleotides with convenient restriction sites as primers for PCR reactions. Five plasmids were prepared, pLM3496, pLM3497, pLM3697, pLM3698 and pLM3691. They contain exact complete copies of genomic segments S and M in plasmid pT7T3 19U and three variants of segment L sequence. The sequences start at the first nucleotide of the SP6 RNA polymerase transcript.32P-UTP and products were analyzed by electrophoresis in agarose gels.Nucleocapsids of Φ2954 were prepared from purified virions stripped of their lipid-containing membranes by treatment with two percent Triton X-100 . TranscrJQ, XQ, YS and FD devised, carried out and analyzed the experiments described in this report. LM conceived the project and drafted the manuscript. All authors read and approved the final manuscript.


Good recruitment is integral to the conduct of a high-quality randomised controlled trial. It has been suggested that recruitment is particularly difficult for evaluations of surgical interventions, a field in which there is a dearth of evidence from randomised comparisons. While there is anecdotal speculation to support the inference that recruitment to surgical trials is more challenging than for medical trials we are unaware of any formal assessment of this. In this paper, we compare recruitment to surgical and medical trials using a cohort of publicly funded trials.n = 114). Comparisons were made by using the Recruitment Index, a simple measure of recruitment activity for multicentre randomised controlled trials. Recruitment at the centre level was also investigated through three example surgical trials.Overall recruitment to trials was assessed using of a cohort of publicly funded trials ) versus ) days per recruit for the medical group ; p = 0.828). For the trials where the comparison was between a surgical and a medical intervention, the Recruitment Index was substantially higher ) versus ; median difference 25.9 ; p = 0.291) for the other trials.The Recruitment Index was found to be higher, though not statistically significantly, in the surgical group (There was no clear evidence that surgical trials differ from medical trials in terms of recruitment activity. There was, however, support for the inference that medical versus surgical trials are more difficult to recruit to. Formal exploration of the recruitment data through a modelling approach may go some way to tease out where important differences exist. However, many trials struggle to recruit to their original target in terms of both time and budget •Level 1 — randomised comparisons of surgical procedures which differ only in a minor way (e.g. a comparison of two methods of suturing) [7]•Level 2 — randomised comparisons of different forms of surgery which differ in a significant way in terms of the overall approach and skills required  [8]•Level 3 — randomised comparisons of some form of medical management versus a surgical intervention  A surgical trial can be defined as a trial undertaking a randomised comparison of a surgical procedure against some form of control. Various options are available for the control, including where viable, a surgical placebo. In general, surgical trials can be considered to fall into three levels of increasing difficulty to conduct:Issues of patient preference and clinical equipoise are likely to be greatest where the difference between comparisons is greatest While there is anecdotal speculation to support the inference that recruitment to surgical trials is more challenging than for medical trials, we are unaware of any formal assessment of this. The aim of this work was to use empirical data to assess whether there is evidence that recruitment to surgical trials is more difficult than for medical trials. Two aspects were considered, overall recruitment to the trial  through the assessment of a cohort of trials, and recruitment at the centre level through three example surgical trials.2We assessed recruitment to surgical trials in general by testing two pre-specified hypotheses on data from a cohort of publicly funded trials. We firstly hypothesised that surgical trials would be more difficult to recruit to and therefore they have a higher level of recruitment activity than medical trials (Hypothesis A) and secondly that the surgical trials of greater complexity (as defined earlier) would similarly lead to a higher level of recruitment activity (Hypothesis B).To test Hypotheses A and B, we used a simple measure of recruitment activity for multicentre randomised controlled trials, the Recruitment Index For example, a trial which recruited 100 patients in 200 days over 5 centres would have a RI of 10 (average number of centre recruitment days per recruit). We calculated the RI for a cohort of publicly funded trials to measure the level of recruit activity for surgical trials. The RI was calculated on the basis of the actual number recruited as opposed to the number completing the trial protocol which was not consistently recorded. We tested for an overall difference between surgical and medical trials to assess Hypothesis A. Level 3 trials versus the remaining trials was tested to assess Hypothesis B.For hypothesis generating purposes, and to illustrate complexities of multicentre recruitment within surgical trials, two hypotheses were tested on three example surgical trials. First, we hypothesised that late starting centres would recruit less than early starting centres (Hypothesis C). Second, we hypothesised that the rate of recruitment within centres would reduce during the course of the trial (Hypothesis D).U test at the 5% significant level in SPSS All hypotheses were tested using a Mann–Whitney 33.1The STEPS project carried out a review of publicly funded trials from two UK funding bodies, the UK NHS R&D National Methodology Programme and the UK Medical Research Council (MRC) 3.2Centre level recruitment data was available for three surgical trials, one representing each of the three levels of surgical trials. Basic information on the three surgical trials used to assess multicentre recruitment is given in 44.1The cohort included trials from a variety of clinical areas  and setting . Trial varied greatly in their target recruitment between 60 and 66,000. The number of centres, number of participants recruited and the time recruiting (start of recruitment to end of recruitment) are given in n = 18, median = 38.0 IQR ) versus ) days per recruit for the medical group ; p = 0.828). For trials which compared a surgical against a medical comparison (level 3), the Recruitment Index was substantially higher ) versus ; median difference 25.9 ; p = 0.291) for the other trials.The Recruitment Index was found to be higher, though not statistically significantly, in the surgical group (4.2The results for Hypotheses C and D are given in All 3 tended towards a reduction in the rate of recruitment within centre (Hypothesis D) with one significant at the 5% level and one just failing to be so. Substantial reductions in the number of participants recruited were observed between the first half of centre's recruitment period and the second half.5In general, there was no clear evidence that surgical trials differ from medical trials in terms of recruitment activity. There was, however, support for the inference that complex (level 3) surgical trials are more difficult to recruit to. We suggest that the 3 levels of surgical trials is a useful paradigm for understanding the variation in required recruitment activity between surgical trials.The Recruitment Index is a simple measure of the recruitment rate for a multicentre trial. We found that the index varied greatly between trials and it might be the case that a more nuanced measure may be more sensitive to differences between trials. For example, extending the measure to the centre level might provide a more accurate picture of recruitment as centres may have staggered start dates.The exploratory centre level analysis illustrated that recruitment is a complex process within a trial and emphasised the variation in recruitment both between and within centres. Haidich and Ioannidis Patient preference is often the most notable reason for recruitment being difficult to surgical trials There were a number of limitations to our study. To enable calculation of the Recruitment Index we used the number of participant randomised. This ignored the quality of data on those included in the trial. The need for long-term follow up to evaluate surgical interventions has been highlighted and consideration of retainment of participant was not assessed 5.1We found no clear evidence to support the assertion that recruitment to surgical trials in general is more difficult than other clinical areas. However, complex  trials appear substantially more difficult to recruit to. Formal exploration of the recruitment data through a modelling approach may go some way to tease out where important differences exist and could inform future trial design.The first author was supported by a Medical Research Council UK Fellowship.


Sir,Two of the statements within the abstract of this paper  of patients’ – the time after operation of this measurement is not stated. Press release and press articles uniformly assume that it was at 12 months.maintained in 70% by 12 months’ – the patient population on which this was measured is not stated in the abstract. Press release and press articles assume that it was the whole group (and wrongly deduce that only 30–40% of the measured group were impotent at 12 months).‘Potency was First, ‘92% no evidence of disease etc’: The statement appears as a summary of the results. The data for this measurement point are not explicitly presented in the abstract or paper. This makes it impossible to be certain of the time of measurement.The two abstract claims (with ambiguous wording in bold) are these: −1 (units of μg ml−1 appear in the abstract but nowhere else). It is particularly unfortunate that early data have been highlighted without it being noted that it is early data. In addition, the use of such early data as an ‘overall’ summary of performance does not seem to be appropriate, particularly as the later data (78% of 83 patients show no evidence of disease at 12 months) are also given in the abstract, and thus seem to be contradicted by the summary.From the size of the patient group  this would appear to be a very early measurement point (shortly after the operation). This can be inferred from the data in Table 3 (p.22) and the Kaplan–Meier curves (p 23). These show data for fewer patients at each successive milestone , with data for only 155 patients at 3 months. It is not evident which test is used for ‘evidence of disease’ at this early measuring point – possibly PSA⩽0.5 ng mlSecond, ‘70% maintain potency etc’: It is slightly difficult to follow from the paper how many were in the group assessed, possibly due to a typo. However, the statement appears to be based on results from 34 patients at 12 months, of whom 16 were potent at 12 months whilst 24 out of 51 patients were potent before the procedure. Thus, 16 out of 24 gives 66.7% maintaining potency (p 21). The text of the paper mentions that potency questionnaires were available for 49 patients at 12 months (p 20); only 34 (or is it 51?) appear in the data set analysed at 12 months (p 21).The rates of return of questionnaire between the groups ‘potent before procedure’ and ‘impotent before procedure’ are not addressed but could potentially introduce some bias. Even if data are available for every patient who has reached the 12-month milestone, the lack of information on Viagra use (before and after) would make it impossible to assess how much impotency has been caused by the procedure. In all, it appears very unsafe to extrapolate the potency results from this small sample to the whole test population. The authors do not do so; the error of the press in doing so might have been avoided if the authors had specified the sample size in the abstract and presented more comment on the reliability of these specific data.Both these claims and their subsequent misrepresentations in the press have been used to make invalid comparisons with other more conventional treatments. Patients volunteering for HIFU should do so in the light of full and correct information. I would urge the authors to make public the necessary clarifications.


Currently the only tumour marker to have a well-defined and validated role in the management of ovarian cancer is CA125. Changes in the level of CA125 can be used as a reliable indication of response or progression according to various criteria, but it does not yet have a clear place in diagnosis or prognosis. Its value as part of a screening tool and during routine follow-up remain the subject of ongoing trials. Other markers remain experimental and do not have a well-defined contribution to make at present. © 2000 Cancer Research Campaign


A growing number of studies of auditory processing are being carried out in awake, behaving animals, creating a need for precisely controlled sound delivery without restricting head movements. We have designed a system for closed-field stimulus presentation in freely moving ferrets, which comprises lightweight, adjustable headphones that can be consistently positioned over the ears via a small, skull-mounted implant. The invasiveness of the implant was minimized by simplifying its construction and using dental adhesive only for attaching it to the skull, thereby reducing the surgery required and avoiding the use of screws or other anchoring devices. Attaching the headphones to a chronic implant also reduced the amount of contact they had with the head and ears, increasing the willingness of the animals to wear them. We validated sound stimulation via the headphones in ferrets trained previously in a free-field task to localize stimuli presented from one of two loudspeakers. Noise bursts were delivered binaurally over the headphones and interaural level differences (ILDs) were introduced to allow the sound to be lateralized. Animals rapidly transferred from the free-field task to indicate the perceived location of the stimulus presented over headphones. They showed near perfect lateralization with a 5 dB ILD, matching the scores achieved in the free-field task. As expected, the ferrets’ performance declined when the ILD was reduced in value. This closed-field system can easily be adapted for use in other species, and provides a reliable means of presenting closed-field stimuli whilst monitoring behavioral responses in freely moving animals. This is easily attainable in anesthetized and un-anesthetized head-restrained preparations , which fAlthough auditory detection and discrimination tasks can be carried out successfully in head-restrained animals , this isAlthough closed-field presentation is very commonly used when recording from auditory neurons in anesthetized animals, this is much more difficult in the awake condition due to variability in the placement of earphones both during and between recording sessions. Moreover, the potential stress induced by fitting them and the resulting tactile stimulation of the face and ears may further compromise the experiment. Animals can be trained to accept such manipulations, but this is time consuming and normally requires a larger cohort of animals to undergo initial training in order to screen their suitability for wearing such devices .A potential way of reducing these problems is to position earphones via a permanent implant mounted on the animal's skull, but this approach has so far been restricted to animals, such as cats  and barnTo address these issues in the ferret, a species that is widely used in behavioral studies of auditory processing e.g. , we have2Mustela putorius furo) from our breeding colony were used in this study.All procedures involving animals were performed following local ethical review committee approval and under licence from the UK Home Office in accordance with the Animal Scientific Procedures Act (1986). Seven female adult pigmented ferrets  cage with a solid plastic floor (55 cm × 43 cm × 41 cm) enclosed with acoustic foam . Each animal was trained to make behavioral responses by inserting its nose into a poke-hole containing a spout that delivered a specified amount of water . There were three such spout locations, a ‘center spout’ in the middle of one of the short sides of the chamber and two ‘response spouts’, one of which was located on each the lateral sides. Infrared LEDs were used to produce beams of light spanning the entrance to each poke-hole, the status of which was monitored continuously by photodiodes connected to a real time processor . Poking behavior readily interrupted these beams, enabling responses to be registered. All electronic circuits were constructed using standard components  and a design similar to that used by All stimuli were broadband noise bursts of 200 ms duration, generated afresh on each trial at sound levels varying (in 5 dB steps) from 70 to 80 dB SPL using Tucker-Davis Technologies System 3 hardware. We used 200 ms as this matches the latency of head-orienting movements in the ferret , avoidin2.3Naïve animals took about a week to learn the two-alternative forced-choice task. During the initial training, the animal had to insert its nose into the center spout poke-hole for 200 ms after which a broadband noise burst was played from one of the speakers. In order to receive a reward, the animal approached the source of the sound and placed its nose into the underlying poke-hole containing the response spout. Once the animal learnt the mechanics of this task and performed ∼100 trials on each session, we increased the delay at the center spout  and started training the animals by playing broadband noise from both speakers simultaneously but with a difference in level of 30 dB between them. The animal then had to judge the perceived location of the sound by licking the response spout on the side of the loudest sound. To avoid a bias towards one speaker location, an incorrect response was followed by a number of correction trials (same stimulus and location) until the animal responded correctly. Once the animals attained a stable performance of >95% correct for several days, they were judged trained and ready for implantation of the cranial support.After implantation, the animals were tested again for their ability to discriminate between the same free-field noise bursts, and were then presented with these stimuli over headphones using Panasonic headphone drivers  across which the interaural level difference (ILD) was varied, as outlined in Section 2.42 were monitored and maintained. Once the animal was stabilized, the following drugs were administered intramuscularly: atropine  to reduce secretions; buprenorphine  and meloxicam  to provide perisurgical analgesia. We administered atipamezole  subcutaneously to reverse the effect of medetomidine once the animal was switched to isoflurane anesthesia. Finally, local anesthetic  was applied to the stereotaxic pressure points and carbomer  to the eyes.After overnight food deprivation, the animals were anesthetized by an intramuscular administration of a mixture of 0.022 mg/kg medetomidine hydrochloride  and 5 mg/kg ketamine . The left radial vein was cannulated for drug and fluid  administration, the trachea was intubated for mechanical ventilation and the anesthesia was switched to isoflurane 0.5–1.5% . Throughout the procedure the temperature, ECG and end tidal COOnce the head was fixed in the stereotaxic frame, a midline incision, typically 30–40 mm in length, exposed the dorsal part of the skull and the temporal muscles were partially displaced laterally. The exposed skull surface was cleaned mechanically of any soft tissue and etched using a 1% citric acid solution. In two animals, up to four steel self-tapping screws were attached to the skull. In the remaining five animals, no screws were used.2, expanding 20 mm rostrally from the back of the skull with a lateral width of 15 mm centered at the midline. Once the layer of dental adhesive, prepared following the manufacturer's instructions, had formed a skin, the first layer of bone cement (1–2 mm thick) was poured over it. Additional bone cement was then applied to raise the implant while reducing its area up to final dimensions of ∼1.5 mm × 10 mm at the height of the original level of the skin (∼4 mm at the midline). This approach maximized the bonding area of the implant, whilst minimizing the region to be externalized above the scalp. Consequently, we were able to reposition the temporal muscles without having to trim them. Due to the highly exothermic curing of the bone cement, the implant was built in small layers, allowing complete hardening and cooling of the previous layer before applying the next one to avoid heat damaging the underlying bone.A thin layer (∼1 mm thick) of dental adhesive  was applied to the area of the skull where the cranial pedestal  is determined by the transfer functions of the head (Fhrtf), the ear canal (Fec), and the probe-tube microphone (Fm),Fcfr) is determined by the transfer functions of the earphone (Fe), the ear canal (Fec), and the probe-tube microphone (Fm),Fs) can be obtained by dividing the Fourier transform of the free-field response by that of the closed-field response,Acoustical measurements were carried out in an anechoic chamber using a ferret cadaver that underwent the same implantation procedure as that adopted for the animals that were used for behavioral testing. The ferret cadaver was situated at the center of the anechoic chamber and a pair of polythene tubes  were inserted at the entrance of the ear canal and connected to a pair of Sennheiser microphone capsules  via customized connectors. The transfer function of each earphone was measured, flattened up to 35 kHz, and calibrated to 85 dB SPL. Test stimuli were 1000-ms bursts of broadband noise presented at 85 dB SPL and were presented unilaterally over headphones attached to the implant. All stimuli were generated and recorded using an RP2 running at 100 kHz, controlled via customized Matlab code. Interaural attenuation was calculated by comparing the acoustical responses measured at each ear via implanted probe-tube microphones at the entrance of the ear cannal. The same probe-tube microphones were additionally used to record the acoustical response to a 1000-ms burst of broadband noise flattened up to 20 kHz, and presented at 85 dB SPL from a location 1 m directly in front of the animal. We then attempted to replicate this response using appropriately filtered closed-field stimuli. The Fourier transform of the free-field response  no cleaning or special care was needed apart from the occasional hair trimming around the implant to keep it visible. The ready acceptance by these two animals to wear the headphones, together with the superior strength of those initial implants compared to the ones used in acute recording experiments, where fixation screws only are used, encouraged us to abandon the use of screws in the remaining animals. The five animals in which no screws were used were implanted in July 2009 and, eight months later, are still yielding useful data without observing any weakening of the bond between the pedestal and the bone, indicating that the dental adhesive is sufficient by itself for providing long-lasting attachment of the implant.We built the pedestal out of bone cement in which two hexagonal nuts (3 mm thread (M3) and 6 mm long) were embedded to allow fixation to the central block of the earphone holder . To ensuTo increase the area of adhesion, the temporal muscles were partially detached from the midline and retracted temporally so that a thin layer of bone cement could be applied to spread the load. However, the pedestal was built up in height and externalized only near the midline . This al3.3Several stimulus presentations were made to measure the interaural attenuation while wearing the headphones. Between presentations the headphones were removed and reattached to the chronic pedestal to account for the possibility of changes due to the repositioning of the headphones. Interaural attenuation was typically around 40 dB up to the maximum frequency tested (35 kHz), with a minimum value of approximately 20 dB A. AlthouAlthough attachment of the headphones to the cranial implant via two bolts ensured that the earphones were reliably positioned over the ears, it was still important to assess the consistency of the probe-tube microphone measurements with repeated attachment of the headphones. Under conditions approximating normal use, these measurements showed that the overall sound level recorded in each ear typically remained consistent to within 1 dB (SD = 1.1 dB).To illustrate the level of stimulus control made possible by this system, and to demonstrate its suitability for virtual acoustic space techniques, we used probe-tube microphones to record the acoustic response to a free-field stimulus and attempted to replicate this response using closed-field stimuli that had been appropriately filtered . This shows that the fidelity of our sound delivery system is sufficient to support performance of an ILD task that is as good as that observed under free-field conditions, and suggests that, in the closed-field condition, the ferrets were lateralizing the stimulus on the basis of the ILDs rather than monaural differences in sound level.The animals were initially trained using randomly interleaved ILDs of ±15 dB. Their performance on this fixed-ILD lateralization task steadily improved over the course of a few weeks, most likely as they became acquainted with wearing the headphones, and reached a consistent level of performance of >98% correct. They were then tested with a range of smaller ILDs, and an example of the data and the fitted psychometric function obtained at an early stage of training from one animal is shown in 4The detachable headphones that we have developed allow sounds to be presented dichotically to freely moving animals, thereby facilitating the behavioral measurement of sensitivity to different sound localization cues. We have shown that ferrets adapt quickly to wearing the headphones and perform to the level expected when ILDs are provided over the headphones, indicating that the animals are readily able to transfer the spatial behavior learnt from free-field to closed-field stimulation.4.1One of the main issues when designing the headphones was the welfare cost for the animal. This was reduced in a number of ways by (1) minimization of the size and weight of the headphones and cranial support, (2) removing the headphones to facilitate normal behavior when not in use, (3) the ease and consistency of headphone positioning and attachment, and (4) limiting the invasiveness of the surgery required for implantation.The total weight borne by the animals was ∼23 g , but <5 g when the headphones were removed. Once adjustments are made for head size, this design should be suitable for a number of other species, such as non-human primates, cats and rats. All the ferrets used in this study adapted quickly to wearing the headphones and, beyond the first 1–2 min following attachment did not try to remove them. No obvious postural change was observed as a result of wearing the headphones, which has so far been limited to about one hour daily divided into two or three sessions. Importantly, it was not necessary to use a commutator to enclose the ceiling-mounted cables as we have observed no instances of an animal becoming caught or entangled in them. This obviously has considerable implications for the cost of this setup. Although the method of tethering the animal might be more of an issue in a larger testing arena, boxes of the approximate size used in this study are perfectly adequate for measuring the discrimination abilities of ferrets in a range of auditory tasks .The modest size of the cranial pedestal meant that chronically implanted animals could be housed in the same conditions and shared cages with other ferrets, without fear of damage to the implant. Antibiotic treatment is normal practice in animals with chronic implants e.g. . HoweverAlthough our headphone system still requires surgical intervention for building the chronic implant, the surgery time has been shortened by moving away from traditional methods of skull fixation, and using a dental adhesive instead. Because of the thinness of the skull, adhesive resins have been adopted for attaching cranial implants in small animals , whereasBecause we have not directly compared the strength of the cranial implant in the presence and absence of self-tapping screws, it is unclear how much additional stability would be provided by the use of screws. For our purposes, however, self-tapping screws were unnecessary, but it is difficult to extrapolate to other preparations because of variability in (1) the composition and size of the implants, (2) the forces the implants are exposed to, and (3) the characteristics of the bone supporting the implant, all of which will ultimately depend on the species and degree of restraint required. Although there are data on the shear bond strength of different dental adhesives, including the one used in this study, to dentine , we are 4.2It is possible to measure the spatial sensitivity of auditory neurons by situating earphones close to, but not actually touching, the opening of the animal's ear canals , therebyThe close proximity of the sound source to the head maximizes the acoustic shadowing effect , with thIn humans, dichotic sounds are increasingly lateralized as the ILD is increased up to a value of ∼10 dB . Similar


Graph theoretical methods are extensively used in the field of computational chemistry to search datasets of compounds to see if they contain particular molecular sub-structures or patterns. We describe a preliminary application of a graph theoretical method, developed in computational chemistry, to geographical epidemiology in relation to testing a prior hypothesis. We tested the methodology on the hypothesis that if a socioeconomically deprived neighbourhood is situated in a wider deprived area, then that neighbourhood would experience greater adverse effects on mortality compared with a similarly deprived neighbourhood which is situated in a wider area with generally less deprivation.We used the Trent Region Health Authority area for this study, which contained 10,665 census enumeration districts (CED). Graphs are mathematical representations of objects and their relationships and within the context of this study, nodes represented CEDs and edges were determined by whether or not CEDs were neighbours (shared a common boundary). The overall area in this study was represented by one large graph comprising all CEDs in the region, along with their adjacency information. We used mortality data from 1988–1998, CED level population estimates and the Townsend Material Deprivation Index as an indicator of neighbourhood level deprivation. We defined deprived CEDs as those in the top 20% most deprived in the Region. We then set out to classify these deprived CEDs into seven groups defined by increasing deprivation levels in the neighbouring CEDs. 506 (24.2%) of the deprived CEDs had five adjacent CEDs and we limited pattern development and searching to these CEDs. We developed seven query patterns and used the RASCAL  program to carry out the search for each of the query patterns. This program used a maximum common subgraph isomorphism method which was modified to handle geographical data.Of the 506 deprived CEDs, 10 were not identified as belonging to any of the seven groups because they were adjacent to a CED with a missing deprivation category quintile, and none fell within query Group 1 . Only four CEDs fell within Group 2, which was defined as having four affluent adjacent CEDs and one non-affluent adjacent CED. The numbers of CEDs in Groups 3–7 were 17, 214, 95, 81 and 85 respectively. Age and sex adjusted mortality rate ratios showed a non-significant trend towards increasing mortality risk across Groups .Graph theoretical methods developed in computational chemistry may be a useful addition to the current GIS based methods available for geographical epidemiology but further developmental work is required. An important requirement will be the development of methods for specifying multiple complex search patterns. Further work is also required to examine the utility of using distance, as opposed to adjacency, to describe edges in graphs, and to examine methods for pattern specification when the nodes have multiple attributes attached to them. Geographical epidemiology has a long history . One of An alternative approach is to decide a priori on some geographical pattern of interest based on a hypothesis, and then examine disease rates in areas with and without those patterns to see if they are consistent with the prior hypothesis. Standard geographic information systems (GIS) software may be used to identify such patterns and then to investigate the health experience of populations in these areas of interest. However, if the pattern is complex, then specifying the pattern of interest could also become quite complex within standard GIS packages.An alternative approach which may offer some potential is the use of methods based on graph theory. Graph theoretical methods are extensively used in the field of computational chemistry to search datasets of compounds to see if they contain particular molecular sub-structures or patterns. We have previously described an adaptation of these methods for use with geographical datasets to identify pre-specified geographical patterns -5.In this paper, we describe a preliminary application of this methodology to geographical epidemiology in relation to testing a prior hypothesis. We tested the methodology on the hypothesis that if a socioeconomically deprived neighbourhood is situated in a wider deprived area, then that neighbourhood would experience greater adverse effects on mortality compared with a similarly deprived neighbourhood which is situated in a wider area with generally less deprivation.We used the Trent Region Health Authority area for this study. It had a population of approximately 5 million people. We used census enumeration districts (CED) as a proxy for neighbourhood areas, of which there were 10,665 in the Trent Region. CEDs were the lowest level of 1991 census geography at which detailed population information was available in England and Wales.Mortality data were provided by the Office for National Statistics and included all deaths from 1988 to 1998. The population denominator data were based on the 1991 CED level mid-year population estimates by five-year age band and sex, corrected for under-enumeration . For yeaThe Townsend Material Deprivation Index was used as an indicator of neighbourhood level deprivation . It is aGraph theory is a branch of mathematics which is concerned with the study of 'graphs', which are mathematical representations of objects and their relationships. The graph is a collection of points (referred to as nodes or vertices) connected by lines (referred to as edges). The nodes may have various attributes attached to them and the edges may represent adjacency, distance or some other connection between pairs of nodes. Within the context of this study, the nodes represented CEDs and the edges were determined by whether or not CEDs were neighbours (i.e. they shared a common boundary).The overall area in this study was represented by one large graph comprising all the CEDs in the region, along with their adjacency information. The information relating to the graph was held in a space-separated text file. The file contained three parts. The first part held, on one line, the total number of CEDs, the maximum number of neighbouring CEDs and the number of variables describing attributes attached to the CEDs. The second part held, for each CED, one line containing the CED number, CED code, the deprivation quintile and the number of adjacent CEDs. The third part held, for each CED, one line containing the CED number and the CED number for each adjacent CED.An extract from the data file is shown below, displaying the format of the text file:10665   22   2 (Part 1).10000   38PMFF03   4   6 (Part 2).10000 9998 9999 10001 10002 10003 10004 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (Part 3)Part 1 shows there were 10665 CEDs within the data file, a possible maximum of 22 adjacent CEDs, and that there were two variables describing attributes (deprivation category and number of adjacent CEDs) for each CED. Part 2 shows that the CED 38PMFF03 was numbered 10000, had a deprivation quintile of 4 and had 6 neighbouring CEDs. Part 3 shows the CED number and the numbers of the six neighbouring CEDs. Because the maximum number of neighbouring CEDs in this dataset was 22, the graph theory based program expected 22 numbers to follow each CED number in part 3. The CED 38PMFF03 had only 6 neighbouring CEDs so 16 zeroes were included to ensure that the CED had the 22 expected values.For this demonstration study, we defined deprived CEDs as those in the top 20% most deprived in the region, i.e. quintile category 5. We defined CEDs in quintile categories 1 and 2 as affluent CEDs and CEDs in quintile categories 3 and 4 as non-affluent and non-deprived CEDs. We then set out to classify the deprived CEDs into seven groups determined by the deprivation levels in the surrounding areas, using the classification below:Group 1   All affluent surrounding areasGroup 2   75% – <100% affluent surrounding areasGroup 3   50% – <75% affluent surrounding areasGroup 4   < 50% affluent, <50% deprivedGroup 5   50% – <75% deprived surrounding areasGroup 6   75% – <100% deprived surrounding areasGroup 7   All deprived surrounding areasWe then examined the distribution of the 2094 CEDs in quintile category 5 in terms of the number of adjacent CEDs. Table To operationalise the seven-group classification in relation to five adjacent CEDs, we produced seven query patterns. Figure We used the RASCAL  program to carry out the search for each of the query patterns . This prThe version of RASCAL we used contains an algorithm that makes use of a mathematical structure known as a modular product graph to reduce the computational expense (i.e. time) of maximum common subgraph isomorphism detection . Within We grouped the population and death counts by age, sex and seven-category deprivation group. We used Poisson regression analysis in SAS, with correction for overdispersion, to examine the association between deprivation and mortality. We entered age, sex and group (1–7) as categorical variables. Results are presented as rate ratios with 95% confidence intervals.The number of deprived CEDs with five adjacent CEDs within each group identified by running the RASCAL program is displayed in table Table As there were 85 CEDs in Group 7, we then split it into two further groups based on the CEDs surrounding the five adjacent CEDs, i.e. the second order neighbours. We defined Group 7b as the situation where each adjacent CED had at least one deprived adjacent CED. This query pattern is shown in figure We found that the basic graph theory method we used to identify neighbourhoods which were surrounded by varying levels of deprivation showed that there was some evidence of a trend towards higher mortality in neighbourhoods surrounded by deprived areas, although this was of borderline significance (p = 0.07). Methodology based on graph theoretical methods available in computational chemistry may be a useful addition to methods available for geographical epidemiology but there are a number of problems which need to be overcome first.A significant issue is the specification of the search pattern. In our example, we set out to examine mortality rates is deprived CEDs surrounded by CEDs with varying levels of deprivation. For the purpose of this exploratory demonstration project, and to keep the task manageable, we restricted the search patterns and analysis to deprived CEDs with five adjacent CEDs. We chose this category because it was the category with the largest number of deprived CEDs. Even so, the category only comprised 24.2% of deprived CEDs and for a more complete analysis, we would need to specify search patterns for all of the other categories.Expansion of the search to include deprived CEDs with different numbers of adjacent CEDs raises a further issue. If we have a deprived CED with two adjacent CEDs, both of which are deprived, the effect on the central CED may be less marked than the effect on a CED surrounded by seven deprived CEDs. A further related issue which we have not taken into account is the size of the neighbouring CEDs. The size may be conceptualized in terms of geographical area, the population count or a combination. The graph theory method allows a variety of attributes to be attached to nodes, in this case the CEDs. However, the specification of search patterns which take into account not just the deprivation level but also the population and geographical size would be much more complex than the search patterns we have specified in this demonstration project.We investigated extending pattern searching to second order neighbours for the deprived CEDs with five deprived neighbours in order to examine the feasibility of specifying second order neighbours. We found that this increased the complexity considerably with a whole range of patterns that could be specified and we settled for a pattern that was simple to specify but which was not very specific. We did not specify the number of second order neighbours the first order CEDs could have in addition to having one second order deprived CED. A complete specification of second order neighbours for our example would have involved specifying a large number of patterns.We did not find clearer evidence for the hypothesis that a neighbourhood surrounded by deprived neighbourhoods would be more likely to have higher mortality rates than a similarly deprived neighbourhood surrounded by less deprived neighbourhoods. This may be explained partly by the limitations described above, such as not taking the geographical size and population of adjacent CEDs into account when specifying the search patterns. In addition, there are other potential explanations. Our study may not have had sufficient power, particularly as there were no CEDs which fell into Group 1 and only four which fell into Group 2. The latter demonstrated the problem with small numbers where the rate for the group was substantially influenced by the high rate for one of the four CEDs. We used CEDs from the UK 1991 census as proxies for neighbourhoods. Whilst some of these may have been reasonable representations of neighbourhoods, others may well not have been. It could also be argued that the concept of neighbourhoods is a complex issue which is unlikely to be adequately represented by geographical boundaries which are in turn usually defined for other administrative purposes.The method we have used is an adaptation of search algorithms for 2D graphs, that is, where edges are defined in terms of adjacency. Graph-theoretical methods have been used in computational chemistry for several years and a suite of programs have been developed to address increasingly complex search requirements. These include 3D graphical methods where edges may be defined in terms of distance but we have not as yet explored the utility of 3D graphs and search patterns for geographical epidemiology.Whilst we have described a preliminary application of graph theory methods within the health geography field, it is important to recognise that these methods have been used in a wide variety of disciplines ranging from physics and mathematics to biology and sociology ,12. ContGraph theoretical methods developed in computational chemistry may be a useful addition to the current GIS based methods available for geographical epidemiology but further developmental work is required. An important requirement will be the development of methods for specifying complex search patterns. Further work is also required to examine the utility of using distance, as opposed to adjacency, to describe edges in graphs, and to examine methods for pattern specification when the nodes  have multiple attributes attached to them.The authors declare that they have no competing interests.RM, PAB and PW jointly conceived the idea for adaptation of the methodology for use in geographical epidemiology. CC carried out the analysis supervised by RM. SR contributed information regarding technical details of the methodology used. RM drafted the paper with contributions from the other authors. All authors read and approved the final manuscript.