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DoOR 2.0 - Comprehensive Mapping of Drosophila melanogaster Odorant Responses

Odors elicit complex patterns of activated olfactory sensory neurons. Knowing the complete olfactome, i.e. the responses in all sensory neurons for all relevant odorants, is desirable to understand olfactory coding. The DoOR project combines all available Drosophila odorant response data into a single consensus response matrix. Since its first release many studies were published: receptors were deorphanized and several response profiles were expanded. In this study, we add unpublished data to the odor-response profiles for four odorant receptors (Or10a, Or42b, Or47b, Or56a). We deorphanize Or69a, showing a broad response spectrum with the best ligands including 3-hydroxyhexanoate, alpha-terpineol, 3-octanol and linalool. We include all of these datasets into DoOR, provide a comprehensive update of both code and data, and new tools for data analyses and visualizations. The DoOR project has a web interface for quick queries (http://neuro.uni.kn/DoOR), and a downloadable, open source toolbox written in R, including all processed and original datasets. DoOR now gives reliable odorant-responses for nearly all Drosophila olfactory responding units, listing 693 odorants, for a total of 7381 data points.

Results
Different types of new data. With this update of the DoOR.data package we introduce three classes of new data: (1) Better precision raw data from datasets that were already included. When we published the first version of DoOR, not all raw data from the considered publications was available. In many cases we had to estimate quantitative responses from graphical plots. In the meantime we received the raw data for many of these studies from the authors and updated the existing DoOR datasets. This situation is improving: publishing data that underlies the plots of a paper in the supplements becomes a widespread practice, giving increasingly access to raw data in (a) Each point in the matrix represents an odor-responding unit combination. Colors indicate whether that combination is new in DoOR (red), was updated (blue), unchanged (green) or is still missing (grey). Response units were sorted according to the numbers of odorants they were tested with, odorants were sorted accordingly (see Tables S2, S3 and S4 for responding unit and odorant names respectively). (b) Visualization of ensemble responses. Responses elicited by propanoic acid mapped onto a representation of the Drosophila antennal lobe model from Grabe et al. 17 . Glomerulus names are shown in the top panel, the corresponding receptor names are shown in the bottom panel. DoOR 2.0 contains mappings of 13 IR innervated glomeruli that were still unmapped (dark grey) in DoOR 1.0. The two dark grey glomeruli VP2 and VP3 are non-olfactory. the community. (2) Raw data from new publications: Many new studies have been published since the first DoOR release. These studies added important new data to the Drosophila olfactome (Fig. 1a). Importantly, they deorphanized most of the remaining OR response profiles for which no ligand was known previously. (3) Data recorded by us that is first published along with this paper. Here we present the response profiles of five OSN classes, measured via calcium imaging. Our set of ~100 odorants, added a total of 529 new odorant responses to DoOR. One of these response profiles deorphanizes the Or69a sensory neurons, others contribute to existing profiles (Fig. 2). See Table S1 for an overview of all studies that contributed to the DoOR project. We note, with respect to points 1 and 2, that many but not all colleagues were willing to share (published) odorant response data.
We added original data for the following receptors ( Fig. 2 and Table S5): For Or10a OSNs methyl salicylate, ethyl benzoate and butyric acid elicited the strongest responses in our hands. About one third of the tested odorants led to a reduction of calcium concentration (inhibition). In total our dataset added 52 odorants to the known response profile. Or10a is expressed in ab1D neurons, their axons innervate glomerulus DL1. The cells co-express the gustatory receptor Gr10a 8 . For Or42b our data added 76 substances. 3-hexanone, ethyl propionate and ethyl (S)-(+ )-3-hydroxybutyrate were the three strongest ligands in this dataset. The receptor is chirality selective: the stereo isomer ethyl (R)-(− )-3-hydroxybutyrate did not activate these neurons. Or42b is expressed in ab1A neurons, their axons innervate glomerulus DM1. Or47b responded mainly with inhibition, confirming previous reports 3, 25,26 . We added 61 new odorant responses to DoOR and observed weak inhibitions for most substances. Benzaldehyde, furfural and acetic acid produced stronger inhibitions. Excitatory responses were weak as compared to other receptor cells. The strongest responses in our set were to (S)-carvone and propanoic acid, but these are not the best ligands. A stronger ligand, methyl laurate, has been recently discovered by Dweck et al. 27 , the dataset is included in DoOR. Or47b is expressed in at4 neurons, their axons innervate glomerulus VA1d. Or56a expressing ab4B OSNs responded best to geosmin, confirming the deorphanizing data by Stensmyr et al. 28 . In addition, stimulation with (1R)-(− )-fenchone and alpha-ionone also evoked lower but reliable signals. We also observed inhibitory responses: 2,3-butanedione and acetic acid produced the strongest decreases in fluorescence. In total we added 82 responses to the response profile. Or56a is expressed in ab4B neurons, their axons innervate glomerulus DA2. The cells co-express the receptor Or33a 8 . For Or69a no ligands were known previously. We recorded responses to 106 odorants. Or69a OSNs responded particularly broad, showing activity towards most of the odorants in our set: the receptor kurtosis was − 0.36. We found the strongest responses for ethyl 3-hydroxyhexanoate, alpha-terpineol, 3-octanol and linalool. Or69a is expressed in ab9 neurons, their axons innervate glomerulus D. Overall, across the five characterized receptors, we analyzed the response profiles with respect to chemical class (Fig. 2). It is apparent from the figure that chemical class is not a good response predictor: all colors intermingle across the entire odor-response range. In other words, it is not useful to characterize individual receptors as "alcohol receptor", or "ester receptor". Deorphanized OSNs. With the new datasets included, DoOR response profiles are now existing for all known OSNs except for Ir40a, and all antennal lobe glomeruli except VA7m have been assigned to a sensory neuron ( Fig. 1b and Table 1). All other glomeruli without cognate receptor gene in the previous 2010 version of DoOR have been deorphanized now. They are innervated by IR expressing OSNs housed in coeloconic sensilla or in the sacculus 12 . We updated the "OSN to glomerulus" mappings and the glomerulus nomenclature according to Silbering et al. 12 and the recently published in-vivo atlas of the Drosophila AL by Grabe et al. 17 . Ligands have been published for IRs 12 , and are included in DoOR. Response profiles for five OSNs measured via calcium imaging and added to DoOR. Bars represent mean calcium signals (n = 3 − 16 and 122 − 296 for controls) measured from five different GAL4 driver lines in response to a set of 100 odorants. Colors indicate the chemical classes the different odorants belong to. Shaded areas indicate half maximal and half minimal response ranges respectively. Or56a was recorded using a different reporter (GCaMP3 vs. GCaMP1.3); the different scale is due to the reporter, and does not indicate different receptor calcium response properties. Mineral oil solvent responses were subtracted. Number of odorants in the dataset is given as n. All odorant names and response values are given in Table S5. Updated DoOR mappings. There were multiple cases where response profiles could not be assigned unambiguously to a single receptor already in DoOR 1.0. These included cases where the receptor of the measured OSN was not known (e.g. OSN ac1A), or where more than one functional receptor was expressed in an OSN (e.g. ab5B, which expresses Or47a and Or33b). In these cases we assigned the response profile to the OSN name. While some of these cases have been resolved in the meantime (unknown partners were mostly IRs), others have to remain. For example, when a sensory neuron expresses two odorant receptors, it is necessary to measure each receptor and the neuron separately, generating three datasets (for more examples, see below). Consequently, we had to expand this naming scheme, assigning some response profiles to the sensillum and others to the glomerulus they were measured in. Due to these difficulties in nomenclature we refer to the different origins of DoOR response data as "responding units" throughout the text. In most -but not all -cases a "responding unit" consists of an unambiguous mapping of receptor cell in a given sensillum, receptor gene/protein, and glomerulus in the antennal lobe. All "responding units" are listed in Table 1, together with the relevant information. In many cases electrophysiological recordings from coeloconic sensilla could not be mapped to the individual OSN, because spike amplitudes and/or shape were not discrete enough to perform a separation. For example, single sensillum recording (SSR) data from Silbering et al. 12 was integrated as summed OSN responses for the individual coeloconic sensilla ac1-4. Marshall et al. 29 were able to separate the unit with the strongest amplitude (the A neuron) but summed the remaining. We considered this data as e.g. "ac1A" (the strongest, unambiguous) and "ac1BC" (the other two, not separable). Mapping responses recorded from glomeruli to the corresponding IR is also not always straight forward due to complex innervation patterns. For example, OSNs that express Ir64a project from chamber III in the sacculus to the two glomeruli DC4 and DP1m 30 . As a mapping to the IR name would be ambiguous and the OSN names for ac sensilla are not well defined, we extended our nomenclature and introduced concatenated names of the receptor & glomerulus (Ir64a.DC4 and Ir64a.DP1m). Ir75d is expressed in three different OSNs housed in the three sensilla ac1, ac2 and ac4, they all target the VL1 glomerulus. Assuming that the IR is the main determinant of the OSN response, we mapped recordings from the VL1 glomerulus to Ir75d. We also used the published IR response profiles to estimate putative sensillum/receptor/glomerulus relationships (see below).
We updated existing names in two instances. We renamed Gr21a to Gr21a.Gr63a as neither receptor alone is functional and none is known to be a co-receptor 31 . We renamed ab3B to Or85b as the co-expression of Or98b is not clear and no response profile for the latter is existing 7 . Or23a and Or83c as well as Or2a, Or19a and Or43a were initially described as being expressed in the trichoid sensilla at2 and at3 7 . Ronderos et al. 32 describe Or23a and Or83c OSNs as being housed in an intermediate sensillum and thus rename at2 to ai2. Dweck et al. 33 renamed at2 and at3 to ai1 and ai2 respectively. Since these two proposed new nomenclatures are conflicting we decided to reduce confusion and to keep the old at1-at4 nomenclature.
Merging algorithms. We rewrote and optimized large parts of the DoOR code, mainly to increase computational speed. The logic of the core algorithms for merging several datasets into a single consensus response profile remained unchanged. Pairwise merging was based on the assumption that the same monotonic relationship between odorant responses for a given responding unit applies for all data sets: A better ligand A should always elicit a stronger response as compared to a weaker ligand B, regardless of the recording technique used. Pelz et al. 2006 34 have shown that this assumption is valid for calcium responses and extracellular recorded action potentials in Drosophila OSNs. The merging procedure consisted of the following steps: datasets were rescaled to the range [0, 1] and merged pairwise by calculating the best fitting function on odorants recorded in both studies. Odorants measured only in one of the studies were subsequently projected onto this function. The sequence of merging was determined by iteratively finding the pairs that produced the best fit 4 . The "best fit" was quantified as the fit yielding the lowest "mean orthogonal distance" (MD, see Methods). Where feasible (  ≡ , , n 10 3 628 800 datasets permutations) we computed all possible merging sequences and selected the one with the lowest MD to all original datasets. We were able to test all possible permutations for all datasets except Or22a.

InChIKeys as new default odorant identifiers.
With this version of DoOR we switched from CAS numbers to InChIs (International Chemical Identifier) and InChIKeys respectively as main chemical identifiers used in DoOR 35 . InChIs are unique chemical identifiers derived from the chemical structure of a compound. InChIs are free to use and the algorithm for generating them is freely available under an open source license (http:// www.inchi-trust.org/downloads/). Another advantage is that InChIs are human readable and thus their correct use can be verified. As compared to InChIs, CAS numbers are ambiguous: they are assigned to substances rather than compounds. This can result in several CAS numbers for the same chemical. Isopentyl acetate for example, an odorant with a banana like smell for humans, was tested by many studies included in DoOR. PubChem lists 152 synonyms, including "isopentyl acetate", "isoamyl acetate" and its IUPAC name "3-Methylbutyl acetate". Among the synonyms are also two different CAS numbers, 123-92-2 as well as 29732-50-1 which both map correctly to isopentyl acetate but might have created two separate entries in DoOR. Conversely, the InChI algorithm always produces the standard InChI InChI= 1S/C7H14O2/c1-6(2)4-5-9-7(3)8/h6H,4-5H2,1-3H3 and the corresponding InChIKey MLFHJEHSLIIPHL-UHFFFAOYSA-N. As InChIs can be quite long, within the DoOR algorithms we use InChIKeys for all computations. InChIKeys are the 27 character long hashed version of each InChI. While only InChIKeys are used for our DoOR algorithms, we included additional information as a service to the users: we included the name, CID (PubChem Compound Identification) and CAS (Chemical Abstracts Services) identifiers and also added SMILES (simplified molecular-input line-entry system), another structure based chemical identifier.
Redundancy in nomenclature can create duplicates. In fact, even in published sets, we found several cases where the same odorant appeared multiple times in a single dataset, sometimes with different chemical names. Possible explanations could be that the different instances of a chemical were provided from different suppliers. In these cases we merged the entries by calculating the mean responses. Whenever we performed such a merge, we noted that in the dataset.info data frame. New tools. With DoOR 2.0 we provide several new tools. These include seven new functions for data visualization, e.g. a function for mapping odorant responses on the AL model of Grabe et al. 17 (dplot_ALmap(); Fig. 1b), a function for generating tuning curves (dplot_tuningCurve(); Figs 4 and 3) and several functions to visualize response profiles or to compare responses across responding units. We added a tool for sensillum identification based on physiological measurements (e.g. single sensillum recordings; identifySensillum()) and a tool to find odorants that sparsely activate a given responding unit (privateOdorant()). Additionally we provide several helper functions, for example to translate chemical identifiers (transID()). A complete list of new functions is available in the detailed documentation that is provided as R-vignettes with the DoOR.functions package and available on the DoOR web page.
Contribute to DoOR. The source code of the two DoOR packages (DoOR.data and DoOR.functions) is now available via GitHub (https://github.com/Dahaniel/DoOR.data & https://github.com/Dahaniel/DoOR.functions). This allows to download pre-release versions of DoOR. Everybody (the community) can now contribute feature requests, bug reports and improved code. Package releases will be available via Zenodo (zenodo.org) with individual DOIs assigned, thus all DoOR.data and DoOR.functions versions will be citable. The most recent DoOR version will also be made available via the CRAN R-package repository for easy installation from within R.
We encourage all users to get access to the full DoOR.data and DoOR.functions, because they offer several important features, among which direct computer-readable access to all used datasets, routines for back-calculation of consensus responses onto particular datasets, the possibility to add, include or test own datasets and many ways to easily visualize data via the DoOR plotting functions. However, we have seen that many users value DoOR for its ease to get immediate responses to quick questions, such as "what is the best ligand for receptor X", "which are the receptors responding to odorant Y", or "which glomerulus is innervated by OrZ". For all of these uses, and graphical displays, we implemented a web interface as a service to the community at http:// neuro.uni.kn/DoOR. The interface was improved with respect to DoOR 1.0, adding sortable tables and changes in the graphical display of the antennal lobe (now based on Grabe et al. 17 ).
Broadly and narrowly tuned responding units. How large the set of odors is that a given responding unit is sensitive to can be quantified as its sparseness. Several sparseness measures exist and two commonly used ones are lifetime sparseness 36 (LTS) and lifetime kurtosis 37 (LTK; Equation 1). We chose LTK as a sparseness measure because in contrast to LTS it allows for negative values (inhibitory responses), which are frequent in Drosophila OSNs. We implemented both statistics in the sparse() function in DoOR.functions. We computed LTK across all DoOR responding units that contained at least 50 odorant responses. We arbitrarily defined the threshold of 50 responses to exclude bad LTK estimates calculated on response profiles where only few odorants were measured. We found responding units to be widely distributed across the LTK scale, with many being broadly tuned (low or negative kurtosis; Fig. 3a,c) and less that responded only to a few specific ligands (high kurtosis; Fig. 3a,b). We found the highest LTK values (most narrowly tuned receptors) for Or82a (LTK = 63.88, n = 180, narrowly tuned to geranyl acetate), ac2A (LTK = 39.12, n = 84, narrowly tuned to putrescine), Or49b (LTK = 35.87, n = 164, narrowly tuned to 2-, 3-, and 4-methylphenol), Gr21a.Gr63a (LTK = 23.57, n = 52, specifically activated by CO 2 ) and ab2B (LTK = 20.9, n = 101, tuned to ethyl 3-hydroxybutyrate and cyclohexanol; Fig. 3a,b). At the lower end of the scale we found ac3B (LTK = − 0.92, n = 98), Or35a (LTK = − 0.44, n = 123; expressed in ac3B OSNs and also measured individually in the empty neuron system), the newly deorphanized Or69a (LTK = − 0.26, n = 107) and Or85f (LTK = 0.17, n = 114) (Fig. 3a,c). The lowest LTK value resulted for ab4B (LTK = − 1.53, n = 182), but see below.
A low LTK value can also indicate that the best ligands for this responding unit is still unknown. For example, Or47b had a low lifetime kurtosis of 0.35 in DoOR 1.0 (where it seemed to be broadly tuned) because at that time only weak responses were known. Dweck et al. 27 discovered Or47b to be narrowly tuned to the single compound methyl laurate, for their dataset we calculated the high LTK value of 33.12 (i.e. narrow tuning; Fig. S1a). When adding datasets with newly discovered single ligands of narrowly tuned OSNs to DoOR, these responses get systematically underestimated. The reason is in the mathematical model used: merging functions are calculated on the overlapping range of two datasets. If one of the two datasets contains an extremely good ligand, and the other does not, that ligand is, from a statistical point of view, and outlier, and cannot be considered for the merging function. We add these outliers using a linear function with slope 1, added to the fitting function outside of the overlap region. For good ligands, this function creates a systematic underestimation in the consensus set. The situation becomes unfortunately bad when the overlap between two studies (i.e. the odors in common) is low. The Or47b dataset from Dweck et al. 27 , for example, was excluded from the default merge because it overlapped with all other datasets only by three odorants (the minimum criterion in DoOR is five), and thus the resulting consensus spectrum did not contain the best ligand methyl laurate. Consequently, LTK was low (LTK: 0.25, Fig. S1a). When including the Dweck et al. 27 dataset by manually adjusting the minimum criterion to three overlapping odors, the resulting LTK value increased to 3.4. This was still lower than the LTK value of 33.12 calculated for the original Dweck 27 dataset (Fig. S1b), due to the necessity of mapping to a function with slope 1. We observed a similar effect for ab4B (Or56a): the LTK value of − 1.53 increased to 89.35 when we merged only the studies that included its best ligand geosmin ( Fig. 3c and S1).
We did not apply any manual selection of source datasets for the pre-computed consensus matrices included in DoOR.data, thus Or47b and ab4B (Or56a) have a low LTK in these matrices. As published datasets will appear, and will be included into DoOR, the consensus spectrum will better reflect the new ligands. We have added a  comment to this respect on the homepage. However, we encourage the users to install the DoOR packages and to adjust the merging parameters to their individual needs.
Odorants activated responding units with differing specificity. We calculated the kurtosis from the perspective of the odorant (population kurtosis, PK). This yields a quantification of how specific or unspecific a given odorant activated the population of responding units (Fig. 4). Considering only odorants that were tested with at least half of the responding units present in DoOR (39) we found odorants to be continuously distributed along the PK scale, with some odorants activating small subsets of responding units and many eliciting broad ensemble responses (Fig. 4a). We found the following odorants on the upper end of the PK scale: CO 2 activated Gr21a.Gr63a, geranyl acetate activated Or82a, water activated Ir64a.DC4, (1R)-(− )-fenchon activated Or85e and methyl salicylate activated Or10a the strongest (Fig. 4a,b): these are candidates for a "labeled line" coding logic. The broadest ensemble responses were elicited by 2-heptanone, hexanol, isopentyl acetate, Z3-hexenol and 4-methylphenol (Fig. 4a,c).
Mapping IRs to their corresponding OSNs. It is difficult to map IR responses to OSNs, because the two available data sources are difficult to disentangle: on one hand single sensillum recordings with spikes that are difficult to sort (with exceptions, in some studies the largest spike amplitude could be separated), on the other hand single IR calcium imaging data, and the difficulty that individual IRs are expressed in several OSN types. See Table 1 for responding units and their relationship to IR/OSN/sensillum/glomerulus. By correlating response profiles from SSR recordings with response profiles from IR calcium data (using the mapReceptor() function from DoOR), we have created hypotheses about their respective mappings.
We considered correlations that were significant (p < 0.05) and relevant (correlation coefficient above 0.75). With these criteria we found that glomerulus VC5 (Ir41a) responses correlated with high significance to the DoOR responding units ac2 (summed OSN data; r = 0.8, p-value = 2.3 * 10 −6 , n = 24) and ac2A (r = 0.76, p-value = 4 * 10 −3 , n = 12). For DP1l the situation is more complex: Two classes of OSNs express Ir75a. Neurons from ac2 sensilla that target glomerulus DP1l express only Ir75a. Neurons from ac3 sensilla that target glomerulus DL2 additionally express Ir75b and c 12 . In our analysis data originating from DP1l correlated with high significance to the ac3A responding unit in DoOR (r = 0.82, p-value = 7.2 * 10 −7 , n = 24), indicating that ac3A neurons expresses Ir75a/b/c and that Ir75a accounts for large parts of the response profile. This supports the notion from Yao et al. 38 that the B neuron expresses Or35a. Together the situation appears to be the following: ac2A neurons express Ir75a and target the DL2 glomerulus; ac3A neurons express Ir75a/b/c and target the DL2 glomerulus; ac3B neurons express Or35a (and Ir76b) and target the VC3 glomerulus (Table 1).
While these correlations match published IR-sensillum expression patterns 12 , we note that Ir64a.DC4 correlated with high significance to ac3A (r = 0.81, p-value = 2 * 10 −6 , n = 24) which would contradict that Ir64a is expressed in sacculus neurons 16,30 : more experimental data will be needed here.

Discussion
Animals can code for millions of odors with a limited number of olfactory receptors. Even though estimating the exact capacity of olfactory systems is a matter of fierce debate [39][40][41] , it is clear that the combinatorial nature of olfaction lies at the basis of this astounding capacity: it is the pattern of activity across receptor neurons that gives the brain the necessary information about the chemicals in its environment. In a combinatorial world (receptor ON or OFF) the theoretical capacity of n receptor types would be 2 n , with 50 receptors in Drosophila that would be 2 50 , corresponding to approx. 10 15 . Since olfactory coding is not binary (i.e. receptors can have all intermediate and also negative activity values), the number might even be higher. On the other hand, the capacity might be smaller, since the code is redundant, and has also to accommodate temporal complexity, concentrations, and mixture analysis, making a prediction of the exact number difficult. Nevertheless, it is apparent that understanding the olfactory code is only possible if the response to a substance is known for all sensory neurons. Given the large number of receptors, and the large number of possible stimuli, this is a daunting task, and no task that a single research group, not even a consortium, could accomplish. Therefore, we have created a technology that allows to merge data from different groups, recorded with different techniques, into a consensus database. The first version has been used by the community for five years now, with great success. We present the second version, with more data (including previously unpublished data) and better tools, in this paper.
Every database is a service to the community that needs long-term care and participation. In order to keep its value we need to continuously update and improve data and code and for this rely on the support from the community. For DoOR 2.0 we received considerable help from many colleagues who supplied us with the raw data of their publications, and even with unpublished datasets. Some datasets however could not be obtained (lost data, crashed disks, refusals to reply to emails). DoOR version 2.0 has many improvements over DoOR 1.0. DoOR is now hosted on GitHub so the most recent code and data are always accessible. At the same time releases with individual DOIs will be available via Zenodo and CRAN. GitHub also eases bidirectional communication, users can send feature requests, report bugs and hints to missing data via the GitHub issue tracker system. InChIKeys are the new unique identifiers used in DoOR, and we added several new functions, e.g. for calculating kurtosis (sparse()) or identifying a recorded OSN based on its odorant specificity (identifySensillum()). Our goal is to move DoOR increasingly into a common tool used and shaped by the community, rather than just provided by us.
This new version contains additional data for new odorants and additional receptors. DoOR now includes 11 new studies contributing 15 new datasets. 467 new odorants were added, 13 glomeruli and five additional response profiles were deorphanized. In total 2894 responses of new odorant-responding unit combinations were added.
Scientific RepoRts | 6:21841 | DOI: 10.1038/srep21841 Where are the remaining major lacks in the dataset now? From a neuro-circuitry point of view, the most important lack is in glomerulus VA7m, which has not yet been linked to a receptor. Also, the area innervated by IR receptors in the antennal lobe and their mapping to sensilla and response profiles remains understudied. More data in the next few years will elucidate the missing information. From an olfactory point of view, many odorant responses are still missing (see gray area in Fig. 1a). But the number of potentially interesting chemical compounds that may have an odor is virtually infinite, and therefore just adding new compounds will have only limited effect on our understanding of the system. Thus, the major question for the future years will be with respect to those receptors which do not yet have very strong best responses (e.g. Or2a, Or23a and ab10B; see Table 1 for a full list). It is conceivable that we are missing the best ligands for these receptors, and that finding a stronger ligand will help us understand the importance of that responding unit within the olfactory code, and for the animal's ecological niche. These studies will also help us in defining how odors are encoded in the first place, i.e. how the brain extracts information from the activity patterns across receptors. When does the animal interpret activity in a receptor with low kurtosis (e.g. geosmin in ab4B) as that substance being present, when does it interpret activity in the same receptor as a result of another odorant with weaker affinity? The answer most likely is found in the ensemble response across receptors, and can only be found if we know that representation. The value of DoOR lies in the overview of the Drosophila olfactome that helps to understand the nature of combinatorial coding. It enables analyses as calculating the kurtosis of ensemble responses elicited by individual odorants or mapping unknown response profiles. It is a resource for modeling, for selecting experimental parameters and can be used for extrapolating new response patterns.
Nevertheless DoOR is -and always will be -incomplete, not only in the sense that some odorants will always be missing. Some aspects of olfactory coding are not yet covered, others are impossible to cover in such a consensus approach. Aspects that might be covered in future versions include odorant concentrations. Merging responses of different odorant concentrations across labs is difficult because it is difficult if not impossible to measure absolute concentration in a controlled way. For one, the absolute odorant concentrations reaching the animal depends on the vapor pressure of a compound. Concentrations are also influenced by how a stimulus is presented. Within studies extremely potent substances are often used in higher dilutions, so that we had to exclude these in some of the DoOR datasets -an unfortunate aspect, because these are, after all, the best ligands. While it is impossible to correctly integrate absolute concentrations of stimuli across studies, it is mathematically possible to integrate concentration-response curves, should they be published more often. In this case, we would add separate datasets per concentration/dilution into DoOR. Similarly saturating and adapted responses created by strong ligands are problematic as they lead to flattening of the odorant response patterns and create distortions in the mapping function. As an example: butyl acetate and ethyl hexanoate elicit equally strong responses from Or22a expressing OSNs when tested at a 1:100 dilution 3 but the OSNs are sensitive to an almost three log steps lower concentration of ethyl hexanoate as compared to butyl acetate (quantified as the dose eliciting the half maximal response in Pelz et al. 34 ).
Other features that cannot be consensualized in a database include temporal dynamics of odorant responses. However, such information could be implemented in future DoOR releases by enabling to store full time traces as meta links to the individual response points in the matrix, allowing the user to have access to all response time-courses. Similarly, mixture responses or responses to complex odor stimuli are unlikely to enter DoOR due to the many parameters they depend on, but a future version of DoOR might still act as a repository for such information.
There are many other online databases that collect information across studies to provide important tools for neuroscience research. To name a few, FlyBase (http://flybase.org/), and the Virtual Fly Brain (http://www. virtualflybrain.org/) focus on genetic or morphological aspects of Drosophila; Sense Lab (https://senselab.med. yale.edu/) offers four different olfactory databases offering e.g. odorant activity maps of mouse olfactory bulbs and genetic codes of olfactory receptors of many species; PubChem (https://pubchem.ncbi.nlm.nih.gov/) and ChemSpider (http://www.chemspider.com/) provide detailed information on millions of chemical compounds. All these databases complement each other and the value of each individual database increases with mutual links so that additional information on odorants, genes or morphological structures is only a click away. Currently we link from the individual records of our DoOR web page to FlyBase, the Virtual Fly Brain and PubChem. We plan to increase the linked databases in the future.
DoOR facilitates a variety of analyses on the Drosophila olfactome, allowing to ask important questions. As an example, we searched for odorants that elicit narrow activation patterns across responding units (Fig. 4b). These odorants are likely of special importance for Drosophila, since narrow response patterns are computationally easy to distinguish from patterns elicited by other odorants. One example of such an important odorant for Drosophila is CO 2 , which activates a single type of OSN and innately mediates aversion 42 . Water had the second highest kurtosis value in our analysis. Water is a very important stimulus for every animal. However the activated Ir64a.DC4 is likely an acid sensor, and whether the water response might be resulting from the pH < 7 of the distilled water remains to be investigated (Ai et al. 54 ; Ana Silbering, personal communication). We also provide a sensillum identification tool, identifySensillum(): by inserting odor-response values of a recorded unit, the tool provides plausible responding unit and sensillum candidates. We hope this tool will be helpful for electrophysiologists or other experimenters as a tool for reliably identifying recorded units in physiological experiments.
The increasing number of narrowly tuned OSNs being described in Drosophila in the recent years 27,28,42 has sparked a debate about whether effective coding of odorants works via a set of narrowly tuned labeled line OSNs, or whether the predominant nature of olfactory coding is combinatorial 27,28,[42][43][44] . Drosophila also has many OSNs that are broadly tuned to many different odorants, such as Or35a (in ac3B) or Or69a (in ab9) (Fig. 3a,b). Both strategies have advantages. On the one hand being extremely sensitive to ecologically relevant substances is a prerequisite for finding proper food, mating partners or egg-laying sites. On the other hand, broadly tuned OSNs that sample all across the chemical space ensure that animals are not anosmic to new substances and can adapt Scientific RepoRts | 6:21841 | DOI: 10.1038/srep21841 to new or changing environments. Indeed, the Drosophila olfactory system is able to detect and distinguish substances that do not play a role in a fly's daily life, like explosives, drugs and breast cancer metabolites 29,45 . It may well be that the same glomerulus that is highly specialized for a particular odorant in a low concentration/high sensitivity mode, participates in an across-glomeruli combinatorial representation in a high concentration/low sensitivity situation 46 .
We put this service into the hands of our colleagues and hope to provide a useful tool for the exploration of the olfactory code and for designing future experiments. We strongly rely on your support and feedback and are happy to include new data and process feature requests and bug reports. We also note that the DoOR framework is in no way restricted to Drosophila but can be used for different species right away if sufficient odor-response data is available. With the advances in in-vitro screenings of human olfactory receptors 47,48 , a human DoOR might soon be possible.

Material and Methods
Animals. For  Flies were kept at 25 °C in a 12/12 light/dark cycle at 60-70% RH. Animals were reared on standard medium (100 mL contain: 2.2 g yeast, 11.8 g of sugar beet syrup, 0.9 g of agar, 5.5 g of cornmeal, 1 g of coarse cornmeal and 0.5 mL of propionic acid).

Odorant preparation.
Odorants were purchased from Sigma-Aldrich in the highest purity available. Pure substances were covered with Argon to avoid oxidation. All odorants were applied at 10 −2 diluted in 5 mL mineral oil (Sigma-Aldrich, Steinheim, Germany). Odorants were prepared in 20 mL head space vials covered with pure nitrogen to avoid oxidation (Sauerstoffwerk Friedrichshafen GmbH, Friedrichshafen, Germany) and immediately sealed with a Teflon septum (Axel Semrau, Germany). A list of all odorants and the measured responses is given in Table S5.
Calcium imaging. Calcium imaging was performed on two setups which consisted of a fluorescence microscope (BX50WI or BX51WI, Olympus, Tokyo, Japan) equipped with a 50× air lens without cover slip correction (Olympus LM Plan FI 50× /0.5). Images were recorded with a CCD camera (SensiCam, PCO, Kelheim, Germany) with 8 × 8 pixel on-chip binning, which resulted in 80 × 60 pixel sized images. We recorded each stimulus for 20 s at a rate of 4 Hz using TILLvisION (TILL Photonics, Gräfelfing, Germany). A monochromator (Polychrome II or Polychrome V, TILL Photonics, Gräfelfing, Germany) produced excitation light of 470 nm wavelength which was directed onto the antenna via a 500 nm low-pass filter and a 495 nm dichroic mirror, emission light was filtered through a 505 nm high-pass emission filter.

Stimulus application.
A computer-controlled gas chromatography auto sampler (PAL, CTC Switzerland) was modified and used for automatic odorant application. A head space of 2 mL was injected in two 1 mL portions at time points 6 s and 9 s with an injection speed of 1 mL s −1 into a continuous flow (60 mL min −1 ) of purified air. The stimulus was directed at the antenna of the animal via a Teflon tube (inner diameter 2 mm, length 39.5 cm, with the exit positioned ~2 mm from the antenna). Stimuli arrived at the antenna with 750 ms delay due to delays in the autosampler and the flow. Therefore, stimulus onset was determined as 6.75 s and 9.75 s.
Four to eight odorants were presented in a row (one block, ISI > 2 min) interspaced by solvent control, room air control and an receptor specific reference odorant. The reference odorants were Data analysis: calcium imaging. We analyzed calcium imaging data using custom written routines in IDL (ITT VIS, USA) and R 51 .
Recorded movies were manually corrected for lateral movement artifacts. Then, an area of interest was defined for the parts of the antenna that showed fluorescence increase upon stimulation. Time traces were averaged across this area. We included all measurements into the analysis as long as animals showed stable responses to the reference odorant.
Relative percentage fluorescence change was calculated as ∆ / = (( − )/ ) × F F F F F 100 i 0 0 with F i being the fluorescence at frame i and F 0 being the mean fluorescence of 5 s before stimulus onset.
To correct for the photo-bleaching of the dye, we fitted an exponential decay function of the form A * exp −x/B + C to each response trace using the nls() function in R. Because some odorant responses would not reach baseline within measurement time, the decay rate parameter B was estimated from the median mineral oil control trace within each animal. We omitted 750 ms at the beginning of the time-trace and 11 s after stimulus presentation. The pre-stimulus part of the recording was weighted 100 fold 52 .


extfieldsofstudy: Biology Medicine

text:
Yuanansuchus maopingchangensis sp. nov., the second capitosauroid temnospondyl from the Middle Triassic Badong Formation of Yuanan, Hubei, China

A second species of Yuanansuchus, Y. maopingchangensis, is erected for new temnospondyl material from the Maopingchang site, Yuanan County, Hubei Province, China. These specimens are from the same horizon that produced Y. laticeps, the type species. Y. maopingchangensis shares the following features with Y. laticeps: postorbital portion of skull occupied more than 1/3 of skull length; tabular horn directed laterally; lateral line sulci continuous, well impressed; supraorbital sensory canal enters lacrimal; preorbital projection of jugal less than half length of snout; and vomerine plate short. However, Y. maopingchangensis differs from Y. laticeps in having an elongated skull, frontal extending posteriorly to the level of the posterior orbital margin, closed otic fenestra, cultriform process of parasphenoid extending to the level of the anterior margin of the interpterygoid vacuities, and absence of fodina vomeralis. Analysis of a new capitosaur phylogenetic data matrix, containing 56 characters and 29 species, confirms that the two species from Yuanan are sister taxa and that Capitosauroidea can be divided into two major clades: one including Parotosuchus, Eryosuchus, Calmasuchus and Cherninia, and another including Mastodonsaurus, Yuanansuchus, Stanocephalosaurus, Procyclotosaurus, Paracyclotosaurus, Antarctosuchus, Eocyclotosaurus, Quasicyclotosaurus, Tatrasuchus and Cyclotosaurus.

INTRODUCTION
Temnospondyls are the largest and most speciose group of amphibians. Within this group, the stereospondyl clade Capitosauria produced some of the largest species and dominated many Mesozoic aquatic ecosystems (e.g., Schoch, 2008). Capitosaur research has a long history, and more than 120 nominal species have been erected within this group (Damiani, 2001;Fortuny, Galobart & De Santisteban, 2011;Liu & Wang, 2005;Sidor, Steyer & Hammer, 2014;Sulej & Niedźwiedzki, 2013). The taxonomy of Capitosauria was poorly understood for a long time, but recent systematic revisions of the group by Schoch & Milner (2000) and Damiani (2001) have led to a consensus regarding the taxonomic status of most previously named genera and species. Meanwhile, large-scale cladistic analyses of Capitosauria have been undertaken by Schoch (2000) and Damiani (2001). To resolve the position of Yuanansuchus laticeps, Liu & Wang (2005) added Y. laticeps and Quasicyclotosaurus campi to the matrix of Damiani (2001). In later works, researchers modified the character list and data matrix of Damiani (2001) for their own studies (Fortuny, Galobart & De Santisteban, 2011;Schoch, 2008;Sidor, Steyer & Hammer, 2014), and they produced the phylogenetic hypotheses that have varied widely in such particulars as the position of Edingerella, the proximity of Eryosuchus to Mastodonsaurus, and the content of the subgroup Capitosauroidea (sensu Schoch, 2013) ( Fig. 8 of Schoch (2008) vs. Fig. 7 of Fortuny, Galobart & De Santisteban (2011)).
Capitosaurs are widely distributed on the land masses derived from the Pangean supercontinent, and are known from many well-preserved specimens (e.g., Damiani, 2001;Schoch & Milner, 2000). However, this group is poorly represented in east Asia (Ingavat & Janvier, 1981;Li, Wu & Zhang, 2008), a region from which only two highly informative capitosaur specimens have been described: the posterior part of a skull almost identical to the corresponding cranial region of Cyclotosaurus posthumus was reported from Thailand (Ingavat & Janvier, 1981), and the nearly complete skull of a taxonomically novel capitosaur was reported from the middle Triassic Badong Formation of China (Liu & Wang, 2005). The notably broad Chinese skull was collected in Maopingchang Township, Yuanan County, Hubei Province in 2001, and was named Yuanansuchus laticeps (Liu & Wang, 2005). In 2011, a mandibular fragment was reported from the Lower Triassic of Japan, implying that the dispersal of stereospondyls into China occurred before the latest Early Triassic (Nakajima & Schoch, 2011).
In 2011, my team made a second trip to the Maopingchang locality and collected a considerable number of fossils. In this paper, several new temnospondyl specimens are described and assigned to a new species, Yuanansuchus maopingchangensis. Y. maopingchangensis represents the second temnospondyl species from Maopingchang. To evaluate the phylogenetic relationships of these Chinese capitosauroids, a new character list is established based on a revision of previously established characters, and new matrices including both Y. laticeps and Y. maopingchangensis are constructed.

GEOLOGICAL SETTING
Outcrops of the Triassic Badong Formation are widely distributed in Hubei, Chongqing, and Hunan. The Badong Formation conformably overlies Lower Triassic shallow marine carbonates of the Jialinjiang Formation, and in most places underlies an Upper Triassic terrestrial coal series. The complete Badong Formation is generally divided into five members (numbered I to V), but whole five members only present in a few areas such as Badong, Sangzhi. In Yuanan, only memebers I to IV are present as showing by the geological map and lithological column (Fig. 1). Vertebrate fossils have been collected from the purplish-red calcareous siltstone and mudstone of Member II in both Hunan and Hubei (Liu & Wang, 2005;Zhang, 1975). In Yuanan, the bonebed lies in the middle portion of the Member II (Fig. 1). Member I contains a rich bivalve fauna, which is generally regarded as Anisian in age. Member III yields the bivalves Plagiostoma striatum and Placunopsis plana, the ammonoid Progonoceratites, and the conodont Neospathodus kockeli, and is accepted as Anisian in age although Zhang, Chen & Palmer (2003) argued that it could instead be Ladinian. Member II has also been suggested to be Anisian in age, based on its fossil flora (Meng, Zhang & Xu, 1995), so the fossil tetrapods from Member II are accepted here as Anisian, possible late Anisian, as also suggested by magnetostratigraphic work (Huang & Opdyke, 2000). Member II has been interpreted as comprising tidal flat sediments (Meng, Zhang & Xu, 1995), but the sediments at the Maopingchang locality are more likely to represent a floodplain facies. No tidal-related structures were observed in the field.
The specimens described in this paper were excavated from a hill 300 m away from the type locality of Yuanansuchus laticeps (Fig. 2). The new locality is approximately the same stratigraphic level as that of Y. laticeps on the section. This fossiliferous layer is less than 10 m in thickness. Among the bones collected at this new spot, four skulls, one left lower jaw, three interclavicles, and nine clavicles are of temnospondyl origin. They represent at least six individuals, based on the number of clavicles. In addition to the temnospondyl fossils from the type locality of Y. maopingchangensis, vertebrae with high neural spines were also discovered and almost certainly belong to an archosaur similar to Lotosaurus from Sangzhi County in Hunan (Zhang, 1975). Nomenclatural acts-The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. A ZooBank LSID (Life Science Identifier) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub: D68F2D09-0AC0-4AE1-ACE1-EFD0C5680916. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS. TEMNOSPONDYLI Zittel, 1887-1890STEREOSPONDYLI Zittel, 1887-1890CAPITOSAURIA Yates & Warren, 2000, sensu Schoch, 2008CAPITOSAUROIDEA Säve-Söderbergh, 1935sensu Schoch, 2013 Yuanansuchus Liu & Wang, 2005 Revised diagnosis (modified from Liu & Wang, 2005)-Postorbital portion of skull occupied more than 1/3 of skull length; tabular horn directed laterally; lateral line sulci continuous, well impressed; supraorbital sensory canal enters lacrimal; preorbital projection of jugal shorter than half length of snout; vomerine plate short; interclavicle wider than long; ventral part of blade of clavicle with convex medial margin.

Description
The following description, except where specified otherwise, is based on the holotype; the other two known skulls are used as a supplementary source of information.
The holotype is a slightly distorted skull missing the posterolateral corner of the right cheek (Fig. 3). As preserved, the skull has an anteroposterior midline length of 275 mm and a width of 215 mm, but the complete skull was probably about 23 cm wide. This skull is slightly longer than IVPP V 13463, the holotype of Yuanansuchus laticeps, but is also much narrower. The posterior part of the skull was exposed and damaged by weathering prior to excavation of the rest of the specimen, so that the occipital and otic regions are poorly preserved. The skull roof is generally well preserved, but white plaster has been inserted between the skull roof and palate to reinforce the skull. None of the marginal or palatal teeth are preserved.
In dorsal and ventral views, the outline of the skull is parabolic and elongate, with straight lateral margins. The outline is very similar to that of the skull of Quasicyclotosaurus campi (Schoch, 2000). The preorbital region is low in profile and relatively broad. The snout is long and slender compared with that of Y. laticeps. The two sides of the skull are  asymmetrical. This anomaly can only partly be explained by distortion, and it seems likely that the preserved shape is similar to the original one. The midline suture between the frontals lies considerably further to the left than that between the nasals, so that the left nasal has a substantial contact with the right frontal. In most capitosaurs, the midline is nearly straight. Other differences in suture pattern exist between the left and right sides of the skull; for example, the right frontal is distinctly longer than the left one.
The dermal sculpturing is well preserved on the skull roof ( Fig. 3) and largely consists of a pattern of deep pits, which grade into alternating ridges and grooves near the lateral margins of the skull and the nasal surface. The lateral line sensory sulci are continuous and obvious on the skull roof. The infraorbital sulcus (IS) has a Z-shaped lacrimal flexure, extends backwards along the lateral skull margin on the maxilla, and seems continuous with the jugal sulcus (JS) to the squamosal. The supraorbital sulcus (SS) is better-defined than the IS, runs across the prefrontal and the lacrimal, nearly contacts the IS, and extends onto the nasal. The SS seems to not meet its counterpart across the midline within the premaxilla. Posteriorly, the SS extends across the frontal and postfrontal, and intersects with the temporal sulcus (TS) on the postorbital. The TS extends downwards on the jugal to meet the IS, and extends posteriorly onto the supratemporal.

Skull roof
A small interpremaxillary foramen lies on the midline, close to the anterior border of the premaxillae. The nares are elongated and large (the left measuring 314 mm in length, and the right 275 mm), and are situated near the anterior margin of the skull. The external naris is bordered anteriorly and laterally by the premaxilla, dorsally by the nasal, and posteriorly by the maxilla. The posterolaterally directed suture between the premaxilla and the nasal touches the medial margin of the external naris, and the right suture is displaced anteriorly relative to the left one. In the dorsal view, the maxilla is restricted to the skull margin and does not extend posteriorly to the anterior margin of the orbit. No septomaxilla is evident. The nasal contacts the maxilla posterior to the external naris. The lacrimal contacts neither the orbit nor the naris.
The two large orbits are both ovoid, but differ in size, the right orbit being longer (43 mm in length) and more anteriorly positioned than the left (38 mm in length). Their sizes are slightly larger than those of Y. laticeps, which are 35 mm in length. However, both orbits are situated relatively far anteriorly, lying close to the mid-length of the skull roof. In this characteristic Y. maopingchangensis resembles Y. laticeps, Mastodosaurus giganteus and some juvenile capitisauroids (such as Watsonisuchus aliciae) (Liu & Wang, 2005;Schoch, 1999;Warren & Hutchinson, 1988). The orbits are almost dorsally directed and are raised above the rest of the skull roof, indicating an aquatic or semiaquatic lifestyle in which the animal habitually positioned itself near the water surface. The highest point on the skull roof is located on the postfrontal, just posterior to the orbit. The prefrontal is relatively long (measuring about one quarter of the midline length of the skull), tapers to an anterior point, and is elevated just anterior to the orbit. The frontal participates in the orbital margin, as is the case in many other capitosaurs (Damiani, 2001;Schoch, 2008) but not in Y. laticeps (Liu & Wang, 2005). The frontal is subequal in length to the nasal, although the left frontal is shorter than the right. However, in contrast to the condition in all other capitosaurs, the frontal extends posteriorly beyond the posterior margin of the orbit (Schoch, 2008;Schoch & Milner, 2000). The postfrontal is a nearly quadrangular in shape, and forms the posteromedial margin of the orbit. The postorbital is wide, and its lateral margin extends beyond that of the orbit. However, the postorbital does not extend as far anterolaterally as in many other capitosaurs, but narrows anteriorly and makes only a small posterior contribution to the orbital margin. The jugal forms most of the lateral border of the orbit, and has a long anterior process that extends far anteriorly to the orbit and reaches nearly the same level as the tip of the prefrontal. However, the length of the preorbital part of the jugal is nevertheless less than half that of the snout. Although the suture between the jugal and quadratojugal is unclear, the former is definitely one of the longest bone in the skull roof.
The skull table is very long for the anteriorly positioned orbit. The ratio of postorbital portion to the whole skull is 0.38. This ratio is similar to the ratio in Y. laticeps, bigger than in Mastodonsaurids (∼0.3), much bigger than in other capitosaurids (<1/4) (Liu & Wang, 2005;Fortuny, Galobart & De Santisteban, 2011;Schoch & Milner, 2000;Sidor, Steyer & Hammer, 2014). The parietal foramen is round and small, with a diameter of 6.5 mm, and lies on the midline near the anteroposterior midpoint of the parietals. The parietal is short, and is similar in length to the postparietal, the supratemporal, and the tabular. The postparietal is relatively long, its length exceeding its width. The posterior margin of two postparietals is incompletely preserved but looks transversely aligned as in Y. laticeps and Mastodonsaurus giganteus (Liu & Wang, 2005;Schoch, 1999). The tabular is short and wide, and its lateral process is only slightly posteriorly directed. As a result, the posterior margin of the skull roof is nearly straight in dorsal view as in Y. laticeps, rather than distinctly concave as in most capitosaurs (Schoch, 2008). The tabular contacts the squamosal, enclosing an otic fenestra and preventing the supratemporal from entering the dorsal margin of this opening. The otic fenestra is more or less rounded as in Eocyclotosaurus, not ovoid as in Quasicyclotosaurus and Cyclotosaurus; it embays the squamosal more deeply than the tabular as in Quasicyclotosaurus, Eocyclotosaurus, and Cyclotosaurus mordax (Schoch, 2000;Schoch & Milner, 2000). The squamosal is a large bone in the posterolateral part of the skull roof. The posterior margin of the squamosal is poorly preserved, but this bone does extend far enough posteriorly to overhang the occiput. The posterior margin of the postparietal, tabular and squamosal appears thick (∼7 mm) relative to the total height of the occiput (47 mm).

Palate
The palate is moderately vaulted, with the lateral edges slightly below the level of the middle portion. The maxilla bears more than 30 marginal tooth sockets, but no teeth are preserved. The anterior palatal vacuities are paired, oval in outline and completely separated by the premaxilla and the vomer, as in Eocyclotosaurus wellesi, Mastodonsaurus giganteus, and Paracyclotosaurus crookshanki (Damiani, 2001;Schoch, 1999;Schoch, 2000). A small fossa lies on the midline, crossing the suture between the premaxilla and the vomer. This structure may be a fossa subrostralis media, a feature present in Eocyclotosaurus woschmidti (Kamphausen, 1989).
The vomerine plate is longer than wide. The plate is in contact with the premaxilla anteriorly, the maxilla laterally, and the palatine posterolaterally, and it sends out a long posteromedial process that runs lateral to the cultriform process. The vomerine tusk pair is large, and situated at the anterolateral corner of the vomer. A triangular depression lies on the midline, behind the fossa subrostralis media. The transvomerine tooth row is not preserved, but the protruding base shows it is slightly convex posteriorly for the presence of the depression as in Edingerella madagascariensis (Maganuco et al., 2009). However, this depression is not observed in smaller specimen IVPP V 22629. The left side of the plate is poorly preserved, but on the right side a depression is situated posterior to the transvomerine tooth row.
The choana is elliptical in outline, and relatively long, as in most capitosauroids. It is bordered by the maxilla laterally, the vomer medially, and the palatine posteriorly. The interpterygoid vacuities are well-developed, measuring nearly 50% of the midline length of the skull, and are approximately as wide posteriorly as anteriorly. Each vacuity is bordered laterally by the palatine and pterygoid, and medially by the vomer and parasphenoid. The orbits are located at the level of the posterior half of the vacuities, as in Yuanansuchus laticeps. Apart from a small portion of the right orbit, they can be seen through the vacuities in their entirety.
The palatine forms a slender posterior process that meets the ectopterygoid laterally and the pterygoid posteriorly. The palatine tusk pair is not preserved, and even the shapes of their alveoli are unsure. The right ectopterygoid is a slender elongate bone situated medially to the maxilla. Shallow tooth sockets lie on the lateral margin of the bone, medial to the maxillary teeth. However, the part of the palate forming the lateral margin of the left interpterygoid vacuity is so mediolaterally compressed that even the suture between the maxilla and the ectopterygoid is unclear.
Although the middle portion of the vomerine plate is not well-preserved, the length of the midline suture between the two vomers shows that the parasphenoid extends anteriorly to around the level of the anterior margin of the interpterygoid vacuities. The anterior tip of the parasphenoid is far more posteriorly positioned than in most Capitosaurians, including Y. laticeps. The parasphenoid seems smoothly sutured with the vomer, and the fodina vomeralis should be absent here, in contrast to Yuanansuchus laticeps, The cultriform process is a flat bar which narrows laterally in the vicinity of its midpoint, and whose triangular base is smoothly continuous with the vaulted basal plate of the parasphenoid. The width of the basal plate is subequal to its length. The crista muscularis is a transversely oriented ridge located on the posterior rim of the basal plate. The ventrally positioned 'pockets' mentioned by Watson (1962) are indistinct and widely separated. The parasphenoid contacts the pterygoid laterally at an anteroposteriorly long suture, the length of the suture is slightly greater than the width of the corpus of the parasphenoid.
The palatine ramus of the pterygoid tapers anteriorly, and contacts the posterior margin of the palatine along a short suture located roughly at the level of the anterior margin of the orbit. The corpus of the pterygoid curves ventrally towards its lateral side; its lateral margin is granular and forms a roughened area. The quadrate ramus has a ventral oblique surface and an tall dorsal oblique ridge (crista obliqua), and extends posterolaterally to form a sutural contact with the main body of the quadrate. The palatine and quadrate rami of the pterygoid participate in medially delimiting the subtemporal fenestra, which is bordered posteriorly by the quadrate and laterally by the quadratojugal and maxilla. Both quadrates are preserved. This bone is nearly triangular in palatal view, and the medial side which contacts the pterygoid is much wider than the lateral side which contacts the quadratojugal. The ventral condyle for the articular is saddle-like. The quadrate bears a boss (hyoid tubercle) above the medial part of the condyle. The occipital condyles are separated from one another and are formed by the exoccipitals, which are in sutural contact with the parasphenoid anteriorly. The occipital condyles are positioned posteriorly to the level of the quadrate condyles. A long, slender, unidentified piece of bone is preserved on the left ventral margin of the skull. It could be part of a rib. The anterior end of this element covers the posterior portion of the upper alveolar row.
The skull of IVPP V 22629 is nearly complete, with a midline length of 21 cm and a width of approximately 15 cm (Fig. 4). The long axis of the orbit measures 27 mm, and the short axis measures 20 mm. Another skull (IVPP V 22630) is slightly smaller (Fig. 5). Both specimens are similar to the holotype in their general features, but differ from the holotype in that the frontal merely comes close to the orbit rather than entering the orbital margin. The frontal extends only to the level of the posterior margin of the orbits, rather than continuing posteriorly beyond this level as in the holotype. In IVPP V 22629, only one vomerine tusk socket lies anterior to the choana, and there is no socket lying posterior to the choana (Fig. 4B). In IVPP V 22629, the area of the contact between the vomer and the cultriform process is poorly preserved, and the anterior extent of the cultriform process is uncertain.
The occipital region is better preserved in two specimens. The occiput is relatively low because the occipital condyle is nearly situated at the same dorsoventral level as the quadrate condyle, as in Yuanansuchus laticeps (Liu & Wang, 2005). The posttemporal fenestra is triangular. The foramen magnum is wide. The right stapes is preserved in situ in both specimens, and the one in IVPP V 22630 is complete. The stapedial footplate is large, and the stapedial shaft is slab-like as in Tatrasuchus wildi (Schoch, 1997). The foramen for nerves IX-X (the glossopharyngeal and the vagal nerves) is visible on the lateral side of the exoccipital.

Lower jaw
IVPP V 22631 is a robust and long left lower jaw, missing the anterior tip and the postglenoid area (PGA) (Fig. 5). The intact specimen is estimated to have measured 27 cm in length and 65 mm in height. The preserved anterior tip of the ramus (approximately the midway between the mandibular symphysis and the forward edge of the adductor fossa) has a height to width ratio of approximate 1.5. The height to width ratio immediately behind the postglenoid ridge approximately is 1.7. These ratios are similar to those of Stanocephalosaurus birdi (Jupp & Warren, 1986). The lower jaw increases in height posteriorly until the posterior end of the coronoid, then begins to decrease in height on the lateral side of the adductor fossa. As a result, the dorsal margin of the lateral wall of the adductor fossa is strongly convex. The lateral surface of the angular shows well-developed radial ornamentations, which become less evident on the postsplenial. A distinct mandibular sulcus extends posterodorsally across the lateral surface of the angular, beginning at the ventral margin.
The dentary bears more than 20 tooth sockets, which are anteroposteriorly compressed. Only two broken teeth are preserved on the dentary. The middle and posterior coronoids both bear a single row of teeth, but only a few conical teeth are actually preserved. The medial side of the postsplenial bears a small anterior Meckelian foramen, posterior to which is an additional tiny foramen. The ratio of the length of anterior Meckelian foramen to the adductor fossa length is less than 0.1. The posterior Meckelian foramen is bordered by the postsplenial anteriorly and ventrally, the prearticular dorsally, and the angular posteroventrally. It is elongated and much larger than the anterior Meckelian foramen, but still far less than half as long as the adductor fossa. The center of posterior Meckelian foramen lies anterior to the vertical line passing through the anterior-edge of the adductor fossa. The medial wall of the adductor fossa is incomplete, but the original height of medial wall could be deduced from preserved portion. The height of the medial wall should be slightly greater than 2/3 height of the lateral wall at the middle of the adductor fossa length. The prearticular extends anteriorly beyond the anterior margin of the posterior Meckelian foramen, and contacts the postsplenial below the posterior coracoid. The hamate process of the prearticular is not complete, but still rises above the level of the glenoid fossa. Most of the glenoid facet lies above the level of the dorsal surface of dentary.
Although the postglenoid area is nearly completely missing, the following features on this area still can be deduced from the preserved specimen: the prearticular does not extend into the PGA, and the angular does not participate in the PGA. So the PGA should be type I of Jupp & Warren (1986) for this specimen.

Postcranial skeleton
Many isolated bones have been discovered at this locality, but only a limited number have been successfully excavated because they are quite fragile and the matrix is very hard. Among the elements that have been recovered, three interclavicles and nine clavicles have been identified as temnospondyl in origin and tentatively referred to Y. maopingchangensis (see 'Discussion').
The interclavicle is well preserved in IVPP V22632 (Fig. 6). It is rhomboidal in shape, lacking a distinct anterior stylus, and is much wider than long. In contrast, the interclavicle is longer than wide in Mastodonsaurus giganteus (Schoch, 1999). On the dorsal side, this bone bears three converging low ridges (Figs. 6B and 6C). The anterior two ridges lie in the anteroposteriorly middle portion of the bone, and the posterior one is longitudinally aligned. The anterior and posterior margins of the interclavicle are broad and nearly straight. The ventral surface displays the characteristic temnospondyl dermal sculpture pattern, with furrows and ridges radiating outwards from the ossification center.
Among the nine clavicles, only two right clavicles are nearly complete (Fig. 6). The ventral blade of the clavicle is slightly longer than wide, and shows a radial ornamentation on the ventral surface. The medial margin is convex, making the blade fanlike in shape rather than triangular. The dorsal process is posterodorsally directed and similar to that of Stanocephalosaurus pronus (Schoch & Milner, 2000;Howie, 1970). The lateral surface of the dorsal process combines with the ventral blade to form a prescapular cavity, and the dorsal process tapers to form a sharp dorsal tip.

DISCUSSION
The specimen IVPP V 22628 should represent an adult stage of the animal for the large size of the skull, the oval cross-section of the marginal teeth, ornament of coarse pits extending to sutural boundaries, well-ossified exoccipitals and quadrates (Steyer, 2003;Warren & Hutchinson, 1988). Specimens IVPP V 22632, 22633.1, and 22634.1 also should represent an adult stage for central reticulated ornamentation on their ventral surfaces, turning into radial ornamentation at their peripheries (Steyer, 2003).
The only previously reported tetrapod species from the Maopingchang locality is Yuanansuchus laticeps. The specimen IVPP V 22628 is different from the only known specimen of Yuanansuchus laticeps, and cannot be referred to this species. IVPP V 22628 differs from Y. laticeps in possessing the following features: elongated skull, large orbit, frontal enters the medial border of the orbit, closed otic fenestra, absence of fodina vomeralis and cultriform process of parasphenoid extends to the level of the anterior margin of the interpterygoid vacuities. Particularly considering that IVPP V 22628 is similar in skull length to the holotype of Y. laticeps, the differences between the two specimens are too great in magnitude to represent intraspecific variation, implying that IVPP V 22628 represents a new taxon. This specimen has a laterally directed tabular horn, as in the holotype of Y. laticeps, and the lateral orientation of the horn is a unique feature not known in other capitosaurs. Although such capitosaurs as Eryosuchus and Mastodonsaurus have been described as having laterally directed tabular horns (Damiani, 2001;Schoch, 2008), their tabular horns in fact differ from those of the two Chinese taxa in being directed posterolaterally.
The two small skulls collected from the same small hill (IVPP V 22629, 22630) are very similar to IVPP V 22628 in most features but differ in that the frontal is excluded from the orbital margin in both specimens. As shown by Kamphausen (1994), however, frontal asymmetry can be present even in a single skull, so these two specimens are referred to Y. maopingchangensis here. The lower jaw and postcranial bones are tentatively also referred to Y. maopingchangensis because they came from the same small spot and same horizon where the skulls described in this paper were found. All of the cranial material from this spot is referable to Y. maopingchangensis. Because postcranial morphology is relatively stable within most small temnospondyl clades although the ossification varies during ontogeny (Pawley, 2007;Pawley & Warren, 2005), the postcranial features observed in the new Maopingchang specimens probably characterize the entire genus Yuanansuchus rather than only Y. maopingchangensis.

Phylogenetic analysis
To assess the phylogenetic position of Yuanansuchus maopingchangensis, I incorporated this taxon into the data matrices of Schoch (2008) and Sidor, Steyer & Hammer (2014), both of which represent modifications of that of Damiani (2001). The original dataset of Damiani (2001) included 47 characters. Fortuny, Galobart & De Santisteban (2011) analyzed a modified version of this matrix, which excluded Damiani's (2001) characters 22 and 34 but added eight new characters. Subsequently, Sidor, Steyer & Hammer (2014) added Antarctosuchus to the matrix of Fortuny, Galobart & De Santisteban (2011). When I ran the matrix of Sidor, Steyer & Hammer (2014) using the same software (PAUP 4.0 beta 1.0 for PC) (Swofford, 2001) and settings specified in the paper, the results differed from those reported by Sidor, Steyer & Hammer (2014) in that A. polyodon was found to be the sister taxon of Mastodonsaurus giganteus rather than that of Paracyclotosaurus crookshanki, although the parameters two equally most parsimonious trees were the same (145 steps, a consistency index of 0.40, and a retention index of 0.70). Except with respect to the position of Antarctosuchus, however, the two trees are topologically identical with each other and with the cladogram published by Fortuny, Galobart & De Santisteban (2011). However, this could be due to the type error for the coding of character 50. If the coding is changed to 1, the result is same as the original paper. The two species of Yuanansuchus were added to this dataset (see Appendix S1), which was analyzed in PAUP 4.0 beta 1.0 for PC using the same settings as previous analyses. This analysis recovered four most parsimonious trees, the strict consensus of which is almost identical to Fig. 7 of Sidor, Steyer & Hammer (2014) except the position of Chinese clade comprising Y . laticeps and Y. maopingchangensis (Fig. 7). Schoch (2008) revised the characters of Damiani (2001) and added 19 further characters and used a slightly different list of taxa. He obtained results quite different from those of Fortuny, Galobart &De Santisteban (2011) andSidor, Steyer &Hammer (2014), one key difference being that Edingerella was recovered as a trematosaur rather than a capitosaur by Schoch (2008). However, when Schoch's (2008) small matrix of 25 taxa was analyzed in PAUP 4.0 beta 1.0 for PC, the shortest trees were found to be only 152 steps in length, whereas the single most parsimonious tree reported by Schoch (2008) was 162 steps. The strict consensus of the trees obtained is similar to the topology depicted in Schoch's (2008) Fig. 8, but includes the monophyletic clade (((Cyclotosaurus (Eocyclotosaurus, Quasicyclotosaurus)), Mastodonsaurus), Paracyclotosaurus). The matrix of Schoch (2008) including Yuanansuchus laticeps resulted in an unresolved strict consensus tree.
To resolve the interrelationships of Capitosauria, a new data matrix was constructed using a revised list of 56 characters (Appendix S1). The codings of the characters were mostly adopted from previous analysis, though the matrix incorporates a few novel codings based on examination of specimens, photos and the literature (Table 1). Y. maopingchangensis was coded primarily based on the holotype, and secondarily with reference to the other specimens described in this study. The matrix was analyzed with TNT 1.1 (Goloboff, Farris & Nixon, 2008), and all characters were equally weighted and left unordered. The holotype only dataset recovered two most parsimonious trees with 185 steps in length; while the composite dataset recovered three most parsimonious trees with 186 steps in length, two have same topology as the previous (Fig. 8). One tree (Fig. 8A) is similar in some degree to its topology to that depicted in Fig. 7A of Fortuny, Galobart & De Santisteban (2011). Both topologies posit the two Chinese taxa as a monophyletic clade, supporting referral of the new species to Yuanansuchus. This clade is supported by the following synapomorphies in all shortest trees: (character 4) laterally directed tabular horn; (character 7) lateral line sulci continuous, well impressed; (character 10) supraorbital sensory canal entering larimal; (character 13) preorbital projection of jugal shorter than half the length of snout. Yuanansuchus does not form a clade with Eocyclotosaurus plus Quasicyclotosaurus as suggested by Liu & Wang (2005). The positioned recovered for this clade is more basal than that recovered for Y. laticeps by Liu & Wang (2005) and Schoch (2008), but still falls on the branch of capitosauroid phylogeny that includes Cyclotosaurus. Schoch (2013) Figure 7 Cladogram. Cladistic relationships of Yuanansuchus within capitosaurian temnospondyls recovered from a cladistic analysis of 29 taxa and 53 characters, strict consensus of four equally most parsimonious trees of 154 steps. See Appendix S1 for the data.
containing Parotosuchus nasutus and Cyclotosaurus robustus. Here, Capitosauroidea can be divided into two clades: a relatively small one containing Parotosuchus, and a much larger one containing Cyclotosaurus. Capitosauroidea includes Edingerella, Watsonisuchus, and Xenotosuchus in two most parsimonious trees (Figs. 8A and 8C) but not in the other (Fig.  8B). In the phylogenies of Fortuny, Galobart &De Santisteban (2011) andSidor, Steyer &Hammer (2014), Parotosuchus and Cyclotosaurus are positioned close to one another, resulting in a restricted Capitosauroidea that excludes Paracyclotosaurus and many other taxa recovered as capitosauroids in the present analysis. The present analysis did not recover Figure 8 Cladogram. Cladistic relationships of the capitosaurian temnospondyls based on 29 taxa and 56 characters. Three most parsimonious trees (A, B, C) were recovered from the analysis when Yuanansuchus maopingchangensis is coded for all known specimens, while two of them (A, B) were obtained when Y. maopingchangensis is coded for only holotype.


Genetic Variants of CD40 Gene Are Associated with Coronary Artery Disease and Blood Lipid Levels

Objectives. The present study aimed to evaluate the effect of CD40 and CXCR4 genes polymorphisms on CAD susceptibility and the blood lipid levels and history of cardiovascular risk factors in a Chinese Han population. Materials and Methods. A total of 583 unrelated patients with CAD and 540 controls were recruited. Two tag SNPs (rs4239702 and rs1535045) at the CD40 locus and one tag SNP (rs2228014) at the CXCR4 locus were genotyped using the SEQUENOM Mass-ARRAY system. Results. After adjusting the risk factors, the frequency of rs1535045-T allele was also higher in patients than controls. Haplotype analysis showed that the rs4239702(C)-rs1535045(T) haplotype was associated with CAD. People with rs4239702-TT genotype had higher blood lipid levels in case group while it was not in the control group. History of cardiovascular risk factors showed no association for the three SNPs in case group and control group. Conclusions. rs1535045 in CD40 gene is likely to be associated with CAD in the Chinese Han population. rs4239702(C)-rs1535045(T) haplotype was associated with CAD. Only in CAD patients, the blood lipid level of patients with rs4239702-TT genotype was higher than other patients. CXCR4 gene may not relate to CAD.

Introduction
Coronary artery disease (CAD) is a complex disease and it has become the leading cause of death of human diseases worldwide [1][2][3][4]. There is no doubt that a genetic factor, which accounted for 30%∼60% of the risk of CAD [5,6], and the traditional risk factors, such as age, male gender, smoking, drinking, obesity, hypertension, hypercholesterolemia, and diabetes mellitus, contribute to the pathogenesis of CAD.
Vascular function is an important pathophysiological factor in cardiovascular diseases, and is influenced by many factors. Evidence suggested that inflammation may also be a risk factor for CAD [7]. Inflammation in the vessel wall plays an essential role not only in the initiation and progression of the atherosclerotic lesion, but also in the destabilization and acute rupture of plaques that occur during acute myocardial ischemic events [8][9][10]. Atherosclerotic lesion and acute myocardial ischemia share the same mechanism with CAD.
Thus inflammation is considered to be one of the mechanisms of CAD.
As a consequence of the expanding research, numbers of inflammatory marker tests ordered by clinicians for cardiovascular disease (CVD) risk prediction have grown rapidly. Up to now, several new nontraditional markers such as CD40-CD40 ligand system and chemokine receptors seem to be significant features of cardiovascular diseases [11,12].
It is now generally accepted that CD40-CD40 ligand (CD40L) interaction is a main determinant of the proatherogenic phenotype [13]. Studies suggested that patients with unstable CAD had an elevation of serum-soluble CD40L levels [14][15][16]. The genetic variants of CD40 gene may influence the efficiency of CD40 gene translation and may determine an individual's susceptibility on acute coronary syndrome [17,18]. CXCR4 is required for neovascularization and is also involved in inflammation. Patients with CAD were observed a reduced expression of CXCR4 gene [19].

BioMed Research International
However, the relationship between CD40 and CXCR4 gene polymorphisms and CAD is poorly understood until now. In this study, we strived to investigate the potential genetic role of CD40 and CXCR4 in the susceptibility of CAD in a Chinese Han population. In order to take into consideration traditional risk factors affecting genetic analysis in CAD, the present study was therefore undertaken to investigate the combined effect of the two genes and some common risk factors on the development of the disease.

Subjects.
A total of 583 unrelated patients with CAD from the First Hospital of Jilin University, Changchun, China, were recruited between 2008 and 2012. All patients had been examined by standardized coronary angiography according to the Seldinger's method, and the results were judged by at least two independent cardiologists. According to the coronary angiographic results, patients diagnosed with CAD had at least one or more major coronary arteries with 50% or greater stenosis. Control subjects ( = 540) were randomly selected from the same geographical area in the routine check-up as part of annual body examination, including an electrocardiogram (ECG), chest X-ray, and serum analysis. They were classified as healthy subjects based on their physical examination coupled with the absence of personal or family history and other reasons to suspect CAD. Individuals were excluded from having congenital heart disease, cardiomyopathy, and liver and renal disease.
The demographic and clinical characteristics data were collected through a review of the medical records. All the subjects were given an informed consent and were well told of the study protocol. The study was approved by the ethics committee of Jilin University, Changchun, China. Peripheral venous blood was collected in tubes containing disodium-EDTA (ethylenediaminetetraacetic acid) as an anticoagulant and stored at −80 ∘ C until genomic DNA extraction.

Genotyping.
To perform genetic analysis, we selected two tag SNPs (rs4239702 and rs1535045) at the CD40 locus and one tag SNP (rs2228014) at the CXCR4 locus as genetic markers. The selected SNPs were restricted to frequency of minor allele >10% in the HapMap-CHB database (http://www .hapmap.org/). Genomic DNA used for SNPs genotyping was extracted from peripheral blood lymphocytes using a DNA extraction kit (TianGen, Beijing, China). SNPs were genotyped using SEQUENOM Mass-ARRAY system with amplification primers and extension primers described in Table 1. For quality control, genotyping was performed without knowledge of the case or control status. 30 random samples were tested in duplicate by different persons, and the reproducibility was 100%.
2.3. Statistical Analysis. SPSS16.0 for windows was used for analysis. Data were expressed as percentages of total for categorical variables, or median ± QR and mean ± SD for continuous variables. The statistical analysis on the characteristics of the subjects was performed with Student's -test for the continuous variables with normal distribution (BMI), TC, and TG, while skewed distribution variables were compared by the Mann-Whitney test (age, WHR) and Pearson 2 test for the categorical variables.
The statistical differences between case and control in genotype and allele frequencies were assessed by 2 -test. The logistic regression models were performed to adjust the risk factors and calculate the odds ratios (ORs) and 95% confidence intervals (CIs). The statistical analysis on the association of the genotypes with blood lipid level was performed with test and multiple comparisons with Dunnett test. Genotype association for history of cardiovascular risk factors, including diabetes mellitus and hypertension, was performed with 2 test.
The Haploview program (version 4.1) was applied to estimate the linkage disequilibrium (LD) measures ( and 2) between paired SNPs. Haplotype analysis was performed with the unphased program (Version 3.0.12). Table 2 lists the demographic and clinical characteristics of the 583 CAD patients and 540 control subjects. Compared with the control group, the CAD group had more smokers and more individuals with hypertension and with diabetes mellitus. CAD patients also had higher waist-to-hip ratio (WHR). There was no significant difference in the mean age, sex, body mass index (BMI), TC, and TG. Tables 3 and 4 summarized the allelic and genotypic frequencies of the SNPs in Chinese CAD patients and controls. As shown, the frequency of rs1535045-T allele was higher in patients than controls (OR = 1.21, 95% CI 1.01-1.45, < 0.05); after adjusting the risk factors (smoking, hypertension, diabetes mellitus, and WHR), the OR is 1.27 (95% CI 1.01-1.59). But there was no significant difference in distribution of genotypic and allelic frequencies of rs4239702 and rs2228014 and in distribution of genotypic frequencies of rs1535045 between cases and controls.

Haplotype Analysis.
Analysis with Haploview showed that rs4239702 and rs1535045 were located in a similar LD block ( = 1.0).
We used unphased program to analyze the relationship between the haplotypes of the CD40 gene and CAD. rs4239702(C)-rs1535045(C) haplotype was selected to be the reference for testing the overall association. As shown in Table 5, there was no significant difference between cases and controls of the overall association, but the rs4239702(C)-rs1535045(T) haplotype was associated with CAD (OR = 1.246, 95% CI 1.014-1.531, < 0.05). Tables 6 and 7 analyzed the genotype association of the 3 SNPs with blood total cholesterol (TC) and blood triglyceride (TG) levels in CAD patients and controls. Table 6 showed that CAD patients with rs4239702-TT genotype had higher total cholesterol   level than patients with rs4239702-CC genotype ( < 0.05), while, in control group, there was no difference between genotypes and blood total cholesterol level ( > 0.05). Table 7 showed that, in control group, people with rs4239702-TT genotype had the lowest blood triglyceride level than people with rs4239702-CT genotype or rs4239702-CC genotype ( < 0.05), while, in CAD patients, there was no difference between genotypes and blood triglyceride level ( > 0.05).

Genotype Association for History of Cardiovascular Risk
Factors. Tables 8 and 9 analyzed the genotype association of the 3 SNPs with diabetes mellitus and hypertension in CAD patients and controls. Neither of them showed association for the three SNPs.

Discussion
Coronary artery disease is a complex disease, which may be due to genetic and environmental factors interaction.
Previous studies have suggested that the smoking, drinking, obesity, hypertension, diabetes mellitus, and circulating blood lipid levels were independent risk factors for cardiovascular diseases [20][21][22]. In this case-control study, we matched cases and controls by age and sex. Several classical risk factors for CAD were detected as well. There were no significant differences in the BMI, TC, and TG between the cases and controls. That may be caused by our small sample size or by patients taking medicine for lipid-lowering therapy. However, compared with controls, the CAD group had more smokers, more individuals with hypertension and with diabetes mellitus, and higher WHR. Our results confirmed that smoking, hypertension, diabetes mellitus, and central obesity are related to CAD. Inflammation has been found to be involved in the formation, development, and outcome of CAD. Traditional inflammatory biomarkers such as tumor necrosis factoralpha (TNF-) [23] (Mizia-Stec et al., 2003), C-reactive protein (CRP) [24], and interleukin-6 (IL-6) [25] have been shown to predict cardiovascular events. More recently, several new nontraditional biomarkers, such as CD40 and CXCR4, have been introduced in the clinical practice.
CD40 is a 50-KD integral membrane protein of tumor necrosis factor receptor family. CD40L had been found positively expressed in the atheromatous plaque. Increasing evidence shows that the CD40-CD40L system plays a critical role in atherosclerotic disease progression and plaque destabilization [13]. Studies showed that polymorphisms in CD40 may influence individuals' susceptibility to the development of cardiovascular events [18,26]. Studies had reported the relationship between rs1883832 at CD40 locus and acute coronary syndrome [27,28]. In this study, we selected two different tag SNPs (rs1535045 and rs4239702) at the CD40 locus to analyze the relationship between the two different tag SNPs and CAD in Chinese Han population. We found that the frequency of rs1535045-T allele in cases was higher than that in controls ( < 0.05). It indicates that T allele of rs1535045 at the CD40 locus may be a susceptive factor for CAD in Chinese Han population. There are no statistical significant differences of genotypic frequencies of rs1535045 between cases and controls, but they still seemed to have the tendency ( = 0.056). Because our sample size is not BioMed Research International 5      large enough, this result may be a false-negative result. In the further study, we should increase the sample size to verify it. There was no significant difference on genotypic and allelic frequencies of rs4239702 between cases and controls before or after adjustment for traditional risk factors, which suggests that the genetic variant of rs4239702 may be not associated with CAD. Results of haplotype analysis showed that the frequency of rs4239702(C)-rs1535045(T) haplotype was associated with CAD, and it may be a risk factor of CAD in Chinese Han population (OR = 1.246, 95% CI 1.014-1.531, < 0.05). CXCR4, which is highly expressed in circulating endothelial progenitor cells (EPC), together with its ligand chemokine stromal-derived factor-(SDF-) 1 is essential to normal cardiovascular development [29]. EPC from patients with CAD or diabetes have been shown to be functionally impaired [30,31], and the CXCR4 importantly modulates the migratory and angiogenic capacities of human EPC [29]. Based on that, we can assume that CXCR4 may be associated with CAD. In this study, through analyzing the relationship between the tag SNP (rs2228014) at CXCR4 locus and Chinese CAD patients, we had the hypothesis inspected. Unfortunately, we did not find any significant difference on genotypic and allelic frequencies of rs2228014 between cases and controls, which suggested that the genetic variant of rs2228014 at CXCR4 locus may be not associated with CAD in Chinese Han population.
Plasma lipid levels have been proved to be a considerable risk factor of CAD. Previous study had indicated that plasma inflammatory factors level was related to plasma lipid levels [32]. But inflammatory genes' potential impact on serum lipids in CAD patients has not been adequately studied. In this study, in order to assess whether the 3 polymorphisms had any effect on lipid, we used plasma lipid levels as quantitative traits to test the association for the three SNPs. The results showed that CAD patients with rs4239702-TT genotype had higher total cholesterol level than patients with rs4239702-CC genotype, while, in control group, there was no difference between genotypes and blood total cholesterol level; people with rs4239702-TT genotype had lower blood triglyceride level than people with rs4239702-CT genotype or rs4239702-CC genotype in control group, while, in CAD patients, there was no difference between genotypes and blood triglyceride level. It was observed that only patients with rs4239702-TT genotype at CD40 gene locus had higher blood lipid levels. It is tempting to speculate that, only in CAD patients, there were some factors interacting with rs4239702-TT genotype. Because of the interaction, patients with rs4239702-TT genotype had higher blood lipid level than other patients. But it is not clear what factors interact with rs4239702-TT genotype and how they interact with it. However, our study could not obtain the data of medications for lipid-lowering therapy, which might affect the serum lipids of subjects, to speak volumes for the impact of CD40 gene on serum lipids. In the further study, we should collect data of the detailed lipid profile and lipid-lowering agents to prove the effect of CD40 gene on serum lipids and select this as a new research aspect to explore the mechanism why genetic variants on CD40 gene can affect the occurrence of CAD.
Blood pressure and blood glucose levels associated with inflammation had been proved by many researches [33,34]. In CAD patients, many of them were taking medicine to control blood pressure or blood glucose levels, so, in this study, we analyzed genotype association of the 3 SNPs with history of diabetes mellitus and hypertension, instead of blood pressure and blood glucose levels. As results showed, neither of them was associated with the three SNPs.
In conclusion, we found that rs1535045-T allele at the CD40 locus may be a susceptive factor for CAD in Chinese Han population. Variant of rs2228014 at CXCR4 locus is not associated with CAD. rs4239702(C)-rs1535045(T) haplotype was associated with CAD. Only in CAD patients, the blood lipid level of patients with rs4239702-TT genotype was higher than other patients.