C. elegans nervous system contain multi-neuron computational modules that perform stereotypical functions? We attempt to answer this question by searching for recurring multi-neuron inter-connectivity patterns in the C. elegans nervous system's wiring diagram.Does the Our statistical analysis reveals that some inter-connectivity patterns containing two, three and four (but not five) neurons are significantly over-represented relative to the expectations based on the statistics of smaller inter-connectivity patterns.C. elegans nervous system. These modules may appear in other species and need to be investigated further.Over-represented patterns (or motifs) are candidates for computational modules that may perform stereotypical functions in the  There is little doubt that neurons are elementary building blocks of the nervous system . It is lCaenorhabditis elegans nervous system. We choose C. elegans as a model organism because its wiring diagram is largely known, including the identities of all 302 neurons and most synapses between them [In this paper, we search for anatomically defined multi-neuron modules in the een them -6. Our aeen them ,8. If a N-neuron modules, we sort all N-neuron combinations into classes defined by their inter-connectivity pattern and count the number of combinations in each class. By comparing these counts with the mean counts from random networks, constructed based on our expectations, we detect significantly over-represented patterns, or motifs. In order to avoid assigning significance to a N-neuron pattern just because it contains N-1-neuron motifs we incorporate the N-1-neuron statistics into the expectations used to search for N-neuron motifs [N = 2) and then increasing the number N of neurons included in the pattern sequentially up to quintuplets (N = 5).To search for n motifs . To do tC. elegans nerve ring (a large fraction of the nervous system) assembled in two datasets [C. elegans neurons. Two given neurons may be connected by more than one synapse, which we call the multiplicity of connection. However, the small size of the dataset compels us to use the binary representation of these connections (connected or unconnected). In order to obtain binary connectivity matrices, we threshold the multiplicity of connections at various values Θ: Pairs having less than Θ synapses are considered unconnected while those having at least Θ synapses are considered connected. Such procedure is justified because more than a single synaptic contact may be necessary for an observable physiological effect of one neuron on another. Since we do not know the physiologically relevant count of synapses, we repeat our calculation for 1 ≤ Θ ≤ 7.We look for motifs in the wiring diagram of the datasets . Datasetdatasets ; for detC. elegans. When one pre-synaptic neuron makes contact with two adjacent processes of different neurons (send_joint in Durbin notation [Unfortunately, datasets 1 and 2 contain a caveat of synaptic ambiguities, which arises from the limitations of EM in notation ), it is notation ).C. elegans neurons into three classes: unconnected, uni-directionally connected and bi-directionally connected, and compare the number of doublets in each class to that expected in a random network  of the k Figure . The ranN>2), these constraints become less stringent because the number of patterns increases (see below).We find that the number of doublets in each class deviates from the mean of the random matrix counts, as shown in Figure C. elegans, such over-representation was reported previously on a qualitative level [C. elegans can provide a clue to the functional significance of the over-representation of reciprocally connected doublets.We repeat the above calculations for other datasets and threshold values and consistently find the significant over-representation of bi-directionally connected doublets (data not shown). In ve level . Interesve level -13. ThisC. elegans neuronal network account for the over-representation of the reciprocally connected doublets? Indeed, about two thirds of C. elegans neurons have a bilaterally symmetric partner. If connections between these pairs obeyed bilateral symmetry then they could not be uni-directional, creating a bias in favor of bi-directional connections. To see whether this is the case, we calculate the percentage of bi-directional connected doublets, which consist of a bilateral neuron pair. We find that these percentages are small: 7.1% and 5.5% in datasets 1 and 2, respectively. Therefore, bilateral symmetry is not sufficient to explain the observed result.Can bilateral (left-right) symmetry of the C. elegans has been explained [The over-representation of reciprocally connected doublets in xplained  as a conxplained  supportsxplained ,14. Adjaxplained ,14.C. elegans remains unclear. It could be that the number of neuronal pairs, which can be adjacent, is limited by physical constraints. This would restrict the adjacent pairs only to the ones that need to connect for functional reasons. Indeed, volume exclusion explains neuron dimensions in the cortical column  neighborhoods [Although correlation between adjacency and connectivity may account for the over-representation of reciprocally connected doublets, why such correlation would exist in  column  when many different classes are evaluated simultaneously. This situation is known as multiple hypothesis testing and requires an adjustment of the raw p-values (see Methods).Are these differences between triplet counts in actual and random networks significant? One might answer this question by calculating, for each class, a significance single-step min P procedure [p-values for every class and threshold represent the probability of finding a random matrix R, in which at least one class i has smaller  raw p-value than that found for a given class and threshold in the actual network. This measure can be calculated by counting the number of random matrices, which have a smaller  raw p-value (in at least one class) than that in the actual network for a given class and threshold. By dividing this number of matrices by the total size of the random matrix ensemble, we estimate the multiple hypotheses testing corrected significance measure Pm for each class and threshold, Figure We chose to perform multiple hypothesis testing adjustment by controlling the family-wise error rate, i.e. the probability of mistakenly reporting at least one non-over-represented pattern, by using the rocedure ,17. The Pm, one of the most consistently over-represented motifs is the feedforward loop (triplet pattern #10), previously noticed in C. elegans [According to the significance measure,  elegans ,18 and o elegans ,8. For t elegans  and 3. CC. elegans nervous system as a three-layer feedforward network, where sensory neurons synapse mostly on interneurons, and interneurons synapse on other interneurons or motorneurons, this could explain the over-representation of the feedforward loop. We argue that this is not the case for two reasons. First, the feedforward loop is also over-represented among interneurons . But even if these factors are found, the characterization of the network in terms of over-represented motifs remains valid. The over-representation of the feedforward loop still requires a functional explanation just as the bi-directionally connected doublet does. In gene transcription regulation networks, the feedforward loop was proposed to carry out information processing functions such as filtering out fluctuations and responding only to persistent stimuli . FeedforC. elegans nervous system. Indeed, the bilateral symmetry implies that pairs of bilaterally symmetric neurons are also connected symmetrically, meaning that triplets containing such a pair are likely to be symmetric. However, we find that the fraction of triplets #12 and #14 containing a bilaterally symmetric pair of neurons and an unpaired neuron is rather small (between 10% and 20% in datasets 1 and 2). This suggests that the bilateral symmetry of the nervous system is not sufficient to explain the over-representation of pattern #12 and #14.In addition to the feedforward loop, we find that two other (both symmetric) patterns are consistently over-represented: pattern #12 and pattern #14 Figure . For theC. elegans network are small, we cannot verify that they are consistently over-represented. Further work on larger datasets will show whether these patterns may be viewed as motifs.Just like in any other screening algorithm, our criteria for outliers are somewhat subjective and the goal is to draw attention to interesting candidates. We limit our discussion to over-represented patterns #10, #12 and #14 because in our judgment they are most robust outliers based on the several criteria used. The reader may judge that some other patterns are over-represented as well. For example, patterns #15 and #16 are significantly over-represented for small thresholds Figure . BecausePm must be less than 0.1 for at least one threshold per pattern, while the number of quadruplets in the actual network must be at least 5. The last condition excludes patterns that may appear as over-represented due to very small quadruplet counts.We classify all connected quadruplets into 199 classes and count the number of quadruplets in each class. Then we compare the actual counts of quadruplets in each class to the mean counts of quadruplets in a random matrix ensemble. In this case, random matrices preserve the numbers of uni-directional and bi-directional connections for each neuron and, in addition, the numbers of triplets (see Methods). Because of the large number of quadruplet classes, we show results Figure  only forWe find that quadruplet pattern #45 is consistently over-represented . Can we One could propose that symmetric patterns should be over-represented because of the bilateral symmetry of the nervous system. We think that this argument by itself cannot explain the observed over-representation for two reasons. First, the fraction of feedforward quadruplets containing two bilaterally symmetric neuron pairs in motif 45 is rather small (less than 10% in dataset 1 and less than 14.3% in dataset 2). Second, many symmetric patterns are not over-represented, such as, for example, patterns 25, 30, 31, 35, 43, 44 and 65 . We find that 14% to 37% of the feedforward quadruplets do not fit into this proposition because either they contain a feedback connection or all neurons belong to the same layer Table . After cIt is possible that some other factors (in addition to feedforwardness) account for the reported quadruplet over-representation. Just as argued in case of triplets, discovering these factors would be complementary to the characterization of the over-represented motif. It would be particularly interesting to determine the functional role of these motifs. Again, we arbitrarily limit our discussion of over-represented quadruplets to pattern #45. The reader may judge that some other patterns are over-represented and deserve attention (e.g. patterns #36, 50). This is why in Figure C. elegans network. Therefore, absence of significantly over-represented quintuplets in C. elegans does not rule out the existence of five-neuron modules that can be detected as motifs by applying our algorithm to larger networks.We classify all connected quintuplets into 9364 classes  and count the actual number of quintuplets in each class. We compare these counts with the mean of the random matrix ensemble. In this case, the random matrices preserve the numbers of uni- and bi-directional connections for each neuron and, in addition, keep the numbers of all triplets and quadruplets in a 10% range of the actual network. We do not find any significantly over-represented quintuplets. This could happen because there are no significantly over-represented quintuplets with a given number of quadruplets. Alternatively, this could happen because specifying the numbers of triplets and quadruplets constrains the number of quintuplets in any random matrix the size of the C. elegans wiring diagram to the mean counts of the appropriate random matrix ensemble, we find several over-represented motifs. First, we find that bi-directionally connected doublets (out of three possible doublet classes) are over-represented, given the number of connections on each neuron is fixed. Second, several triplet classes (out of thirteen possible connected patterns) are over-represented, given the actual number of bi-directional  connections for each neuron. Third, we find that several quadruplet classes (out of 199 connected patterns) are over-represented, given the numbers of triplets are preserved in addition to previously listed constraints. We find no over-represented quintuplet classes. Some of these results, such as the over-representation of the feedforward loop and the feedforward quadruplet, have been reported previously [C. elegans nervous system, and they need to be investigated further.By comparing counts of multi-neuron patterns in the eviously ,8,18. Thsingle-step min P procedure to the absolute value of the deviation of counts from the mean. We found that the feedforward loop is not significantly under-represented, yet many other patterns, such as weakly connected triplets were significantly under-represented  was small . To deteormation ).N-neuron motif then it would appear as an N + 1-neuron motif. If, on the other hand, the motif is connected in many different ways in different instances, their significance will be washed out. Thus our approach may hierarchically detect modules with recurring input/output sites, growing them out of smaller patterns. A second justification for looking at N-neuron patterns is that the nervous system is capable of performing many different functions under different circumstances and neurons active in one case may be silent in another. Therefore, in any particular case, many of the anatomical inputs to the module may remain silent and can safely be ignored. This speculation may be verified experimentally by simultaneous monitoring of neuronal activity in different neurons.Our motif search algorithm is different from previous attempts to find modules . For exaC. elegans nervous system, whose wiring diagram is largely known. Unfortunately, C. elegans has some disadvantages when it comes to the interpretation of the results: the polarity of a synapse (excitatory vs. inhibitory) in C. elegans is often unknown; electrophysiological investigations are still difficult in C. elegans [C. elegans. The modules we identify in C. elegans may be a general property of the nervous system, and, once identified, can be searched for in other species. Finally, the algorithm itself can be applied to other networks [The strategy and algorithms we described in this paper can be applied to incompletely mapped networks because a highly significant pattern is also likely to be over-represented in a sub-network. However, the statistical power of our algorithm increases with the knowledge of the wiring diagram. Therefore it was natural to choose the  elegans ; and the elegans  will elinetworks  once theAs in any other theoretical analysis, we made several simplifications. For example, we assumed that the strength of synaptic connection between a pair of neurons is characterized by its multiplicity (i.e. the number of synapses between that pair). This assumption may be questioned if synapses implementing high-multiplicity connections are weaker than those implementing low-multiplicity connections, as known to happen in nematodes . Yet, thC. elegans nervous system. These patterns, called motifs, are candidates for computational modules that may perform stereotypical functions. It would be interesting to determine what these functions are and whether these motifs appear in other nervous systems.We have shown that certain neuronal connectivity patterns are significantly over-represented in the C. elegans synaptic connectivity data into a binary matrix A, called Adjacency Matrix or Connectivity Matrix, in which an entry Aij is 1 if there is a connection from neuron i to neuron j and 0 otherwise. The order in which neurons are assigned to rows in this matrix is not important for our calculations. The multiplicity of synapses between two given neurons is mapped to a binary value by applying a threshold to the data. We assume a synaptic connection of threshold Θ from neuron i to neuron j if neuron i makes at least Θ synapses on to neuron j. Adjacency matrices that we used are available in the Supplementary Information [We transformed the ormation  and 3.N-neuron patterns, we took all different N-neuron subsets and characterized their connectivity. Second, we took all possible N-neuron subsets out of the neighborhood of a neuron x. This neighborhood is defined by all neurons that can be reached from x, if the directed connectivity matrix is made undirected. In both cases it is crucial for the run time of the algorithm to detect the pattern class from these connectivity sub-matrices as quickly as possible. We realized this by defining a function that maps each possible N-neuron sub-matrix to a unique integer value. Then we classified all the sub-matrices based on the function value and a pre-calculated lookup table, which identifies the pattern class from the function value.We implemented two strategies for counting the number of triplets, quadruplets and quintuplets in a given connectivity matrix. First, to obtain the count of all x is called out-degree of x. The number of neurons providing synaptic input to neuron x is called in-degree of x. In the binary matrix representation of a network as described above, the out-degree of a neuron x can be calculated as the sum of row x, the in-degree as the sum of column x.The number of neurons that receive synaptic input from a given neuron C. elegans network. This choice of random matrices is motivated by the observation that the distribution of in-degrees and out-degrees in C. elegans is significantly different from Poisson, which is expected for a randomly generated matrix without any correlations (Erdõs-Rényi random graph)  Figure .C. elegans network. It reconnects all incoming connections of that neuron to other neurons, as long as their out-degree does not exceed that in the C. elegans network. It reconnects all reciprocal connections of that neuron to other neurons with available unconnected reciprocal connections. We also implemented a second algorithm to verify the robustness of our results. This algorithm [N = 3. We keep the number of incoming and outgoing uni-directional connections as well as the number of reciprocal connections for each neuron the same. One of the implemented algorithms starts with an empty matrix. Then it randomly selects a neuron and does three things. It reconnects all outgoing connections of that neuron to other neurons, as long as their in-degree does not exceed that in the lgorithm ,10 will N = 3 as described above, we use the Simulated Annealing algorithm [C. elegans.N = 4. For comparing the count of quadruplets, we construct random matrices that keep the same not only in-degree and out-degree of uni-directional and bi-directional connections for each neuron but also the count of the 16 different 3-neuron pattern in the whole matrix. Starting from a random matrix for lgorithm  by swappN = 5. For the analysis of the quintuplets, we modified the Simulated Annealing algorithm to match the count of all 4-neuron patterns to the real network. With this algorithm we could only find random matrices for which the relative difference between the count of each pattern in the random matrix and the real dataset was less than 10%.p-value, which is the probability that a fair coin would have that or greater deviation. For example, the probability of getting 62 or more heads is about 1% and the corresponding p-value = 0.01. Now consider testing simultaneously 100 different coins by tossing each 100 times. Analyzing these 100 experiments for outliers reveals that one coin yielded 62 heads. Does this mean that this specific coin is unfair? Not necessarily. Even if all the coins are fair, such a seemingly unlikely result will be observed approximately once when examining 100 coins. In other words, the p-value estimated for a single coin is an underestimation of the true p-value when 100 coins are examined simultaneously. This situation is called multiple hypotheses testing and requires a modification of the p-value definition.Here we illustrate the issue of multiple hypothesis testing by considering a classical coin-tossing example. Imagine determining whether a given coin is fair (i.e. yielding heads with probability 1/2) or not by tossing it 100 times and recording the number of heads. If the number of heads is not too different from 50, we expect that the coin is fair. The significance of the deviation in the number of heads from 50 is characterized by the N-neuron patterns in the i-th class in the actual network A and a random network R is given by: cN,i (A) and cN,i (R). Then the raw p-value is defined by:Assume the number of pi = Pr(cN,i (Rk) ≥ cN,i (A), Rk ∈ {R}).m = 13 patterns for N = 3, m = 199 patterns for N = 4 and m = 9364 patterns for N = 5), there is an increased probability of finding an over-represented pattern by chance. We correct for that by calculating a multiple hypothesis testing corrected p-value for each pattern and threshold. This p-value, Pm, reflects the probability that one random matrix Rk0 out of our random matrix ensemble {R} will have at least one pattern, i, which has smaller  raw p-value than the given pattern in C. elegans. This is known as the single-step min P procedure and controls for family-wide error rate [single-step min P adjusted p-values are defined by:Because we look for over-representation of all connected patterns in parallel  ≥ cN,j (Rk0)).where Pm for a pattern i we perform the following procedure:To determine   out of the ensemble we calculate  between  and all other random matrices in this ensemble for each pattern i:1. For all random matrices .p-value for  as a minimum of these values across all patterns i: .2. We then derive the raw i the observed count in a random matrix Rk out of our ensemble {R} is greater than the count in the C. elegans network :3. We calculate the probability that for a given pattern single-step min P adjusted p-value Pm for a given pattern i as:4. Last, we calculate the single-step max T adjusted p-value [In addition, we verified our results with the alternative  p-value ,17 .We used data from , which pA document containing supplementary information and data not presented in the paper. See also Click here for fileDescription of the files containing triplet lists and used data sets. See also Click here for fileZip file containing files mentioned in Additional file 2Click here for file


Caenorhabditis elegans NHR gene nhr-49 yielded worms with elevated fat content and shortened life span. Employing a quantitative RT-PCR screen, we found that nhr-49 influenced the expression of 13 genes involved in energy metabolism. Indeed, nhr-49 served as a key regulator of fat usage, modulating pathways that control the consumption of fat and maintain a normal balance of fatty acid saturation. We found that the two phenotypes of the nhr-49 knockout were linked to distinct pathways and were separable: The high-fat phenotype was due to reduced expression of enzymes in fatty acid β-oxidation, and the shortened adult life span resulted from impaired expression of a stearoyl-CoA desaturase. Despite its sequence relationship with the mammalian hepatocyte nuclear factor 4 receptor, the biological activities of nhr-49 were most similar to those of the mammalian PPARs, implying an evolutionarily conserved role for NHRs in modulating fat consumption and composition. Our findings in C. elegans provide novel insights into how NHR regulatory networks are coordinated to govern fat metabolism.Mammalian nuclear hormone receptors (NHRs), such as liver X receptor, farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs), precisely control energy metabolism. Consequently, these receptors are important targets for the treatment of metabolic diseases, including diabetes and obesity. A thorough understanding of NHR fat regulatory networks has been limited, however, by a lack of genetically tractable experimental systems. Here we show that deletion of the  Caenorhabditis elegans gene nhr- 49 causes worms to accumulate fat and die younger; but these two phenotypes are a result of distinct and separable pathwaysDeletion of the  Due to their ability to interact with fatty acids and other lipids, nuclear hormone receptors (NHRs), including peroxisome proliferator-activated receptors (PPARs), liver X receptor, and farnesoid X receptor, are important regulators of mammalian fat metabolism; accordingly, these small-molecule-gated transcription factors have been favored targets for therapies designed to combat diabetes, obesity, and atherosclerosis ,2,3. AltCaenorhabditis elegans, has facilitated the elucidation and interpretation of biological networks, particularly in the context of the whole animal. However, despite the fact that C. elegans contains 284 NHR genes, compared to only 48 in mammals, there is a notable absence of worm NHRs orthologous to mammalian receptors involved in fat metabolism [C. elegans and Drosophila lineages as these organisms simplified their body plans [The study of invertebrates, such as tabolism . In factdy plans . ConceivC. elegans employs NHRs to regulate fat metabolism, the evolution of the C. elegans NHR family is intrinsically interesting. Only 15 of the 284 worm NHRs are members of the broadly conserved subfamilies found in mammals and other metazoans [C. elegans “nematode-specific” HNF4-like receptors. It will be interesting to determine if these divergent NHRs have adopted novel regulatory functions or whether they carry out physiological tasks similar to those of other metazoan NHRs.Whether or not etazoans ,8. The retazoans . Althougetazoans , virtualC. elegans. In this study, we present a physiological characterization of nhr-49 (K10C3.6), one of the C. elegans receptor genes most closely related to the mammalian HNF4. Our findings reveal a surprising role for this HNF4-like receptor in the regulation of fat storage and metabolism.Elucidating the physiological responsibilities of even one of the worm HNF4-like receptors will likely advance our understanding of NHR-regulated gene networks and provide insight into the evolution and diversification of the NHR family in nematodes. To this end, we have been systematically investigating the function of HNF4-like receptors in C. elegans HNF4-like receptors in worm development and longevity, we found that interference of nhr-49 resulted in dramatically reduced life span (C. elegans strain, nhr-49(nr2041), which harbors a deletion in the nhr-49 gene encompassing part of the DNA binding domain and more than half of the ligand binding domain (LBD); this deletion likely results in complete loss of function [nhr-49(nr2041) worms lived only 6–8 d as adults, significantly shorter than the 15 to 18-d life span of N2 wild-type (WT) animals (nhr-49 deletion did not noticeably affect development or fertility, nhr-49(nr2041) worms experienced rapid decline in function beginning around day 3 of adulthood, when vacuoles appeared in the intestine and gonad (nhr-49(nr2041) animals were significantly smaller than WT, vacuoles were ubiquitous, and there was widespread gonadal necrosis. By days 5–7, the gonad had completely deteriorated, and the worms died shortly thereafter. Thus, nhr-49 function is not required for development or fertility, but is clearly essential for normal longevity. Even though the increased vacuole formation was consistent with reported aging characteristics [nhr-49(nr2041) reflects accelerated aging or an unrelated pathology.In an RNA interference (RNAi) screen designed to identify the role of ife span A. For fufunction . At 23 ° animals A. Althound gonad B. By dayeristics , we havenhr-49 was recently identified in a C. elegans genomewide RNAi screen as one of 112 genes that, when knocked down by RNAi, resulted in abnormally high Nile Red fat staining [nhr-49 also yielded a high-fat phenotype, we used a similar Nile Red assay to visualize fat content in nhr-49(nr2041). Indeed, we found that nhr-49(nr2041) animals stained more brightly with Nile Red than did WT worms (nhr-49(nr2041) animals was most pronounced in the L3 and L4 stages of larval development; by day 2 of adulthood there was only a slight change in Nile Red staining, suggesting that the effects of nhr-49 on visible fat content may vary during development (data not shown).WT worms C. The dinhr-49(nr2041) mutant led us to suspect that nhr-49 might regulate genes involved in energy metabolism. To test this hypothesis, we identified from a survey of the C. elegans genome 65 genes predicted to participate in fatty acid synthesis, β-oxidation, desaturation, elongation, and binding/transport, in addition to 16 genes expected to function in glycolysis, gluconeogenesis, and glucose transport, and eight genes involved in the glyoxylate pathway (nhr-49(nr2041) mutant worms. For the purpose of our survey, gene expression was measured in all four larval stages .Our screen revealed that deletion of  pathway . Similarnhr-49 deletion displayed the most dramatic effects on two metabolic pathways, mitochondrial β-oxidation and fatty acid desaturation, as the expression of multiple genes in each of these pathways was significantly compromised by loss of nhr-49 function , F09F3.9, and an acyl-CoA synthetase gene (acs-2) (F28F8.2), were expressed at significantly lower levels in nhr-49(nr2041) animals throughout development  worms might be high in fat due to the reduced expression of these key β-oxidation enzymes. To test this hypothesis, we used RNAi to reduce separately the expression of acs-2, F09F3.9, and ech-1 in WT animals. Indeed, we found that RNAi of either acs-2 or ech-1 resulted in worms with elevated Nile Red fat staining  mutant worms are likely to be high in fat due to the impaired expression of these genes.Three genes predicted to participate in mitochondrial β-oxidation, elopment A. acs-2 staining B. acs-2  storage . We concacs genes in C. elegans, nhr-49 affected only the expression of acs-2, suggesting that nhr-49 may only influence a subset of mitochondrial β-oxidation pathways in worms. To determine if acs-2 was expressed in a specific set of tissues, we fused the full-length acs-2 gene and promoter to gfp (acs-2::gfp); injection of this construct into WT worms revealed that the ACS-2::green fluorescent protein (GFP) was widely expressed in many cell types, including intestine, hypodermis, pharynx, body wall muscle, and several neurons (nhr-49(nr2041) worms, indicating that the effects of nhr-49 on acs-2 expression were widespread  animals (acs-2 expression is reduced in nhr-49(nr2041) such that it becomes a rate-limiting factor in the consumption of fat; thus, high fat levels can be overcome simply by increasing acs-2 expression. Overexpression of acs-2 in WT animals did not affect fat content, however, indicating that WT levels of acs-2 expression are not rate limiting. These data further support the conclusion that reduced expression of acs-2 is a significant contributor to the high-fat phenotype of the nhr-49(nr2041) mutant.Strikingly, overexpression of the ACS-2::GFP fusion was able to suppress the high-fat phenotype of  animals F. These nhr-49 also promoted the expression of three C. elegans fatty acid desaturases (see fat-5 (W06D12.3) and fat-7 (F10D2.9) expression was dramatically lowered in nhr-49(nr2041) worms (>30-fold) in all four larval stages, whereas fat-6 (VZK8221.1) expression was marginally reduced (approximately 2-fold) only in L3 and L4 animals , and fat-6 and fat-7 are stearoyl-CoA desaturases (SCDs), preferentially functioning to desaturate stearic acid (C18:0). Mammalian SCDs have been shown to be important for maintaining an appropriate level of fatty acid desaturation, vital for modulating membrane fluidity and for controlling lipid metabolism [C. elegans, SCDs are also involved in catalyzing the first step in the synthesis of polyunsaturated fatty acids (PUFAs) [Studies in yeast have shown that the ty acids . fat-5 itabolism ,16. Furt (PUFAs)   to determine whether overall fatty acid desaturation and PUFA synthesis were altered in nhr-49(nr2041) animals. The most significant consequence of nhr-49 deletion on fatty acid composition was a marked increase in the abundance of stearic acid (C18:0) and a corresponding decrease in the level of oleic acid (C18:1n9) (Because the expression of these three C18:1n9) B. This rC. elegans we found that the ratio of stearic acid to oleic acid (C18:0/C18:1n9) was increased from 1.9 in WT animals to 4.3 in nhr-49(nr2041) .In mammals, a primary role of SCD is to control the ratio of fully saturated stearic acid to monounsaturated oleic acid (C18:0/C18:1n9), and this ratio has been employed as an indicator of the activity of the mammalian SCD1 ,16. In C(nr2041) . A similing RNAi . This smC. elegans SCDs are also important for the synthesis of PUFAs; however, we did not observe any measurable changes in PUFA abundance in nhr-49(nr2041) animals . Although we did not observe a significant consequence of fat-5 RNAi on fatty acid desaturation, fat-7 RNAi altered fatty acid composition in a manner very similar to that of nhr-49 deletion  to 8.0. fat-7 RNAi animals also exhibited a slight lowering of PUFA abundance. The stronger effect of fat-7 RNAi on fatty acid composition suggests that fat-7 expression was reduced to levels even lower than those observed in nhr-49(nr2041). Because fat-7 shares significant homology (approximately 85% nucleotide identity) with fat-6, it was possible that fat-7 RNAi also partially interfered with fat-6 expression; however, we found by QRT-PCR that the overall levels of fat-6 mRNA were not noticeably reduced by fat-7 RNAi. (data not shown).To determine which of the Δ9-desaturases accounted for the influence of deletion . fat-7 Rnhr-49 influences fatty acid composition in C. elegans. Although it appears that the primary effect of nhr-49 deletion is a modulation of stearic and oleic acid levels, not PUFA abundance, we cannot rule out the possibility that nhr-49 influences PUFA synthesis in a more localized manner, such that the changes in PUFA composition are not detectable by our whole-worm analysis.Therefore, we conclude that by strongly promoting SCD expression, nhr-49-dependent genes identified in this study, we found that interference of only fat-7 resulted in a shortened life-span phenotype similar to that of nhr-49(nr2041)  mutant, including widespread vacuole formation and germ line necrosis (Using RNAi to knock down the 041) see . fat-7 Rnecrosis .fat-7 RNAi on life span was even more potent than nhr-49 deletion, reducing adult life span to 3–5 d, as opposed to the 5- to 7-d life span of nhr-49(nr2041)  mutant. In fact, we observed a striking correlation between fat-7 activity, as determined by the ratio of stearic to oleic acid in our various mutants, and life span  lived approximately 10–12 d, nhr-49(nr2041) animals (stearic/oleic = 4.3) lived approximately 6–8 d, and fat-7 RNAi animals (stearic/oleic = 8.0) lived approximately 3–5 d.Interestingly, the effect of 041) see . This isspan see . WT animfat-7 expression is significantly compromised by nhr-49 deletion, and because lowered fat-7 expression can lead to shortened life span, we suggest that the reduced life-span phenotype of nhr-49(nr2041) likely reflects, at least in part, diminished fat-7 expression.Because nhr-49 stimulates fat consumption, and by enhancing fat-7 expression, nhr-49 modulates fat desaturation and ensures normal longevity. We next set out to determine whether nhr-49 regulates these two pathways independently.By promoting β-oxidation gene expression, acs-2 or ech-1 was sufficient to cause a high-fat phenotype, acs-2 or ech-1 interference did not significantly impact life span, fatty acid composition, or desaturase gene expression, demonstrating that the effects of nhr-49 deletion on fat-7 expression and life span were not a downstream consequence of compromised β-oxidation or increased fat content  mutant using RNAi, indicating that fat-7 hinders the expression of ech-1 and acs-2 through a mechanism that is independent of nhr-49 (see fat-7 reduced fat content in nhr-49(nr2041) worms, demonstrating that the stimulation of fat storage by fat-7 is also not dependent upon nhr-49  were attributable to deficiencies in two different metabolic pathways, fatty acid β-oxidation and fatty acid desaturation. The high-fat phenotype is explained by lowered expression of β-oxidation enzymes, and the shortened life-span phenotype results from reduced expression of the fat-7 SCD. Interestingly, these pathways are linked by an apparent feedback mechanism, as fat-7 also serves to inhibit the expression of fatty acid β-oxidation genes , indicating that acs-2 expression is rate-limiting in nhr-49 deletion mutants, thus leading to reduced fat breakdown and excessive fat accumulation. Acyl-CoA synthetases activate fatty acids for many different metabolic pathways; in particular, mitochondrial acyl-CoA synthetases are likely to activate fatty acids for transport into the mitochondrial matrix, where the fatty acids are then subject to β-oxidation [acs-2 expression, nhr-49 likely acts by increasing the flow of fatty acids into the mitochondria for β-oxidation  to multiple mammalian acyl-CoA synthetases, mammals contain a particular homolog with approximately 41% identity to the C. elegans ACS-2 protein; this mammalian ACS protein is yet to be characterized. It will be interesting to determine whether the mammalian counterpart of acs-2 also serves as an important focal point for gene networks that regulate fat utilization in mammals.Although nhr-49 stimulates expression of a carnitine palmitoyl transferase (F09F3.9). Carnitine palmitoyl transferases execute the step immediately downstream of acyl-CoA synthetase in mitochondrial β-oxidation, directly shuttling activated acyl-CoAs into the mitochondrial matrix. Thus, nhr-49 appears to target multiple enzymes involved in transporting fatty acids across the mitochondrial membrane for β-oxidation . Thus, we have demonstrated that nhr-49 strongly promotes the expression of three separate enzymes predicted to participate in mitochondrial β-oxidation. Consequently, we suggest that nhr-49 plays a prominent role in promoting the breakdown of fatty acids in mitochondria, and that, through control of β-oxidation, nhr-49 influences overall fat storage.In contrast, we found that RNAi of nhr-49 helps to maintain the balance of saturated and monounsaturated fatty acids. By stimulating the expression of three fatty acid Δ9-desaturase genes, nhr-49 facilitates the conversion of saturated fatty acids to monounsaturated fatty acids, and consequently, nhr-49(nr2041) mutant animals are high in saturated fat and lower in monounsaturated fat. In C. elegans, the FAT-6 and FAT-7 SCDs may also be involved in the synthesis of PUFAs [fat-7 expression, either by nhr-49(nr2041) deletion or by fat-7 RNAi, did not significantly affect PUFA synthesis. This result suggests that a normal level of FAT-7 expression is not necessary for PUFA synthesis, likely because FAT-6 is the principle enzyme in this pathway, or because FAT-6 is capable of substituting for reduced FAT-7 activity. In either event, we propose that the primary effect of nhr-49 on fatty acid composition is a modulation of the ratio of C18:0 to C18:1n9 fatty acid  is due, at least in part, to reduced expression of fat-7. The strong correlation between the increase in C18:0 saturated fatty acid and the shortening of life span is compelling. Several reports in mammals have described changes in fatty acid composition during aging, and it has been argued that an alteration of fatty acid saturation ratios in mitochondrial membranes may affect the rate of aging [C. elegans have identified fat-7 as one of the targets of the mechanisms that extend longevity through the insulin pathway [nhr-49(nr2041) and fat-7 RNAi animals results from an improper ratio of saturated and monounsaturated fats, and if the mechanistic cause of the reduced life span is related to accelerated aging or to another pathology.Our results also indicate that the short life-span phenotype of nhr-49nr41 is dueof aging ,16. Alth pathway . Thus, inhr-49 independently promotes the expression of genes in fatty acid β-oxidation and desaturation, we found that downstream components of these pathways do indeed communicate with each other. By inhibiting the expression of genes involved in β-oxidation, including the nhr-49-dependent genes acs-2 and ech-1, fat-7 works against the actions of nhr-49, acting to hinder fat consumption .Although it is clear that sumption . Thus, oon genes . Thus, wfat-7 is to convert C18:0 to C18:1n9 fatty acid, it is tempting to speculate that one of these two fatty acid species serves as a signal to modulate the feedback effect of fat-7 on fatty acid β-oxidation . All of the other RNAi constructs were obtained from the Ahringer RNAi library [All of the RNAi constructs used in the NHR screen were created by cloning full-length NHR cDNAs into the vector L4440 (Andy Fire, Stanford University). The  library .nhr-49(nr2041) and WT life-span assays were carried out at 23 °C on OP50 bacteria.Approximately 30–40 starved L1 worms were transferred to RNAi bacteria (HT115 transformed with RNAi clone) and life-span assays were carried out at 23 °C as described previously . nhr-49(Nile Red fat-staining assays were carried out as described previously .http://www.genome.ad.jp/kegg/pathway.html). Other genes were identified using BLAST searches designed to find C. elegans open reading frames that were highly related to known mammalian glucose and fat metabolism enzymes, or to plant glyoxylate cycle genes.Most of the fatty acid and glucose metabolism candidate genes examined in this study were initially identified using the KEGG pathway database (http://www.cbs.dtu.dk/services/TargetP/) [http://psort.nibb.ac.jp/form.html).For prediction of β-oxidation enzyme subcellular localization, we used the following online prediction tools. TargetP server version 1.01 (argetP/)  and PSORC. elegans N2-Bristol (WT) and nhr-49(nr2041) were grown at 23 °C on high-growth plates seeded with OP50 bacteria. Gravid adults from 30 10-cm plates were bleached, and embryos were dispersed onto 15-cm nematode growth media (NGM)-lite plates seeded with OP50. The worms were distributed as follows: for L1 harvest, 80,000 embryos/plate; for L2 harvest, 40,000 embryos/plate; for L3 harvest, 20,000 embryos/plate; and for L4 harvest, 10,000 embryos/plate. The remaining embryos were saved for embryonic RNA preparation. Worms were harvested at the appropriate stage, washed twice with M9, and frozen in liquid nitrogen. For RNAi experiments, 10,000 embryos were placed onto NGM-lite plates containing 8 mM IPTG and 100 μg/ml carbenicillin seeded with control bacteria (HT115 transformed with empty L4440 vector) or nhr-49 RNAi bacteria (HT115 transformed with L4440-nhr-49). Worms were harvested at the L4 stage of development, washed twice with M9, and frozen in liquid nitrogen. For RNA preparation, worms were thawed at 65 °C for 10 min, and RNA was isolated using the Tri-Reagent Kit (Molecular Research Center, Cincinnati, Ohio, United States). Isolated total RNA was subjected to DNAase treatment and further purification using RNAeasy .Both TaqDNA Polymerase  and consisted of the following reaction mixture: 0.3 μM primers, 1/500th of the cDNA reaction , 125 μM dNTPs, 1.5 mM MgCl2, and 1X reaction buffer (20 mM Tris pH 8.4, 50 mM KCl). 0.15 μl (0.75 units) of TaqDNA Polymerase was used for each reaction. Formation of double-stranded DNA product was monitored using SYBR-Green (Molecular Probes, Eugene, Oregon, United States). All QRT-PCR reactions were carried out and analyzed on a DNA Engine-Opticon 2 . Data were collected using RNA from at least three independent C. elegans growths. To determine the relationship between mRNA abundance and PCR cycle number, all primer sets were calibrated using serial dilutions of cDNA preparations. Primer sets were also calibrated by performing QRT-PCR reactions on serial dilutions of C. elegans genomic DNA. Relative abundance is reported as the mRNA abundance of each gene relative to the mRNA abundance of several control genes, which are expressed at constant levels throughout development.cDNA was prepared from 5 μg of total RNA in a 100-μl reaction using the Protoscript cDNA preparation kit (New England Biolabs, Beverly, Massachusetts, United States). Primer pairs (primer sequences available upon request) were diluted into 96-well cell culture plates at a concentration of 3 μM. Next, 30-μl PCR reactions were prepared in 96-well plates. Each PCR reaction was carried out with nhr-49(nr2041) experiments, worms were grown at 23 °C on a lawn of OP50. For RNAi, 10,000 embryos were placed on control bacteria, or on fat-5, fat-7, ech-1, acs-2, or nhr-49 RNAi bacteria. Fatty acid extract was prepared as described previously [Fatty acids were isolated from 10,000 L4 animals grown on a single 15-cm NGM-Lite plate. For WT and eviously . GC/MS snhr-49GFP plasmid, a genomic fragment including 3.5 kb of the nhr-49 upstream sequence and 88 bp of the first exon of nhr-49 was ligated into the L3691 GFP expression vector (a gift of A. Fire). Germ-line transformation was performed following standard procedures [nhr-49GFP was injected at the following concentrations: 1 ng/μl, 5 ng/μl and 20 ng/μl along with the co-injection marker pRF4 rol-6(su1006) injected at 60 ng/μl. At all three Pnhr-49GFP concentrations, we were unable to obtain stably transmitting lines. Animals used for GFP imaging were derived from the F1 generation and were mosaic, hosting the Pnhr-49GFP transgene in only a subset of tissues.To make the Pocedures . Pnhr-49acs-2::gfp, a 4-kb genomic fragment, including the full-length acs-2 gene plus 2 kb of upstream sequence, was cloned into the vector L3691. Germ-line transformation was performed using 50 ng/μl of acs:2::gfp and 50 ng/μl pRF4 rol-6(su1006). For N2/acs-2::gfp and nhr-49(nr2041)/acs-2::gfp, over ten independent transmitting lines were obtained and analyzed by GFP microscopy. GFP reporter expression was observed using a Zeiss Axiovert S100 fluorescence microscope (Carl Zeiss, Thornwood, New York, United States) under a 40X objective.To construct acs-2::gfp was injected into nhr-49(nr2041) and WT animals along with 50 ng/μl of pRF4 rol-6(su1006). Transgenic adults, selected for rolling behavior, were placed on Nile Red plates and assayed for fat content as described above. pRF4 rol-6(su1006) was injected into nhr-49(nr2041) in combination with unrelated plasmids to demonstrate that the lowered fat content was not a result of hosting a transgenic array or caused by expression of the rol-6(su1006) marker gene.For rescue experiments, 50–100 ng/μl of Table S1t, the number of cycles required for amplification of a particular target gene from a cDNA preparation. Changes in gene expression in the nhr-49(nr2041) mutant were measured as ΔCt. ΔCt are obtained by subtracting nhr-49(Ct) from N2(Ct) and adjusting for RNA concentration differences with a correction factor (CF). The CF was determined by aligning the data to a set of standard genes. Thus the equation for ΔCt is as follows:Data are shown as Cnhr-49 dependent if the Ct was >1.0 in multiple developmental stages (and was greater than the standard error). nhr-49 targets are marked with an X. The QRT-PCR results of the major nhr-49 targets, acs-2, ech-1, fat-5, and fat-7, were confirmed with a second primer pair.Genes were designated as (100 KB XLS).Click here for additional data file.


Inflammatory cytokines and infiltrating T cells are readily detected in herpes simplex virus (HSV) infected mouse cornea and trigeminal ganglia (TG) during the acute phase of infection, and certain cytokines continue to be expressed at lower levels in infected TG during the subsequent latent phase. Recent results have shown that HSV infection activates Toll-like receptor signaling. Thus, we hypothesized that chemokines may be broadly expressed at both primary sites and latent sites of HSV infection for prolonged periods of time. Real-time reverse transcriptase-polymrease chain reaction (RT-PCR) to quantify expression levels of transcripts encoding chemokines and their receptors in cornea and TG following corneal infection. RNAs encoding the inflammatory-type chemokine receptors CCR1, CCR2, CCR5, and CXCR3, which are highly expressed on activated T cells, macrophages and most immature dendritic cells (DC), and the more broadly expressed CCR7, were highly expressed and strongly induced in infected cornea and TG at 3 and 10 days postinfection (dpi). Elevated levels of these RNAs persisted in both cornea and TG during the latent phase at 30 dpi. RNAs for the broadly expressed CXCR4 receptor was induced at 30 dpi but less so at 3 and 10 dpi in both cornea and TG. Transcripts for CCR3 and CCR6, receptors that are not highly expressed on activated T cells or macrophages, also appeared to be induced during acute and latent phases; however, their very low expression levels were near the limit of our detection. RNAs encoding the CCR1 and CCR5 chemokine ligands MIP-1α, MIP-1β and RANTES, and the CCR2 ligand MCP-1 were also strongly induced and persisted in cornea and TG during the latent phase. These and other recent results argue that HSV antigens or DNA can stimulate expression of chemokines, perhaps through activation of Toll-like receptors, for long periods of time at both primary and latent sites of HSV infection. These chemokines recruit activated T cells and other immune cells, including DC, that express chemokine receptors to primary and secondary sites of infection. Prolonged activation of chemokine expression could provide mechanistic explanations for certain aspects of HSV biology and pathogenesis. Th1 cells) specific for infection-induced inflammation, while others including CCR3 and CXCR4 are on cells (e.g., Th2 T cells) associated with allergic inflammation. Certain receptors are expressed by specific subsets of a given cell type. For example, CCR6 is highly expressed on Langerhans-like (CD34+) DC that migrate to skin, but not on monocyte-derived DC that migrate to non-skin tissues  starting from 5.5 × 104 copies for tk and ICP0 gene levels. The HSV tk and ICP0 primer/probe sets gave linear amplification curves over 4 logs of template concentrations until the limit of detection within the linear range was reached at 55 DNA copies for tk and 550 copies for ICP0 (not shown). At these limits of detection, the threshold cycle (CT) value, which indicated the PCR cycle at which a significant increase in amplification was first detected, was 39.2 for tk at 55 DNA copies and 36.5 for ICP0 at 550 DNA copies.To characterize the range over which the HSV ® assays [Using 2-fold dilutions of uninfected mouse TG cDNA, we observed that the primer/probe sets for host genes listed in Table ® assays -24 for ltk and ICP0 gene transcripts. In infected corneal tissue, HSV tk and ICP0 transcripts were readily detected at 3, but not at 10 or 30 dpi where CT values = 40 (indicating no measurable RNA) , we collected corneas and TG during acute 3 and 10 dpi) and latent (30 dpi) phases. To monitor viral gene expression in infected mice, we tested tissue samples for 0 dpi andtk RNA peaked at 3 dpi then dropped precipitously (200-fold) to low but readily detectable levels by 10 dpi. At 30 dpi, we detected very low or undetectable tk RNA expression in infected TG. In the experiment shown in Fig. tk expression in infected TG at 30 dpi, resulting in a relative expression value of 0.0002. In an independent experiment, we measured a CT of 38.1 for tk RNA in 30 dpi TG; however, a CT value of 40 was measured in two additional experiments (not shown). CT values for all reactions without RT were 40, indicating no DNA contamination. Thus, while tk expression in latent TG was at the limit of detection for our assay, our ability to detect tk expression in some but not all latent TG was consistent with previous reports in which very sensitive RT-PCR assays were used to detect tk (and ICP0) gene transcripts in some but not all TG during latent infection [tk nucleic acid per PCR reaction. Our present assay for tk transcripts is at least 50-fold less sensitive than that used by Kramer and Coen [In infected TG, nfection ,25. In tICP0 RNA levels were similar to tk in that they peaked at 3 dpi in cornea and TG  DCs, CCR7 which is primarily expressed on resting T and B cells and mature DCs that home back to lymphoid tissues, and CXCR4 which is broadly expressed on many immune and non-immune cell types (Table We next used TaqMani.e., >3-fold) at 3 and 10 dpi  and latent (30 dpi) phases  for up to 150 dpi in infected TG, indicating that latent virus persists well beyond 90 dpi in this mouse model. Induction of all RNAs in our panel persisted for at least 62 dpi; furthermore, all but CCR3 and eotaxin-1 were also induced at 90 dpi  for Exps. #2, #3, and #4 following the manufacturers' suggested protocols. Design of the PCR primers and TaqMan® probes for mouse chemokine and chemokine receptors was done using Primer Express (Applied Biosystems) software. Primer and probe sequences are listed in Table ® probes for the housekeeping control genes rodent GAPDH and 18S rRNA were purchased from Applied Biosystems. Real-time quantitative RT-PCR assays were performed with reagents recommended by the manufacturer (Applied Biosystems) using an ABI PRISM 7700 Sequence Detection System instrument. Briefly, 0.5 μL (approximately 300 pg) of cDNA was added to 25μL reactions containing 12.5 μL of PCR Universal Mix (Applied Biosystems), 600 nM F primer, 600 nM R primer, 200 nM FAM-labeled TaqMan probe, 200 nM rodent GAPDH F primer, 200 nM rodent GAPDH R primer, and 100 nM rodent GAPDH TaqMan® probe. The number of PCR cycles needed for FAM or VIC fluorescence to cross a threshold where a statistically significant increase in change in fluorescence (CT=threshold cycle) was measured using Applied Biosystems software. Relative RNA expression was determined using the formula Rel Exp= 2-(ΔΔCt) × 1000 where ΔΔ CT= -(CT gene of interest-CT rodent GAPDH in a no-template control sample) (the ΔΔ CT method, Taqman® Bulletin #2: Relative Quantitation of Gene Expression, Applied Biosystems, updated 2001, ). To assure that GAPDH RNA levels were not affected by HSV infection and thus a good control, we repeated most analyses using 18S rRNA as an internal control. In all cases tested, induction measurements (HSV+/mock) were indistinguishable whether 18S or GAPDH were used (not shown). Control reactions lacking RT were used to test for the presence of contaminating HSV or mouse DNA, and in all cases either no or low (relative to when RT was present) levels of amplification were measured (not shown). Purified HSV-1 genomic DNA was kindly provided by Jean Pesola.Total RNA was purified from tissues using RNA STAT-60 (Tel-Test, Friendswood, TX), followed by secondary purification and DNAse I treatment using RNeasy columns (Qiagen). cDNA was synthesized using the Omniscript Reverse Transcriptase Kit (Qiagen) for Exp. #1 or TaqManThe author(s) declare that they have no competing interests.W. Cook, R. Walker and T. Burwell performed the RT-PCR analyses of chemokine transcripts. M. Kramer and H. Holman performed the animal infections and provided tissues for transcript analysis. D. Coen and D. Knipe participated in the design of experiments, oversight of the conduct of the experiments, and in the interpretation of the results.


The rapid increase in published genomic sequences for bacteria presents the first opportunity to reconstruct evolutionary events on the scale of entire genomes. However, extensive lateral gene transfer (LGT) may thwart this goal by preventing the establishment of organismal relationships based on individual gene phylogenies. The group for which cases of LGT are most frequently documented and for which the greatest density of complete genome sequences is available is the γ-Proteobacteria, an ecologically diverse and ancient group including free-living species as well as pathogens and intracellular symbionts of plants and animals. We propose an approach to multigene phylogeny using complete genomes and apply it to the case of the γ-Proteobacteria. We first applied stringent criteria to identify a set of likely gene orthologs and then tested the compatibilities of the resulting protein alignments with several phylogenetic hypotheses. Our results demonstrate phylogenetic concordance among virtually all (203 of 205) of the selected gene families, with each of the exceptions consistent with a single LGT event. The concatenated sequences of the concordant families yield a fully resolved phylogeny. This topology also received strong support in analyses aimed at excluding effects of heterogeneity in nucleotide base composition across lineages. Our analysis indicates that single-copy orthologous genes are resistant to horizontal transfer, even in ancient bacterial groups subject to high rates of LGT. This gene set can be identified and used to yield robust hypotheses for organismal phylogenies, thus establishing a foundation for reconstructing the evolutionary transitions, such as gene transfer, that underlie diversity in genome content and organization. The study demonstrates that single-copy orthologous genes are resistant to horizontal transfer and can be used to generate robust hypotheses for organismal phylogenies The availability of complete sequences of genomes for clusters of related organisms presents the first opportunity to reconstruct events of genomic evolution. By comparing related genomes and inferring ancestral ones, we can identify events, such as specific chromosomal rearrangements, gene acquisitions, duplications, and deletions, that have produced the observed diversity in genome content and organization. The Bacteria offer the most immediate opportunities for such reconstruction, because many clusters of related genomes are now available and because the genomes are small and contain relatively little repetitive sequence, reducing computational complexity. Among bacterial groups, the γ-Proteobacteria presents the most intensively studied and sequenced cluster of genomes with varying degrees of relatedness.Intertwined with the problem of reconstructing genomic change is the problem of inferring phylogeny. Evading this issue is particularly difficult in the Bacteria. First, using complete genomes to obtain a robust phylogeny for all bacteria has presented problems due to the age of the group and the resulting loss of phylogenetic signal. Furthermore, lateral gene transfer (LGT) occurs in bacteria and has been claimed to be rampant for all classes of genes, potentially resulting in a diversity of phylogenetic histories across genes and complicating, or completely defeating, attempts to reconstruct bacterial evolution at both deep and more recent evolutionary depths . AlthougFor purposes of reconstructing genomic change, what we seek is the organismal phylogeny—that is, the topology that traces the history of the replicating cell lineages that transmit genes and genomes to successive generations. The organismal phylogeny provides the backdrop against which events of genomic change, including LGT, have occurred. High incidence of LGT may cause the organismal phylogeny to be elusive, because we do not know which genes represent the true history of the cell lineages.The gene most used for reconstructing organismal phylogeny is the small subunit ribosomal RNA (SSU rRNA), which has been argued to rarely undergo transfer among genomes . But eveEscherichia coli and Salmonella sp.) for which knowledge of gene function is more complete than for any other cellular organisms. The potential obstacles to phylogenetic inference that are found across the Bacteria are certainly present in the γ-Proteobacteria. In particular, LGT is known to be extensive in this group, based on studies of genome composition , tend to place Wigglesworthia and Buchnera as sister taxa  topology, using the Shimodaira–Hasegawa (SH) test, as recommended by Pseudomonas conflicts with all widely supported topologies; it is placed as sister-group to Vibrio (BioB) or as sister-group to the enterics Escherichia, Salmonella, and Yersinia (MviN). Although initial examination of the topologies obtained from these genes suggests more than a single LGT  as well as all other topologies tested. We have investigated these two genes to determine whether the incongruence is likely to result from LGT. The proteins correspond to biotin synthase (BioB) and to the virulence factor MviN. The trees obtained using ML for each alignment are shown in rrection B. In eac genomes A. MoreovEscherichia, Vibrio, Salmonella, Yersinia, and Pseudomonas, bioB is flanked by bioF, also involved in biotin biosynthesis. To determine whether bioF could have been transferred with the bioB gene, we built a tree based on the protein translation of bioF using all species except Buchnera, Haemophilus, and Pasteurella, which lack this gene. The tree obtained did not show any unexpected position of Pseudomonas, indicating that only bioB has been horizontally transmitted. A possible explanation, consistent with the flanking position of bioB and bioF in the Pseudomonas genome, is that the original bioB gene was replaced through homologous recombination in a common ancestor of the included Pseudomonas species. Similar comparisons for mviN did not illuminate its history in Pseudomonas, as the flanking genes differed from those in other species. The observations for mviN are consistent with a transfer event from an enteric species to a new genomic position in a Pseudomonas ancestor.In The general lack of conflict observed among the 203 remaining families was not due to the absence of phylogenetic signal in the gene alignments because most genes did conflict with several other topologies see . We inteWigglesworthia and Buchnera were rejected by the majority of the alignments. In the best-supported topology  . Thus, this grouping is probably not an artifact of the biased composition of the endosymbiont genomes.All topologies separating logy see , Wiggleslmonella . Becauselmonella , and alslmonella . To test lysine) . Using tP. aeruginosa genome. Because the γ-Proteobacteria has been the bacterial group most often cited as showing high rates of LGT, this finding is unexpected. However, we note that the evidence for LGT from sequence features and comparisons of genome content . Thus, our finding that very few orthologs have been exchanged within the group and that none show evidence of having been imported from other bacterial lineages is relevant for the understanding of long-term bacterial evolution.One possible explanation for the lack of observed events of orthologous replacement might be that these are sufficiently rare that significant frequencies are encountered only when considering deeper phylogenetic levels. However, the group studied here, though recent enough to allow accurate phylogenetic reconstruction, is old. Indeed, the divergence of different l record , and theIt has been proposed that LGT may be more frequent within clusters of related bacteria and even that phylogenetic groupings, such as the γ-Proteobacteria, may reflect boundaries to LGT rather than recent shared ancestry of lineages . Such a Wigglesworthia and Buchnera have sometimes been considered as closely related and sometimes not, based on relatively weak phylogenetic evidence provided by the SSU rRNA alone. Our confirmation of their close relationship raises the question of whether their common ancestor was an endosymbiont with a reduced genome or a free-living bacterium (perhaps one with a host-associated lifestyle that promoted formation of intimate symbiosis). Because Buchnera and Wigglesworthia do not share any genes absent from the other species, no particular genes can be implicated as conferring a predisposition to symbiosis, a result that eliminates some hypotheses about how symbiosis originates. Furthermore, although emphasis has previously been placed on the close relationship of Buchnera with E. coli, our results shows that the phylogenetic relationship is equally high with other enterics, such as Yersinia pestis, which indeed shares as many ortholog families with Buchnera as does E. coli. This knowledge of relationships to other genomes allows more accurate reconstruction of ancestral genome content and of the chromosomal deletions and rearrangements occurring during the evolution of reduced symbiotic genomes . To identify genes likely to have been transmitted vertically through the history of the γ-Proteobacteria, we first eliminated proteins annotated as elements of insertion sequences or as bacteriophage sequences, since they are likely to be subject to lateral transfer. Such sequences were present in most genomes but lacking in a few .Click here for additional data file.The GenBank accession numbers discussed in this paper are NC_000907, NC_000913, NC_002488, NC_002505, NC_002506, NC_002516, NC_002528, NC_002663, NC_003143, NC_003197, NC_003902, NC_003919, NC_004088, and NC_004344.


Combining bright light exposure and physical exercise may be an effective way of relieving depressive symptoms. However, relatively little is known about individual factors predicting either a good response or treatment failure. We explored background variables possibly explaining the individual variation in treatment response or failure in a randomised trial.Participants were volunteers of working-age, free from prior mental disorders and recruited via occupational health centres. The intervention was a randomised 8-week trial with three groups: aerobics in bright light, aerobics in normal room lighting, and relaxation/stretching in bright light. Good response was defined as a 50% decrease in the symptom score on either the Hamilton Depression Rating Scale (HDRS) or 8-item scale of atypical symptoms. Background variables for the analysis included sex, age, body-mass index, general health habits, seasonal pattern, and sleep disturbances.2 = .02). The number needed to treat (NNT) for light was 3.8.Complete data were received from 98 subjects (11 men, 87 women). Of them, 42 (5 men, 37 women) were classified as responders on the HDRS. Overall, light had a significant effect on the number of responders, as assessed with the HDRS (XWe investigated the effect of bright light and exercise on depressive symptoms. Problems with sleep, especially initial insomnia, may predict a good response to treatment using combined light and exercise. Bright light exposure and physical exercise, even in combination, seem to be well tolerated and effective on depressive symptoms. At northern latitudes reduced vitality, increased appetite and sleep complaints are common symptoms during wintertime, even among people considered healthy . AtypicaExercise would seem to be the ideal treatment for depression: available, affordable, with minimal side effects. Unfortunately, many exercise treatment studies suffer from methodological weaknesses and lack of adequate follow-up to determine long-term efficacy. In their systematic review, Lawlor and Hopker even concluded that the effectiveness of exercise in reducing depressive symptoms cannot be determined because of a lack of good quality research, though exercise was found to be more effective than no treatment, and as effective as cognitive therapy . FortunaAdding light exposure to exercise for the treatment of depression and depressive symptoms seems a promising intervention. Combinations of bright light exposure and physical exercise have achieved beneficial effects on mood in trials on healthy adult populations ,13. NatuIt remains to be determined, however, whether it is possible to define a subgroup of people who are especially likely to benefit from this kind of intervention, and what, if any, are the individual factors predicting a good response to light or exercise, or their combination? And on the contrary, another important subgroup to identify, in determining the efficacy of any intervention, is the subjects who drop out of the study prematurely. Several predictors of response to light treatment in winter depressives have been identified: the ratio of atypical to classical symptoms of depression  and hypeIn the present study we tried to address this question: who will benefit from light, exercise or their combination? A variety of background variables were investigated: age, seasonality, depressive and atypical symptoms, treatment adherence, fitness, body mass index, self-perceived quality of sleep, and alcohol consumption. We did not measure diurnal variation or personality dimensions.Adult volunteers of working age were invited to participate in a study of light and exercise via occupational health centres. The enrolled subjects were randomly allocated to three intervention groups: aerobics training in bright light (>2500 lx, measured at eye-level), similar training in the normal lighting of the gym (400–600 lx), and relaxation and stretching sessions in bright light. The training or relaxation sessions (45 minutes each, starting at 7:30 a.m. or 8:30 a.m. Monday through Friday, and at 10:00 a.m. or 11:00 a.m. on Saturdays) were scheduled two times a week over eight weeks. Study was conducted between November 25, 1997, and January 25, 1998. The length of daylight on these dates was 6 h 48 min and 7 h 23 min, respectively.Both the aerobics training and relaxation groups were led by 3 physiotherapists, each of whom supervised one third of each group's training or relaxation sessions. The training and relaxation sessions were structured to maintain treatment consistency. In the aerobics groups, the intensity of the training was checked with a heart rate monitor, the target rate being 120 to 150 beats per minute. Stretching/relaxation training was designed to avoid raising the pulse. All training sessions were in the same gym, the ceiling of which was equipped with 30 extra light fixtures with cool-white (6000 K) fluorescent lamps (F58W/186, Sylvania, Germany), which were turned on for the bright light groups.Mood during the study period was recorded using the Structured Interview Guide for the Hamilton Depression Rating Scale – Seasonal Affective Disorders Version Self-Rating Format (SIGH-SAD-SR) , which iAt the start of the study and at weeks 4 and 8, all subjects were weighed to assess body-mass index (BMI). Before and after the study period all subjects in the Aerobics training groups participated in a 2-km walking test, which predicts maximal oxygen uptake using a model with age, sex, walking time, BMI, and heart rate at the end of the test as variables . At baseAll subjects returned a written informed consent prior to participation. The ethics committee of the National Public Health Institution approved the study protocol.All analyses were done on SPSS for Windows (Release 11.5.1)-statistical package (SPSS Inc., Chicago, Illinois).Participants were classified as dropouts if they, for any reason, did not finish the eight-week study protocol. Analysis of variance (ANOVA)-models were used to compare the baseline characteristics of dropouts with those who completed the study. In the models, the baseline characteristic was the dependent variable, and the outcome (dropouts vs. completers) and treatment group (Light & Exercise, Exercise, Light) were factors. The Outcome × Treatment group interaction was also tested in each model. Background characteristics examined were age, GSS, BMI, fitness, and alcohol consumption, all as continuous variables. Baseline HDRS and ATYP were included, as well as percentage of sessions attended. From the BNSQ, following variables were chosen: initial, middle, and late insomnia, and quality of sleep.Treatment benefit was defined separately as response and, with stricter criteria, as remission.The main outcome measures were changes in the HDRS, ATYP and the SIGH-SAD-SR over the 8-week study period. A 50 % decrease on the HDRS, ATYP or the SIGH-SAD-SR total score was used to divide subjects into responders and non-responders. To assess the effect of baseline characteristics, logistic regression models were formulated, with the defined clinical response as the dependent variable. Light therapy, physical exercise, and sex were constant in the models. Other independent baseline variables in the analysis were age (over/under 40 years), seasonality (from the SPAQ), initial, middle and late insomnia, quality of sleep, feeling tired after waking (from the BNSQ), serum total cholesterol levels, current smoking, physical training, and consumption of alcoholic beverages (from the FINRISK questionnaire). All categorical variables were dichotomised for the analysis. The best-predicting co-variates were found by backward step-wise selection. Analysis of variance (ANOVA) was applied to compare means between groups, and associations were analysed by calculating partial correlation coefficients, after controlling for age and sex. Pearson chi-square (two-sided) was used when applicable.Stricter criteria were applied for the definition of remission. To increase clinical meaningfulness and to avoid 'flooring effect', subjects with low symptom scores were excluded from these analyses.and a score of less than eight at the end of the study period.Only subjects with a baseline HDRS of eight or higher were included. Remission was defined as at least a 50% reduction on the HDRS during the trial, and a final SIGH-SAD-score of eight or less.Subjects with a baseline SIGH-SAD-total score of fourteen or more were included. Remission was defined as at least a 50% reduction on the score during the eight-week study, Dropouts for description).All analyses were done 'intention-to-treat', i.e. dropouts were classified as treatment failures. With remitted subjects, we desisted from using logistic regression because of lower number of subjects, which would limit the number of variables. Instead, the background variables were examined one-by-one with ANOVA-models (see 2 = .04).Complete data were received from 98 subjects  (r = -.23, p = .03), and high levels of serum total cholesterol (r = -.21, p = .04).2 = .9) were classified as responders. Their mean age (s.d.) was 41.3 (9.5) years, ranging from 26 to 58. Thirty-five (83%) had had light therapy, 24 (57%) had been in the aerobic exercise groups, and 17 subjects (40%) in the combined group. Overall, light had a significant effect on the number of responders, as assessed with the HDRS (X2 = .02). The number needed to treat (NNT) for light was 3.8.Based on the HDRS, 42 subjects (5 men, 37 women, XOn the basis of the ATYP scores, 51 subjects (8 men, 43 women) were classified as responders. Their mean age (s.d.) was 41.9 (9.8) years, ranging from 26 to 63. Thirty-seven (73%) had received light therapy, 30 (59%) had been in the exercise groups, and 16 (31%) in the combined group.2 = .03). Based on these figures, the NNT for light was 3.8.Response on the SIGH-SAD-SR was negatively associated with baseline self-reported alcohol consumption (r = -.26, p = .01). There were 45 responders (5 men, 40 women) on the SIGH-SAD-SR, with a mean age (s.d.) of 41.5 (9.5) years, ranging from 26 to 58. Thirty-seven (82%) had received light therapy, 26 (58%) had done aerobic exercise, and 18 (40%) had been in the combined group. The effect of light therapy was significant , 8 (20%) in the light & exercise group, 13 (31%) in the exercise group, and 5 (12%) in the light group (X2 = .1). Thirteen subjects (50%) had received light therapy (X2 = .05). Table There were 26 (21%) subjects classified as dropouts  were considered remitted on the HDRS after the study period. Nine had been in the light & exercise group, 5 in the exercise group, and 11 in the light group (X2 = .5). Thus, 20 of the subjects had received bright light therapy (X2 = .3) and 14 had been in the exercise groups (X2 = .5). Proportion of sessions attended had a significant impact on remission status (F = 8.66, p = .004). The BNSQ initial insomnia-variable was influenced by treatment group assignment (F = 3.83, p = .03).A total of 74 subjects  were included in the analyses. Twenty-five subjects  were considered to be in remission on the SIGH-SAD-scale. Seven subjects had been in the light & exercise group, 1 in the exercise group, and 8 in the light group (X2 = .08). Fifteen subjects (94%) had been in groups with bright light exposure (X2 = .03), and eight (50%) in the exercise groups (X2 = .4). In the ANOVA-models, remission was significantly influenced only by the GSS (F = 5.23, p = .03).Sixty-seven subjects  were included in the analyses. After the trial, sixteen of them  predicted a poorer response on all the assessed scales. One explanation to this might be the negative effect of alcohol on circadian rhythms and especially on sleep ,32.The dropout rate was relatively low (combined 21%), and did not differ significantly between the treatment conditions. There seemed to be a trend towards subjects in groups receiving bright light therapy adhering to the study more closely than subjects in the exercise group. Subjects might have felt bright light therapy is a novel, more attractive treatment option than plain, 'old-fashioned' exercise. We tried to minimise this problem by emphasising 'exercise trial' in leaflets provided to possible volunteers. It was not possible to avoid this problem altogether, as reflected by those four subjects who dropped out of the study after hearing they had been randomised into the exercise group. When these four subjects, now counted as dropouts, are excluded, the dropout rates of the light & exercise and exercise groups are virtually identical, and similar to the rates reported previously . The droAn interesting finding is that completers were in a better physical condition in the pre-intervention fitness test than were dropouts. Regular exercising may be a representation of self-motivation trait, which would increase adherence to a therapeutic exercise program . A majorUsing the strict remission criteria yielded few results. Attendance to treatment sessions did predict remission on the Hamilton scale , but post-hoc analyses showed that this was caused by dropouts, which were automatically labeled as treatment failures.A lower GSS was predictive of remission on the SIGH-SAD total score. Again, post-hoc analyses showed this effect was caused by dropouts with higher than average scores.In previous research on the use of light therapy, atypical depressive symptoms have been predictive of treatment response and remission. This effect was not seen in the present study. We did separate analyses for hypersomnia, hyperphagia, increased appetite / carbohydrate craving, and reduced vitality, but none of these individual atypical symptoms predicted remission either.The study subjects were not patients with clinically diagnosed depression, but volunteers with varying degrees of depressive symptoms. This poses a major question of definition of treatment response and remission, and also measurement of depressive symptoms, e.g. using scales originally designed for the follow-up of depressed in-patients . Our priMore studies, with expanded methodology, are clearly needed to shed light on the individual factors separating responders and non-responders in exercise trials, with or without bright light. Interesting research subjects would be circadian phenotyping , and moWe investigated the effect of bright light and exercise on depressive symptoms in working-age men and women, free from mental disorder and/or psychotropic medication. We found that problems with sleep, especially initial insomnia, may predict a good response to treatment using combined light and exercise. Regular intake of alcoholic beverages (over 7 drinks per week) seems to have an opposite effect. Bright light exposure and physical exercise, even in combination, seem to be well tolerated and effective on depressive symptoms, but more research is needed to confirm these findings.None declared.SL, TP, and JL planned the study protocol and supervised the study. JH planned the statistical analyses. SL, TP, and JH analysed the data. SL and TP wrote the manuscript, which was commented by JH and JL. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Asthma is a complex genetic disease with more than 20 genome-wide scans conducted so far. Regions on almost every chromosome have been linked to asthma and several genes have been associated. However, most of these associations are weak and are still awaiting replication.In this study, we conducted a second-stage genome-wide scan with 408 microsatellite markers on 201 asthma-affected sib pair families and defined clinical subgroups to identify phenotype-genotype relations.The lowest P value for asthma in the total sample was 0.003 on chromosome 11, while several of the clinical subsets reached lower significance levels than in the overall sample. Suggestive evidence for linkage (p = 0.0007) was found for total IgE on chromosomes 1, 7 and again on chromosome 11, as well as for HDM asthma on chromosome 12. Weaker linkage signals could be found on chromosomes 4 and 5 for early onset and HDM, and, newly described, on chromosome 2 for severe asthma and on chromosome 9 for hay fever.This phenotypic dissection underlines the importance of detailed clinical characterisations and the extreme genetic heterogeneity of asthma. Many chromosomal regions have been shown to be linked or associated to asthma and asthma-associated traits in humans . More reThe mainstay of all genetic studies has been genome-wide linkage scans in families with at least two asthma-affected siblings. Based on a previous analysis of a genome-wide scan of asthma ,8 with i97 families consisting of at least two children with confirmed clinical asthma were collected during the first stage of the German genome scan [Children with premature birth, low birth weight and any severe pulmonary disease other than asthma were excluded as prematurity (and or associated low birth weight) may be a major non-genetic risk factor for the development of pulmonary symptoms. Another 4 families were excluded after genotyping because of Mendelian errors. The additional 104 families comprised 452 individuals  asthma was large, consisting of 134 families all with at least one positive skin prick test reaction or one increased specific IgE.Almost all family members participated in a skin prick test (SPT). We selected an "HDM SPT positive" group of 33 families with at least 3 family members showing a skin prick test reaction ≥ 3 mm. House dust mite allergy is quite common in asthmatic patients, and a known trigger factor for asthma attacks. Some studies have successfully used the combination with asthma as lead trait ,10. As tSeveral interview questions related to asthma symptoms, diagnostic findings and therapy. We used information about asthma attack frequency, actual medication and emergency hospital visits for a severity index with 5 levels. This index is based on subjective patient information, current/previous therapy and influence on quality of life table . "SevereAll participants were asked to specify the months when asthma attacks occurred. 39 "winter-type" families had one affected child with symptoms only in the cold months and his or her sibs not a fully "summer-type". In contrast 35 "summer-type" families were found with one affected child with summer attacks and his or her sibs not purely "winter-type". Attacks year-round usually were seen with severe disease only. Sporadic attacks usually did not have any seasonal preferences while both the intrinsic, usually infection-related "winter-type", as well as the extrinsic, hay fever related "summer-type", appeared to be concordant within families. This observation may already support the hypothesis of different genes in different subtypes.In 170 of 201 families, both parents were of German descent ("German"). In this subgroup, we tried to reduce the genetic heterogeneity by leaving out the remaining 31 families, of which 5 were from Sweden and 5 from Turkey.DNA was isolated from peripheral white blood cells using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer's recommendation. For genotyping we used almost the same microsatellite marker as in the first scan ,8. The fMarker amplification was performed in microtiter plates, either in 96-  or in 384-well format (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA). Fragment analysis of PCR pools was conducted on an ABI 3700 DNA sequencer and genotypes were scored using GENESCAN and GENOTYPER (ABI) software. In the second scan, 98% of all possible genotypes could be unequivocally determined.Genotyping data were transferred to a SQL 2000 database, pre-checked with previously developed routines and exported in ASCII format for multipoint linkage analysis with MERLIN 0.9.3 . Allele Figure Chromosomes are arranged by number from p-ter to q-ter with distance in centimorgans on a linear scale. Table chromosome 1, two linkage areas for total IgE could be found at about 55 cM (p = 0.00009) and at 188 cM (p = 0.0007), which reached the threshold for suggestive linkage (p = 0.0007) [chromosome 2 at about 64 cM (p = 0.002) evidence was found for a locus for severe asthma and for house dust mite sensitive asthma. Early onset, together with HDM, is represented both on chromosome 4 at about 195 cM (p = 0.002) and on chromosome 5 at about 55 cM (p = 0.002).On  0.0007) . On chrochromosome 7 (65 cM, p = 0.0004) and chromosome 11 , all with suggestive linkage according Kruglyak-Lander.Three loci for total IgE regulation were found on chromosome 9 at about 104 cM (p = 0.002) there is evidence for a locus linked to allergic rhinitis plus asthma ("summer-type"), having an effect mainly in families of German descent. Lastly, a locus showing suggestive linkage could be identified on chromosome 12 (95 cM, p= 0.0002) for house dust mite sensitised asthmatic patients.On Several linkage regions in asthma families could be found in this extended sample, although again no linkage with P < 0.003 could be found with any marker for asthma. Asthma is apparently of such a heterogeneity that even investigating affected sib pairs from more than 200 families failed to yield significant results. Nevertheless, the quantitative trait IgE in these families, both as a discrete and continuous variable, led to three suggestive linkage findings on chromosomes 1, 7, and 11, which have all been discussed in prior publications table .Moreover, the strategy of phenotypic dissection proved to be quite successful, though the sample size of subsets seems to be very small. The size of the sample in a linkage study is known to be one of the most important factors for a significant finding , making In particular, the subgroups segregated by high disease severity, sensitisation by HDM, early onset and population origin attained better lower significance levels than the main trait table . So far,Sample recruitment with restricted phenotypic requirements proved to be successful for some other traits as well. Early onset definitions have been used for cancer studies and metabolic disorders to increase the underlying genetic component ,28. SepaRecently, evidence was shown for asthma genes on chromosomes 2 , 14 [4],Frequent replication, however, may point toward lead genes. Most regions listed in table The locus on chromosome 12 with suggestive linkage for HDM in our study is known from many other reports. Also our first scan showed weak evidence for linkage in this region at D12S351 (p = 0.01) , whereasOn chromosome 2, we found a locus in families with severe asthma that has not yet been described in other studies. A p-value of 0.007 has already been found in our first genome-wide scan [The German "summer-type" locus on chromosome 9 is also not frequent in other studies. This might be due to the fact that many, but not all, genes are necessary for a common trait. Our first asthma scan has already shown linkage in this region with a p-value of 0.007 for D9S1784 [We conclude that this phenotypic dissection is a useful tool to detect linkage in a heterogeneous disease like asthma because some of the sub phenotypes reached even better significance values than the main trait. We show that the precision of the phenotype can be more effective than expanding the sample size only. Unfortunately, large sample sizes are needed to assure at least moderate sample size in subsets. Grouping by early onset or disease severity could be applied to almost every complex disease, but for a more specific dissection, clinical expert knowledge is required. A prior analysis of clinical data can help to identify symptom clustering, which -if consistent within families- can reduce genetic heterogeneity.The author(s) delcare that they have no competing interests.MW, JA, and CS were involved in the study design, JA organised the sample collection, conducted the genetic analysis, and prepared the manuscript, MW organized funding and supervised the study. YAL and PN organised and helped with genotyping and revised the article critically for important intellectual content. SL, FR and MW carried out the statistical analysis and made substantial contributions to its conception and design, CS, DB, FF, HJ, JK, AK, AS, MS, WW, PW assisted in the recruitment of families and examined patients and therefore made substantial contributions to the acquisition of data, asthma severity grades are part of the thesis of CS. GS was responsible for the IgE analysis.The pre-publication history for this paper can be accessed here: