A novel algorithm (ChipStat) is presented for detecting gene-expression changes from Affymetrix microarray data. The method is used to identify changes in murine mammary development. We describe a novel algorithm (ChipStat) for detecting gene-expression changes utilizing probe-level comparisons of replicate Affymetrix oligonucleotide microarray data. A combined detection approach is shown to yield greater sensitivity than a number of widely used methodologies including SAM, dChip and logit-T. Using this approach, we identify alterations in functional pathways during murine neonatal-pubertal mammary development that include the coordinate upregulation of major urinary proteins and the downregulation of loci exhibiting reciprocal imprinting. Theseocations . The latocations .The ability to sensitively detect changes in gene expression is crucial for a transcript-level analysis of developmental processes and other processes involving changes in the relative sizes of cellular compartments. Early attempts to limit the false-positive rate of microarray studies focused on the magnitude of fold-change in gene expression . For stBecause of the variety of techniques used to measure gene expression, it has become commonplace to utilize simple, numerical estimates of gene expression as the starting point for such identification. One major drawback to this approach has been that individual probe cell information from Affymetrix microarrays is routinely discarded. This issue has only recently begun to be addressed -10, and An additional compromise has been driven by the practical difficulties of performing large numbers of microarray experiments. Given limited samples, permutation of the existing experimental dataset, rather than use of independent sets of control samples, has been widely used to estimate the statistical significance of differential gene expression . AlthougThe impetus for the work described here is the desire to sensitively identify coherent patterns of gene expression during mammary gland development. At 2 weeks of age, the female FVB mouse mammary gland exists as a rudimentary epithelial tree embedded at one end of a fat pad composed of adipose tissue and fibroblasts. Previous work has demonstrated a fundamental transition in the composition of the mammary adipose compartment from brown fat to white fat during early development . By 3 weWe describe here a novel algorithm for sensitively detecting gene-expression changes using information derived from individual probe cell hybridizations to Affymetrix oligonucleotide microarrays. In addition to modeling the predicted behavior of this algorithm, we have generated an independent cohort of control samples derived from the murine mammary gland that can be used to empirically calibrate its statistical behavior. We have then used this algorithm to analyze a biological transition in early murine mammary gland development in order to compare the sensitivity of this approach to other commonly used algorithms. In conjunction with a second novel algorithm, we have developed an aggregate approach to the reliable detection of differential gene expression that yields substantially improved sensitivity across a range of false-positive rates and have applied this approach to the analysis of early murine mammary gland development.t test, have been used in conjunction with microarray datasets to detect changes in gene expression  [A variety of traditional statistical methods, such as the  example ). Given ys (SAM) , expliciTo test this approach, we developed a novel analytical algorithm that is based on identifying individual differences at a given statistical significance between corresponding probe pairs. To a first approximation, the signal on any given probe cell can be modeled as:S = M + E(b) + E(p) + E(h), E ~ NWhere S is the signal detected on the microarray, M is the average message level in a given experimental state, E(b) is noise due to biological variation between animals or animal pools, E(p) is the noise due to variations in sample measurement, and E(h) is the noise inherent in hybridization to oligonucleotide features on the array. The goal of our analysis was to identify a method that would allow us to reliably distinguish significant differences in M under particular experimental conditions.t-test or other measurement based on the average of the probe set would be expected to perform as well as an algorithm based on individual probe-pair comparisons. In contrast, if most noise in the measurement of true transcript level exists at the level of hybridization to a given oligonuclotide (E(b) + E(p) << E(h)), then the independent measurements of probe-pair differences more closely approximate independent measurements of differences in gene expression. In the most extreme case - if E(h) is sufficiently larger than E(b) + E(p) - each oligonucleotide in the probe set could be considered as an independent measurement of gene expression and the probability of observing a given number of probe pairs changing under the null hypothesis would be determined by the binomial distribution.Given this model, we reasoned that the relative magnitude of E(b) + E(p)  compared with E(h) (the hybridization noise) should determine whether comparisons between individual probe pairs would be useful. If the bulk of noise in our microarray data was due to factors influencing the level of transcript available for measurement  + E(p) >> E(h)), then individual probe-pair measurements should only reflect the pre-hybridization bias in transcript availability. In this case, the P less than a fixed value (hereafter denoted pps). To avoid making assumptions about equal variance in both groups, a heteroscedastic t-test is used. We would expect that probe sets in which larger numbers of individual probe pairs show a significant change in the same direction are more likely to be measuring differentially regulated genes. Thus, for any given probe set, the number of probe pairs (0-16) changing in a given direction with P less than pps is tabulated and used as a measure of the significance of change in gene expression. We simulated the expected behavior of this algorithm under the null hypothesis (no difference in gene expression) across various ratios of E(b) + E(p) and E(h) . Results are shown in Figure To explore this possibility, we implemented an algorithm, hereafter designated ChipStat, that takes corresponding probe pairs across two comparison groups and tests them for statistical significance with  pairs 0-6 changinP value associated with this change in limiting cases. If E(h) >> E(b) + E(p), the binomial distribution can be used to calculate the resulting significance ; however, the relative contributions of E(h), E(b), and E(p) to the total error function are not known a priori.Although this approach provides a statistical methodology for identifying changes in gene expression, it is only possible to directly calculate a pps and the number of probe pairs changed was tabulated. Results are shown for pps = 0.05 , and the cumulative distribution of false positives as a function of An ideal method for identifying differentially regulated genes would maximize the number of genes identified while maintaining a low fixed number of expected false positives. We have previously shown the utility of testing the statistical overlap of discrete gene lists with biologically relevant annotation in order to identify functional pathways during murine mammary gland development . This mapps = 0.05), and the number of statistically significant increases was measured as a function of the number of genes expected to appear on the list by chance. Results are shown in Figure pps = 0.05 with 8/16 probe pairs increasing, where around five genes are expected to be identified by chance, we find that the measured number of differentially regulated genes is around 160. This corresponds to a false-positive rate of approximately 3% . It is also apparent , values of 0.04-0.05 appear appropriate across most highly-significant P values. A marked decrease in sensitivity for a given false-positive rate is noted both at low (0.01) and high  values of pps.To determine whether the sensitivity of this algorithm could be further optimized, similar analyses were performed at various values of s Figure . These dpps = 0.04) from 2 to 5 weeks of mammary gland development were identified, and analysis of the control data suggested that fewer than 10 increases would be expected by chance at this significance level (corresponding to P < 7.7 × 10-4). Manual inspection of the resulting list revealed the presence of a number of genes known to be upregulated during this developmental transition, including cytokeratin 19 (Krt1-19), cytokeratin 8 (Krt2-8), and κ casein (Csnk). However, to avoid bias toward previously studied genes or known genes with high fold change, genes were randomly selected from subsets of this list corresponding to high-stringency (P < 2.2 × 10-4), low-stringency with high fold change , and low-stringency with low fold change . Results from northern blot analyses using probes for these randomly selected genes are shown in Table Although the use of negative-control samples provides a definitive method for evaluating the behavior of our statistical algorithms, we independently verified these results using northern blot hybridization. Genes differentially expressed  was from the low-stringency group and was predicted to show only a 1.37-fold change. In contrast, northern hybridization confirmed the differential expression of other genes with only modest fold-changes . As the genes tested were not biased toward higher fold change (only 2/75 genes with fold change > 3 were randomly selected for northern confirmation), our data demonstrate the ability of ChipStat to reliably detect the types of small, reproducible changes in gene expression that are necessary for whole-organ analysis.Of note, the single gene that did not show a significant change . Results are shown in Figure t-test, results obtained using calculated P values are generally within 5% of comparable results using empirically calibrated P values. Logit-T and dChip appear much less sensitive when using reported P values, although both of these techniques show improvement when calibrated using the control dataset. Of particular note, logit-T performs only slightly less well than ChipStat when calibrated against our control distribution, consistent with the fact that it was the only other algorithm considered that performs probe-pair-level comparisons when testing for differential gene expression.SAM, dChip, the 2 comparisons in general for equal groups of N arrays, it is easily feasible for three-sample versus three-sample comparisons. We have designated this approach Intersector. Significantly, the control data previously generated to calibrate ChipStat also allow us to determine the empirical false-positive rate for Intersector as a function of the number of 'increase' calls and to perform direct comparisons with other algorithms.Although the Affymetrix Microarray Suite (MAS) software utilizes probe-level information in identifying differentially expressed genes, its use has been restricted to single-array comparisons. As a result, it has been widely recognized that this approach generates an unacceptably high number of false-positive results. The use of replicate samples, however, might be expected to lower the false-positive rate while achieving a higher sensitivity. We therefore combined pairwise comparisons between triplicate data points in two different groups  and determined differential expression based on the Affymetrix call  for these comparisons. A similar technique, in which a simple majority cutoff (5/9 changes) was considered to denote significant change, has recently been described . AlthougThe performance of the Intersector algorithm in comparing 2- versus 5-week mammary gland gene expression is shown in Figure Given substantial differences between the types of probe-pair comparisons performed by ChipStat and MAS, we next wished to ascertain if these algorithms identify the same sets of upregulated genes. Direct comparison requires that the analyses result in comparable false-detection rates. We therefore compared the lists at thresholds corresponding to approximately 2.5 genes expected by chance, and the closest available threshold with each algorithm was chosen. The resulting thresholds were Intersector (MAS4) 7/9 (1.75 expected by chance), Intersector (MAS5) 8/9 (2.8 expected by chance), and ChipStat (.04) 8/16 (2.68 expected by chance). Notably, examination of these lists demonstrates that each algorithm  detects a discrete set of genes that are not detected by the others Figure . This isα-, β-, and γ-casein; previous work in our lab has demonstrated that these genes are differentially regulated with expression at 5 weeks greater than that at 2 weeks (data not shown).To confirm this result, five out of the 13 genes uniquely identified by ChipStat were randomly chosen for confirmation. One of these genes was undetectable by northern blot hybridization, and the remaining 4/4 showed differential expression in the predicted direction (5 weeks > 2 weeks)  and Intersector  were combined, and aggregate lists of genes identified by both algorithms were tabulated . The results demonstrate that a combination of these two approaches can lower the expected false positive rate while maintaining a high sensitivity. For example, the combination of ChipStat  and Intersector  detects 209 increasing probe sets with only 3.4 expected to increase by chance . A comparison of the false-positive rates for single  and combined (ChipStat and Intersector) approaches is shown in Figure Given the presence of genes uniquely identified by Intersector or ChipStat at a given false positive rate and the feasibility of performing Intersector analysis on small numbers of replicates, we next explored whether a combination of these approaches could further improve overall detection. To test this, all possible pairwise threshold combinations of ChipStat  allows no choice between approximately three and approximately seven expected false positives , the combined approach provides a smoother continuum of values. More important, these data show that, for certain targeted numbers of expected false positives, a combination of ChipStat and Intersector can provide improved performance in gene detection compared with either algorithm alone.The goal of these methodological developments has been the elucidation of biological mechanisms underlying mammary gland development and carcinogenesis. We therefore used the hybrid ChipStat/Intersector lists representing early mammary gland development as a basis for further exploration of developmental processes during this time period. A complete list of genes differentially expressed between 2- and 5-week murine mammary gland was compiled using the techniques described above. The results are listed in Additional data file 2.P less than 0.05 was used. Results are shown in Table To identify coherent functional patterns of gene expression during neonatal development through the onset of puberty, statistically significant associations between Gene Ontology (GO) categories  and listTo provide a crude check on the reliability of these results in addition to the confirmation previously performed, gene lists were examined for association with previously described biological processes. In addition to individual genes that are consistent with epithelial proliferation and differentiation (discussed above), several statistically associated categories represent pathways that have been previously described in the mammary gland during this developmental window . These iMup1, Mup3, Mup4, Mup5) account for four of the five most highly upregulated genes from 2 to 5 weeks. Large quantities of MUPs are synthesized in the male liver and excreted in the urine, where they bind pheromone and play a role in signaling for complex behavioral traits [Slp (sex-limited protein), which also shows sex-restricted expression in the male liver and - like Mup expression - is normally repressed by Rsl [Interestingly, 'pheromone binding' and 'odorant binding' categories are also associated with upregulated expression at the onset of puberty. Genes within these categories are primarily members of the major urinary protein (MUP) gene family, and MUP transcripts  and Igf2, are also reciprocally imprinted neighbors, suggesting the possibility of a common regulatory mechanism for altering expression from loci exhibiting this genomic organizational structure .The ability to reliably detect changes in gene expression is critical for the analysis of experimental microarray data. This problem assumes particular importance when analyzing complex mixtures of cells, such as those derived from a whole organ during ontogeny. The challenge can be most clearly seen by considering a small subpopulation of cells that demonstrate a marked change in gene expression. If the expression of this gene is uniform and low throughout the rest of the tissue, the biologically relevant change within a few cells will appear as a low fold change in organ-wide gene expression. A variety of such nonabundant yet developmentally critical cell types have been described. For example, the proliferative capacity of small structures in the mammary gland known as terminal end buds gives rise to the extensive ductal structure that is elaborated during puberty . More ret-test, have been used to identify significant changes in gene expression using replicate microarray data. Given the substantial economic investment required for large microarray experiments, attempts have also been made to improve detection of differentially regulated genes through better estimates of the null distribution using permutation analysis; the use of software incorporating such methods, such as SAM [A variety of nonparametric and parametric statistical tests, including variants of Student's h as SAM , has bech as SAM ) has attWhile much work has focused on such use of probe-level analysis for estimating gene expression ,9, the at-test comparisons between probe pairs, and the number of probe pairs that change greater than a significance threshold are tabulated. A greater number of consistently changing probe pairs should indicate that the difference is less likely to be due to hybridization noise, and thus this number relates the overall probability that the probe set is measuring a true change in gene expression. The processing time for the ChipStat algorithm scales as a linear function of the number of replicates processed (O(N)), and thus it is feasible to apply this approach to much larger numbers of samples.The ChipStat algorithm uses heteroscedastic To assess the statistical significance of ChipStat results, it was necessary to empirically measure the underlying null distribution. While the recent availability of a number of publicly available Latin square datasets representing measurements of spiked-in control samples has greatly facilitated measurements of this sort , these dTo generate these samples, mammary glands from six independent cohorts of mice were harvested. These data provide a true, gold-standard negative control within a representative mammalian experimental system, and we anticipate that their public availability will be similarly useful to the broader scientific community in analytical development and validation. Furthermore, the use of this dataset as an empirical calibration control for ChipStat argues that these results will be valid independent of the adequacy of the statistical noise model used. It is worth noting that the use of pooled groups of animals is likely an important parameter, as single-animals groups, for example, would be expected to exhibit increased biological variability and thus decrease the proportional contribution of hybridization noise.t-test, SAM, and dChip) based on aggregate gene-expression measures  known to contain a substantial number of increasing transcripts. Consistent with our hypothesis that probe-level comparison analysis should improve sensitivity, ChipStat was able to substantially outperform a variety of methods  that is also based on probe-level t-test comparisons for identifying differentially expressed genes [t-tests performed on log-transformed PM probe data. ChipStat differs from this approach in several significant respects. These include the use of a fixed P value threshold for pairwise probe comparisons and the use of the degree of reproducibility across the entire probe set as an indication of statistical significance. Results from the empirical control data suggest that ChipStat performs slightly better than logit-T in most cases within our biological system. Interestingly, however, the advantage of ChipStat over logit-T was more modest than the advantage over SAM, dChip, and the t-test; as logit-T also uses probe-level comparisons, this result is consistent with our overall observations regarding the increased power of probe-based analysis. It is also worth noting that the nominal P values derived from both logit-T and dChip substantially underestimated statistical significance prior to correction with our control data, suggesting that, for example, the median P value cannot be used to directly assess significance without such correction.Recently, Lemon ed genes . The logOne additional difference between ChipStat and logit-T stems from the use of mismatch (MM) probe cells (ChipStat) and log-transformed data (logit-T). As currently implemented, the ChipStat algorithm compares differences in probe pair (PM - MM) values rather than in PM values alone. Interestingly, the use of PM values within the ChipStat algorithm does not result in superior performance (data not shown), and log(PM) data yield performance that is roughly comparable to PM - MM (data not shown). Further work will be required to determine if the log(PM) approach can be adapted to improve the performance of ChipStat.2 pairwise comparisons are required for equal groups of N replicates ), this method is not easily scalable to larger numbers of samples. In contrast, ChipStat scales linearly with N, and the use of the heteroscedastic t-test also makes it possible to precompute results for a  baseline control population against which multiple comparisons will be performed. While both approaches are feasible for triplicate comparisons, extension of Intersector to much larger numbers is unlikely to be practical.The Intersector algorithm tabulates MAS calls from all pairwise comparisons across replicate groups. As we have shown, this algorithm provides the most sensitive method for detecting gene expression changes at low false-detection rates. However, it suffers from several substantial drawbacks. First, the proprietary nature of the Affymetrix algorithm and its associated decision matrices limits the ability to automate the analytical process. Additionally, because NA third disadvantage to the Intersector approach stems from the lack of a detailed model for its underlying statistical framework. Both ChipStat and Intersector, as currently described, require the use of control samples to generate an estimate of statistical significance. Thus, extension of these results to encompass either a substantially different experimental system or larger numbers of replicates will require the generation of new empirical significance curves. In the case of large numbers of replicates, however, the cost of generating such data is likely to remain prohibitive at least in the near future. Statistical simulations of ChipStat behavior may, however, provide a mechanism for extending the current control curves for larger datasets. This approach would rely on the relative estimates of E(b) + E(p) and E(h) obtained by fitting the current empirical curve derived from six samples. While a small number of probe sets change more often than would be predicted by simulation, it should be possible to conservatively estimate an upper bound to the P value curve by overestimating the relative contribution of E(b) + E(p) vs. E(h). Further experimental work will be required to confirm this possibility.One caveat to this approach is that the simplified statistical model that we have used to illustrate the theoretical advantages of probe-level comparisons does not account for the variability in the behavior of specific probe cells that exist on Affymetrix arrays. In contrast, the model-based approach to signal estimation implemented in dChip explicitly incorporates such variability and has been shown to provide a good fit to empirical measurements of array data ,32. It iIn this context, it is worth noting that the ability to perform our current validation and to tune both Intersector and ChipStat was critically dependent on the gene-expression datasets derived from our independent cohorts of control animals. One might naively assume, in the absence of true negative control data, that robust changes in gene expression should be detected by simply taking the Affymetrix MAS calls and requiring that they consistently demonstrate increases for all pairwise comparisons. In contrast, our data show that the Intersector algorithm can achieve increased sensitivity while retaining an appropriately low (and defined) false-positive rate. Both the Intersector and ChipStat algorithms can be tuned using negative control data for sensitivity versus false-positive rate, depending on the type of analysis and application-specific tolerance for false-positive calls. Furthermore, these algorithms can be combined to further improve their sensitivity. As we have demonstrated that each of these algorithms detects a population of probe sets not identified by the other at a comparable stringency, this combined approach may yield the best result. Given these considerations, we favor the use of the hybrid ChipStat/Intersector approach for small number of replicates (around three), with ChipStat alone being useful for large numbers of replicates. Although ChipStat shows greater sensitivity than logit-T at moderate numbers of false positives , their comparable performance at high stringency  suggests that the overlap in genes identified by these two techniques may also be of interest.An additional piece of evidence for the utility of our approach is provided by the statistical association of GO annotation with lists derived from ChipStat/Intersector, dChip or logit-T. At the level of significance tested (3.4 genes per list expected by chance), ChipStat/Intersector lists were statistically associated with a greater number of GO terms than were lists derived from dChip or logit-T. Furthermore, as would be predicted from the fact that logit-T is also a probe pair-level comparison method, logit-T lists are associated with GO terms at a level that is intermediate between dChip and ChipStat/Intersector. One of the terms associated only with the ChipStat/Intersector list of downregulated genes is 'mitochondrial inner membrane'; this example is particularly noteworthy in light of previous work demonstrating a presumptive role for enzymes of fatty acid oxidation in adaptive thermogenesis during the neonatal period [It should be noted that these results depend on the level of significance chosen for generation of the original lists, and an increase in the total number of differentially expressed genes identified may actually decrease the statistical significance of a given association if it does not result in the detection of more genes within the category in question. Despite some caveats as to the generalizability of these results, our data demonstrate that the improved sensitivity of ChipStat/Intersector can measurably influence the ability to interpret patterns of biological activity.For the FVB murine mammary gland, the period from 2 to 5 weeks of age encompasses critical developmental milestones that include the suckling-weaning transition as well as the profound hormonal changes that characterize the onset of puberty and its consequent rapid ductal epithelial proliferation. Our present work has more completely characterized changes in the transcriptome that occur during early murine mammary gland development than have previous reports. A total of 213 upregulated and 130 downregulated probe sets were identified under conditions designed to yield a low expected false-positive rate (3.4 probe sets expected to change by chance per list).Four out of five of the most highly upregulated transcripts through the onset of puberty are members of the MUP family of odorant-binding proteins. MUPs are lipocalins that can bind hydrophobic molecules such as pheromones, and they have previously been shown to play a role both in the delivery of signals within the urine as well as in the reception of these signals on the nasal epithelium  downregulation from 2 to 5 weeks of murine mammary gland development. As this gene was first identified as a preadipocyte transcript that is downregulated during subsequent differentiation [Meg3/Gtl2, a noncoding RNA that is reciprocally imprinted with Dlk1 [Dlk1-Meg3/Gtl2 regulation has been compared with Igf2-H19, another tandem pair of reciprocally imprinted genes in which one member produces a noncoding RNA [Igf2 and H19 are also downregulated during this time period, suggesting the hypothesis that a common regulatory mechanism exists for the tandem control of both imprinted genes at these loci. It will be particularly important to determine whether there is functional significance to this Igf2-H19 regulation, or whether it reflects the epiphenomenal byproduct of a mechanism designed to downregulate Dlk1 during adipocyte development.Delta-like kinase (Dlk1) is a member of the epidermal growth factor (EGF) superfamily  that is ntiation , we hypoith Dlk1 . This Dlding RNA ,39. Intet-test, SAM, dChip, and logit-T. However, its lack of scalability, along with the baseline time required for processing, make it unsuitable for larger numbers of replicates. ChipStat, in contrast, provides comparable sensitivity with triplicate samples and has the capability of handling much larger numbers of replicates in order to improve the reliable dectection of small changes in gene expression. Both algorithms provide a substantial increase in the ability to sensitively detect statistically significant changes in gene expression within the context of the whole mammary gland.We have developed two novel algorithms for the analysis of Affymetrix oligonucleotide microarray data. We have validated these algorithms by using empirically derived distributions from control animals to calibrate their statistical significance. These control data, which reflect both experimental and biological sources of variability likely to be representative of many mammalian experimental systems, should facilitate further work in this area. For triplicate samples, Intersector appears to provide the most sensitivity at a given threshold of statistical significance, and its performance is substantially superior to other widely used methods including the We have applied these techniques to the analysis of genomic patterns during early murine mammary gland development. In addition to detecting patterns reflecting known biology, we have noted the coordinate upregulation of a class of molecules not previously known to be differentially regulated in the mammary gland. We also suggest that peri-pubertal changes in the mammary gland may utilize mechanisms for tandem upregulation of multiple imprinted regions. Our observations suggest a variety of future directions for functional validation and demonstrate the utility of coupling sensitive detection of differential gene expression with pathway analysis for the elucidation of biological patterns during organogenesis.The third, fourth and fifth mammary glands were harvested from FVB mice at the indicated time points. Samples from 2 and 5 weeks of age reflect triplicate pools of 10 animals at each time point . In addition, tissue from 18 control animals was harvested when they were 6 weeks and 4 days old. These control animals also carry a transgenic construct consisting of the murine mammary tumor virus (MMTV) promoter upstream of the reverse tetracycline transactivator (rtTA) and had been given 2.0 mg/ml doxycycline in drinking water for 96 h before harvest. This line (previously designated MTB) has been previously described, and no developmental abnormalities have been noted . All aniAll tissue was snap frozen after removal of the lymph node present in the fourth gland, and total RNA was isolated by homogenization in guanidinium isothiocyanate and subsequent centrifugation through a cesium chloride cushion as previously described . Northerμg total RNA was used for each hybridization. RNA was visualized by gel electrophoresis to ensure its integrity before analysis. Biotinylated cRNA was generated and hybridized to Affymetrix MG_U74Av2 arrays according to the manufacturer's instructions. To scale between chips, these expression values were rank ordered, and the median approximately 96% were averaged. Chips were scaled relative to each other to equalize this average value. All Affymetrix control probe sets were eliminated from analysis, yielding data from a total of 12,422 probe sets. Datasets are publicly available as CEL files designated MTB_ [Approximately 15-20 ted MTB_   was tabulated. We consider that a greater number of probe pairs changing in a given direction indicates a greater probability that the gene detected by the probe set is differentially expressed. If the bulk of the noise within the array data derives from pre-hybridization experimental factors  + E(p); see Results section for definition), the expectation is that all probe pairs would change coordinately. That is, if there are 16 probe pairs in the probe set, we would expect (for E(b) + E(p) >> E(h)) that under the null hypothesis (no change in gene expression) either 0/16 or 16/16 probe pairs should change significantly . Conversely, if the bulk of the noise derives from hybridization to individual probe cells  + E(p) << E(h)), then the number of probe pairs r that change within a given probe set of size t can be approximated by the binomial distribution:To detect differentially regulated genes, we implemented an algorithm (ChipStat) that takes identical probe pairs across two comparison groups and performs a heteroscedastic However, under experimentally realistic conditions, neither of these limiting cases is likely to apply. Therefore, to empirically determine the null distribution using six independent, biologically identical control populations, all pairwise three by three combinations were compared and the number of probe pairs changing was tabulated. To determine the expected number of changes per probe set when fewer than 16 probe pairs are available, these analyses were repeated after randomly discarding 1, 2...15 probe pairs. In this way, a similar statistical estimate was obtained for the 602 probe sets on the MG_U74Av2 array that have fewer than 16 probe pairs per probe set. A conservative simplification of these data was performed by rounding up the significance of changes in these 602 probe sets to the nearest appropriate bin in the 16 probe pair per probe set curve. A Microsoft Windows-compatible application implementing the ChipStat algorithm is freely available for academic use .pps for various relative proportions of E(b) + E(p) and E(h). Briefly, a random test dataset was generated in which equal gene expression was perturbed by Gaussian noise (representing E(b) + E(p)). Each expression value was then independently perturbed 16 times (representing 16 probe pairs/probe set) by another Gaussian noise function (representing E(h)), and comparisons were tabulated using the ChipStat algorithm. This simulation was implemented in C and the source code is available [On the basis of the simplified statistical model described, a Monte Carlo simulation was implemented to determine the number of expected false-positive values as a function of vailable . All valxi is the number of times i probe pairs increased significantly and μi and σi represent the mean and standard deviation from the Monte Carlo simulations.with respect to (E(b) + E(p)) / E(h) where A separate algorithm (Intersector) uses pairwise calls of differential gene expression derived from Affymetrix Microarray Suite (MAS) analysis. All pairwise comparisons were performed  using the manufacturer's default settings, and the number of 'increases' or 'marginal increases' was tabulated. Similarly to the ChipStat method described above, the null distribution was generated by tabulating results from all 20 distinguishable 3 vs 3 combinations of the six control samples. Results were obtained using both MAS version 4 (MAS4) and MAS version 5 (MAS5), as indicated in the text and figures.t-test or SAM [Tests for differential gene expression using a homoscedastic t or SAM  were pert or SAM . Becauset or SAM  was perft or SAM , and a Pt or SAM  was perfP < 0.05 was used to identify statistically significant associations.Associations between GO  annotatipps = 0.05) and Intersector  lists of significantly increasing probe sets was generated. Rows indicate the threshold number of probe pairs (0-16) significantly increasing from ChipStat, and columns indicate the threshold number of Increase or Marginal Increase calls (0-9) identified by Intersector. (a) Number of increasing probe sets in 2- vs 5-week murine mammary gland. Selected results correspond to values plotted on the y axis of Figure b) Average number of increasing probe sets using all 3 × 3 combinations of 6 negative control samples. Selected results correspond to values plotted on the x axis of Figure pps = 0.05, 6/16 probe pairs increasing and Intersector 7/9 increases + marginal increases, were used to identify lists of probe sets that are up- and downregulated from 2 to 5 weeks of FVB female murine mammary gland development. Additional data files The following additional data are available with the online version of this paper. Additional data file pairs 0-1 signific calls 0- identifiA table showing ChipStat and Intersector in combinationClick here for additional data fileA table showing differential gene expression in 2- vs 5-week murine mammary gland using a hybrid ChipStat/Intersector approachClick here for additional data fileControl file 1 containing CEL data from Affymetrix MG_U74Av2 oligonucleotide microarrays hybridized to RNA from the third to fifth mammary glands harvested from independent pools of three female MTB transgenic mice at 6 weeks 4 days old after 96 hours of doxycycline treatmentClick here for additional data fileControl file 2 containing CEL data from Affymetrix MG_U74Av2 oligonucleotide microarrays hybridized to RNA from the third to fifth mammary glands harvested from independent pools of three female MTB transgenic mice at 6 weeks 4 days old after 96 hours of doxycycline treatmentClick here for additional data fileControl file 3 containing CEL data from Affymetrix MG_U74Av2 oligonucleotide microarrays hybridized to RNA from the third to fifth mammary glands harvested from independent pools of three female MTB transgenic mice at 6 weeks 4 days old after 96 hours of doxycycline treatmentClick here for additional data fileControl file 4 containing CEL data from Affymetrix MG_U74Av2 oligonucleotide microarrays hybridized to RNA from the third to fifth mammary glands harvested from independent pools of three female MTB transgenic mice at 6 weeks 4 days old after 96 hours of doxycycline treatmentClick here for additional data fileControl file 5 containing CEL data from Affymetrix MG_U74Av2 oligonucleotide microarrays hybridized to RNA from the third to fifth mammary glands harvested from independent pools of three female MTB transgenic mice at 6 weeks 4 days old after 96 hours of doxycycline treatmentClick here for additional data fileControl file 6 containing CEL data from Affymetrix MG_U74Av2 oligonucleotide microarrays hybridized to RNA from the third to fifth mammary glands harvested from independent pools of three female MTB transgenic mice at 6 weeks 4 days old after 96 hours of doxycycline treatmentClick here for additional data fileCEL file 1 of data from Affymetrix MG_U74Av2 oligonucleotide microarrays hybridized with mammary gland RNA from independent pools of 10 female FVB mice harvested at 2 weeks of ageClick here for additional data fileCEL file 2 of data from Affymetrix MG_U74Av2 oligonucleotide microarrays hybridized with mammary gland RNA from independent pools of 10 female FVB mice harvested at 2 weeks of ageClick here for additional data fileCEL file 3 of data from Affymetrix MG_U74Av2 oligonucleotide microarrays hybridized with mammary gland RNA from independent pools of 10 female FVB mice harvested at 2 weeks of ageClick here for additional data fileCEL file 1 of data from Affymetrix MG_U74Av2 oligonucleotide microarrays hybridized to mammary gland RNA from independent pools of 10 female FVB mice harvested at 5 weeks of ageClick here for additional data fileCEL file 2 of data from Affymetrix MG_U74Av2 oligonucleotide microarrays hybridized to mammary gland RNA from independent pools of 10 female FVB mice harvested at 5 weeks of ageClick here for additional data fileCEL file 3 of data from Affymetrix MG_U74Av2 oligonucleotide microarrays hybridized to mammary gland RNA from independent pools of 10 female FVB mice harvested at 5 weeks of ageClick here for additional data file


Based on a semester-long seminar on the topic, this book aims to fill a gap in the current engineering curricula by taking a wide-angle view at the process of engineering design rather than focusing on a more narrow and in-depth approach. As part of Artech House Publishers' technology management and professional development library, this is an excellent introduction to the topic and may serve for later reference.The author, Peter. H. Sydenham is the inaugural professor and head of the University of South Australia's School of Electronic Engineering, co-founder of the Australian Centre for Test and Evaluation, and a director of Global Systems Engineering Consulting Pty Ltd. This brief biographical sketch explains his qualification to write a book of such practical value and dimension, and why the book is pedagogically sound.The book, according to the preface, aims at those readers who are engineers "who have, or aspire to team leadership or want to take on increased team interfacing responsibilities". As such it builds on and expands what the author perceives to be too little taught in the regular university courses, and covers what is lacking: "breadth of the knowledge now needed to be an effective engineering designer."Twelve chapters offer a step-by-step introduction to the topic. The first chapter, "Systems Thinking and Systems Engineering", gives an overview and the "philosophical" background, while the last looks at "Change and Future Trends". The remaining ten chapters cover the practical aspects of the whole process of systems engineering design with a more hands-on approach, including basics of supporting knowledge such as staff management from planning over recruitment through training to promotion or termination of contract (chapter 3) or information technology support. As such, chapters 2–4 cover the more general aspects, while chapters 5–9 cover the design process from idea to evaluation. Chapter 10 intersperses legal issues, while chapter 11 covers the prototype build, i.e. the step from abstract design to material product.This is an introductory textbook, and as such, it does a very good job of the task at hand. The language is clear, the case examples are well chosen, and the message is conveyed without fail. Some issues are discussed somewhat at length; for example, the basic IT issues are explained in a very detailed way, but one might assume that the basics would be self-evident for today's user: the target audience will have grown up in the age of the personal computer and the internet and as such, will neither question the computer's usefulness nor be overly naïve with regards to the many IT bugs one has to deal with.The illustrations are about as clear as can be-some, in fact, are not clear at all but as they are meant to demonstrate the complexity of the issue at hand, the message gets conveyed the way it should be.How does this general book relate to the specialist field of biomedical engineering? To quote from p. 12: "Researchers in the life sciences were driven by a need to better understand how nature works and controls itself. Out of this pioneering work emerged general systems theory cybernetics, self-organizing systems, automation, automaton systems, organizational science, operations research, systems science, and more-topics with which engineers are not usually that familiar". This lack of familiarity becomes very evident when young engineers join research teams with a focus on applications for the life sciences, such as biomechanical engineering, but also within the broad field of bioinformatics, and they usually take more time to acquire and apply that holistic view of their work than life scientists need to acquire and implement detailed and circumscript engineering knowledge to accomplish their tasks. As such, this book can be recommended to engineers working in biomedicine even at the outset of their careers as it may draw their attention to the importance of this view of things in the new field they enter. Competing recent titles include works by Blanchard . A more One point that a next edition seriously needs to address is the text editing. Using a word processor does not guarantee a perfect text: this one has a lot of extra words, and as many words missing. As faulty as the text editing is the punctuation check. These errors occur as often as once per page, and force the reader to repeat the study of entire paragraphs several times because, more often than not, it is the missing preposition or punctuation that poses a serious threat to understanding. The publishing house would do well in employing an old-fashioned human text editor to spare the reader such nuisance.In summary, this is a basic textbook to project design management for aspiring team leaders, not only in engineering but for any scientific and some business projects as well. Well written, this is a recommended introduction to the matter and may serve as a reference book and reminder even to the experienced team leader.Adrian Mondry is at Bioinformatics Institute, Singapore Mondry@hotmail.com.


Recent literature has called for humanistic care of patients and for medical schools to begin incorporating humanism into medical education. To assess the attitudes of health-care professionals toward homeless patients and to demonstrate how those attitudes might impact optimal care, we developed and validated a new survey instrument, the Health Professional Attitudes Toward the Homeless Inventory (HPATHI). An instrument that measures providers' attitudes toward the homeless could offer meaningful information for the design and implementation of educational activities that foster more compassionate homeless health care. Our intention was to describe the process of designing and validating the new instrument and to discuss the usefulness of the instrument for assessing the impact of educational experiences that involve working directly with the homeless on the attitudes, interest, and confidence of medical students and other health-care professionals.The study consisted of three phases: identifying items for the instrument; pilot testing the initial instrument with a group of 72 third-year medical students; and modifying and administering the instrument in its revised form to 160 health-care professionals and third-year medical students. The instrument was analyzed for reliability and validity throughout the process.A 19-item version of the HPATHI had good internal consistency with a Cronbach's alpha of 0.88 and a test-retest reliability coefficient of 0.69. The HPATHI showed good concurrent validity, and respondents with more than one year of experience with homeless patients scored significantly higher than did those with less experience. Factor analysis yielded three subscales: Personal Advocacy, Social Advocacy, and Cynicism.The HPATHI demonstrated strong reliability for the total scale and satisfactory test-retest reliability. Extreme group comparisons suggested that experience with the homeless rather than medical training itself could affect health-care professionals' attitudes toward the homeless. This could have implications for the evaluation of medical school curricula. In 2003, the Department of Health and Human Services reported that there are between two to three million people in the United States who experience homelessness each year . AccordiThe disinclination of the homeless to seek care may be due in part to the way in which many health-care workers respond to them. A less investigated but possibly equally important circumstance is the attitudes that health-care professionals have toward the homeless. As members of the larger society, these professionals often harbor the same preconceived ideas and biases toward the homeless that the rest of society does. Such judgmental attitudes can, and often do, emerge during the provider-patient encounter, thus limiting the effectiveness of medical treatment of the homeless .As a countermeasure to prevailing negative attitudes and widespread stigmatization toward marginalized persons, recent medical literature has drawn on a well-known practice in the social sciences ,11 and hAlthough it would be helpful to have a better understanding of physicians' current opinions about homeless patients, only two instruments have been designed and validated to measure attitudes toward homeless persons: the Attitudes Toward the Homeless Inventory (ATHI) , and theThe purpose of our study was to develop and validate a new measure, the Health Professionals' Attitude Toward the Homeless Inventory (HPATHI), an instrument that could be used to assess United States medical students' and physicians' attitudes toward homeless persons and to measure their level of interest and confidence in their ability to deliver health-care services to the homeless population. The objectives of this study were to: 1) describe the process of designing and validating the new instrument, and 2) discuss the usefulness of the instrument for assessing the impact of educational experiences working with the homeless on the attitudes, interest, and confidence of medical students and other health-care professionals.The development and validation of the HPATHI occurred in three phases. The first phase consisted of the identification of items for the inventory; the second phase involved a pilot test of the initial instrument with a group of medical students; and the final phase entailed modifying the instrument and administering it in its revised form to our target population – health-care professionals, including primary care physicians, primary care residents, and third-year medical students.Throughout this three-phase process, we sought to ascertain the reliability and validity of the HPATHI. Three types of validity were tested. Content validity was evaluated using the Delphi technique , which sattitudes toward the homeless; b) interest in working with the homeless; and c) confidence in one's ability to work with the homeless.In Phase 1, a group of physicians and nurse practitioners, all experts in homeless health care, was recruited through snowball sampling  to serveDuring Phase 2, we administered the first draft of the HPATHI to a convenience sample of third-year medical students enrolled at Baylor College of Medicine (BCM) in Houston, Texas. These students were selected because they showed an interest in working with the homeless by registering for an elective course that allows students to provide health care to the homeless in a clinic setting . Of the In Phase 3, we prepared Web-based versions of the HPATHI and of the ATHI and sent the Internet link to our target population with a standard invitation to complete both instruments. Primary care physicians who serve as faculty in the BCM Department of Family and Community Medicine, family practice residents in the BCM Department of Family and Community Medicine, general internal medicine residents in the BCM Department of Family and Community Medicine, and students from the BCM Medical School served as a convenience sample of individuals completing the instrument over a six-month period. Subsequent data analysis consisted of:1. Conducting an exploratory factor analysis using a Promax rotation to examine the structure of the HPATHI.2. Estimating the internal consistency reliability of the online version of the HPATHI using Cronbach's alpha coefficient;3. Determining the concurrent validity of the HPATHI against the ATHI;4. Comparing extreme groups on their responses to the HPATHI. The extreme groups were determined according to their level of training  and according to their experience in working with the homeless (<1 month vs. >1 year).® (V8.2 and V9.1) for Phase 3.This study was approved by the BCM Institutional Review Board for educational research with human subjects. Data analysis was conducted using SPSS Version 12.0 statistical software for Phases 1 and 2 and SASOf the 23 panel members who received the first list of 24 statements, 16 (70%) reviewed them, rank ordered them, and generated an additional 26 statements. We rearranged the combined list of 50 statements according to the proposed ranking  and returned them to the 16 panel members for a second ranking. Based on their responses, six statements were eliminated and the remaining 44 were returned to the 16 panel members for a third ranking. Nine panel members returned responses after this iteration, and we eliminated nine additional items according to their recommendations. The remaining 35 items then constituted the first draft of the instrument we named the Healthcare Professionals' Attitude Toward the Homeless Inventory (HPATHI). Although seven panel members did not participate in the third iteration, their rankings in the second iteration corresponded to the final list of items.The sample population of third-year medical students completed a pilot administration of the 35-item HPATHI; the subset of students who responded two weeks later underwent a second administration of the same instrument. Table Cronbach's alpha coefficient for the first administration was 0.87; the test-retest reliability coefficient (Pearson r) was 0.69. Through an item analysis we discarded 12 items that were either highly correlated with other items, and were thus considered repetitious, or that had item-scale correlations less than 0.20.One hundred and sixty health-care professionals  from one academic institution completed the HPATHI; 147 of them also completed the ATHI. Table The exploratory factor analysis , using a Promax rotation to account for the relationship among the factors, yielded a three-factor structure that explained 39% of the variance of the data. Factor 1 consisted of items 12, 13, 14, 17, 18, 19, 20, 21, 22, and 23 and was labeled Personal Advocacy; factor 2 consisted of items 1, 3, 4, 7, 8, 9, and 15 and was labeled Social Advocacy; and factor 3 consisted of items 5, 6, 10, and 11 and was labeled Cynicism. Table The HPATHI was further shortened to 19 items by the deletion of four more items, which either were not represented in the three-factor structure  or had an adverse effect on the subscale's reliability (item 15). The three subscales were also significantly related to each other: factor 1 had Pearson's r correlations of 0.47  with factor 2 and 0.43  with factor 3; and factor 2 had a Pearson's r correlation of 0.48  with factor 3. Table The Pearson's correlation coefficient between the HPATHI and the ATHI was 0.68 for the HPATHI's total scale  Table . For theUsing the Delphi method to select the survey items helped ensure the HPATHI's content validity. Not only were the items selected and validated by homeless health-care experts, but the items chosen for inclusion in the HPATHI were based on findings from current literature on the issue. In addition, by correlating the HPATHI with the ATHI, we were able to demonstrate satisfactory concurrent validity. Construct validity for the HPATHI  was determined by the extreme group comparisons, the item analyses, and the factor analysis.Results of the extreme group comparisons showed that experience with the homeless, rather than medical training, is a significant factor that correlates with health-care professionals' attitudes towards the homeless and their interest in working with the homeless population. Moreover, individuals who had more extensive experience with the homeless showed more positive attitudes toward and interest in homeless patients. Therefore, increasing the opportunities to provide direct patient care to the homeless might improve the attitudes of health-care professionals toward this group.personal advocacy, which contains the items that reflect a personal commitment to work with the homeless; social advocacy, which consists of the items that reflect society's responsibility to care for the homeless population; and cynicism, which encompasses the items that reflect a negative attitude and a sense of futility in working with the homeless.The factor analysis suggested that we do have potential subscales in the instrument that may offer meaningful information regarding the general attitudes of health-care professionals who work with the homeless. The three subscales appear to represent: A limitation of this study is that only 160 health-care professionals participated in the online administration of the HPATHI. This small sample size limits the effectiveness of the factor analysis. Moreover, our sample focused only on the medical profession as represented by medical students and primary care residents and physicians. The participation of a larger number of health-care professionals from other medical specialties would strengthen the study.The development of the HPATHI has many implications for and applications to future research on homeless health care, although its most valuable use may be to assess the attitudes toward homeless persons of medical students, residents, and practicing physicians. Throughout its various iterations, the instrument demonstrated strong internal consistency reliability for the total scale and satisfactory test-retest reliability. The scales identified by the factor analysis also showed satisfactory internal consistency reliability. The information collected from the expert panel and the literature search on attitudes toward the homeless and their health-care status proved to be an excellent framework for determining which statements to retain for the final instrument. The inter-item correlations and the correlations among the subscales indicate that the items are measuring similar underlying constructs within an overall theme – the attitudes of health-care professionals toward the homeless.Despite the limited sampling, the research process has demonstrated that the HPATHI is a reliable and valid instrument that has the ability to assess the attitudes of health-care professionals toward the homeless population. We believe that the instrument may also be used in the future within the academic framework of medical schools to determine if attitudinal changes are affected by training experiences occurring with the homeless. To determine whether this is the case, we intend to administer the HPATHI as a pre/post-test survey to students who enroll in the homeless health-care track. Moreover, over the next year, we plan to keep on assessing the attitudes of health-care professionals toward the homeless by having new groups respond simultaneously to the ATHI and the HPATHI, as was done in Phase 3 of the original study. Additionally, we intend to include participants from other medical schools in the United States and to expand our sample to other health-care professionals who traditionally work with the homeless, to further test the instrument's overall validity.The author(s) declare that they have no competing interests.DB contributed to the conception, design, and acquisition of data, and drafted and revised the article. FMM contributed to the conception, design, and acquisition of data, helped draft the article, and carried out the data analysis. SK contributed to the conception, design, and acquisition of data, helped draft the article, and carried out the data analysis. DR contributed to the draft of the manuscript and critically revised it for important content. DLC contributed to the conception, design, and acquisition of data. AM contributed to the conception, design, and acquisition of data. RJV contributed to the interpretation of the data and critically revised it for important content. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:


Gastrointestinal symptoms are common in patients with Chronic Fatigue Syndrome (CFS). The objective of this study was to determine the frequency of these symptoms and explore their relationship with objective (radionuclide) studies of upper GI function.Thirty-two (32) patients with CFS and 45 control subjects completed a questionnaire on upper GI symptoms, and the 32 patients underwent oesophageal clearance, and simultaneous liquid and solid gastric emptying studies using radionuclide techniques compared with historical controls.The questionnaires showed a significant difference in gastric (p > 0.01) symptoms and swallowing difficulty. Nocturnal diarrhoea was a significant symptom not previously reported.5/32 CFS subjects showed slightly delayed oesophageal clearance, but overall there was no significant difference from the control subjects, nor correlation of oesophageal clearance with symptoms. 23/32 patients showed a delay in liquid gastric emptying, and 12/32 a delay in solid gastric emptying with the delay significantly correlated with the mean symptom score (for each p ≪ 0.001).GI symptoms in patients with chronic fatigue syndrome are associated with objective changes of upper GI motility. Chronic Fatigue Syndrome (CFS) is a descriptive term used to define a classifiable pattern of symptoms that cannot be attributed to any alternative condition . It can Many with CFS have gastro-intestinal (GI) symptoms, which are often unrecognised as being part of CFS. The commonest of the upper GI symptoms include fullness and bloating after a small meal, abdominal distension, nausea, and loss of appetite. Lower GI tract symptoms have considerable overlap with irritable bowel syndrome .The hypothesis explored in this paper is that symptoms of possible upper gastrointestinal origin are more common in patients with CFS and are related to upper gastrointestinal motility as assessed by radionuclide methods.Consecutive patients with CFS who met the Fukuda criteria  for CFS oesophageal" , "gastric": , "other" frequency of bowel actions, consistency of stools, presence or absence of diarrhoea, urgency and timing.Symptoms were divided into "Symptoms were scored. 0, none, 1, mild (symptom could be ignored), 2, moderate , 3, severe, (symptom influenced daily activities). A mean symptom score (maximum score 3) for the 6 gastric symptoms, and 3 oesophageal symptoms was obtained.The volunteer control subjects who completed the questionnaire were in regular full time employment, with no history of excessive fatigue, on no GI medication, and had no previous GI surgery.99mTc-sulfur colloid-chicken liver, and the liquid meal consisted of 150 ml of 10% dextrose in water containing with 20 MBq of 67Ga-ethylenediaminetetraacetic acid (EDTA). All medication  was discontinued for 24 hours prior to each study. The test was performed at 10 am (after an overnight fast) and monitored for at least two hours with the subject in the sitting position with the scintillation camera behind. The study commenced with a standardised oesophageal clearance study (solid bolus) followed by eating the solid meal and then immediately drinking the glucose solution. Each study was continued for at least 2 hours. Oesophageal clearance was expressed as time to 95% clearance (ref range < 93 sec) [Details and normal ranges of this double isotope test have been previously published . The sol2, and Gastric emptying indices compared with historical normal range (t test comparison of means), and correlated with the mean symptom score (± SD).The GI questionnaires were compared between CFS and control by ChiThe Study was approved by The Human Research Ethics Committee of the Royal Adelaide Hospital and informed consent given by the subjects.Thirty-two 32) CFS patients (22F), with a mean age of 38.5 years had gastric emptying studies. Forty-five (45) control subjects undertook the questionnaire. The demographic details of the controls vs. patients are shown in table  CFS patiThe overall, grouped gastric emptying studies of CFS subjects showed no significant slowing of oesophageal clearance p = 0.45 from the control population, and no significant correlation between emptying and oesophageal symptom score r = 0.15) although 5 of the symptomatic and 2 of the asymptomatic subjects 7/32 (22%), were slower than the 95% confidence limits,  declare that they have no competing interests.RB examined the patients and analysed the clinical data, BC performed the Nuclear Medicine studies, drafted the manuscript and performed the statistics. Both read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Plasmodium falciparum malaria in Sudan has been in process of change since 2003. Preceding the change, this study aimed to determine which artemisinin-based combination therapies is more effective to treat uncomplicated malaria in Malakal, Upper Nile, Sudan.The treatment for P. falciparum infections in children aged 6–59 months, in a period of 42 days after treatment.Clinical trial to assess the efficacy of 2 antimalarial therapies to treat A total of 269 children were followed up to 42 days. Artesunate plus Sulfadoxine/Pyrimethamine (AS+SP) and Artesunate plus Amodiaquine (AS+AQ) were both found to be efficacious in curing malaria infections by rapid elimination of parasites and clearance of fever, in preventing recrudescence and suppressing gametocytaemia. The combination of AS+SP appeared slightly more efficacious than AS+AQ, with 4.4% (4/116) versus 15% (17/113) of patients returning with malaria during the 6-week period after treatment . PCR analysis identified only one recrudescence which, together with one other early treatment failure, gave efficacy rates of 99.0% for AS+AQ (96/97) and 99.1% for AS+SP (112/113). However, PCR results were incomplete and assuming part of the indeterminate samples were recrudescent infections leads to an estimated efficacy ranging 97–98% for AS+SP and 88–95% for AS+AQ.These results lead to the recommendation of ACT, and specifically AS+SP, for the treatment of uncomplicated falciparum malaria in this area of Sudan. When implemented, ACT efficacy should be monitored in sentinel sites representing different areas of the country. Plasmodium falciparum resistance against the two most commonly used antimalarials: chloroquine (CQ) and sulfadoxine/pyrimethamine (SP). CQ resistance in the northern and central part of the Sudan is near 50% [in vivo studies documenting of 0–11% resistance in Khartoum and the eastern part of the country [The health situation in Sudan continues to be affected by long-lasting conflict and related humanitarian emergencies such as food crises and epidemics. Malaria is one of the major causes of morbidity and mortality. In Sudan, an estimated 7.5 million patients suffer from malaria each year and 35,000 die from this disease, which accounts for up to 20% of hospital deaths . The pronear 50% , but wasnear 50% . Resista country  and vary country -6. AmodiP. falciparum malaria in Sudan at the time of this study was still CQ as first-line and SP as second-line. A change in protocol was however in progress, and the Sudanese Health authorities had indicated that artemisinin-based combination therapy (ACT) was the preferred new first-line treatment [The nationally recommended treatment protocol for reatment . The Worreatment ,11.in vivo therapeutic efficacy of the first two combinations were studied in Malakal town, Upper Nile State.In line with recommendations for several other African countries, two ACTs were proposed for Sudan: Artesunate (AS) +SP or AS+AQ. The third option, used in other African countries, Artemether-Lumefantrine (Coartem) was not seen as a viable option at the time, because of its cost. To provide information on the efficacy of ACT treatment in Sudan, the P. falciparum infection with a density of 2,000 to 200,000 parasites/μl but no signs of severe malaria , which is an area is of medium to high malaria endemicity (main transmission season: June-November). This open-label study was done in Malakal paediatric hospital supported by Médecins Sans Frontières, from September 2003 to January 2004. The methodology used was in accordance with standard WHO procedures for antimalarial drug efficacy assessment in high transmission settings ,13. ChilThe sample size of at least 116 inclusions per arm was calculated to detect a 12% difference between the two arms, assuming a 97% efficacy at day 28 for the AS+SP arm  and 85%msp1 and msp2 loci was used to distinguish re-infections from recrudescences [Slides were examined by two microscopists independently and 20% of slides were cross-checked in an external laboratory . The density of parasites was determined by simultaneous count of white blood cells and parasites, assuming a standard density of 8,000 WBCs per μl . Haemoglescences ,15. In cThe research protocol was reviewed and approved by the Sudan Research Directorate, the Malaria, Leishmaniasis and Schistosomiasis Directorate, as well as by the Ethical Review Board of MSF . During recruitment of the children in study, informed written consent was obtained from their parents/guardians.A total of 269 children were recruited during the 5-month study period. Of these, 134 were treated with AS+AQ and 135 with AS+SP. Baseline characteristics were similar in both treatment groups .Both artemisinin-combitherapies were highly effective to treat up Table . At day PCR analysis was unable to generate results for a large part of cases  parasitaemia in the AS+AQ and AS+SP groups, respectively. Gametocytes were found in 20% (52/262) of children on inclusion. During follow-up, 78% of these carriers at enrolment showed gametocytes again on one or more follow-up days (32/41), whereas 22% of the children without gametocytes at inclusion were later found to be gametocytemic (42/192). There was no difference between the age, temperature, parasitemia, haemoglobin on admission of the two groups, neither in rates of ACPR at study-endpoint. Gametocyte carriage was similar in both treatment groups and all gametocytes disappeared gradually from the blood during the first weeks after follow-up, and cleared by day 21 in 95% of cases  at day 1 and day 2 was 86% and 97% in the AS + AQ group and 83% and 93% in the AS+SP, and at day 3 all but one (on AS+SP) were free from fever. Average haemoglobin levels for the AS+AQ and AS+SP treatment groups increased from under 8.0 g/dl to near 10 g/dl 6 weeks after treatment. The proportion of children classified as moderately anaemic (haemoglobin from 5 to 8 g/dl) reduced from 51% to 11% during follow-up period, similar in both arms. No serious adverse events were observed or reported on routine clinical examination during follow-up and none of the failures developed complicated malaria.This is one of the first studies into the clinical outcomes of ACT combination therapy in Sudan and provides useful information for decision-makers working to ensure effective antimalarial protocols in this part of the country.P. falciparum malaria in this area of the Sudan. AS+SP appears to be the better treatment option on the basis of non-PCR corrected responses, showing a lower percentage of patients returning with parasitaemia. The PCR analysis indicates the true efficacy is comparable between both treatments (near 99%), but is, however, limited by a high proportion of indeterminate cases. More realistically, an efficacy between 97–98% can be expected for AS+SP and 88–95% for AS+AQ, acceptable levels after the long follow-up of 42 days after treatment. A rapid parasite clearance and fever reduction was found following treatment with both ACTs. The rise in haemoglobin values and the reduction of the proportion of (moderately) anaemic children after treatment confirms that the malaria parasites were effectively removed from the blood and red blood cell levels rose after treatment.Both of the artemisinin-based combination therapies tested here were found to be highly efficacious in the treatment of uncomplicated The ACTs tested also had an effect on gametocytes. In general, the gametocidal action of AS appears to work through preventing the development of new gametocytes rather than clearance of existing ones . In our The main limitations of the study were that the number of patient exclusions were higher than anticipated (15%) due to concomitant febrile illnesses and loss to follow-up, as well as the lack of results for PCR-analysis, caused by samples missing or an inability to provide sufficient DNA on amplification due to low densities in post-treatment samples. Increasing the number of inclusions compensated losses to follow-up. Repeated attempts at PCR-analysis for problematic samples were only partly successful and, therefore, lead to extrapolation about the findings for the missing samples.P. falciparum and SP has shown high efficacy in various areas of northern Sudan [At time of writing, a change of national protocols towards ACT in northern Sudan is in preparation – coordinated among health authorities, NGOs and other relevant actors. The first line treatment recommended country-wide is AS+SP, based on reported high efficacy of SP. Northern Sudan is the only country in Africa, which has chosen the option AS+SP . On the rn Sudan ,4,23. Itrn Sudan . The useAn advantage of AS+SP over AS+AQ is that it is more convenient to administer, as SP is given as a single dose and can be administered under observation in a health facility, whereas AQ requires 3 days to complete a course. SP tablets are also easier to take (AQ has a bitter taste). Blister packs of AS+SP, which combine the two drugs and clearly indicate daily tablets to be taken, are currently available for different age categories in Sudan. AS+SP is cheaper than AS+AQ.Implementation of the new national protocol with AS+SP will hopefully take place as quickly as possible to prevent rising morbidity and mortality. Vulnerable displaced populations in epidemic-prone areas and areas of high perennial malaria transmission should be prioritized. Introduction of ACT will have to go hand in hand with laboratory confirmed diagnosis (microscopy or rapid diagnostic tests) to prevent unnecessary use of valuable drugs (thus minimizing drug pressure) and ensure that non-malarial cases are appropriately treated. The change in guidelines should also filter through all health service providers, including the private sector and drug vendors to decrease the potential risk of SP monotherapy and incomplete dosages. The change will initially require more funds to be made available for malaria treatment. The international community – including many NGOs, the WHO, donors, and the Global Fund – has shown willingness to support countries to change antimalarial protocols ,25.At present, MSF has already started to pilot AS+SP treatment in its project areas in Northern Sudan, i.e. Darfur, Upper Nile and Gedaref, on behalf of the Ministry of Health. The implementation of treatment as well as the future efficacy of AS+SP should be monitored carefully in a number of dispersed sentinel sites, as there is a possibility that SP resistance may further rise before the combination has been made available countrywide.IB was responsible for overall supervision, data analysis and writing of the paper, RA for field-implementation, communication as well as data analysis and writing, MB for preparation and set-up of study, support and advocacy. PH was responsible for microscopy laboratory work and quality. EA participated as field supervisor of clinical work and data gathering. EBH and MAF were responsible for day-to-day study procedures and supervision of clinical teams. JM did the PCR analysis and FC was involved in design of study, data analysis and writing of manuscript. All authors read and approved the final manuscript.


Despite intense effort the treatment options for the invasive astrocytic tumors are still limited to surgery and radiation therapy, with chemotherapy showing little or no increase in survival. The generation of Serial Analysis of Gene Expression (SAGE) profiles is expected to aid in the identification of astrocytoma-associated genes and highly expressed cell surface genes as molecular therapeutic targets. SAGE tag counts can be easily added to public expression databases and quickly disseminated to research efforts worldwide.SAGE Genie [in silico search for candidate therapeutic targets by comparing astrocytoma to normal brain transcription. Real-time PCR and immunohistochemistry were used for the validation of selected candidate target genes in 2 independent sets of primary tumors.We generated and analyzed the SAGE transcription profiles of 25 primary grade II, III and IV astrocytomas . These pA restricted set of tumor-associated genes was identified for each grade that included genes not previously associated with astrocytomas , with a high percentage of cell surface genes. Two genes with available antibodies, Aquaporin 1 and Topoisomerase 2A, showed protein expression consistent with transcript level predictions.This survey of transcription in malignant and normal brain tissues reveals a small subset of human genes that are activated in malignant astrocytomas. In addition to providing insights into pathway biology, we have revealed and quantified expression for a significant portion of cell surface and extra-cellular astrocytoma genes. Astrocytomas are the most frequent malignant primary brain tumors in adults. Clinically, this group of tumors can be divided into four World Health Organization (WHO) grades. Pilocytic astrocytomas (WHO grade I) are generally slow growing and non-infiltrative pediatric tumors, which are rarely fatal. For the infiltrating astrocytomas, survival decreases with increasing grade. Grade II astrocytomas patients survive an average of over 5 years, but survival drops to 3 years for anaplastic astrocytomas (grade III). Grade IV astrocytomas (glioblastoma multiforme or GBM) account for about half of all astrocytic tumors, with a median survival of less than a year. Effective treatment options for the invasive grade II to IV tumors are still limited to surgery and radiation therapy, with most chemotherapy regimens showing little or no increase in survival.Several recent gene expression profiling studies of human astrocytomas have been able to distinguish between various grades of astrocytomas and between astrocytomas and other human glial tumors, and to identify new molecular classes within histological grade -7. This [In this study, we sought to create a public and comprehensive gene expression resource for astrocytomas, with the primary intention of aiding searches for new therapeutic targets in malignant astrocytomas. For this purpose we produced and analyzed in detail 25 gene expression profiles of primary astrocytic tumors  using Serial Analysis of Gene Expression (SAGE) . Complet were useAstrocytic tumor samples from 21 adults and 4 children were obtained from the Duke Brain Tumor Bank. All samples were classified based on histology according to the World Heath Organization grading criteria. Pediatric normal cortex (15 months) was a gift of Dr. Rachel Myerowitz and normal pediatric cerebellum was from the Maryland Brain Bank. Normal adult cortex and cerebellum were rapid autopsy samples obtained from the Duke Alzheimer's Brain Bank. Total RNA from normal substantia nigra was obtained from Clontech . PolyA+ RNA from normal adult leukocytes was obtained from Stratagene , as noted for each library's information at the SAGE Genie Website [Nla III as the anchoring enzyme and BsmF I as the tagging enzyme using a micro-SAGE protocol. The SAGE library clones were partially arrayed at Lawrence Livermore National Laboratories and inserts were purified and sequenced at the BC Cancer Agency Genome Sciences Centre or arrayed and sequenced at Agencourt Bioscience Corporation.SAGE libraries from 25 selected astrocytomas: 8 grade II astrocytomas, 10 grade III anaplastic astrocytomas and 7 grade IV glioblastomas (primary GBM) were constructed using ) was used to extract SAGE tags from the original sequence files, remove duplicate ditags, remove linker sequences, remove one base pair variations of linker sequences and tabulate the occurrence of each tag. Tag sequences, tag counts and gene associations were stored in a Microsoft Access relational database for subsequent selection of tags with a particular profile. A total of 2,605,122 tags were obtained with an average of 102,988 tags per library. Normal neural tissue tags included a total of 443,560 tags from normal brain [[The SAGE 2000 software version 4.12  in combination with microwave treatment. Sections were then blocked in PBS/0.5% Triton X-100 containing 2.5% normal horse serum for at least 30 min at room temperature and incubated with primary antibody overnight at 4°C. The primary antibodies used were mouse monoclonal anti-aquaporin1, at a 1:60 dilution  and mouse monoclonal anti-Topoisomerase II Alpha used at a 1:30 dilution . Sections were developed with a DAB  substrate, counterstained with hematoxylin and mounted with Cytoseal-60.Formalin-fixed 5-μm paraffin-embedded sections were stained with various antibodies using the biotin/streptavidin RTU Vectastain Universal Quick kit  as previously described . ShortlyThe tissue micro array used in these studies contained cores from 20 glioblastomas, 20 anaplastic astrocytomas, 20 infiltrating astrocytomas and 20 oligodendroglial lesions and was prepared according to methods described by Kononen and Kallioniemi . Microsc, where the libraries can be downloaded or viewed online using SAGE Genie tools [This report describes the comprehensive generation of expression profiles of three astrocytic tumor grades based on Serial Analysis of Gene Expression (SAGE). The main goals were to identify genes not expressed in normal brain tissue and genes highly expressed in the more aggressive astrocytomas encoding cell surface or extra-cellular matrix related proteins that could be of potential therapeutic interest. We generated SAGE profiles on 8 infiltrating astrocytomas, 10 anaplastic astrocytomas and 7 glioblastoma samples. Combined with two glioblastoma profiles [ie tools .in silico selection using SAGE profiles revealed tumor-associated genes that can be found in a different set of primary tumors implying a possible role for these genes in tumor development and increasing their value as putative therapeutic targets.In order to identify tumor-associated genes, we sought highly expressed transcripts in each grade of astrocytoma that were not expressed in 7 normal brain tissues. This also helped control for contaminating normal cells within the tumor sample. Tags expressed in a normal leukocyte SAGE library were also included in the analysis so they could be subtracted to reduce the chances of identifying transcripts from white blood cells that frequently infiltrate these tumors. Initially we selected for tags with an average expression of at least 3 per 100,000 tags. Subsequently, we selected for tags expressed at less than 2 counts per 100,000 in each of the 8 normal libraries , reducing the number of tags to less than 100 per tumor type  was used as a final filter to identify membrane, cell surface and cell adhesion related genes . Both anaplastic astrocytomas and glioblastomas show high expression of VAPB and anaplastic astrocytomas express caveolin1 Table . It has CHI3L2) or YKL-39 (Table CHI3L2 is a chondrocyte growth related gene and is an antigen found in rheumatoid arthritis [Neuromedin B. This neuropeptide has been implicated as an autocrine growth factor in lung cancer cells [ABCC3 in the real-time PCR analysis as a positive control.One of the other genes expressed in common between the three astrocytomas is chitinase 3-like 2 (rthritis  and osteer cells  that binSMOC-1 [Another relatively unknown gene is the recently characterized SMOC-1 , which wSMOC-1  had incrSMOC-1 .AQP1 in human brain tumors was described in a limited array study of 4 Glioblastomas [Aquaporin 1 is an integral membrane protein important in the regulation of water transport in various epithelial and endothelial cell types . The ovelastomas  and it hlastomas . AlthougThymidilate synthetase and Topoisomarese 2A were over-expressed in glioblastoma as well as in our previous study of medulloblastoma . ConsideSAGE Genie, this data can be used as a standard to determine gene expression in astrocytomas. Further evaluation by in vitro and in vivo studies will be necessary to establish the role of these over-expressed genes in brain tumor development and progression.In summary we have identified a number of new tumor-associated genes for three different grades of astrocytic tumors, and helped re-confirm in a larger set of samples several previously known astrocytoma genes. Despite the high heterogeneity among gliomas, a small set of genes is consistently observed at high levels in more than a third of each grade of astrocytoma studied. Many other cell surface, extra-cellular matrix or cell adhesion genes have been identify as potential targets for cancer therapy in astrocytic tumors. Although the therapeutic value of these markers is speculative at this point, by integrating this data onto the commonly used gene expression resource, KB performed computational analyses, generated experimental data, participated in the design of the study and drafted the manuscript. JBE generated experimental data. CBE and GJR participated in the study design and manuscript editing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Immunor (IM28), an analog of dehydroepiandrosterone (DHEA), inhibits human immunodeficiency virus type-1 (HIV-1) by inhibiting reverse transcriptase. We assessed the ability of IM28 to inhibit the cell-cell fusion mediated by HIV envelope glycoprotein in an in vitro system. For this purpose, we co-cultured TF228.1.16, a T-cell line expressing stably HIV-1 glycoprotein envelopes, with an equal number of 293/CD4+, another T cell line expressing CD4, and with the SupT1 cell line with or without IM28.In the absence of IM28, TF228.1.16 fused with 293/CD4+, inducing numerous large syncytia. Syncytia appeared more rapidly when TF228.1.16 was co-cultured with SupT1 cells than when it was co-cultured with the 293/CD4+ cell line. IM28 (1.6 – 45 μg/ml) completely inhibits cell-cell fusion. IM28 also prevented the development of new syncytia in infected cells and protected naive SupT1 cells from HIV-1 infection. Evaluation of 50% inhibitory dose (IC50) of IM28 revealed a decrease in HIV-1 replication with an IC50 of 22 mM and 50% cytotoxicity dose (CC50) as determined on MT2 cells was 75 mM giving a selectivity index of 3.4These findings suggest that IM28 exerts an inhibitory action on the env proteins that mediate cell-cell fusion between infected and healthy cells. They also suggest that IM28 interferes with biochemical processes to stop the progression of existing syncytia. This property may lead to the development of a new class of therapeutic drug. The human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein is composed of two subunits: a surface glycoprotein gp120) and a trans-membrane glycoprotein gp41). These two subunits interact with each other in a non covalent manner. Gp120 is critical for attachment to host cell CD4 receptors, whereas gp41 contains the fusion sequence. HIV and simian immunodeficiency virus (SIV) require a co-receptor in addition to CD4 for entry into cells. Primary HIV can use a broad range of co-receptor molecules, including CCR1, CCR2b, CCR3, CCR4 and CXCR4 1. These . The abi0 and a tWe determined the concentrations of various drugs required to inhibit and to partially inhibit the fusion of TF228.1.16 and 293/CD4+ Table . InfecteIn the presence of dexamethasone Figure  or IM28 To further characterize the biological effect of the drug, the 50% inhibitory dose (IC50) and the cytotoxic dose (CC50) of IM28 were evaluated and the selectivity index which is the CC50/IC50 ratio was determined. The decrease in HIV-1 replication was obtained with an IC50 of 22 mM and the CC50 as determined on MT2 cells was 75 mM giving a selectivity index of 3.4.IM28 is a potent new derivative of DHEA that can stop the replication of HIV-1 by inhibiting its reverse transcriptase activity . Here, wAlthough the precise site at which IM28 acts to inhibit cell-cell fusion remains unknown, our results suggest that IM28 fights the HIV-1 virus at a new site. It is possible that this drug interacts with phospholipase A2 (PLA2), which plays an important role in the entry of HIV virus in the host cell ,19. IndeIn conclusion, our data show that IM28, a potent new analog of DHEA, is able to prevent and to inhibit cell-cell fusion, an important step at the beginning of HIV infection of naive cells, this drug seems to display the required properties for an anti-HIV drug.Three cell lines were used: TF228.1.16, which is a BJAB cell line that stably produces functionally active HIV-1 envelope protein (BH-10 clone of HIV-1 LAI) . 293/CD4DHEA, dextran-sulfate (DXSF), dexamethasone, suramin, heparin, the mannose-specific lectin concanavalin A (ConA) and Rowell Park Memorial Institute (RPMI)-1640 medium were purchased from Sigma-Aldrich . Cells were cultured in complete medium containing L-gltamine, penicillin, streptomycin and fetal calf serum. All these reagents were purchased from Invitrogen . IM28 was produced from DHEA as specified in its data sheet .4) were combined with an equal number of TF228.1.16 cells in a 24-well plate and incubated overnight at 37°C in a humidified incubator with 5% carbon dioxide as described by Moore et al. 1993 [Cultured 293/CD4+ cells in complete medium were harvested by trypsinization. These cells  followed by Dunnett' test. All analyses were performed using the Graph-Pad PrismD M coordinated and participated in the design of the study, statistical analysis and the drafting of the manuscript. V P-M carried out and participated in the biological tests. M-Y A carried out and participated in the biological tests. B O carried out and participated in the biological tests. E M participated in the design of the study, carried out the biological tests and participated in the drafting of the manuscript.


Analysis of growth and division often involves measurements made on cell populations, which tend to average data. The value of single cell analysis needs to be appreciated, and models based on findings from single cells should be taken into greater consideration in our understanding of the way in which cell size and division are co-ordinated. Examples are given of some single cell analyses in mammalian cells, yeast and other microorganisms. There is also a short discussion on how far the results are in accord with simple models. What is the point of single cell studies of the cell cycle? The simple answer is that they provide extra information that is not available from studies of cell populations. Without them a cell biologist can be misled.Schizosaccharomyces pombe with which I have worked since the mid-1950s. It was then a fairly obscure organism for physiological studies though it had a good genetic background found by U. Leupold in Bern [It is easiest for me to start with the theme of the extensive results on single cells of the fission yeast One the early results on its growth came from a single cell study by Bayne-Jones and Adolph . Here I Tetrahymena and Chlorella by periodic changes in their environment. Good synchronous cultures would mean that powerful biochemical techniques, often enzyme activity assays at that time, could be applied in a cell cycle context. In the next 15 years, induction synchrony was somewhat improved but the cell cycles were always and inevitably distorted. Methods were also developed to select out a fraction of an asynchronous culture in one stage of the cycle and grow it up separately . They produce less distortion but a much lower yield than induction.This period of the fifties was when attention in this field was largely focused on the successful synchronisation of Because of what can be measured in synchronous cultures, they are the natural choice for the molecular biologist. But it is as well to remember their limitations. The distortions after induction have been mentioned, but even with selection synchrony there are problems. The main one is that they are, in practice, not all that synchronous. The selected cells come from more than a very narrow region of the cycle. Some of the variation can be reduced by a correction for asynchrony  but therReturning to single cell analyses of fission yeast, volume growth was followed in finer detail by Mitchison and Nurse . One paret al. [A much more detailed analysis of populations of single cells followed on films was made by Sveiczer et al.  on fissiSingle cell studies have their problems. We have been lucky in using yeasts that are not apparently affected by growing on warm agar pads under a coverslip. They show "balanced growth", a property in which there is no change in extensive properties between successive cycles  and thatThe cells also have to keep still or be followed, a problem discussed below. We have not found ways of sticking yeast to glass (e.g. with lectins) that permit "normal" growth. Cells may also need a continuous supply of fresh medium, probably for oxygenation. Various types of microscopic mounting chambers have been described in the last 50 years or so, e.g. , but fewEscherichia coli. The limitations of synchronous cultures in hiding the fine detail of increases in volume or area could well mean that single cell studies might reveal more than an exponential increase. There might even be something like the two linear patterns that were popular models in earlier work with this bacterium [Many experimental studies on cell growth kinetics can be tedious; single cell studies are no exception. Here, however, modern automation is beginning to have very promising prospects. Anyone who has spent a day on a yeast film re-focusing the microscope every 5 min will welcome auto-focusing devices that are now available. Analysis has also become much easier with electronic imaging followed by image analysis programmes, and perhaps presentation on spreadsheets. It is now possible to have a programme that requires some hand work in the initial setting up under the microscope but will then run automatically, measuring cell length and diameter. This has been done for fifty or more single cells of fission yeast – a long way from the early days of using a ruler to measure the length of yeast cells on projected film images. Another point that should be raised here is that the new technology could profitably be applied to the growth of acterium .Amoeba proteus in an important classic paper by Prescott [Acanthamoeba and CO2 production in fission yeast , in botAnother interesting single cell method was the colorimetric enzyme assay of single yeast cells in microdrops . This miOne of the advantages of single cell work with yeasts is that they stay still on an agar pad so they can be followed for a couple of cycles before overlapping spoils the image. This is not true of many mammalian cells, which move around on the substrate. One solution to this problem comes in the work on fibroblasts (mouse L cells) described in Zetterberg , KillandWhat are needed now are techniques that combine the subtlety and precision of single cell measurements with the new techniques of automation. A promising start was made by Zicha and Dunn , and theAmoeba proteus mentioned above. The results showed that the increase of single cell "dry mass" fell in a reverse exponential, with a rapid increase at the start of the cycle falling to zero towards the end. This, of course, is lethal for anyone who believes that a rising exponential is the paradigm for the cell cycle.Organisms which tend to be forgotten about these days are those lower eukaryotes that make poor material for molecular biologists because of inadequate genetic backgrounds. One important set of results are those from the early pioneer work of Prescott  on AmoebTetrahymena pyriformis has a long and distinguished history in the cell cycle with its early induction synchrony. But in the 1960s there was a burst of studies on selected single cells or small groups. The growth patterns were often not well defined but it seems that absolute measurements of volume and of respiration rate were a better fit to linear growth . Such aPhysarum polycephalum is a myxomycete of considerable importance in some earlier work on cell cycle control. It is effectively a big single multinucleate cell with complete natural synchrony in nuclear division. It does not show exponential increase in macromolecular synthesis. For instance, there are two peaks in the rate of protein synthesis, one in the S period and the other in G2 .It would appear that there are no universal patterns of growth in these lower eukaryotes.My title makes mention of "some models". Let me be clear that there are two quite different types of cell cycle models. One type includes detailed mathematical and molecular models dealing with discrete periodic events like mitosis (e.g. ). These However, what I am concerned with here are much earlier and much simpler models, not of periodic events in the cycle like DNA synthesis, but of continuous growth. Here the two dominant models were, for simplicity, an exponential pattern of increase and a linear one. My own view  of the e