Tools performing such analyses are often restricted to a category score based on a cutoff in the ranked list and a significance calculation based on random gene permutations as null hypothesis.Ranked gene lists from microarray experiments are usually analysed by assigning significance to predefined gene categories, ), to compare different scores and null hypotheses in gene category analysis, using Gene Ontology annotations for category definition. When a cutoff-based score was used, results depended strongly on the choice of cutoff, introducing an arbitrariness in the analysis. Comparing results using random gene permutations and random sample permutations, respectively, we found that the assigned significance of a category depended strongly on the choice of null hypothesis. Compared to sample label permutations, gene permutations gave much smaller p-values for large categories with many coexpressed genes.We analysed three publicly available data sets, in each of which samples were divided in two classes and genes ranked according to their correlation to class labels. We developed a program, Catmap (available for download at In gene category analyses of ranked gene lists, a cutoff independent score is preferable. The choice of null hypothesis is very important; random gene permutations does not work well as an approximation to sample label permutations. These gene annotation analyses can unravel new information about pathways and cellular functions responsible for different phenotypes. Computational tools aiding in this process have recently been developed -8, most p-value [In microarray analyses such as clustering, which provide defined subsets of genes with no internal ranking, it is natural to base the score on the number of category genes in the relevant subset. However, ranking of genes appear in many techniques for microarray analysis, such as correlation of gene expression to target profiles  and scorp-value ,8, whichp-value , investip-value for the assigned score, a set of gene lists, ranked according to a chosen null hypothesis, are needed. The simplest choice of null hypothesis is just random gene permutations, and for some rank-based scores, the p-value can then be calculated analytically, without explicitly performing the permutations. However, the random gene permutations null hypothesis assumes independence of gene expression over biological samples, and the p-value is thus a combination of the p-value of how important the category is and the p-value for the genes of the category being coexpressed. When category genes behave similarly over a wide range of experimental conditions, the coexpression does not indicate relevance of the category for the question under study. In many analyses, a more appropriate null hypothesis is therefore sample label permutations, in which a set of ranked gene lists are generated based on the gene expression correlations to randomly permuted target values of the samples. This approach accounts for correlations between category genes and gives p-values that are bounded from below by the number of possible permutations of the samples in the data set. The latter is particularly important in data sets with few samples. Despite this, publicly available tools for gene annotation analysis are restricted to gene permutations [To calculate a utations -8.p-values, that would otherwise have been computationally infeasible. The input to the program is two files and some arguments. The first file contains the biologically relevant ranked list of genes and, if needed, additional ranked gene lists drawn from the null hypothesis. The second file contains the categories and their corresponding genes. The input arguments can either be specified on the command line or in a settings file, and are as follows: 1) a choice between the cutoff score the Wilcoxon rank sum score; 2) a choice of null hypothesis, which can be either the above mentioned user-supplied ranked lists or random gene permutations; 3) the number of permutations used in multiple category testing. If zero, no multiple category testing is performed.We present a program, Catmap, for gene category analysis based on ranked gene lists. The program uses either the number of genes above a cutoff or the Wilcoxon rank sum as score, and the significance of the score can be calculated from a user supplied set of ranked lists, thus allowing for sample label permutations. Furthermore, the program calculates corrections for multiple category testing, using permutation results to assess an effective number of independent categories, which enables Catmap to estimate very small multiple category p-value, the multiple comparison p-value, the false discovery rate, the ROC area , the number of genes in the category, and the 25th, 50th, and 75th percentiles of the ranks. The other output file, the companion file, contains all the categories, with all the genes and their ranks listed below. Each line contains a gene and its rank. The program can be downloaded at [The output of Catmap is two files. The main output file contains all the categories, one on each line ordered according to their significance. The line of a category contains the oaded at , where fet al. [p-values from cutoff independent scoring, showing that the p-value depends strongly on the choice of cutoff. This is further illustrated by the very different cutoffs at which the minimized cutoff-based p-value was obtained. A table with all categories is provided as a supplement [see We analysed the breast cancer data set of van 't Veer et al.  with a cment see .p-values, using the Wilcoxon rank sum and the minimized cutoff-based p-value, respectively. The p-value based on the Wilcoxon rank sum was most often larger than the minimal cutoff-based p-value. Since the latter is biased by a minimization process, it must be interpreted as a score, rather than a p-value, thus requiring additional analyses to find statistical significance [Compared to the variations between the cutoff-based alternatives, the results shown in Table ificance ,8.p-values based on gene permutations tend to be lower than those based on sample label permutations. For categories with small p-values, there are remarkable differences, in particular for large categories with more than 20 genes. Since the gene permutation null hypothesis assumes independent genes, we expect a GO category whose genes are uncorrelated to have roughly the same p-value under the two different null hypotheses, whereas a significant category whose genes are highly correlated will get a lower p-value using the gene permutation null hypothesis. To illustrate this coexpression effect, we picked two large categories, "carboxylic acid metabolism" and "M phase", which are encircled in Figure et al. [p-values for the two null hypotheses, while "M phase" has a p-value of 10-7using gene permutations but the much higher p-value of 3 · 10-2 using sample label permutations. As seen in Figure Using the Wilcoxon rank sum, we compared the results of different null hypotheses. Three publicly available data sets were examined ,13,20. AIn Table p-value used in FuncAssociate [individual p-values for categories, but ranks categories based on the chosen score. Nevertheless, they give results similar to those obtained with the Wilcoxon rank sum and gene permutation. This is expected, since the minimized p-value is calculated with gene permutations, and the score adopted in GSEA [p-value, based on gene permutations, would do. It should be noted that GSEA, FuncAssociate, and iGA calculate multiple hypotheses corrected p-values, but these do not change the ranking of categories.Table ssociate  and iGA ssociate . These t in GSEA  ranks cap-value score on one hand, and the Wilcoxon rank sum on the other, in the treatment of categories for which only a subset of genes have expressions correlating significantly with the question under study. The important genes being in the top of the ranked list will give the category a good score with all three score functions, provided the remaining, seemingly insignificant, genes are distributed in the ranked list as expected by random. However, if these less important genes lie higher in the list than expected by random (though not high enough to affect the Kolmogorov-Smirnov or min-p scores), the category will be considered more important by the Wilcoxon rank sum. Reversely, if the less important category genes prevail in the bottom of the list, the Wilcoxon rank sum score function will deem the category as unimportant, while the other two scores will give the category a high significance, based on the top ranked genes alone. Whether seemingly insignificant genes being ranked better or poorer than explainable by random expectations should be observed or ignored is of course a matter of taste, and a possibility is to use several score functions, that may complement each other. The differences are, however, much smaller than those related to choice of null hypothesis, as revealed in Table There is a possible difference . Furthermore, for all data sets and ontologies studied, Neff was approximately half of the total number of categories. If this is a general feature for GO categories, the simple Bonferroni correction would not be totally unreasonable for small p-values.For all 3 sub-ontologies, the effective number of categories, r et al.  the numbp-values were found for 1000 random gene lists.Figure et al. [It should be noted that whenever several ranked lists are examined as part of a project, this additional source of multiple hypotheses testing should also be corrected for. An example of such a correction, for cutoff-based score functions, is presented by Corà et al. .p-values of biologically relevant categories depend strongly on the choice of cutoff. The cutoff independent Wilcoxon rank sum score overcomes the problem, representing an alternative to the Kolmogorov-Smirnov score [p-value [We developed a computer program for calculating the significance of gene categories in a ranked list of genes. Corrections for multiple category testing can be performed by the program. To investigate the properties of different scores and null hypotheses, we analyzed three publicly available data sets ,13,20. Cov score -17 and t[p-value . The ranp-values and the ranking of categories depend strongly on the choice of null hypothesis. Compared to sample label permutations, gene permutations gave much smaller p-values for large categories with many coexpressed genes.Though sample label permutations in many situations represent a better null hypothesis than gene permutations, available gene annotation analysis tools are restricted to the latter. Our implementation allows for both null hypotheses, and we find that both the The implemented algorithm treats the categories sequentially and independently. As score function for category relevance, the program uses either the Wilcoxon rank sum or the number of genes above a given cutoff in the ranked list. The latter is implemented for method comparison and for the case of a defined subset of relevant genes, without internal ranking.For the case of the Wilcoxon rank sum, the user can supply a set of ranked lists distributed according to an appropriate null hypothesis, or request random permutations of genes as the null hypothesis. In the latter case, the significance of the score is calculated analytically by the program, using either an exact calculation by an iterative method, a Gaussian approximation, or a continuous volume approximation. The program chooses method based on a balance between accuracy and computation time. Details are presented in supplementary information see .p-value of category relevance is determined with Fisher's exact test [For the case of the cutoff-based score function, the act test , correspN independent categories are tested simultaneously, family-wise error rate simply means calculating the probability,When pmultiple (q) = 1 - (1 - q)N,     (1)p-value below any given number q by chance. For correlated categories, we make the assumption that the same functional form describes pmultiple (q), with N replaced by an effective number of independent categories Neff. We find Neff by generating a number, K, of ordered lists under the null hypothesis and calculating the p-values of all categories. We fit Neff using the maximum likelihood estimationthat at least one category has a pk is the minimal p-value for the k'th ordered list.where j highest ranked categories is found by counting the number of p-values from K permuted lists lower than the p-value of the j:th category and divide this number with K · j. For the case of sample label permutations, when a user supplied set of ranked gene lists are used to represent the null hypothesis, the first K lists are used to find Neff and false discovery rates, and the remaining lists are used to calculate p-values for each of the K lists.The false discovery rate for the The algorithm is implemented in the Perl program Catmap.pl and is released under the GNU General Public License (GPL). Catmap.pl, together with user instructions, is available for download at .Using Catmap, we analysed three publicly available data sets with gene annotations from the Gene Ontology.et al. [The data set of van 't Veer et al.  consistset al. , and renet al. .et al. [The data set of Golub et al.  consistset al. [The data set of Alon et al.  consistset al. [All genes were first mapped to corresponding UniGene clusters . For theet al.  this mapet al. ,20, the et al. , and comTB and MK implemented the algorithms in Catmap. All authors participated in the design of the study, prepared, read, and approved the final manuscript.supplement to Table 1.Click here for filesupplement to Table 2.Click here for filep-values for the Wilcoxon rank sum score.Click here for file


A great deal of research on the prefrontal cortex (PF), especially in nonhuman primates, has focused on the theory that it functions predominantly in the maintenance of short-term memories, and neurophysiologists have often interpreted PF's delay-period activity in the context of this theory. Neuroimaging results, however, suggest that PF's function extends beyond the maintenance of memories to include aspects of attention, such as the monitoring and selection of information. To explore alternative interpretations of PF's delay-period activity, we investigated the discharge rates of single PF neurons as monkeys attended to a stimulus marking one location while remembering a different, unmarked location. Both locations served as potential targets of a saccadic eye movement. Although the task made intensive demands on short-term memory, the largest proportion of PF neurons represented attended locations, not remembered ones. The present findings show that short-term memory functions cannot account for all, or even most, delay-period activity in the part of PF explored. Instead, PF's delay-period activity probably contributes more to the process of attentional selection. Persistent activity of neurons in an area of the frontal lobe - the prefrontal cortex - is often proposed to underlie short- term memory. Mikhail Lebedev and colleagues provide an alternative explanation Note, however, that when we use the phrase maintenance memory, many authorities would use “working memory” instead.Once the concept of working memory  became edelay-period activity applies to neuronal activity that follows the transient presentation of an instruction cue and persists until a subsequent “go” or “trigger” signal. The description of delay-period activity in PFdl appeared very early in the history of behavioral neurophysiology . Dimming of the circle signaled that the monkeys should make an eye movement to its current location , for the following reasons. As a key feature of the experimental design, the circle's brightness changed only subtly and remained visible in its new form only briefly. Because the monkeys could not predict whether the circle would brighten or dim and because that subtle, short-lived event provided essential information about the time and target of the response, the monkeys had to attend to the circle intently during the period preceding the trigger signal. As a result of the central fixation requirement, this attention was necessarily covert, although it seems likely that the monkeys would have attended overtly to the circle (i.e., fixated it), had they been allowed to do so. Indeed, the monkeys did so during training. The Discussion takes up the issues of divided attention, multiple motor plans, default motor plans, and other interpretational issues.Brightening of the circle indicated that the monkeys should make a saccade to the circle's initial location on that trial, which the monkeys had to remember in order to perform the task correctly . We can only speculate about the cause of this difference, but reaction times on Rem-trials may have been longer because attention had to be disengaged from the circle's location and reoriented to the remembered one prior to the response. For the “no-memory” condition (not given in p < 0.001). These data are consistent with the idea that each of the two marked locations attracted attention in the no-memory condition, whereas the monkeys directed most of their covert attentional resources to the attended location in the standard version of the task. We acknowledge, however, that there are other interpretations of these data. On control trials, for example, when the saccade was always toward the circle, saccade initiation was approximately 18 ms slower when the circle brightened  than on trials when it dimmed . Thus, factors other than the orientation of attention probably contributed to reaction-time differences.preferred location. The lowest firing rate occurred when the monkey attended to the 270° location, termed the least preferred location.Att),attended-location index remembered-location index  see . A neuroAtt)(I and remembered (RemI) locations. Each data point on the scatter plot represents a single spatially tuned neuron (both monkeys combined). Tuning for the remembered location (red symbols) was both weaker and less frequent than tuning for the attended location (blue symbols). Note that hybrid cells (green symbols) fill most of the space between the other two classes and that relatively few cells represent a single location exclusively. For example, many of the neurons classed as memory cells show some sensitivity to the attended location, albeit not a statistically significant one by the test that we employed. For the entire group of spatially tuned neurons , the mean selectivity indexes (± SEM) for the attended and remembered locations were AttI = 1.84 ± 0.08 (median = 1.39, interquartile range [IQR] = 0.73) and RemI = 1.21 ± 0.02 (median = 1.08, IQR = 0.23), which differed significantly at the p < 0.001 level (Wilcoxon matched-pairs test). p < 0.001).early period) and during the last 800 ms of the delay period, immediately prior to the trigger signal (the late period). . This measure is devoid of any bias caused by a cell's tuning properties in one task period or the other, but it includes the contribution of the spatially untuned cells. When we restricted the comparison to neurons that had any type of significant spatial tuning, in either the early or late periods, the late tuning index (1.76 ± 0.07) continued to exceed the early one (1.42 ± 0.05) significantly (p < 0.001). Most important, we obtained similar results for neurons with significant tuning to the circle's location, which characterizes attention and hybrid cells . We examined whether these results merely reflected the presence of a stimulus in the monkey's visual field and found strong evidence to the contrary. We compared tuning for the circle's location during the 800 ms before the circle started moving  were predominantly attention cells . Neurons dorsomedial to the fundus (n = 412) fell into the three cell classes approximately equally . These regional differences within PFdl were highly significant for each monkey (p < 0.0001, χ2 test). Cells with significant memory signals (memory and hybrid cells, combined) composed 70% of the spatially tuned population in dorsomedial PFdl, but only 20% in ventrolateral PFdl.Based on a cytoarchitectonic analysis conducted on two of the three hemispheres, all of the cells situated ventrolateral to the fundus of the principal sulcus were located within area 46 and none were located in area 12. The area 46/12 architectonic boundary was first described by The right side of Population representations of the attended and remembered locations were further analyzed using a neuron-dropping analysis. Neuron-dropping curves express the strength of spatial tuning as the ability to estimate a spatial variable from the activity of a neuronal ensemble, as a function of ensemble size. We randomly selected an ensemble from the population of recorded PFdl neurons and used a single trial of activity from each cell to estimate both the attended and remembered locations. The findings of the neuron-dropping analysis agree with those from the analysis of single-cell activity and the population histograms and thus provide independent support. However, neuron-dropping analysis offers several advantages over the population histograms, in addition to providing confirmation of those results. In neuron-dropping, the estimation of either an attended or remembered location does not depend on any assumptions about the nature of the spatial tuning curve or the relative importance of very active cells versus those showing less activity. It does not ascribe any special significance to increases in activity relative to baseline (excitation) versus decreases (inhibition) or to the most preferred and least preferred locations. Each cell's activity contributes to the population estimation for all locations regardless of the direction of its modulation relative to baseline and whether that modulation significantly differs from baseline levels. Furthermore, the computation makes no assumption about any relationship between tuning for attended locations and remembered ones. This analysis also has the advantage that its results are expressed as a percentage of correct estimations by the neuronal ensemble, thereby facilitating comparison with the monkeys' performance, which in this experiment always exceeded 75% correct and sometimes approached 100% .RemI > 1.0). The same analysis was applied to the ventromedial and dorsolateral regions within the PFdl, described in the section entitled Histological Analysis, above (not shown). The ventrolateral subpopulation of PFdl neurons see A–4C overWe also used a neuron-dropping analysis to examine the ensemble's properties during response selection and execution. On Att-trials, the estimation of the attended location , while attending covertly to a stimulus located in peripheral visual space, make it unlikely that attention was further divided . Accordinot remembering some location. However, there is no basis for assuming a “memory” of a currently visible stimulus. It seems especially unlikely that the monkeys “remembered” the attended location in the context of the requirement that they centrally fixate while attending somewhere and remembering somewhere else.Second, the monkeys were required to remember the place where the circle first appeared on each trial, and their performance shows that they did so. Did they also “remember” the attended location? There is ample precedent for skepticism about the proposition that monkeys are Third, we cannot rule out the participation of neurons we class as attention or memory cells in a variety of processes involved in preparing or planning the movement or selecting the response target. Prior to the trigger signal, the monkeys may have prepared to make a movement to the remembered location, to the attended location, to both, or to neither. Fourth, we need to consider the possibility that the neural signals we observed reflect the prediction or anticipation of reward. attention has been used to cover many disparate concepts, including the effects of attention on sensory processing and the mechanisms that mediate those influences. We emphasize that the present finding differs from previous ones describing effects of attention on phasic, sensory-like responses. Often called the enhancement effect, the finding that sensory responses are larger when a stimulus or location is more attended was first described for the superior colliculus  in the orientation of spatial attention. In the context of the present results, the finding that inactivation of parts of PFdl  producedThe present results agree better with those of The present study reexamined the interpretation of PFdl's delay-period activity in terms of the maintenance-memory theory. We found that other factors are more important than mnemonic ones. The present results do not argue against a short-term memory function for PF, as one among many contributions to behavior. Nor should they lead to the dismissal of interpretations of some delay-period activity in PF, or some neuroimaging signals from that region, in terms of short-term memory. However, spatial memory signals occur less frequently in PFdl than the maintenance-memory theory predicts. Our data thus accord better with neuroimaging and neuropsychological studies indicating that PF plays a major role in attentional selection, including the monitoring of information and actions .How do our findings mesh with the fact that damage to PF appears to produce deficits in short-term memory, as Although attention could account for many findings about PF, we do not aim to replace one monolithic theory of PF function—the maintenance-memory theory—with an equally monolithic “attention theory.” Delay-period activity appears to reflect the learning and implementation of behavior-guiding rules , categor(Macaca mulatta) to perform the task. Each monkey sat in a primate chair in front of a computer monitor placed 57 cm from the monkey's eyes. We recorded eye position with an infrared oculometer and sampled at 250 Hz. The monkeys pressed a waist-high button with their right hand to start each trial and did not release the button until the end of the trial. Once the monkeys pressed the button, a 0.2° fixation point appeared at the center of the screen. After they had fixated this stimulus for 1.0–1.5 s, a 2° solid, gray circle appeared 8° from the center of the screen in one of four places. We trained two rhesus monkeys After the monkeys learned the task, we implanted recording chambers over the left (monkeys 1 and 2) and right (monkey 1) PFdl. For monkey 1, we used a single-electrode microdrive to obtain single-neuron activity records; for monkey 2, we used a microdrive that independently moved up to seven electrodes. During recordings in monkey 1, we intentionally biased the selection of task-related neurons toward those with delay-period activity. In monkey 2, we recorded the activity of all isolated neurons, regardless of whether they were task related.For histological reconstruction of recording sites, we examined Nissl-stained sections of 40 μm thickness from the right hemisphere in monkey 1 and the left hemisphere in monkey 2.ij,F with rows (i) corresponding to the remembered location and columns (j) to the attended location F is the firing rate on the thl trial for which position i was the remembered location and position j was the attended location, and 1N and 2N are total number of elements in the respective sums. Control trials were excluded from the calculation by not considering the diagonal elements of ijF (i = j, j = k).where We evaluated tuning to the attended location similarly by comparing across-column variability with within-column variability, one row at a time. The strength of representation of the attended location, was quantified as:ij (l).F To classify neurons into those representing remembered versus attended location, we used an 800-ms period preceding the trigger signal. We also evaluated spatial tuning (I) during the final 800-ms period before the circle started to move. .We used two task periods to compute the single trial firing rates We computed the population histograms of n neurons from a population. Then, for a given condition (e.g., a remembered location i of 0° and an attended location j of 90°), we selected one trial of that condition randomly from each neuron . All the other trials for that neuron contributed to a look-up table of firing rates. This look-up table consisted of a matrix of average firing rates ij><F for remembered locations, i, and attended locations, j. The differences between firing rates in the look-up table and the rate on the selected trial were rank ordered, with a smaller rank signifying a closer match. We then summed the ranks ijr across individual neurons and took the remembered and attended locations associated with the lowest combined rank as the population estimation. The estimated remembered location either agreed or disagreed with the actual remembered location of the selected trial, as did the estimated attended location in a separate computation. Repeating this procedure for a given number of neurons, n, more than 2,400 times—each time starting with a randomly selected set of test trials —yielded a percentage of correct estimations of the attended and remembered locations. We then calculated neuron-dropping curves for ensembles of size one to the total number of neurons, but typically the range 1–100 sufficed to capture the main features of the population estimation.Neuron-dropping curves D and S7 To assess the representation of attended and remembered locations during the delays see A–7D, we Figure S1The activity matrix is the same as in (103 KB PPT).Click here for additional data file.Figure S2The activity matrix is the same as in (95 KB PPT).Click here for additional data file.Figure S3The activity matrix in (A) is the same as in (151 KB PPT).Click here for additional data file.Figure S4A table of tuning indexes is given at the top for each of the cell classes (plotted in the bottom part of the figure), combinations of those classes, and other groups of cells as described in the left column. These population averages are divided into two groups of columns, those on the left showing data for the period before the circle began rotating (early) and those on the right showing data for the period after it had stopped and the monkey awaited the trigger signal (late). In the plot, the dashed line shows the median values, the dotted line shows the upper IQR.(56 KB PPT).Click here for additional data file.Figure S5Same PFdl neuron as in (188 KB PPT).Click here for additional data file.Figure S6Same PFdl neuron as in (156 KB PPT).Click here for additional data file.Figure S7Format as in (46 KB PPT).Click here for additional data file.


Drosophila, identifying two mutants. A new reverse-genetics mutagenesis method that uses the crosslinking drug hexamethylphosphoramide to introduce small deletions has been used to generate and screen pools of mutagenized  Drosophila melanogaster. A compatible mutation-detection method based on resolution of PCR fragment-length polymorphisms on standard DNA sequencers is implemented. As the spectrum of HMPA-induced mutations is similar in a variety of organisms, it should be possible to transfer this mutagenesis and detection procedure to other model systems.We report the use of the cross-linking drug hexamethylphosphoramide (HMPA), which introduces small deletions, as a mutagen suitable for reverse genetics in the model organism  Drosophila melanogaster has been the prime genetic model organism for almost a century. This success story is mainly founded on countless so-called forward genetic screens designed to elucidate gene functions on the basis of their mutant phenotypes. Many of those screens reached a scale that has been termed 'saturating' as they identify all nonredundant genes involved in a certain phenotypic trait. However, forward genetic screens are limited in that they are only capable of uncovering functions that are easily measurable or visible. Furthermore, genes having a redundant or nonessential role are less likely to be found by forward genetics.The fruitfly The reverse genetic approach to unravel gene function starts with the DNA sequence. Mutations within the gene are induced and identified by various techniques and only subsequently is the mutant phenotype analyzed . ReverseDrosophila genome by insertion mutagenesis is far from being achieved [Both undirected and directed reverse genetic techniques have certain advantages and drawbacks. Transposon-based mutagenesis tends to be nonrandom because of the occurrence of hotspots for transposon integration. The use of transposable elements of different origin, such as P-elements and piggyBac, which exhibit a different insertion bias, can partly circumvent this problem. However despite large-scale efforts, the ultimate goal of covering the whole achieved ,11. Moreachieved .C. elegans [Drosophila is not feasible.RNAi and small interfering RNA (siRNA) screens provide a powerful tool to dissect the function of genes at a genome-wide scale -14, but  elegans  and for Drosophila allows for generation of both null as well as hypomorphic mutations [Targeted gene knockout in utations . HoweverN-ethyl-N-nitrosourea (ENU) [Random mutagenesis in reverse genetics generally relies on well-established techniques and commonly used mutagens, such as ethylmethansulfonate (EMS) ,17 and Nea (ENU) . Those cea (ENU) , by denaea (ENU) ,17, or bea (ENU) ,20-22. MFast neutrons have also been used to introduce small DNA lesions, which can simply be resolved by agarose electrophoresis after PCR amplification . This kiWe reasoned that it would be worthwhile to establish a generally applicable reverse genetic technique based on an unbiased and practicable random mutagenesis and an efficient mutation-detection performed on standard laboratory equipment. Here we introduce a novel mutagenesis protocol utilizing the cross-linking drug hexamethylphosphoramide (HMPA) , streamlThere are two ways to handle mutagenized progeny. Either large collections are established and maintained, which then are systematically and continuously screened for mutations of interest, or mutagenized progeny are screened directly and only animals exhibiting a desired trait are kept. The first method is in practice an F3 screen, which requires balancing of mutagenized chromosomes and maintenance of many stocks. This approach is far more labor-intensive than a simple F1 screen of progeny and thus is more suited to stock centers. Moreover, balancer chromosomes have many DNA sequence polymorphisms to wild-type chromosomes (our unpublished data), which will interfere with detection of mutagen-induced sequence polymorphisms.To circumvent the inherent problems with balancers, we devised an alternative genetic strategy, which had to fulfill the following criteria. First, mutagenized chromosomes have to be passed on in an unrecombined form such that mutations cannot be lost. Second, the mutagenized chromosomes should be brought into an isogenic background for mutation detection. Third, for economic reasons stock-keeping should be kept at an absolute minimum.yw wild-type laboratory strain but containing the same dominant marker on the two major autosomes. Both chromosome 2 and chromosome 3 are carrying white+ marked P-element insertions, which were chosen because white+ expression is restricted to different subregions of the eye  isogenic to our e Figure . Chromose Figure . Thus, tyw strain background. Thus, the marked autosomes remained unrecombined and could be unambiguously assigned because of the dominant character of the white+ transgenes . However, analysis of X-chromosomal loci would require additional handling of F1 females or mutagenesis of F0 females and hence we did not carry out X-chromosomal screens.The crossing scheme and analysis procedure illustrated was optimized for autosomal genetics. We have generated another strain, KNF307, which in addition carries X chromosomes marked by a characteristic enhancer trap insertion at the et al. [et al. [EMS has been used as a deletion-inducing chemical in large-scale screens , but unbet al.  ranged i [et al. . The cro [et al. . As our N,N-dimethylbenzylamine, which in our hands potentiated the sterilizing activity of HMPA.We modified the original HMPA mutagenesis protocol to administer a shorter, but more intense pulse of HMPA . As HMPA is reported to induce deletions as small as 2 bp and as a mutated allele is diluted 10-fold as a result of our pooling of five flies, we decided to analyze PCR fragments on a sequencer offering maximal resolution and high sensitivity. We have also evaluated the 'poison-primer technique' which is reported to preferentially amplify alleles with a deletion at the poison-primer binding site from large pools . HoweverPCR products were analyzed on either a gel-based or a capillary sequencer . To increase efficiency of mutation detection on gels, we pooled up to three PCR products. These were labeled with different fluorescent tags, partly because they were of similar size Figure .white- mutations at the transgenes on chromosomes 2 and 3. Overall, we found 24 mutations in about 62,700 male and female flies. Two flies were mosaic for the mutations. Given that mosaicism can only be scored in eyes and there only in nonoverlapping expression domains, the mutation rates discussed below may be slightly underestimated , because we did not recover any white- mutation. As sperm development takes up to 10 days [-4 mutations at the white+ loci, which are about twice as large as the vermilion locus. Our mutagenesis procedure involving an overnight incubation with HMPA rather than a 3-day incubation with HMPA and N,N-dimethylbenzylamine is therefore not much less efficient than the original protocol.Aguirrezabalaga et al.  reported 10 days , we alsowhite- mutations between brood 2 and brood 3  of 2,064 bp. We assume that any deletion within the ORF would generate a null phenotype. Only 14 out of 31 HMPA-induced deletions selected at the vermilion locus would have been scoreable by our PCR approach, because the remaining 17 mutations were caused by large deletions affecting both primer-binding sites [The following additional parameters can be utilized to estimate mutant recovery. The ng sites .-4 × 14/31 × (710 - 2 × 20)/2,064)) or one mutation in 6,063 pools, respectively. Taking into account the fact that two mosaic flies may not have transmitted (reducing the mutation rate to 2.0 × 10-4), the estimate would be one mutation in 33,883 flies or one in 6,777 pools.We designed PCR primers for each gene to be scored such that they encompass the first coding exon and the PCR products are between 450 and 807 bp in size. The average weighted length of our PCR fragments was 710 bp (including two primers of 20 nucleotides each). We thus expect one mutation in 30,317 flies . According to the estimate we would have expected three.CG15000, which during the course of this study turned out to be the second exon of the dNAB locus .The first mutation detected was a 41-bp deletion in the first exon of CG17367 on the capillary sequencer , but we have not systematically investigated all of the white- mutations.This study focused on implementing HMPA mutagenesis for reverse genetics. As discussed above, HMPA efficacy has been assessed from mutations at the -3 nucleotide substitutions at the 1 kb awd locus [-4 deletions per 2 kb white+ locus for HMPA), but mutagen dose cannot be increased further because of the concomitant increase in male sterility.While the analysis of PCR fragment-length polymorphisms on our sequencers was very efficient, HMPA mutagenesis turned out to be the limiting parameter. It is about 28-fold less efficient than EMS mutagenesis when it is assumed that all HMPA hits are deleterious . HMPA induced mutations can be detected by fragment-length analysis of primary PCR products on standard sequencers. Hence, screening for small deletions reduces PCR costs by a factor of 2 and spares the effort of secondary assays.Mutant handling is independent of the mutagenesis protocol and may be combined with either EMS or HMPA mutagenesis. For example, TILLING can be performed both on large mutant collections and on a continuous supply of freshly generated mutants.Finally, given the genotoxic properties of HMPA in both prokaryotes and higher eukaryotes ,32, bothy, w; CG31666-white+; CG32111-white+) males were starved for 4 to 6 hours in a plastic bottle containing three layers of water-soaked LS14 filter papers (Schleicher & Schüll). A 1.1 ml sample of HMPA solution  was carefully applied to the filters using a syringe with a long needle (21G2) inserted through the foam stopper. The starved males were exposed to the HMPA solution overnight. Bromophenol blue does not affect mutagenicity detectably, but stains the guts of the flies blue and thus enables mutagen uptake to be monitored and controlled. Freshly eclosed flies do not ingest enough mutagen. HMPA-contaminated plasticware must be disposed of by thermal waste treatment.About 150 1-3-day-old F0 KNF306 . After 2 days males were taken out and crossed to yw virgins in new bottles (brood 2A) and this cross was transferred after 3 days (brood 2B). After another 2 days F0 males were recovered and mated to fresh yw virgins (brood 3A). F1 males of broods 2A, 2B and 3A were collected and mated individually to three yw virgins in about 650 separate crosses per week. Five hundred non-sterile males were removed after 4 to 5 days and five males were pooled for DNA extraction. Fertilized females were returned, and unsuccessful crosses were discarded. If analysis of PCR fragments indicated a primary positive pool, crosses were traced back and kept for further analysis; the other crosses were discarded. From each of the five crosses of primary positive pools a single F2 male or female containing the chromosome of interest as manifested by the typical eye-color pattern was collected for DNA extraction. If PCR analysis yielded a secondary positive result in one of the five F2 flies, a single F2 male containing the chromosome of interest was taken out from the respective cross for balancing .2O) the beads were virtually free of contaminating DNA.) Debris was allowed to settle for about 5 min and each 50 to 100 μl of supernatant were transferred into a 96-well PCR plate. The reactions were incubated in a thermocycler for 30 min at 37°C, and finally for 5 min at 95°C to heat-inactivate proteinase K.DNA was extracted in bulk by squishing pools of each five flies through mechanic force in a vibration mill (Retsch MM30) programmed to shake for 20 sec at 20 strokes per second. Flies were placed into wells of a 96-well deep-well plate. Each well was then filled with 500 μl squishing buffer (10 mM Tris-Cl pH 8.2, 1 mM EDTA, 0.2% Triton X-100, 25 mM NaCl, 200 μg/ml freshly added proteinase K) and one tungsten carbide bead (Qiagen). The deep-well plate was then sealed with a rubber mat (Eppendorf) and clamped into the vibration mill. (Tungsten carbide beads can be recycled: after an overnight incubation in 0.1 M HCl and thorough washing in double-distilled water (ddH2O, 0.6 μl MgCl2 (25 mM), 0.6 μl dNTPs (10 mM), 0.1 μl fluorescently labeled primer 1 (100 μM), 0.1 μl primer 2 (100 μM), 0.12 μl hot-start Taq polymerase (HotStar, Qiagen, 5 U/μl), 3 μl 10× buffer containing MgCl2 (Qiagen). Cycling conditions were 95°C 15 min, 35 × (95°C 20 sec, 60°C 30 sec, 72°C 1 min), 72°C 2 min, 4°C.A Tecan pipeting robot was used for PCR setup. To 5 μl of template DNA, master-mix was added and PCR was performed on an MJR thermocycler that was integrated into the robot. The master-mix per reaction was composed of 20.48 μl ddHThree differently labeled PCR reactions (oligos were 5' labeled with Applied Biosystems' fluorophors FAM, NED and VIC, respectively) were then pooled. To facilitate sizing of fragments we also added ROX1000 size marker (Applied Biosystems) to five DNA pools. Samples of 1.5 μl pooled DNA were mixed with 1.5 μl loading buffer (consisting of one part 25 mM EDTA pH 8.0 with 50 mg/ml blue dextran and five parts HiDi formamide (Applied Biosystems)). The reactions were incubated for 3 min at 95°C, cooled down, and 1.5 μl each were loaded onto a 96-lane ABI 377 sequencer. Run conditions were as follows: 1 h pre-run at 1,000 V, 35 mA, 51°C and 10 h run at 2,400 V, 50 mA, 51°C. Gel images recorded at four different color channels by the GeneScan software were analyzed visually.Slight modifications to this protocol were introduced for analysis performed on an ABI 3730 capillary sequencer. First, DNA was diluted 20-fold before PCR. Second, after PCR, reactions were diluted 100-fold and 2 μl of diluted PCR products were added to each 15 μl HiDi formamide (Applied Biosystems). PCR product was diluted on a Tecan pipeting robot. Diluted DNA was denatured for 2 min at 95°C before analysis. Sample injection (10 sec) and analysis (12,000 scans) was done according to standard protocols. Identification of deletion fragments was then performed by visual inspection of gel-images generated by the Data Collection Software (Array Viewer option, Applied Biosystems). No internal size standard was used, as deletion fragments were identified relative to wild-type PCR product.The following additional files are available with the online version of this paper. Additional data file The time schedule of mutagenesis, fly work, and screeningClick here for additional data fileInformation on the 10 other genes scoredClick here for additional data file


Microstimulation, a technique that activates a cluster of nerve cells by zapping them with a weak electrical current, has helped make causal links between neurons and behavior. For instance, when neurons in an area of the visual cortex that are “tuned” to a particular direction of motion are microstimulated, the way monkeys perceive moving dots on a video screen changes. Microstimulation seems to change what they see. Similar work has also been done for neurons that respond to binocular disparity—the depth-of-field information you gain because each eye has a slightly different view of the world.But many neurons respond, or are tuned, to more than one dimension, leading scientists to wonder how information from these multidimensional neurons contributes to perception—especially when some of that information is irrelevant to a given task. As they report in this issue, Gregory DeAngelis and William Newsome find that neurons tuned to both direction and binocular disparity contribute little to monkeys' perception of motion.The researchers asked three rhesus monkeys to determine the direction a group of dots was moving on a TV screen—a task that can be done regardless of the perceived depth of the dots. The authors had already located two different types of neurons in each of the monkey's brains: sites tuned strongly to direction and multidimensional sites tuned to both direction and binocular disparity. They then determined each site's exact preference: the direction of motion and degree of binocular disparity (if present) that triggered maximum neural activity. The researchers then showed the monkeys several sets of video displays, some with the dots moving in the “preferred” direction and some not. The microstimulation acts somewhat like adding dots in the preferred direction—which confuses monkeys when the real dots are moving against preference and aids them in trials of preferred moving dots. If the behavioral effect of microstimulation—whether it be a help or a hindrance—was significant, it meant that the monkeys were monitoring the activated neurons to perform the task at hand; if there was no change, the stimulated neurons were not being recruited.DeAngelis and Newsome hypothesized that multidimensional neurons  might be ignored during pure motion perception tasks. For two of the three monkeys, this was true. Microstimulation of multidimensional sites had no effect on their behavior, compared to the significant effect of microstimulation of direction-only sites. But for the third monkey, called monkey R, microstimulation of both types of sites had significant effects on his performance. He didn't seem to be ignoring anything. The authors proposed that the monkeys could be using different neural strategies to complete the same task. This conclusion is supported by the fact that monkey R performed better on the task than the other monkeys; he appeared to be recruiting any neuron with applicable information, unlike the others, who seemed to rely on neurons tuned solely to direction of motion. Furthermore, for the few multidimensional sites that affected behavior, their contribution was tempered by how well the depth, or disparity, of the video matched the preference of the stimulated neurons.The results of this paper show that even if neurons carry information that can aid in perceptual decision making, they may not participate, depending on how they are tuned along other (irrelevant) stimulus dimensions. All directional neurons are not created equal—some are more useful than others for a particular task. Whether neurons that respond to a particular stimulus contribute to the task at hand depends on how closely that stimulus hews to the neurons' preference as well as on the subject's learned strategy for performing the task. This neural flexibility, the authors point out, suggests that the brain uses complex, variable strategies to respond to changing environmental stimuli. Techniques like microstimulation will be helpful in drawing the connections between neural activity and behavior.


The problem of training human resources in health is a real concern in public health in Central Africa. What can be changed in order to train more competent health professionals? This is of utmost importance in primary health care.Taking into account the level of training of secondary-level nurses in the Democratic Republic of the Congo (DRC), a systemic approach, based on the PRECEDE PROCEED model of analysis, led to a better understanding of the educational determinants and of the factors favourable to a better match between training in health sciences and the expected competences of the health professionals. This article must be read on two complementary levels: one reading, focused on the methodological process, should allow our findings to be transferred to other problems . The other reading, revolving around the specific theme and results, should provide a frame of reference and specific avenues for action to improve human resources in the health field .The results show that it is important to start this training with a global and integrated approach shared by all the actors. The strategies of action entail the need for an approach taking into account all the aspects, i.e. sociological, educational, medical and public health.The analysis of the results shows that one cannot bring any change without integrated strategies of action and a multidisciplinary approach that includes all the complex determinants of health behaviour, and to do it within the organization of local structures and institutions in the ministry of health in the DRC. A partnership of the Ministry of Health of the Democratic Republic of the Congo (DRC) – more specifically, the directorate that is in charge of health science education – the French-speaking community of Belgium and various education and training associations made it possible to set up and carry out a teaching innovation project to bolster human resources in the health sector. One of the major public health challenges in Africa is to find efficient ways to enhance human resources in the health sector.The goal of the medical policy in the DRC is to promote the health of the population by providing high-quality health care that is complete and integrated and in which the community participates, within the general context of the fight against poverty . With th• restructuring the health system according to political, legislative and administrative orientations as well as updating standards of services;• increasing the availability of resources by implementing an adequate administrative process;• establishing an integrated system of preventive and curative care and health promotion for the target groups;• strengthening the programmes of support to health activities;• coordinating, promoting intrasectoral and intersectoral collaboration and partnership for health;• promoting a suitable environmental framework for health.The Ministry attaches priority importance to delivering high-quality care and health services and by: reaffirming the strategy of primary health care (PHC) as a fundamental option of the national policy on health; reaffirming the health zones' or districts' achieving a minimum package of activities as an operational unit; and regular procurement of essential drugs, including biological products and other laboratory reagents.Training of nurses in the DRC is organized in all provinces of the country and conducted through technical medical institutes (ITM), medical educational institutes (IEM) for the secondary level and higher institutes of medical technology (ISTM) for the higher level. In 1998 there were 308 ITM and ISTM in the country; to date there are 254 schools of nurses  at the secondary level in the country recognized by the DRC government.According to the type of management, these institutions are classified as public, officially agreed and private schools. However, the autonomy of management for all these schools is extensive, given the near inexistence of government subsidy. The infrastructure and quality of training differ widely from one ITM to another.As a rule, the solutions that one observes in public health are located in the area of further training for health professionals. To the extent that further training is important, it is disappointing to ascertain the low yields that these various training courses have in improving the quality of health care and services . There iWhen it comes to basic training, the young nurses who have just graduated from secondary school and make up the critical mass of health professionals in primary health care are commonly required by specific private or church employers to enrol in a full year of training after their academic studies in order to try to fill the gaps between their basic training and health professionals' actual training needs. Confusion on the part of the ministries and other forces involved is attested to by the absence of vision and lack of expertise in educational research and the lack of reforms of the educational curricula in order to keep pace with the fundamental changes that have resulted from the decentralization of primary health care.In addition, several other problems make this succinct analysis more complex. The unemployment rate for the country's nursing school graduates is extremely high, even for those who graduate from the best schools in Kinshasa, the capital. The situation is compounded by the almost total absence of quality management mechanisms, especially when it comes to taking stock of the health workforce that exists. The situation is a complex one in which the expected changes are not clearly discerned.The problem of health sector human resources is vast. The context that interests us in this article is that of the human resources who are in the front line when it comes to grappling with the various communities' health problems. These are the nurses who have completed  secondary school courses. They are the main primary health care professionals in Central Africa, especially in the DRC . This chThe question of research is at three levels: on the theoretical level, this article proposes importing a theoretical model from one field to another; on the methodological level, it uses the action research-like mode of data collection to better establish results; on the empirical level, in the DRC research is unusual ground from which to introduce an innovation.This qualitative research pursues two objectives: to present a methodology  and to study the results of its application . The qualitative hypothesis that subtends this thinking is that using an analytical, systemic planning and strategic synthesis model based on a systemic and participatory approach on various strategic and operational levels will procure the necessary vision for changing the basic education and training of nurses in  secondary schools in the DRC.There is a lack of literature on experiments and experiences that use analytical or planning methods to understand complex social realities and consequently the adoption of strategic plans of action that should result from such experiences. It is thus important for all the players in the process to use the outcomes of the various stages of analysis and planning to produce an appropriate and adapted logical framework. It is necessary, however, to be able to set down on paper the methodology that is used to be able to construct a model that by the end of the process appears obvious for the actors involved, i.e. teachers, internship supervisors and school management, personnel within the ministry's Directorate for Health Education and project officers.Generally speaking, three relatively distinct stages were necessary, as follows:In the DRC, the Ministry of Health and more specifically its directorate in charge of health science education, is responsible for the basic training of secondary school nursing students and further training of health professionals Figure .The situation sometimes varies in neighbouring contexts. Thus in Rwanda, for example, the Ministry of Education is in charge of basic education and training in the technical schools and for the medical professions. In France and Belgium, the general choice was to have the Ministry of Education responsible for most of the training and education of health professionals.We shall not discuss in this article the relevance of the place of oversight for this type of brief for training health professionals. We shall limit our remarks to the need to choose the best place for systemic planning and strategic vision in the existing context. So it is that in the 6th Directorate of the Ministry of Health of the DRC, which is in charge of paramedical secondary education, the need was perceived to develop systemic planning tools that would give a comprehensive, consistent vision of the sector's needs. While the 6th Directorate is indeed the strategic level, there was early involvement of an operational level, in the form of a sample of schools and teachers, and creation of a management unit to guide the implementation of the plans by the 6th Directorate and teachers from the grassroots.It is important to remember that we are talking about systemic and operational planning, not just strategic planning . For theThis article follows on from the given that organizations and human beings are complex, and one way to have public health actions that heed this complexity is to use a systemic approach to analyse them .Various models for a systemic approach exist. The approach that we chose to develop a logical framework for analysing, planning, and synthesising the work of the ministry's directorate in charge of health science education in the DRC is Green and Kreuter's PRECEDE PROCEED model ,7. The PThe PRECEDE PROCEED planning model is welcome because of its multidisciplinary approach, based on the fields of epidemiology, social sciences, behavioural sciences, education and health administration. In a nutshell, the fundamental principles that gave rise to this approach come from the multifactoral nature of all problems. Once this has been posited, all efforts made to act upon behaviour, the environment and social factors must necessarily be multidimensional and multisectoral.The acronym PRECEDE means "Predisposing, Reinforcing and Enabling Constructs in Educational/Environment Diagnosis and Evaluation", while the acronym PROCEED means "Policy, Regulatory and Organizational Constructs in Educational and Environmental Development".The PRECEDE-PROCEED model emphasizes planning interventions by focusing on the expected outcomes of actions based on epidemiological, social, behavioural, environmental, educational, organizational, administrative and political diagnoses of a socio-health and/or educational situation. The stages in the construction of a systemic model for analysing the problem that interests us – health science teaching – are adapted as the process unfolds. One of this method's great potentials is its great flexibility, or its ability to adapt to the specific analyses' needs.A systemic analysis and planning model is built dynamically, in a process that calls for continuous assessment. The model that the ministry's 6th Directorate came up with, and that is presented below, must change with changing knowledge in the area.It is important to stress the qualitative process of continual exchanges and constant observations among the players (teachers, internship supervisors, school management, ministry officers and donors) that made it possible to fill the gaps in the information-gathering process. All these elements are much more difficult to organize in one well-defined stage, but are indeed part of a process that stresses the participatory approach and comes under the third strand of the methodology being discussed. The development of the first model proceeded during the workshop held in Kinshasa at the starting of the project, with the participation of personnel from three pilot schools and the Ministry of Health in October 2002. The three-day workshop, with 40 participants from various institutional levels, permitted the establishment in December 2003 of strategic orientations and guidelines for the continued broadening of the programme.All PRECEDE PROCEED models are built upon the players' actual experiences of the problem to solve. The clarification of the problem itself, which is part of the epidemiological and social diagnosis, comes out of a debate that must be conducted with all the parties concerned. This problem will gradually become more and more clear as its statement shuttles back and forth among the parties until it eventually satisfies the strategic and institutional level that is in charge of the programme and that the problem concerns directly. If, thanks to a resolutely participatory approach, all of the players adopt the use of a systemic approach on a real strategic, managerial and operational level, it will become a solid tool for the entire teaching body concerned.The presentation of results is at two levels: PRECEDE results and PROCEED results. The first are mainly descriptive . PROCEED results are more normative, leading to certain recommendations for practitioners and other actors.To facilitate presentation of the results and understanding of this coherent, overall vision of the interrelated elements, it was considered pertinent to retranscribe the full model as it exists for the 6th Directorate of the DRC's Ministry of Health.To structure the results' presentation, we shall follow the order in which the model's construction progressed. The table must be read from right to left, starting with the epidemiological and social diagnosis, then going on to the behavioural diagnosis and from there to the analysis of the educational and environmental determinants of these behaviours, and then to end with the analysis of the institutional diagnosis see .In the Ministry of Health, all the players are concerned by the mortality and morbidity indicators in the country. For health professionals, the lack of quality of the service provision and care provided by their health system is an obvious cause of the people's lack of confidence in their health system . HoweverWho are these players and what behaviour can explain, through a direct link, the diagnosis of inadequacy? In answering these questions with the players themselves, we discover that there are groups of players that are never clearly identified yet are clearly related to this problem of inadequacy. This is the case, for example, of the donors and nongovernmental organizations (NGOs).Revealing all the groups of players makes it possible to see more easily why importance should be given to a multisectoral approach, especially one that covers teachers and medical and paramedical professionals. If the population is considered a group of players that is separate from the problem at hand, it will not be possible to take it into account in setting up action strategies, to the extent that the aim of such work is to better define people's expectations in terms of the quality of care and arrive at a better understanding of their behaviour.During the discussions, the teachers felt that priority had to be given to separating the group of teachers from that of intern supervisors in order to better highlight the particularities and role of the field training. The school managements revealed their specific role in this problem of mismatches. Indeed, the teachers' and supervisors' behaviour is strongly linked to their own behaviour in dealing with changes . We haveThis diagnosis allows for the factors that are linked to the environment and are direct causes of the epidemiological and social diagnosis. In a context such as that of the DRC, geopolitical and socioeconomic factors head the list, along with the health structures' inaccessibility. To take a more constructive approach without denying reality, it is necessary to focus the analysis of this diagnosis on the more targeted problem of the inadequacies in the training sector. This reveals variables that are more controllable for the levels that are concerned and that everyone agrees are connected to the problem. These are: the learning environment, teaching environment, class hours that facilitate or hamper certain types of learning, etc.The educational diagnosis enables one to home in on the educational and motivational determinants, which must not be overlooked when one goes on to an interventional phase. To the extent that the systemic approach gives significance to each group of players (teachers, learners and others) as well, as is the case in the DRC, it is fully possible to set up a frame of analysis, assessment and action-research that presents the variables and determinants in a PRECEDE model that are specific to each specific group (action-research framework). This is what was done in the DRC to follow the changes in teaching practices, in conjunction with each intervention that was identified, that were made in the specific group of teachers. The results show that it is relatively easy to separate the educational determinants from each other in order to facilitate subsequent reflection about the strategic action to take.The predisposing factors that concern knowledge, experience, attitudes, perceptions and representations have a key place in relation to the behaviours of the players of interest to the Directorate for Education. This construction shows clearly that the training given is usually concerned with knowledge only and generally does not make use of the learners' life experiences.The other important result is to be able to visualize the place of representations in a conceptual framework that will likewise be used for the action. For example, there are the various representations of learning theories when it comes to teaching methods or unfounded beliefs about the quality of care. Specific models exist that enable one to delve much deeper into perceptions and beliefs ,11. ThesThe enabling factors in terms of actual competences (skilfulness, know-how and behaviour) are too often disregarded and underestimated in interventions. Incorporating them in this model thus enables the directorate in charge of this branch of education to check to what extent the projects, programmes and other support measures consider this priority strand in terms of development independence.The reinforcing factors, which are sometimes also referred to as facilitating factors, are the determinants that act upon the positive feedback loops. The importance that all the players give to this type of variable in constructing the model confirmed the need that the directorate had already felt to find means to set up long-range monitoring mechanisms for the various activities engendered by the programme or by some more specific projects.The model contains a certain number of variables. It is clear that it can be enriched in the course of the process through its use and the players' better discovery and gradual appropriation of its features.In terms of results, the institutional diagnosis requires analysing the situation at the organizational level that corresponds to the level of the model's application. This is a national health science education programme under the Ministry of Health. As such, the institutional diagnosis stresses essential strategic variables if one wants to work on a well-knit, comprehensive set of changes. It thus entails the need to analyse the institutional standards when it comes to inspections and assessment, but also those governing health system management and health sector human resource quality management (for example, the existence or lack of a Nursing Board). This is also the level on which we shall discuss how the programme dovetails with other variables and determinants.Before strategic thinking can be put into place based on this situational analysis, it is possible to go on to a more dynamic reading of the relations between determinants and variables. So it is that the DRC's Ministry of Health directorate in charge of health science education foresees a certain number of strategic axes for action. The aim of the action is the problem's translation into an objective form. The directorate thus considered its main goal to be to improve the match between what is learnt in schools and health professionals' needs and the population's expectations.To better understand how the reading of the conceptual model brought us to the action strategies, it is useful to stress an intermediate step that is summarized in Figure A natural adaptation of the PRECEDE model was to define the groups of players by their behaviour. In this way, we obtained a better picture of the division of responsibilities to achieve a common goal and evidence of the need for interdisciplinary work . A comprThe results bring to the fore a number of behaviours that attest to a lack of independence, absenteeism, lack of collaboration, failure to connect theory and practice, a lack of communication, ignorance of the teacher's role, etc., depending on the group. Examination of these results prompts us to stress the importance that must be given to the learning environment and, when it comes to action strategies, the importance to give to a learning environment that is in tune with the strategic axes that are selected, in this part just as in the other parts of the situation's analysis.Given this finding and the need to link the educational and institutional diagnosis with the health problem (seen as an appropriate form for education), one proposed strategic hypothesis is to favour learning techniques that are based on active teaching methods .In going consistently through the various diagnoses and organizational levels, this choice led the education directorate to think about changing its programmes and standards so as to base them on novel teaching concepts such as skills-based learning  and settThe reading of these results in terms of strategic action reveals the need to bolster the analytical and planning process that already exists within the directorate to pay more attention to the educational and environmental determinants for all the target populations concerned. To our mind, the success of the expected changes in terms of narrowing the gap between the "supply" and the "demand" hinges on this.The following diagram summarizes the strategic axes that the Directorate for Health Science Education chose to achieve this objective on the basis of the PRECEDE analysis Figure .This figure reveals four axes to be reinforced:• to reinforce communication and coordination in conjunction with the other reinforcing factors: the pilot schools' teaching method committees, teaching monitoring and feedback, the setting-up of networks, etc.;• to develop methods to enhance the learner's autonomy: active teaching, constructivist approach, interdisciplinary, critical spirit, etc.;• to foster a learning environment that enables the learners to acquire knowledge: library, teaching materials, computer learning, computerized documentation centre, etc.;• to provide institutional and structural support: standards and curricula in tune with teaching and organizational innovations and skills targets that fit health professionals' needs and meet the community's expectations.The discussion will take place on two levels – the operational and the conceptual. On the operational level, we feel it is interesting not to dwell on the presentation of the model as it could have been applied, but on its actual application. The results are presented so as to allow the reader to understand how to organize the problems that are felt to exist in health science education in the DRC.Even if the Directorate for Health Science Education is well aware of its problems, the systemic modelling of the interconnected variables and populations seems to give it a conceptual and operational tool that is useful on various levels, as follows:• tool for dynamic analysis of the situation with regular updates;• tool for systemic planning that also enables the directorate to put forward arguments in dealing with donors and NGOs in the sector;• assessment tool that gives more importance to assessment criteria such as cohesiveness, consistency, relevance, appropriation, and comprehensiveness, i.e., process criteria;• research and evaluation tool that can also promote a more quantitative approach to analysing the relations between variables and various diagnoses or within the same diagnosis;• a dialogue-enhancing tool, for it gives the groups of players involved a vision of the planned change and a common objective.To sum up, this is a tool that provides a certain guarantee that the strategy development process is informed, meets the needs and is complete .The list of these advantages is obviously based on some baseline conditions: a participatory process in which the model is developed and operates and the appropriation of the concepts that subtend the model . Even thOn the conceptual level, the discussion will revolve around Figure We observed through the PRECEDE analysis and then the PROCEED strategic reflection phase that many disciplines converged in order to lead us to this hypothesis and a common objective of needs-matching. Indeed, when action is carried out it will be a matter of achieving a gradual advance that occurs along the  strategic axes defined earlier in this article. Moreover, we are confronted with strategic choices that involve at least three dimensions: a public health approach, an education approach and a sociological approach.These three dimensions are part of the data collection process's success, as well as the success of the strategic choices that follow. This reinforces the fact that the PRECEDE PROCEED model comes from the development of an approach aimed at meeting the need for education and health promotion tools and methods. So it is that we see numerous applications of this process in technical health education establishments that spring from a true systemic analysis of the problems with full mastery of a structuring capacity, unlike some other models such as causal analysis (17).Similarly, we can consider that defining a problem at an institutional and organizational level also requires the identification and involvement of all the parties concerned. We can also consider that the tools that help to understand the relations between elements and insist on a better search for behavioural determinants are prerequisites for organizational learning that has groups of players interact with each other. This is all the more true if the change that is ultimately expected (a match between two sides of the equation) is contingent on changes in the players' behaviour and practices, as is the case of health education.In terms of limits of this research, it targets the analysis of an inadequacy within human resources' management in health, which is that of training of nurses from professional technical levels. Other levels of inadequacies are worthy to be analysed in a complementary way relating to other health professionals, the sectors of health and education planning. The Green model is complementary to the use of methodological dynamic references much as the management of the project cycle focuses on managing interventions or projects whose aim is to contribute to changing a situation from unsatisfactory to satisfactory. Its use within the framework of the project could obtain more means while enabling developments relating to action research. In this context, the contribution from other disciplines, such as psychology, could be reinforced.With regard to the three levels of starting research – the theoretical, methodological and empirical – PRECEDE PROCEED analysis is a model that can be applied to varied situations and problems, although it must be used participatively and proactively in order to enhance its utility in specific circumstances as a personal transfer tool. On the empirical level, the will of all actors – and the Ministry of Health in particular – to have a clear vision of the projected change and manner of reaching that point, while integrating the complexity, was the element carrying the process. We advance the hypothesis that L. Green's systemic approach may become one of a set of active methods, such as problem-based learning, cooperative learning, or even project-based learning, to transfer to learners in nursing schools and sections in the DRC. Indeed, the ability to analyse and synthesize, but also to carry out education and health promotion actions, is essential.DRC: Democratic Republic of CongoPRECEDE: Predisposing, Reinforcing and Enabling Constructs in Educational/Environment Diagnosis and EvaluationPROCEED: Policy, Regulatory and Organizational Constructs in Educational and Environmental DevelopmentThe authors declare that they have no competing interests.FP is responsible for this research. She initiated the project in DRC. She is a specialist in public health and pedagogy. She participated in the design and coordination of the study and drafted the manuscript.JK and DB are two teachers in charge of the reform of the nursing programme in DRC. They set up the collection of data for this study and finalized the analysis.YC and AL participated in the design of the study and the adaptation of de Green's model in the field of nursing training. YC participated in writing the manuscript.MG specializes in management and pedagogy. She conceived the study with FP and participated in its design. DP took part in the elaboration of the methodology. She brought an expertise in health promotion and wrote part of the manuscript.All authors read and approved the final manuscript.Table 1. PRECEDE model for health science teaching in the DRC oversized tableClick here for file


Drosophila has emerged as an invaluable model organism for studying this regulation. Activation of the NF-κB family member Relish by the caspase-8 homolog Dredd is a central, but still poorly understood, signaling module in the response to gram-negative bacteria. To identify the genes contributing to this regulation, we produced double-stranded RNAs corresponding to the conserved genes in the Drosophila genome and used this resource in genome-wide RNA interference screens. We identified numerous inhibitors and activators of immune reporters in a cell culture model. Epistatic interactions and phenotypes defined a hierarchy of gene action and demonstrated that the conserved gene sickie is required for activation of Relish. We also showed that a second gene, defense repressor 1, encodes a product with characteristics of an inhibitor of apoptosis protein that inhibits the Dredd caspase to maintain quiescence of the signaling pathway. Molecular analysis revealed that Defense repressor 1 is upregulated by Dredd in a feedback loop. We propose that interruption of this feedback loop contributes to signal transduction.The innate immune system is ancient and highly conserved. It is the first line of defense and the only recognizable immune system in the vast majority of metazoans. Signaling events that convert pathogen detection into a defense response are central to innate immunity.  Drosphila, Foley and O'Farrell have identified numerous new regulators of the innate signaling response to gram-negative bacteriaBy silencing all conserved genes in As a typical metazoan suffers numerous microbial assaults during its lifespan, survival depends on robust defense strategies. Metazoan defenses are classified as either innate or adaptive. Adaptive immunity is characterized by elaborate genetic rearrangements and clonal selection events that produce an extraordinary diversity of antibodies and T-cell receptors that recognize invaders as nonself. While of profound importance, the adaptive responses are slow and limited to higher vertebrates. In contrast, the machinery of innate immunity is germ-line encoded and includes phylogenetically conserved signaling modules that rapidly detect and destroy invading pathogens . Model oSignaling in innate immunity consists of three steps: detection of pathogens, activation of signal transduction pathways, and mounting of appropriate defenses. The first step is triggered by the detection of pathogen-associated molecular patterns by host pattern recognition receptors . TypicalDrosophila as a model for analyzing innate immune signal transduction had a serendipitous origin. The Toll signaling pathway was discovered and characterized in Drosophila because of its role in specification of the embryonic dorsal ventral axis  pathway, mediates responses to gram-negative bacterial infection in osophila . Althougosophila . Althougosophila . The N-tDrosophila. We developed a cell culture assay that allowed application of this library to a high-throughput RNAi evaluation of Imd pathway activity. This screen identified numerous components of signal transduction , defined a hierarchy of gene action, and identified a novel gene, sickie (sick), required for activation of Relish. Focusing on regulation of the Dredd caspase, we identified a novel inhibitor of Dredd, Defense repressor 1 (Dnr1), which is upregulated by Dredd in a feedback loop that maintains quiescence. We propose that interruption of this feedback loop contributes to signal transduction.Unlike the Toll pathway, which was thoroughly studied in its developmental capacities, analysis of the Imd pathway is relatively recent. Its more complete genetic dissection may well define another conserved and fundamental pathway of immune signaling. Of particular interest, a pivotal step in the Imd pathway—the regulation of Dredd-mediated cleavage of Relish—is not understood. To begin to address this, we developed a powerful RNA interference (RNAi)–based approach to functionally dissect the Imd pathway. In collaboration with others at the University of California, San Francisco, we produced a library of 7,216 double-stranded RNAs (dsRNAs) representing most of the phylogenetically conserved genes of Dipt, that encodes an antimicrobial peptide, Dipt-lacZ. Commercial preparations of LPS contain bacterial cell wall material capable of activating the receptor PGRP-LC and act as gratuitous inducers of antimicrobial peptide genes in Drosophila tissue culture cells  genes, enhanced defense by RNAi (EDRi) genes, and constitutive defense by RNAi (CDRi) genes  genes .In an initial visual screen, dsRNAs that altered the induced or constitutive expression of β-galactosidase were selected as candidate innate immunity genes. We subjected all the initial positives to a more stringent retest where we resynthesized the candidate dsRNAs, retested these under identical conditions, and counted the number of β-galactosidase-positive cells. We defined DDRi dsRNAs as reducing the frequency of Dipt-lacZ-expressing cells to below 40% of LPS-treated controls, EDRi dsRNAs as increasing the frequency of Dipt-lacZ-expressing cells more than 2-fold, and CDRi dsRNAs as inducing Dipt-lacZ-expressing cells to a level equal to or higher than that induced by LPS. About 50% of the initial positives met these criteria, yielding 49 DDRi dsRNAs, 46 EDRi dsRNAs, and 26 CDRi dsRNAs . The entC. EDRiC genes are listed as both EDRi and CDRi . Inactivation of either actin with dsRNA directed to the actin UTRs demonstrated that both actin transcripts must be inactivated for an observable EDRi or CDRi phenotype C.Caenorhabditis elegans, several genes can be inactivated by RNAi in Drosophila without an obvious drop in the efficiency of gene inactivation –tagged Relish . We inactivated sick by RNAi and subsequently tested dsRNAs representing the five CDRi epistatic subgroupings for their ability to activate Dipt-lacZ expression in the absence of Sick  and those that downregulate an ongoing response (EDRis).Interestingly, groups of inhibitors implicate distinct pathways in immune regulation. For example, of the 17 genes that had the strongest EDRi phenotype, four encode splicing factors and four encode products that appear to interact with RNA. This functional cluster suggests that disruptions to some aspect of RNA processing/metabolism can substantially increase the number of S2 cells that activate expression of the Dipt-lacZ reporter in response to LPS exposure. While we do not know how RNA metabolism contributes to this phenotype, the repeated independent isolation of genes lying in a functional cluster reinforces a conclusion that the process is involved. Several other functional clusters were picked up in our screens. Three genes involved in Ras signaling (MESR4, Ras, and Cnk) were identified as EDRi genes. In addition, we noted weak EDRi phenotypes with three additional Ras signaling components (rolled/MAPK, Dsor1, and Pointed). These findings argue that Ras signaling downregulates responses to LPS. This might represent a negative feedback circuit. However, the finding that MESR4 also has a CDRi phenotype suggests that the Ras/MAPK pathway may also impinge on the maintenance of quiescence.(α-Tub84D), a kinesin motor (Klp10A), and microtubule-severing function (CG4448/katanin) were isolated as EDRi genes. Perhaps an event involving microtubule structures helps limit immune responses. The two cellular actin genes (Act5C and Act42A) were individually dispensable, but their joint inactivation produced both EDRi and CDRi phenotypes. A regulator of actin function, SCAR, was also identified as a CDRi, and both actin and SCAR CDRi phenotypes fell into epistasis Group II. This suggests that disruption of the actin cytoskeleton in quiescent cells can activate the immune response in a Relish-dependent fashion. Since S2 cells are induced to phagocytose bacteria, and changes in cell shape are induced in response to LPS, it would not be surprising if cytoskeletal functions contribute to immune responses. Indeed, microarray studies showed induction of numerous cytoskeletal components in S2 cells upon incubation with LPS . This and other tentative indications of involvement of this pathway were either not reproduced or fell below the threshold in retesting. We are left uncertain about SCF contributions to immune induction in our system.A previous conventional genetic screen for mutations leading to constitutive action of the Imd pathway in d Relish . A Skp1/As in the case of the CDRi and EDRi phenotypes, our screen for DDRi phenotypes identified numerous genes falling into functional categories. One potential limitation of our approach for identification of DDRi is that some genes required for ecdysone maturation may be selected as immune deficient. Additionally, one of the largest functional categories was genes involved in translation and included four ribosomal proteins, three initiation factors, two amino acyl-t-RNA synthases, and an elongation factor. It seems likely that RNAi of genes in this category affects translation of the Dipt-LacZ reporter, as opposed to affecting modulation of signaling events. To cull our collection of DDRis of such indirect modulators of the response, we developed a secondary screen that does not rely on de novo gene expression. Based on the previously described phenotypes of Imd pathway members, we reasoned that inactivation of the core components transducing the signal would compromise activation of the Relish transcription factor. To identify DDRi dsRNAs that prevented Relish activation, we prepared a GFP-Relish reporter cell line and rescreened DDRi dsRNAs for loss of GFP nuclear translocation in response to LPS.sick as involved in Relish nuclear translocation in response to LPS. Although cells treated with sick dsRNA failed to mount an immune response, the cells were otherwise healthy through the course of the experiment. Dox-A2 RNAi reduced the survival of cells and was effectively lethal within a few days of the scoring of the immune response. We conclude from this that Sick and Dox-A2 contribute to the central signal transduction process, but it is presently unclear whether Dox-A2 has a significant specific input or if its effects are secondary to a global effect on cell viability.In addition to confirming a requirement for Dredd and PGRP-LC in Relish activation, we implicated a proteosomal regulatory subunit Dox-A2 and identified a novel gene It is notable that only two DDRi genes passed our secondary screen based on GFP-Relish localization. Does this mean that all the other DDRis are not really involved? While we have not yet analyzed all these genes, we suspect that many of them will modify the Imd pathway, either impinging on the pathway at a point beyond Relish translocation, or quantitatively or kinetically modifying Relish translocation in a manner that we did not detect in our screens. Insight into this issue is likely to be derived from further epistasis tests that might place some of these DDRis in the signaling pathway.We identified an unprecedented large number of immune response inhibitors (CDRi genes) in our screens. As there are diverse steps within and potentially outside the Imd signaling pathway at which the CDRi inhibitors might act, we sought to position their actions with respect to known Imd pathway functions by RNAi epistasis tests. By sequential inactivation of known Imd pathway components and CDRi gene products, we tested whether constitutive activation of immune reporters by CDRi dsRNAs depends on steps in the signal transduction pathway. In this way, we defined five distinct epistatic categories of CDRi gene products. The four CDRi genes that continue to activate immune responses despite inactivation of Imd, Dredd, or Relish are likely to act on signal-transduction-independent factors that maintain transcriptional quiescence of Dipt. The largest group of CDRis (12) depends on Relish function but not on upstream activators of Relish. These are likely to include two types of regulators: one type that sets the threshold of response so that basal activity of Relish does not trigger pathway activity, and a second type that contributes to suppression of Relish activity. The latter type of regulator might include inhibitors that impinge on the late steps in the signal transduction cascade. For example, genes whose normal function inhibits the activity of the full-length Relish transcription factor might be required to make the pathway activator dependent, and these would be found in this category. The remaining upstream epistasis groups that rely on additional signal transduction components are strongly implicated as significant contributors to the immune induction pathway. As all of the CDRis induced robust immune responses in the absence of ecdysone (unpublished data), we propose that the CDRis have their input into the Imd pathway at a level that is the same or lower than the level of the input from ecdysone. Given that this is true for all five epistatic groups of CDRis, the result suggests that ecdysone has its input at an early level of the Imd pathway.sick is required for constitutive activation of the responses by inactivation of CDRi genes in Groups III, IV, and V genes but not for the action of CDRi Group II or Group I genes. If we assume a simple linear pathway, this would indicate that Sick functions upstream of Relish and downstream of Imd and Dredd. It is noteworthy that the epistatic data are consistent with molecular data indicating that Sick is required for Dipt-lacZ induction and the nuclear translocation of Relish in response to LPS. This combination of phenotypic, epistatic, and molecular data argues for participation of Sick in the regulated activation of the Relish transcription factor.The identification of five epistasis groups of inhibitors also provides reference points for a second round of epistasis tests that position novel DDRi genes within the Imd pathway. We used this approach to show that the novel DDRi One epistatic group struck us as particularly interesting. While Dnr1 inactivation caused ectopic Dipt-lacZ expression, simultaneous loss of Dredd or Relish restored cells to their resting state. These data indicate that the wild-type function of Dnr1 is to prevent Dredd-dependent activation of Relish. Consistent with this hypothesis, we identified a C-terminal RING finger in Dnr1 with greatest similarity to the RING finger motifs observed in the C-terminus of IAP proteins. In addition to regulating caspase activity, IAPs also regulate their own stability through ubiquitin-mediated proteolysis. Similarly, we observed that mutation of a critical RING finger residue greatly stabilized Dnr1. These features suggest that Dnr1 is a caspase inhibitor, suggesting that it might act directly to inhibit Dredd activity.We observed that exposure of cells to LPS transiently stabilized Dnr1 and that this stabilization directly paralleled the period of Dredd-dependent Relish processing. This suggested to us that Dnr1 stability and accumulation might be regulated by its target, Dredd, a regulatory connection that could establish a negative feedback loop. We confirmed that Dredd activity is required for accumulation of Dnr1. These results suggest that Dredd modulates a RING-finger-dependent Dnr1 destruction pathway .Our results are consistent with a feedback inhibitory loop where Dredd activity promotes accumulation of its own inhibitor ; howeverIn summary, a new and powerful screening approach has provided many candidate regulators of the Imd pathway of the innate immune response, and we suggest that the newly identified contributors Dnr1 and Sick will govern central steps in the regulatory cascade that activates the Relish transcription factor. While our analysis has led to a focus on these two regulators, we suspect that other genes among those isolated will also make important direct contributions to the Imd pathway. Furthermore, some of the groups of genes falling into functional clusters are likely to define physiologically relevant inputs into the induction pathway.A library of DNA templates bearing the T7 RNA polymerase promoter at each 5′ end was prepared from genomic DNA in a two-step PCR protocol. In the first round of PCR, targeted regions of DNA were amplified using gene-specific primers (18–22 nucleotides) with a 5′ GC-rich anchor (GGGCGGGT). Primers were designed to amplify a region of nonintronic genomic DNA between 250 and 800 bp with minimal sequence overlap to all other amplimers. Templates from the first step were amplified in a second round using a universal primer containing the T7 RNA polymerase promoter sequence followed by the GC-rich anchor TAATACGACTCACTATAGGGAGACCACGGGCGGGT. dsRNA was generated from templates in in-vitro transcription reactions for 6 h at 37 °C. In vitro transcription products were annealed by heating to 65 °C and slowly cooling to room temperature. All products were tested for yield and size by gel electrophoresis, with 97% giving satisfactory results.Drosophila Expression System Protocol using hygromycin B  as a selection marker. The Dipt-lacZ plasmid has been described previously  supplemented with 10% heat-inactivated fetal calf serum, penicillin, streptomycin, and hygromycin per well. dsRNA was added to each well at a final concentration of 10 μg/ml. Cells were cultured for 4 d at 25 °C and incubated an additional 24 h in 1 μM 20-hydroxyecdysone (Sigma, St. Louis, Missouri, United States). LPS  was added at a final concentration of 50 μg/ml for 12 h. RNAi protocols were as previously described  with 40,000–50,000 cells in 200 μl of Schneider's escribed .2, 3.5 mM K3Fe(CN)6, 3.5 mM K4Fe(CN)6, and 0.2% X-Gal in DMF). β-galactosidase activity assays were performed as described previously  IMRB microscope. Immunofluorescent images were taken on an Olympus (Tokyo, Japan) IX70 driven with DeltaVision software (Applied Precision, Issaquah, Washington, United States). S2 cells were deposited on Superfrost Plus Gold slides (Fisher Scientific, Hampton, New Hampshire, United States) for immunofluorescence. Cells were fixed for 10 min in 4% formaldehyde (Sigma). Tubulin was detected with mouse anti-α-tubulin (Sigma). DNA was visualized with Hoechst 33258, and actin was visualized with rhodamine-coupled phalloidin (both from Molecular Probes, Eugene, Oregon, United States). Images were processed with Adobe Photoshop 5.5, and figures were assembled with Adobe Illustrator 9.0.g for 3 min, washed in PBS and lysed on ice for 10 min in lysis buffer (0.5 M HEPES [pH 7.5], 150 mM NaCl, 5 mM EDTA, 0.2% NP40, PMSF, leupeptin, pepstatin, NaF, and microcystine LR). Lysate was spun for 10 min at maximum speed, and the supernatant was added to sample loading buffer. Samples were separated by SDS-PAGE and analyzed by Western blotting. Anti-GFP antibody was purchased from BabCO , and HA and actin antibodies were purchased from Sigma.Dnr1-expressing vectors were prepared by cloning Dnr1 cDNA into pAc5/V5HisA (Invitrogen). The C365Y mutant form of Dnr1 was prepared with the Stratagene point mutation protocol using a TTCAATCCGTACTGTCACGTC sense primer and a GACGTGACAGTACGGATTGAA antisense primer. The mutation was confirmed by sequencing. For experiments with z-VAD-FMK, S2 cells were incubated in 100 μM z-VAD-FMK for 4 h at room temperature. Cells were harvested by centrifugation at 1,000