Bats, order Chiroptera, are one of the largest monophyletic clades in mammals. Based on morphology and behaviour bats were once differentiated into two suborders Megachiroptera and Microchiroptera Recently, researchers proposed alternative views of chiropteran classification (suborders Yinpterochiroptera and Yangochiroptera) based on morphological, molecular and fossil evidence. Since genome-scale data can significantly increase the number of informative characters for analysis, transcriptome RNA-seq data for 12 bat taxa were generated in an attempt to resolve bat subordinal relationships at the genome level. Phylogenetic reconstructions were conducted using up to 1470 orthologous genes and 634,288 aligned sites. We found strong support for the Yinpterochiroptera-Yangochiroptera classification. Next, we built expression distance matrices for each species and reconstructed gene expression trees. The tree is highly consistent with sequence-based phylogeny. We also examined the influence of taxa sampling on the performance of phylogenetic methods, and found that the topology is robust to sampling. Relaxed molecular clock estimates the divergence between Yinpterochiroptera and Yangochiroptera around 63 million years ago. The most recent common ancestor of Yinpterochiroptera, corresponding to the split between Rhinolophoidea and Pteropodidae (Old World Fruit bats), is estimated to have occurred 60 million years ago. Our work provided a valuable resource to further explore the evolutionary relationship within bats. Bats belong to the order Chiroptera, one of the largest monophyletic clades in mammals. They constitute ~20% of living mammalian species, arranged in 20 families5et al. claimed that living echolocating bats were monophyleticRecently, O’Leary de novo assembled using trinity packageIn this work, we sequenced cDNAs from 12 bat species and generated 610 million raw reads . After sAlong with 18 previously published mammalian genome sequences, a total of 1470 1:1 orthologous was obtained across 30 species . We performed multiple sequence alignments and our aligned supermatrix included 634,288 amino acids. We first used concatenated nucleotide and protein sequences using maximum likelihood to reconstruct the phylogeny. As with previous worksP-value < 0.05, We obtained a disagreement using nucleotide and amino acid sequences when addressing the position of bats within the superorder Laurasiatheria. The nucleotide tree recovered Pegasoferae group , whereas amino acid tree supported that bats are a sister group to Fereuungulata group  . PreviouTo evaluate the influence of the taxa sampling on phylogenetic reconstruction, analyses were performed from different subsets of taxa. We constructed concatenated trees for different taxa sets that include at least two Old World fruit bat, two rhinolophoid bats and two Yangochiroptera. Phylogeny analyses assigned high support  based on different sampling datasets. As shown in Results of divergence time estimation carried out under the auto-correlated models of molecular clock relaxation are shown in The development of next-generation sequencing technologies have generated many more genome sequences. This has allowed reconstruction of phylogenetic trees based on genome scale data providing a powerful approach to resolve the evolutionary relationships15171812Variation in gene expression and protein sequence can both influence phenotype22Prestin has been shown to have undergone sequence convergence among echolocating mammals. Recent genome analysis demonstrated that adaptive convergences are widespread at both the molecular and morphological levelThe conflict of ‘species tree’ and ‘gene tree’ challenges the traditional methodology of molecular phylogeny. Between-genes phylogenetic incongruences can arise for several reasons, involving convergent evolution in Microchiroptera. The homoplastic signal in morphology within echolocating bats can also be reflected from molecular evidences. For example, the ‘hearing gene’ Taken together, our work generated large-scale transcriptomes of bats and analyzed bat subordinal relationships at genome-wide scale. With increased taxa sampling and sufficient numbers of loci, we can obtain a more reliable phylogeny. These data also provided valuable information for further researches related to molecular evolutionary analyses.All animal experiments were carried out in strict accordance with the regulations for the use of laboratory animals  and were approved by the Animal Ethics Committee of East China Normal University (ID no: 20090219 and 20101002). No endangered or region-protected animal species were included, and no specific permission was required.Murina leucogaster, Myotis ricketti and Scotophilus kuhlii), two from the family Pteropodldae (Cynopterus sphinx and Rousettus leschenaultii), two from the family Hipposideridae (Aselliscus stoliczkanus and Hipposideros pratti), one from the family Rhinolophidae (Rhinolophus pusillus), one from the family Megadermatidae (Megaderma lyra), one from the family Rhinopomatidae (Rhinopoma hardwickei), one from the family Emballonuridae (Taphozous melanopogon), and one from the family Molossidae (Tadarida teniotis)  . These bTotal RNA was extracted from brain tissue using Trizol (Life Technologies Corp) according to manufacturer’s protocols. RNA concentration was determined using a NanoDrop spectrophotometer, and RNA quality was assessed by Agilent Bioanalyzer. New sequence data of each bat brain were generated using the Illumina Hiseq2500 platform. Raw reads were deposited into the Short Read Archive (SRA) database of NCBI under the accession no. SRP062200.de novo assembly the transcriptome of each tissue with default parametersFor each paired-end library, we first removed the adapters of raw reads. Then, the DynamicTrim Perl script in the SolexQA packageExcept for the 12 newly sequenced bat brain transcriptome data, we also downloaded the coding sequences of other 18 mammalian species  from Ensembl database . Only seWe generated two sequence super-matrices by concatenating aligned nucleotide and protein sequences of orthologous genes separately. For each matrix, we conducted the maximum likelihood analyses with model partitioning. The nucleotides substitution model We calculated standard RPKM expression values (that were then log2-transformed) for the orthologous genes. We constructed expression tree using the neighbor-joining approach, based on pairwise distance matrices between samples. The distance between samples was computed as 1-ρ, where ρ is Spearman’s correlation coefficient.P-values at a critical value α=0.05. In addition, we randomly sampled one species for each order within Laurasiatherian  and combined other species to generate nucleotide and protein sequences super-matrices. We repeatedly preformed the same phylogenetic tree reconstruction and AU-test analyses for these matrices.To compare the alternative topologies, approximately unbiased (AU) test statistic was computed using CONSEL packageCoalescent-based analyses were performed using ASTRAL methodTo evaluate the influence of taxa sampling on the phylogeny, several analyses were performed from different subset of taxa using concatenated genes. Because basal lineages of major groups are crucial for phylogeny reconstruction, at least two Old World fruit bats, two rhinolophoid bats and two Yangochiroptera from the taxon set were included for each sampling analysis.The divergence time was estimated using the Bayesian relaxed molecular clock approaches implemented in PhyloBayes, MCMCTree nested in PAML packageHow to cite this article: Lei, M. and Dong, D. Phylogenomic analyses of bat subordinal relationships based on transcriptome data. Sci. Rep.6, 27726; doi: 10.1038/srep27726 (2016).


Aspects of human motor control can be inferred from the coordination of muscles during movement. For instance, by combining multimuscle electromyographic (EMG) recordings with human neuroanatomy, it is possible to estimate alpha-motoneuron (MN) pool activations along the spinal cord. It has previously been shown that the spinal motor output fluctuates with the body's center-of-mass motion, with bursts of activity around foot-strike and foot lift-off during walking. However, it is not known whether these MN bursts are generalizable to other ambulation tasks, nor is it clear if the spatial locus of the activity  is fixed or variable. Here we sought to address these questions by investigating the spatiotemporal characteristics of the spinal motor output during various tasks: walking forward, backward, tiptoe and uphill. We reconstructed spinal maps from 26 leg muscle EMGs, including some intrinsic foot muscles. We discovered that the various walking tasks shared qualitative similarities in their temporal spinal activation profiles, exhibiting peaks around foot-strike and foot-lift. However, we also observed differences in the segmental level and intensity of spinal activations, particularly following foot-strike. For example, forward level-ground walking exhibited a mean motor output roughly 2 times lower than the other gaits. Finally, we found that the reconstruction of the spinal motor output from multimuscle EMG recordings was relatively insensitive to the subset of muscles analyzed. In summary, our results suggested temporal similarities, but spatial differences in the segmental spinal motor outputs during the step-to-step transitions of disparate walking behaviors. Muscle activity during human locomotion is coordinated by tens of thousands of alpha-motoneurons (MNs), organized along the spinal cord Romanes, . Bio-imaIn previous studies, the spinal mapping method was used to investigate development and aging  walking on level ground, and walking on an inclined surface. These tasks may also be relevant to clinical, rehabilitation or sport applications. For instance, toe walking is observed in patients with various neurologic and developmental abnormalities  and foot motion for 8 subjects  during 4 ambulation tasks: walking forward, backward, tiptoe and uphill (20% inclined grade), all at 4 km/hr. These tasks were selected to represent biomechanically distinct walking gaits that were cyclic  and could be performed at fixed speed on a treadmill. The treadmill speed was selected because it was sufficiently fast to distinguish myoelectric activity from the baseline noise , but also slow enough that most subjects could perform all the tasks. However, two out of the eight subjects were not able to walk backward at 4 km/hr. Each walking trial lasted 40 s and was performed barefoot on a standard treadmill. Prior to data collection, the subjects were trained on each task, allowing them time to acclimate to the various walking conditions, and all subjects gave informed consent prior to participation. The protocol was approved by the Ethics Committee of the Santa Lucia Institute.We also collected several additional trials to help identify maximum contraction (MC) magnitude for each muscle EMG. Before collecting the walking data, we asked subjects to perform a set of quasi-static maneuvers against manual resistance. These included: flexing/extending/abducting the toes, plantarflexing/dorsiflexing/inverting/everting the ankle, flexing/extending the knee, flexing/extending/abducting/adducting the hip and flexing the back. Each exercise was performed for 5 s, during which subjects were instructed to perform maximal contractions. Two additional forward walking trials (3 and 5 km/hr) were also recorded and used to help determine MC values.We recorded kinematic data bilaterally at 100 Hz using a Vicon-612 system  with nine cameras. Infrared reflective markers (diameter 14 mm) were place bilaterally over the following landmarks: greater trochanter (GT), lateral femural epicondyle (LE), lateral malleolus (LM), heel (HE), and fifth metatarsophalangeal joint (5MP).We recorded EMG activity by means of surface electrodes from 26 muscles simultaneously on the right side of each subject. These included one muscle from the lower back (erector spinae (ES) at L2 level), two muscles from the buttocks [gluteus maximus (Gmax) and gluteus medius (Gmed)], 11 muscles from the thigh , six muscles from the shank , and six muscles from the foot . The activations of flexor digitorum longus and flexor hallucis longus were indistinguishable in our surface EMG recordings, due to close proximity of the muscles, and thus are reported together. We placed EMG electrodes based on suggestions from SENIAM (seniam.org), the European project on surface EMG. To this end, we located the muscle bellies by means of palpation and oriented the electrodes along the main direction of the fibers Winter, . The plaAll EMGs were recorded at 4000 Hz using a Delsys Trigno Wireless System , except the flexor digitorum brevis which was recorded using a synchronized Delsys Bagnoli System (at 1000 Hz). Due to the recording site of flexor digitorum brevis (on the plantar surface of the foot), the lower-profile Bagnoli electrode was needed. Some electrodes became partially or fully detached during testing, and signals were thus not usable. These EMGs were removed on a subject-specific basis. On average we analyzed 23.4 ± 1.7 muscle EMG from each subject.The beginning of the gait cycle (foot-strike) was defined based on kinematic events. We used vertical height of the right HE marker for forward walking and limb elevation angle  for backward, tiptoe and inclined walking. Similarly, for stance to swing transition (foot-lift) we used limb elevation angle  for forward, tiptoe and inclined walking, and minimum vertical height of the HE marker for backward walking. The usage of these criteria was based on the different kinematic endpoint (foot) behaviors for the various gaits . To reduce residual baseline noise, which appear as offsets in the EMG envelopes, we subtracted the minimum signal from each EMG. This assumes that at some point during walking each muscle is effectively “off” (not actively contracting). Some subjects exhibited artifacts in the foot muscles, generally linked to foot-strike and foot-lift events. In order to remove these artifacts, high-pass filtering of these muscles was performed using a 150 Hz cut-off frequency (rather than 30 Hz). A prior study on cut-off frequency  EMG matrix for each task, where m equaled the number of muscles analyzed. Inter-subject mean (and standard deviation) values for EMG were then computed from these subject-specific data. In addition to calculating the ensemble-averaged EMGs (across strides and subjects), we also present some EMG waveforms of individual strides in order to examine inter-stride variability in the spinal motor output.We divided EMGs into gait cycles based on foot kinematics, then interpolated each stride to 200 time points, and finally averaged across gait cycles . This yielded an . Thus, all EMGs were considered on a scale from 0 to 1, where 0 indicates that a muscle is inactive and 1 represents maximum muscle activation. Across all quasi-static trials (EMGs were low-pass filtered as described previously), we looked at a sliding 1-s window  and computed the average EMG during each. The highest average EMG found during any 1-s window was defined as the maximum quasi-static activation magnitude. Similarly, maximum dynamic activation magnitude was defined for each muscle as the peak stride-averaged EMG across all walking tasks. The normalization constant for each muscle was then defined as the larger of the quasi-static and dynamic activation magnitudes.We normalized EMGs by the MC magnitude across all trials. Normalization was performed to account for the differences in μV magnitudes recorded between muscles. We defined MC magnitude as the muscle's maximum EMG signal from either dynamic  or quasi-static trials  was not used in this study for reconstructing the segmental spinal outputs, which has occasionally been done in the past . This MN pool size normalization primarily affects the boundary segments L2 and S2, which contain 2–3 times fewer MNs than the other segments .The primary assumptions implicit in this analysis are that (1) the rectified EMG provides an indirect measure of the net firing rate of MNs for each muscle  spinal maps, we calculated the spinal motor output for each subject based on stride-averaged EMGs, and then we averaged it across subjects. We computed two summary metrics to describe the spinal maps: Practical considerations limit the number of muscles from which we could record. Thus, there is the potential issue of how the specific selection of the muscles affects the resulting spatiotemporal maps of MN activity. To evaluate the sensitivity of the spinal maps approach we compared the motor outputs obtained from analyzing all 26 muscles with those obtained from subsets of these muscles. Subsets were chosen as follows: (1) the 20 non-foot muscles  and (2) 12 commonly recorded muscles . For forward walking we also made 26 additional comparisons by correlating maps from each unique set of 25 muscles  with the map constructed from all muscles. The correlation coefficient (r) was calculated for each subject and condition. Averaged correlation coefficients were then reported for each comparison.r-values). For instance, to compare segmental activations of individual subjects with those of averaged maps, correlation coefficient was computed for each subject and each segment, and then they were averaged first across subjects for each segment and then across segments. Similarly, to compare the maps obtained by different sets of muscles, correlation coefficient was computed for each segment, and then the data for all segments were averaged. Since correlation coefficients have non-normal distributions, their mean estimates were computed based on the normally distributed, Z-transformed values.To compare activation waveforms we computed linear correlations  ANOVA was used to evaluate differences in the kinematics and the p < 0.006, Figures p < 0.03). The range of hip and ankle angular motion was significantly larger during inclined walking than for the other tasks  demonstrated the major peak of activity around lift-off Rab, . EMG recThe amplitude of EMG signals (in μV) varied considerably across muscles, both during walking and in terms of MC Figures . We founp < 0.01, Figure We observed task-specific spinal motor outputs for each walking condition Figure , althougThe major features observed in the average spinal maps were also present in subject-specific maps. In particular, 6 out of the 8 tested subjects exhibited bimodal (two peaked) motor output profiles for all gaits Figure . The remEMG profiles exhibit stride-to-stride variability related to dynamic stability and walking speed maintenance .We found that the spinal maps were relatively insensitive to the subset of muscles analyzed. Spinal maps computed from 20 and 12 muscle subsets were strongly correlated with the maps computed from the full set of 26 recorded muscles, with average correlation coefficients between 0.98–0.99 and 0.91–0.96, for each task , whereas many animals walk only on their toes (digitgrade gait). In this study we observed roughly a doubling of the intensity of spinal motor output during tiptoe walking Figure , which iThere are several limitations to the spinal mapping approach, many of which have been previously documented (Cappellini et al., In summary, we found that the MN activation patterns exhibited two major bursts during diverse walking tasks, one around foot-strike and the other around foot-lift, but with differences in the segmental level and intensity of the spinal activity. We also found further evidence that spinal MN mapping provides a robust method for estimating spinal motor output, which is relatively insensitive to the subset of EMGs analyzed. We also suggest that spinal motor mapping can be used to assess the recruitment of specific motor pools when using epidural electrical stimulation or corticospinal neuroprostheses for restoring locomotor functions (Capogrosso et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Repeated domains in proteins that have undergone duplication or loss, and sequence divergence, are especially informative about phylogenetic relationships. We have exploited divergent repeats of the highly structured, 50-amino acid WHEP domains that join the catalytic subunits of bifunctional glutamyl-prolyl tRNA synthetase (EPRS) as a sequence-informed repeat (SIR) to trace the origin and evolution of EPRS in holozoa. EPRS is the only fused tRNA synthetase, with two distinct aminoacylation activities, and a non-canonical translation regulatory function mediated by the WHEP domains in the linker. Investigating the duplications, deletions and divergence of WHEP domains, we traced the bifunctional EPRS to choanozoans and identified the fusion event leading to its origin at the divergence of ichthyosporea and emergence of filozoa nearly a billion years ago. Distribution of WHEP domains from a single species in two or more distinct clades suggested common descent, allowing the identification of linking organisms. The discrete assortment of choanoflagellate WHEP domains with choanozoan domains as well as with those in metazoans supported the phylogenetic position of choanoflagellates as the closest sister group to metazoans. Analysis of clustering and assortment of WHEP domains provided unexpected insights into phylogenetic relationships amongst holozoan taxa. Furthermore, observed gaps in the transition between WHEP domain groupings in distant taxa allowed the prediction of undiscovered or extinct evolutionary intermediates. Analysis based on SIR domains can provide a phylogenetic counterpart to palaentological approaches of discovering “missing links” in the tree of life. An increasing amount of whole genome sequence information from diverse organisms offers much promise for determining phylogenetic relationships between known taxa, and resolving the tree of life. However, recent analyses depending on linear sequence data from single gene or genome-scale datasets as indicators of historical relationships between taxa have proved problematic, frequently yielding incongruent trees and equivocal or conflicting phylogenetic hypotheses S. pombe and in protostomes and deuterostomes, in Gly(G)RS and Met(M)RS in protostomes and deuterostomes, but none have been reported in any non-synthetase proteins. An analysis based on intron positions has suggested that the domain was acquired by HRS and GRS early in eukaryote evolution and later spread to EPRS The utility of genome-level characters in inferring phylogenetic relationships is based on the concept that shared genomic changes are indicators of common descent Nematostella vectensis, indicating the evolution of WHEP domains and the non-canonical functionality of mRNA binding in the common cnidarian-bilaterian ancestor Recently, we demonstrated WHEP domains and GAIT element RNA-binding activity in the fused EPRS of the cnidarian Homo sapiens EPRS protein sequence as query. The sequence of EPRS WHEP domains in the elephant shark  was obtained from the elephant shark genome project database. In addition, the linker region that joins ERS and PRS domains of key branch point species, i.e., the primitive vertebrate lamprey (Petromyzon marinus) and the cephalochordate amphioxus (Branchiostoma floridae), were amplified using primers corresponding to flanking conserved regions, and sequenced. Identification as 50-amino acid WHEP domains was confirmed by Pustell dot matrix analysis against human WHEP domains Gallus gallus and Taenopygia guttata) repeats grouped closely with those of the lizard (Anolis carolinensis), supporting a common diapsid ancestor Ornithorhynchus anatinus) had four WHEP domains that grouped with the bird and reptile domains, with the closest mammalian WHEP domains being that of the early-branching marsupial, opossum (Mondelphus domestica). This suggested the relatedness of platypus to reptiles/birds, and supported the phylogenetic position of platypus as intermediate between reptiles/birds and mammals as indicated by the recently sequenced platypus genome Tetraodon nigroviridis, four in Gasterosteus aculeatus, and five in Danio rerio, resulting from lineage-specific duplications  WHEP 3 domains (WHEP 4 in birds and platypus) whereas WHEP 1 was an outgroup to vertebrate (tetrapod) WHEP 1 and 2 domains , the hemichordate acorn worm , and the cephalochordate amphioxus (Branchiostoma florida) showed lineage-specific amplification giving rise to 4 to 6 WHEP domains. Notably, amphioxus WHEP domains clustered with echinoderm and hemichordate domains, suggesting that cephalochordates are more closely related to echinoderms and hemichordates than to urochordates as is generally believed. This phylogenetic position for amphioxus is consistent with recent studies involving multigene datasets The EPRS WHEP domains of the echinoderm sea urchin  forming a strongly-supported separate cluster, whereas the domains from arthropods, annelids, and molluscs clustered together (C. elegans ERS (E6) and the single N-terminal WHEP domain in PRS (P1) formed the outgroup of nematode WHEP domains. This suggests these domains are closest to the EPRS WHEP domains of the last common ancestor of nematodes and arthropods as proposed Drosophila and WHEPs 5 and 6 from the wasp Nasonia vitripennis formed outgroups to all articulata WHEP domains, and showed the closest relationship with C. elegans ERS WHEP E6 and the PRS WHEP domain. The C-terminal WHEP domains of Drosophila and Nasonia EPRS and C. elegans ERS again constitutes a genomic signature of the last common ancestor of all protostomes, and suggests that the other WHEP domains in protostomes were derived by lineage-specific duplications from a single WHEP domain in the EPRS of the last common protostome ancestor.As the analysis of WHEP domain duplication and divergence provided insights into deuterostome phylogeny, a similar analysis was done using seventy-seven WHEP domain sequences from 18 protostomes that included 11 arthropod, two annelid, one mollusc, and four nematode species. Only together . This obB. malawi and L. loa grouped together, supporting a close relationship between these nematode species. Interestingly, Ascaris revealed seven EPRS WHEP domains, whereas the maximum number in EPRS of any other species found in this study is six. However, Ascaris EPRS WHEPs 5* and 6* were atypical and highly degenerate, suggesting genomic instability in that region of the gene. WHEP repeats exceeding six might lead to instability within the EPRS gene, and similarly might have led to fission of C. elegans EPRS, and formation of ERS with six WHEP domains and PRS with one. Similarly, tight grouping of WHEP domains indicated more recent lineage-specific duplications in the mosquito Anopheles gambiae, the wasp Nasonia vitripennis, the water flea Daphnia pulex, and the annelids Capitella capitata and Helobdella robusta. Except for the outgroup WHEP domains 5 and 6 from Nasonia vitripennis, WHEP repeats from the hymenopteran insects  grouped together. Ants exhibited a diversity of WHEP domain repeats; ant species with 2 , 3 (Solenopsis invicta), 4 (Acromyrmex echinatior), and 5 (Camponotus floridanus) WHEP domains formed a separate group with two bee species (Apis mellifera and Bombus terrestris) and showed lineage-specific domain amplification. This group excluded the wasp WHEP domains, suggesting an earlier divergence of ants and bees from wasps in the hymenopteran branch. However, one WHEP domain of the ponerine ant H. saltator grouped with the bee domains, whereas the other WHEP domain formed the outgroup to all hymenopteran WHEP domains. This suggests that H. saltator WHEP domains might be genomic signatures of the last common ancestor of wasps and bees/ants and supports the position of poneroid ants as basal in the ant phylogenetic tree as shown earlier Individual WHEP domains of Schistosoma mansoni, the body louse Pediculus humanus corporis, and the pea aphid Acrythosiphon pisum. Analysis of the pea aphid genome also showed the presence of a second EPRS variant with six WHEP domains. EPRS in the nematode Trichinella spiralis had four highly degenerate WHEP domains, recognizable as repeat domains by low-stringency dot plot analysis, but with little homology with other WHEP domains. Remarkably, the organisms with absent or degenerate EPRS WHEP domains, although belonging to divergent phyla, are all endoparasites or ectoparasites, suggesting a possible link between the absence of these domains and a parasitic life cycle. Because EPRS WHEP domains are involved in the innate immune response in humans, it is possible that these organisms, some of which lack multiple immune system genes Remarkably, we found linked EPRS lacking any WHEP domains in some protostome species, namely, the trematode platyhelminthes Nematostella vectensis as query sequence, showed fused EPRS with one to three WHEP domains in the coral Acropora millepora, sponges Oscarella carmela and Amphimedon queenslandica, placozoa Trichoplax adherens, choanoflagellates Monosiga brevicollis and Salpingoeca rosetta (Proterospongia), and filasterean Capsaspora owczarzaki. A maximum likelihood tree of WHEP repeat sequences from various filozoan taxa including early branching metazoans, choanoflagellates, Capsaspora, and Sphaeroforma arctica, a sister group of filozoans, together with sequences from representative bilaterian taxa was constructed , which formed a sister group to WHEP 1 domain from Monosiga and WHEP 1 and WHEP 3 from the choanoflagellate Salpingoeca rosetta. Remarkably, the other Monosiga WHEP domains assorted between bilaterian and non-bilaterian metazoans; Monosiga WHEP 2 grouped with C. elegans ERS WHEP E6 and the PRS WHEP, forming an outgroup to all nematode WHEP domains, whereas Monosiga WHEP 3 formed an outgroup to all bilateran WHEP domains. This constitutes a genomic signature of a last common ancestor of choanoflagellates and metazoansand supports the phylogenetic position of choanoflagellates as the closest sister group to metazoans with the filastereans as an outgroup to choanoflagellates and metazoans Nematostella which expresses EPRS splice variants with both one and two WHEP domains Inferences regarding the sequence of events leading to the evolution of metazoans from unicellular ancestors require a robust phylogenetic framework of early branching metazoans and their immediate outgroups. However, phylogenetic signals at the base of the metazoan tree have been conflicting, with studies using different sets of DNA and amino acid sequences giving different and inconsistent tree topologies structed . The treCapsaspora has been found to be an outgroup to metazoans and choanoflagellates in several phylogenetic studies, although its exact phylogenetic position in relation to other sister groups of metazoans is uncertain Capsaspora EPRS had two nearly identical WHEP domains suggesting that the fused EPRS originated in a close ancestral organism with a single Capsaspora-like WHEP domain, which underwent a subsequent lineage-specific duplication. We hypothesized that the immediate outgroup of Capsaspora would contain separate ERS and PRS, with a single WHEP domain at the ERS C-terminus or PRS N-terminus, or possibly both, that would have preceded the fusion event. We investigated the genomic sequences of Sphaeroforma arctica, an icthyosporean suggested to be the sister group of filazoa, a clade consisting of filastarea, choanozoa, and metazoa S. arctica ERS harboured a C-terminus sequence recognized as a WHEP domain by dot plot comparison with other EPRS WHEP domains, and by multiple sequence alignment which revealed 46.4% pair-wise identity with Capsaspora EPRS WHEP domains  derived from the ancestral filasterea but contains the “b-like” and “c”-like” WHEP domains derived from choanoflagellates that assorted between the metazoan lineages. A search of newly sequenced metazoan organisms revealed that the deeply-diverging cnidarian Hydra magnipapillata satisfied these criteria, containing a “b-like” WHEP domain grouping with basal metazoans and two “c-like” domains grouping with Monosiga WHEP 2 and C. elegans WHEP domains (two other WHEP domains were highly degenerate). This suggests that EPRS WHEP domains in Hydra are genomic signatures of the predicted ancestor of basal and bilaterian metazoans. Similarly, collapse of bilaterian WHEP domains into a putative species tree reveals evolutionary relationships between the groups and predicts as-yet unknown linking branches . Total RNA from whole adult amphioxus and adult lamprey liver  was reverse-transcribed using oligo(dT) primers and the EPRS linker domain amplified using primers against the conserved enzymatic sequences, and sequenced. EPRS sequence for elephant shark was provided by Byrappa Venkatesh from the Elephant Shark Genome Project at the Institute of Molecular and Cell Biology, A-Star, Singapore (http://esharkgenome.imcb.a-star.edu.sg/). EPRS sequence of Nematostella vectensis was obtained from Stellabase, the Nematostella vectensis genome sequence database. Total RNA from adult Nematostella vectensis anemones  was reverse-transcribed, and EPRS linker domain amplified and sequenced as described Oscarella carmela EPRS sequence was provided by Bernard Degnan, University of Queensland and Amphimedon queenslandica EPRS sequence was obtained from its genome database (www.metazome.net/amphimedon). EPRS sequences for basal metazoans and filasporeans were obtained from the Joint Genome Institute (JGI) and Multicellularity Genome Project at the Broad Institute, Massachusetts Institute of Technology (http://www.broadinstitute.org/annotation/genome/multicellularity_project/MultiHome.html). EPRS sequence of choanoflagellates Monosiga brevicolis and Salpingoeca rosetta were provided by Nicole King, Berkeley University. EPRS linker domain was amplified from cDNA from Monosiga brevicolis (gift from Nicole King) using primers against flanking sequences.EPRS protein sequences for most taxa, unless otherwise stated, were extracted from the National Center for Biotechnology Information (NCBI) databases by BLAST search using protein query against translated nucleotide database (tBLASTn). Most vertebrate EPRS sequences were acquired from the Ensembl databases (www.geneious.com), and dot-plot analysis was performed to identify WHEP domain boundaries. The boundaries were manually applied to designate the 50-amino acid sequence of the WHEP domains. In the case of nucleotide sequence of EPRS linker domains, the sequence was translated in three reading frames and the translated sequences aligned with the human linker sequence to identify the WHEP domains.EPRS protein sequences were pairwise aligned with the human EPRS sequence using Geneious software (www.geneious.com). WAG model with gamma rate variation was utilized. Consensus trees were obtained from the sorted tree topologies after applying a burn-in fraction of 0.25. Maximum likelihood, neighbor-joining, and consensus Bayesian inference trees from each dataset were compared; branches predicted by two or three tree-building algorithms were indicated by * or **, respectively.Phylogenetic analyses were performed using MEGA 5 Figure S1Listing and alignment of all EPRS WHEP domains used in (TIF)Click here for additional data file.Figure S2S. arctica ERS WHEP domain with C. owczarzaki EPRS WHEP domains.Alignment of (TIF)Click here for additional data file.


As Earth's climate warms, the massive stores of carbon found in soil are predicted to become depleted, and leave behind a smaller carbon pool that is less accessible to microbes. At a long-term forest soil-warming experiment in central Massachusetts, soil respiration and bacterial diversity have increased, while fungal biomass and microbially-accessible soil carbon have decreased. Here, we evaluate how warming has affected the microbial community's capability to degrade chemically-complex soil carbon using lignin-amended BioSep beads. We profiled the bacterial and fungal communities using PCR-based methods and completed extracellular enzyme assays as a proxy for potential community function. We found that lignin-amended beads selected for a distinct community containing bacterial taxa closely related to known lignin degraders, as well as members of many genera not previously noted as capable of degrading lignin. Warming tended to drive bacterial community structure more strongly in the lignin beads, while the effect on the fungal community was limited to unamended beads. Of those bacterial operational taxonomic units (OTUs) enriched by the warming treatment, many were enriched uniquely on lignin-amended beads. These taxa may be contributing to enhanced soil respiration under warming despite reduced readily available C availability. In aggregate, these results suggest that there is genetic potential for chemically complex soil carbon degradation that may lead to extended elevated soil respiration with long-term warming. The size of the soil carbon pool exceeds that of atmospheric and terrestrial vegetation carbon pools combined  or unamended Bio-Sep bead  pouches (details in next section). Pouches were deployed as either “surface” bags, or as “subsurface” bags. Surface bags were placed on the surface of the soil under the leaf litter layer. Subsurface bead bags were buried in the soil between the organic horizon and the mineral soil. Where the depth of the organic horizon exceeded 3 cm, the beads were deployed to a depth of 3 cm. Bags were positioned in pairs, such that the lignin amended and unamended bead bags were side-by-side, but the surface and subsurface bead bags were at an approximate horizontal distance of at least 5 cm. We incubated a total of 32 bead pouches at our research site. Bags were deployed on August 5, 2013 and remained buried until October 23, 2013, a total of 11 weeks. This coincides with the seasonal period over which the greatest difference in soil respiration between heated and control plots can be seen  in a Nomex matrix (75%)  following a modified CTAB bead-beating procedure in tubes with three 5 mm glass beads  and to include references cited within papers. We also included other papers that were not included in this search but were already familiar to us. The genera appearing in this search but not found in our dataset were p-value cutoff of 0.1. For data which did not meet parametric assumptions, the function boxcoxfit  of genera with proposed lignin-degraders were pooled by soil effect and warming treatment. OTUs without genus assignment were removed before analysis, and samples were normalized by sum to account for different degrees of genus-level assignment before calculating eff = exp(H) prior to comparison . Looking at the effect of lignin amendment, unamended beads showed approximately 4.5x greater bead weight normalized oxidative enzyme activity than lignin-amended beads . DAPI-based cell counts of the enzyme slurry indicated that the number of cells per gram of beads was unaffected by the experimental factors .In the surface beads, warmed communities showed 4.7x higher cell-normalized oxidative activity compared to controls, while in the subsurface beads there was no warming effect . Those enriched for by lignin amendment included Sphingomonas, Acinetobacter, and Agrobacterium, respectively present at 6.4, 22, and 130x greater relative abundance in lignin than in unamended beads.Sequencing of the 16S ribosomal RNA gene V4 region was used to identify the microbes associated with lignin-amended and unamended beads. Lignin-amended beads recruited many genera containing known lignin-degraders (Table p < 0.05), 10% more even (p < 0.05), and had a phylogenetic diversity that was 28% greater (p < 0.01) than communities from control plots  played a secondary role in shaping overall bacterial beta diversity, with a greater fraction of the variation explained in the lignin-amended (R2 = 0.253) than the unamended beads (R2 = 0.141). We also analyzed factors driving relative abundance of each phylum individually  using step-down regression (Table R2 adj = 0.19) compared to most of the other classes and phyla considered.Bead amendment was the primary force driving bacterial community structure, based on a permutational ANOVA of the UniFrac distances , warming treatment , and soil effect  and soil effect  also played a significant role in structuring the fungal community in unamended beads when examined alone, but not lignin-amended beads.Fungal community structure was also driven by bead amendment  of the 56,835 total OTUs present. Examining the 9924 OTUs shared between lignin-amended and unamended beads and re-rarefying the community, we were able to identify 213 OTUs significantly enriched on lignin-amended beads compared to unamended beads, and 536 significantly enriched on unamended beads compared to lignin-amended beads  and of microbially available C  of the subsurface bead bags were at the mineral-organic interface, it is difficult to directly assess this hypothesis. Factors such as a more stable temperature and moisture in the subsurface beads may have enabled microbes to invest more energy into producing extracellular enzymes, and less into maintenance than was possible for the surface beads. In addition to the showing greater enzymatic response to warming treatment, the surface beads were also home to a less phylogenetically-diverse bacterial community than the subsurface beads. This may in part be due to the increased moisture fluctuations faced by the surface beads, which may reduce diversity of the water-bound bacterial community Lavelle, .Based on the fact that total oxidative enzyme activity was higher in unamended beads than lignin amended beads, it is unclear whether lignin amended Bio-Sep beads successfully recruited a lignolytic community. While it is possible that lignin was depleted on the lignin amended beads at the time of collection, we believe this is unlikely. In a similar experiment using these beads in tropical forest soils, DeAngelis et al.  found thRhodococcus and Pseudomonas but didn't include a number of the better-known lignin degrading genera such as Streptomyces, which are well-known for their biopolymer degrading capacity (Kampfer, Rhodococcus jostii RHA1 and Pseudomonas putida mt-2 are both able to degrade lignin without exogenous hydrogen peroxide and at a rate equivalent to some lignolytic fungi (Ahmad et al., Burkholderia, a cosmopolitan genus known for its diverse metabolism (Garrity et al., Nocardia, one of the better-known lignin degraders (Bugg et al., Sphingobacteriales (Taylor et al., Xanthomonadales (Odier and Monties, Saprospirales, were found to be enriched in lignin compared to unamended beads. Likewise, warming treatment specifically enriched for OTUs in a combination of orders with and without known lignin degraders. For instance, eight members of Acidobacteria were enriched by warming on lignin beads, although to our knowledge direct evidence for their ability to degrade lignin has not yet been reported. It is also possible that their enrichment is due to ability to degrade lignin by-products produced by other lignin-degrading organisms. Dedysh and colleagues isolated Acidobacteria from phenolic-rich peats and mosses using humic acid (Dedysh et al., per se. A recent paper also noted the possibility that organisms in lignin-rich environments may gain the ability to degrade complex carbon sources through rapid horizontal gene transfer (Strachan et al., Lignin amended beads recruited a phylogenetically diverse collection of bacteria, including many in genera where known lignin degraders are found. These genera with significantly higher relative abundance in lignin amended than unamended beads included Kampfer, . RhodocoIn addition to specifically selecting for potential lignin degraders, warming may enhance the ability of the microbial community to degrade chemically complex carbon by increasing overall community diversity. Increased community diversity is associated with the acceleration of multiple ecosystem processes, including decomposition and respiration (Strickland et al., Ultimately, the ability of the microbial community to efficiently convert soil carbon to biomass is the key first step in the release of stored soil carbon (Allison et al., We found evidence for a change in the structure of the bacterial community adapted to degradation of a major litter component, lignin, as we have seen for the “whole-soil” bacterial community after two decades of warming at this site (DeAngelis et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Spinal subdural hematoma is rare and can cause serious neurological symptoms. Sometimes, idiopathic spinal subdural hematoma can spontaneously occur without any identifiable underlying etiologies. In this report, we present such an uncommon case of paraplegia caused by idiopathic spinal subdural hematoma that was successfully managed by laminectomy.A 45-year-old Chinese woman presented with sudden onset of progressive asthenia and numbness in both lower extremities, accompanied by difficulty in micturition. An initial non-contrast spinal magnetic resonance imaging at a local hospital suggested a spinal subdural tumoral hematoma at the T9 level. She was referred to our hospital and an emergency laminectomy from T8 to T10 was performed 22 hours after onset of her initial symptoms. However, nothing but a hematoma was identified during the operation, and a final diagnosis of spontaneous acute spinal subdural hematoma was concluded. She had partial return of sensations and voluntary movement after the operation.On imaging findings, spinal subdural hematoma could manifest as focal and independent from the dura matter, and, therefore, it should be included in the differential diagnosis of medullary compressive lesions. Spinal hemorrhages include epidural, subdural and intramedullary hemorrhage, of which spinal subdural hematoma (SSDH) is the least common form. SSDH usually manifests as a sudden onset of back pain and lower limbs paralysis. On a magnetic resonance imaging (MRI) scan of the spine, SSDH is usually hyperintensive on T2-weighted images and isointensive on T1-weighted images in acute phase -5. The oA 45-year-old Chinese woman presented with sudden onset of progressive asthenia and numbness in both lower extremities, accompanied by difficulty in micturition; she was first sent to a local hospital. Five hours later, she was unable to walk. She reported no fever, back pain, or trauma history. She had no past medical history of note and was on no medication. A general physical examination performed on admission revealed increased heart rate (94 beats per minute) and blood pressure (193/104mmHg). A neurological examination revealed a normal mental state, but grade 0 myodynamia and paresthesia in both lower limbs. An initial non-contrast MRI scan of her spine performed at the local hospital showed a well-demarcated, oval-shaped extramedullary lesion located at the T9 level, ventral to her spinal cord. The lesion appeared isointensive on T1-weighted images and hyperintensive on T2-weighted images, but gadolinium was not administrated in this scan  are important to a patients’ prognosis, special attention should be paid to those patients with SSDH who present untypical symptoms in future clinical practice. Although there are still some debates on the surgical indications for SSDH, we think emergency surgical hematoma evacuation at the local hospital or more prompt referral might be more preferable than simply conservative treatments. In fact, there is no consensus on the optimal management strategy for those patients with SSDH; strategies range from conservative treatments like intravenous methylprednisolone (pulse) therapy , to percAlthough preoperative images of the presented case suggest it to be an extramedullary intradural lesion, for example arachnoid cyst or tumoral lesion with proteinaceous or bloody fluid in it, the intraoperative surgical findings confirmed the diagnosis of idiopathic SSDH. Therefore, SSDH should be included in the differential diagnosis of those lesions that compress the spinal cord.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


The purpose of this study was to present a case of paraneoplastic systemic hypertension and fever in an undiagnosed primary ciliary body carcinoma arising in a painful blind eye.A 37-year-old white man with a history of blindness since childhood was enucleated for neovascular glaucoma because of intractable pain in his right eye. A histopathological examination revealed a ciliary body carcinoma. One year later, an invasive recurrence of his orbit and ethmoid was detected and a debulking procedure was performed. He had untreatable fever and multidrug-resistant systemic hypertension for 3 months before the neoplasm diagnosis. He recovered from fever and systemic hypertension only after tumor excision and relapsed 1 year later when synchronous tumor dissemination was shown through a computed tomography scan. Tumor metastases, despite surgery and chemotherapy, caused his death.Paraneoplastic symptoms such as fever and hypertension may be due to unrecognized ocular malignancy. This case report intends to emphasize the importance of histopathological examination of an enucleated phthisical painful blind eye. Alterations of visual function as a paraneoplastic manifestation of extraocular tumors have been described in the literature. Carcinoma and melanoma-associated retinopathy, cancer-associated cone dysfunction and bilateral uveal melanocytic proliferation are considered paraneoplastic syndromes due to circulating anti-retinal auto-antibodies and immune toxic factors released by the primitive tumors . In thisA 37-year-old white man came under our observation 8 years ago (2007) because of pain and increasing intraocular pressure despite maximal drug therapy in diagnosis of neovascular glaucoma in his right eye (RE). His medical history included blindness since childhood, polar posterior cataract and strabismus surgery in his RE at the age of 5 years. He has always had general good health, his left eye visual acuity was 20/20 and no ocular abnormality was observed. Magnetic resonance imaging (MRI) of his orbit and A-scan ultrasonography showed bulbar subatrophy, vitreous haemorrhage, and choroidal calcifications Fig. . He had After 1 month he underwent enucleation to relieve the pain and a hydroxyapatite prosthesis, 18 mm in size, was implanted. A histopathological examination disclosed an undifferentiated carcinoma arising from the ciliary body. An immunohistochemical study was positive for cytokeratin pool and negative for S-100, HMB45, Melan-a, CD34, CD45, CD3, and CD20 Fig. . He undeParaneoplastic syndromes may be the first signs of malignancy. They are defined as systemic tumor-associated signs and symptoms not directly related to the site of a malignant tumor or its metastases. The symptoms may be endocrine, neuromuscular or musculoskeletal, cardiovascular, coetaneous, hematologic, gastrointestinal, renal, ocular, or miscellaneous. These phenomena are mediated by humoral factors (hormones or cytokines) excreted by tumor cells or by an immune response against the tumor and they could occur in a variety of malignant neoplasms .Paraneoplastic clinical signs are not directly related to the site or local manifestations of a malignant tumor or its metastases. Ocular paraneoplastic syndromes have been described as being due to circulating anti-retinal auto-antibodies and immune toxic factors released by the extraocular primitive tumors. Carcinoma-associated retinopathy, melanoma-associated retinopathy, cancer-associated cone dysfunction and bilateral uveal melanocytic proliferation are considered paraneoplastic syndromes .We report a case of systemic hypertension and fever as paraneoplastic manifestations in a patient affected by primary body ciliary neoplasm.Rare paraneoplastic syndromes, which can produce systemic hypertension, may be associated with hepatocarcinoma or renal malignancies, while it is usually alleged that more than half of patients with cancer will experience fever during the course of their disease . The reaTo the best of our knowledge, paraneoplastic untreatable fever and systemic hypertension have not been related to ocular malignancy.Moreover, previous accurate examinations and radiological imaging failed to show an orbital mass whereas a histopathological examination revealed a primitive ciliary body carcinoma.These tumors arising from the pigment epithelium of the ciliary body are exceptionally rare and benign, but their extension beyond the globe leads to a worse prognosis. It has been previously reported that in phthisical eyes, with or without a history of trauma or inflammation, a malignant transformation may occur probably due to reactive hyperplasia of the ciliary epithelium .In this case we did not consider our patient’s antibiotic-resistant fever and intractable systemic hypertension to be paraneoplastic syndrome which resolved after total surgical removal of the primitive tumor and relapsed with neoplastic recurrences.Physicians should be aware of systemic symptoms such as systemic hypertension unresponsive to drugs and fever in phthisic eye: they may be paraneoplastic signs of ocular malignancy because they are related to paraneoplastic or tumor necrosis.Furthermore, in the presence of a painful blind eye the differential diagnosis between neovascular glaucoma and any form of bulbar tumor has to be considered and histopathological examination should always be performed in order to prevent any malignant dissemination.Written informed consent was obtained from the patient’s next-of-kin for publication of this case report and any accompanying images. A copy of written consent is available for review by the Editor-in- Chief of this journal.


Resting metabolic rate (RMR) is the component of energy expenditure that explains the largest proportion of total daily energy requirements. Since RMR is determined largely by fat-free mass and a low RMR predicts weight gain in healthy adults, identifying the role of muscle atrophy following stroke on RMR may help identify ways to mitigate the development of obesity post-stroke.Thirty-nine stroke survivors with chronic hemiparesis  underwent DXA scans for measurement of body composition, including total, paretic, and non-paretic leg lean mass and fasted, 30-min indirect calorimetry for measurement of RMR.Predicted RMR was calculated by the Mifflin-St Jeor equation, which considers weight, height, and age for both men and women. RMR was 14% lower than predicted . Total , paretic  and non-paretic  leg lean mass predicted RMR.These data indicate that muscle atrophy post stroke may lead to a reduced RMR. This substantiates the need to attenuate the loss of lean mass after a stroke to prevent declines in RMR and possible weight gain common post-stroke. Stroke is the leading cause of long-term disability . We haveTotal daily energy expenditure (TDEE) consists of resting metabolic rate (RMR), the thermic effect of food, and physical activity. Resting metabolic rate (RMR) is the component of energy expenditure that explains the largest proportion of total daily energy requirements. Individuals with a low RMR are at higher risk of significant weight gain, relative to those with a high RMR ,10. AlthThis cross-sectional study included 39 stroke survivors, between the ages of 45–80 years, which were recruited from the Baltimore area for participation in exercise rehabilitation studies. Participants were in the chronic phase of stroke recovery (at least six months prior) and had residual hemiparetic gait deficits. All volunteers signed University of Maryland Institutional Review Board approved informed consent forms.Participants underwent a health history and physical examination, which included height, weight, blood pressure, and a resting electrocardiogram. Dual-energy X-ray absorptiometry (DXA) scans  were conducted to determine total body fat (%) and total, paretic, and non-paretic leg lean tissue mass.Subjects received two weeks of heart healthy diet  counseled by a Registered Dietitian prior to RMR testing. Subjects reported to our lab first thing in the morning following a 12 hour fast. RMR was measured by indirect calorimetry , while participants rested quietly in the supine position under a ventilated canopy for 30 minutes in a thermo-neutral and comfortable environment. Energy expenditure was calculated by the Weir equation  and exprDescriptive statistics were analyzed using SPSS . Results are expressed as mean ± SEM. Student’s t-tests were used to compare predicted to measured RMR. Linear regression was used to assess whether leg lean mass predicts RMR. Statistical significance was set at a two-tailed P < 0.05.Subject characteristics may be viewed in RMR was not associated with younger age  or latency from stroke . However, we observed that total body (r=0.70) and total leg r=0.73,  lean tisChronic hemiparesis is present in nearly 50% of all long-term ischemic stroke survivors . In addiOur observation that RMR is lower than predicted is relevant to stroke recovery since studies suggest that lower RMR is a predictor of greater sequential weight change . FurtherDespite the small sample size, this study is strengthened by the inclusion of a diverse study population, including a wide range of ages (middle aged to elderly), ethnicities, and body composition . Since the incidence of stroke is higher in men than women (aged less than 75 years) , we beliResearch efforts for effective treatment strategies to maintain a healthy body weight focus on diet and exercise programs. While we have previously reported improvements in body composition (muscle hypertrophy and decreased fat mass) following the initiation of exercise during the chronic phase of stroke –34, how In summary, we observed that stroke survivors are at risk for a low RMR in the chronic phase of stroke. It appears that preserving overall lower extremity muscle mass may contribute in the maintenance of RMR after a stroke. This substantiates the need to attenuate the loss of lean mass, possibly with lifestyle interventions, after a stroke to prevent declines in RMR and possible weight gain common post-stroke.


Formica rufa-group) mounds had been identified and correlated with tectonically active gas-permeable faults, mostly strike-slip faults. Linear alignment of RWA mounds and soil gas anomalies distinctly indicate the course of these faults, while clusters of mounds indicate crosscut zones of fault systems, which can be correlated with voids caused by crustal block rotation. This demonstrates that RWA are bioindicators for identifying active fault systems and useful where information on the active regime is incomplete or the resolution by technical means is insufficient.In a 1.140 km² study area of the volcanic West Eifel, approx. 3,000 Red Wood Ant  sites and active tectonic faults. The current stress field with a NW-SE-trending main stress direction opens pathways for geogenic gases and potential magmas following the same orientation. At the same time, Variscan and Mesozoic fault zones are reactivated. The results showed linear alignments and clusters of approx. 3,000 RWA mounds. While linear mound distribution correlate with strike-slip fault systems documented by quartz and ore veins and fault planes with slickensides, the clusters represent crosscut zones of dominant fault systems. Latter can be correlated with voids caused by crustal block rotation. Gas analyses from soil air, mineral springs and mofettes  reveal limiting concentrations for the spatial distribution of mounds and colonization. Striking is further the almost complete absence of RWA mounds in the core area of the Quaternary volcanic field. A possible cause can be found in occasionally occurring H2S in the fault systems, which is toxic at miniscule concentrations to the ants. Viewed overall, there is a strong relationship between RWA mounds and active tectonics in the West Eifel.In a 1.140 km² study area of the volcanic West Eifel, a comprehensive investigation established the correlation between red wood ant mound (RWA;  They are commonly interpreted as a result of magmatic or tectonic processes in the Earth’s upper mantle and lower crust caused by volcanism, crustal updoming, and rift processes  cf. for a gis occurs ,66. Solis occurs ,49. But s occurs  already s occurs .These results suggest that RWA mounds can be used as biological indicators of active, gaspermeable faults ,31,36. TAn area wide GeoBioScience-approach in a 1,140 km² study area in the West Eifel (West Germany) demonstrated the correlation of soil gas anomalies and spatial distribution of red wood ant (RWA) mounds along tectonically active, gas-permeable faults. More specifically, the azimuthal directions in which the RWA mound positions are aligned correspond to those of the main fault directions. The presence and spatial distribution of the RWA mounds at the reference site Oberehe and in the entire study area are valuable biological indicators for a tectonically sheared area. Linear alignments of RWA mounds directly depict the fault zone. RWA clusters are typically considered to represent crosscut zones of different fault systems, reflecting the large-scale fault regime in the smallest space. These RWA mound aggregations can be used to delimit buried small-scale tectonic structures of tectonically active systems especially in those cases, where information about the active tectonic regime is incomplete or the resolution by technical means of other geological and/or geophysical methods is insufficient. Furthermore, soil gas anomalies indicate particularly favorable settlement conditions for RWA. Their mounds can only be mapped where geogenic gas anomalies occur. These results also contribute to complement to further understand settlement processes, mound start-up and spatial distribution of RWA.


Escherichia coli (ETEC) are a leading cause of diarrheal illness in developing countries. Despite the discovery of these pathogens as a cause of cholera-like diarrhea over 40 years ago, and decades of vaccine development effort, there remains no broadly protective ETEC vaccine. The discovery of new virulence proteins and an improved appreciation of the complexity of the molecular events required for effective toxin delivery may provide additional avenues to pursue in development of an effective vaccine to prevent severe diarrhea caused by these important pathogens.Enterotoxigenic  Escherichia coli (ETEC) are a leading cause of diarrheal illness in developing countries where these diverse pathogens account for millions of infections and hundreds of thousands of deaths each year [The enterotoxigenic ach year , particuach year ,3. ETEC While it may be particularly important to prevent cholera-like disease to avoid deaths from diarrheal illness, the association of ETEC with delayed growth  and malnUnderstanding the precise molecular events involved in delivery of ETEC toxins could provide key insights that inform development of more effective vaccines. The recent identification of novel virulence factors required for optimal interaction of these organisms with target epithelial cells suggests that essential elements of toxin delivery are still being defined. Dissection of the details of ETEC pathogen-host interactions has provided additional molecules that can be targeted in new iterations of ETEC vaccines. E. coli is defined by genes encoding one of three toxins: the heat-labile toxin (LT), and the heat-stable toxins ST-Ia (ST-P), or ST-Ib (ST-H). ETEC strains may encode any or all of these toxins each of which has been associated with severe diarrheal illness.The ETEC pathotype of diarrheagenic 5 heterohexameric molecule that shares approximately 85% amino acid identity with cholera toxin (CT). The pentameric B subunit binds to GM-1 gangliosides on the surface of intestinal epithelial cells triggering the internalization of the catalytically active A subunit. Within the cell, the A subunit allosterically activates ADP-ribosylating factors (ARFs) which affect the ADP ribosylation of the intracellular guanine nucleotide binding protein, Gsα, abolishing its GTPase activity, and leading to constitutive activation of adenylate cyclase which increases intracellular cAMP. The resulting intracellular increases in cAMP then activate protein kinase A, in turn phosphorylating the cystic fibrosis transmembrane regulator (CFTR) [+ absorption through Na/H ion exchangers (NHE3) [LT is an ABs (NHE3)  that resST-Ia and ST-Ib are small (18–19 amino acid) peptides with multiple cysteine residues. These molecules are structurally similar molecular mimics of two native eukaryotic gastrointestinal peptides, guanylin and uroguanylin. Both ST-I molecules and their native homologues engage guanylyl cyclase C in the epithelial cell membrane and activate the enzyme activity leading to intracellular increases in cGMP. This cyclic nucleotide also activates protein kinases that phosphorylate and activate CFTR ,11, resuAlthough ETEC are defined as a pathotype by the production of the enterotoxins described above, the pathogenesis of ETEC can best be summarized as the total compilation of virulence features required for effective delivery of these toxins to their cognate receptors on the epithelial surface . In esseHeat-labile toxin is a principal target for ETEC vaccine development. Because of inherent immunogenicity and substantial adjuvant activity  some forTheoretically, neutralization of either the A or B subunit of LT could provide protection, either by engaging the active toxin moiety or by preventing binding of the toxin to its receptor, respectively. Indeed, experiments have demonstrated that these effects appear to be additive in preventing LT holotoxin-induced cAMP activation in target epithelial cells . Mutant While LT antitoxin immunity may afford some degree of protection against strains that produce only this toxin, the majority of strains produce either ST or both toxins, with approximately half of all strains producing only ST , where LST toxoids have faced a number of substantive challenges , includiStudies to date have demonstrated that it is possible, either by chemical conjugation of ST peptides to carrier molecules ,37,38,39Most recent attempts to generate ST antibodies have relied on genetic fusions of ST to other molecules with potent adjuvant activity such as LT. This approach has the distinct advantage of being relatively simple with respect to design and production of potential fusions permitting preclinical testing on a variety of constructs, but cannot achieve the high hapten: carrier ratios anticipated with chemical conjugation methods . NeverthA concerted effort to optimize the approach to formulating either conjugate or genetic fusions is ongoing ,48,49. IAlong with attempts to develop LT-based toxoids, plasmid-encoded fimbrial structures known as colonization factors (CFs), have been central to all efforts to develop an ETEC vaccine, including those currently in advanced stages of clinical development ,22,23,50Escherichia coli in the early 1970s [However, CF-based vaccines have faced a number of obstacles since these structures were identified  soon aftly 1970s ,54. Firsly 1970s  suggest ly 1970s  in candily 1970s .Additional data also suggest that these antigens may not alone be sufficient to stimulate the sustained robust protective responses that will likely be required of an ETEC vaccine. Epidemiologic studies of natural ETEC infections have differed with respect to whether CF immune responses correlate with protection against subsequent clinical illness in young children ,56,57, tThe challenges facing toxoid/CF-based approaches outlined above have stimulated a renewed interest in ETEC pathogenesis and fostered the identification of additional molecules that could be of importance for inclusion as subunits in a recombinant multivalent approach or targeted as part of a live-attenuated vaccine strategy. Outlined below , are demonstrably deficient in adhesion and delivery of LT to target epithelial cells [in vitro [EtpA is in the same family of proteins as filamentous hemagglutinin (FHA), a component of modern acellular pertussis vaccines. Recognition of this similarity to a known protective antigen stimulated initial interest in further molecular characterization of EtpA following its identification in ETEC H10407. Like FHA, EtpA acts as an extracellular adhesin. EtpA acts in a unique fashion by bridging the ends of the long (10 µm) peritrichous flagella of ETEC with one or more host cell surface receptors . Similaral cells . Likewisin vitro . Therefoin vitro , an ETECWhile much of ETEC vaccine development effort has focused on the plasmid-encoded CFs, ETEC are also known to produce other chromosomally-encoded fimbriae known as type 1 fimbriae (T1F) ,66. SimiInterestingly however, recent transcriptome studies of ETEC  demonstrin vivo [in vitro, virulence features that are thought to be important prerequisites for toxin delivery.The majority of human strains of ETEC are motile and can be serotyped with H antisera . Motilitin vivo  and impaEatA. The eatA gene, also discovered [p) acts in part to limit EtpA-mediated adhesion, and prevent these organisms from becoming too sticky. Somewhat counter-intuitively, eatA mutants are retarded in their ability to deliver LT, and antibodies directed against the passenger domain impair delivery of LT [Shigella flexneri [p has recently been shown to cleave MUC2, the major mucin secreted by goblet cells lining the lumen of the intestine [scovered  on the lry of LT . Howeverflexneri , that dontestine . MUC2 nontestine  also resYghJ, an antigen initially discovered by reverse vaccinology approaches with extraintestinal pathogenic E. coli [in vitro and in vivo, and antibodies directed at YghJ impair effective toxin delivery suggesting that this highly conserved antigen common to most ETEC strains examined to date could be a valuable target for vaccine development along with pathotype-specific mucin-degrading proteins like EatA. E. coli , is secr E. coli . YghJ be E. coli . YghJ acE. coli genomes in general [E. coli, such as YghJ and the T1F tend to be more highly conserved. The degree to which these can be targeted without impacting commensal strains of E. coli has not been determined.One potential impediment to effective ETEC vaccine development is the underlying plasticity of  general . As note general ,77. The Escherichia coli continue to present a formidable challenge to attempts to develop a broadly protective vaccine. However, ETEC vaccines to date have focused on a relatively small subset of antigens that are not sufficiently conserved or do not alone offer sufficient protective efficacy to provide broad-based protection from severe diarrhea caused by ETEC. An improved understanding of the molecular events involved in ETEC interactions with the intestinal epithelium that are required for efficient delivery of its enterotoxins has provided additional targets for interdiction that can complement existing strategies as well as emerging toxoid-based approaches to toxin neutralization. These novel targets and an abundance of genomic, and immuno-proteomic data on the horizon should inform a rational approach to designing next generation broadly protective ETEC vaccines.The enterotoxigenic


Background. Our purpose was to investigate the association between the surgical approach for Todani type IV cysts and subsequent malignancy rate. Methods. The records of patients who received cyst excision from 1994 to 2013 were analyzed retrospectively for the following data: demographics, presenting symptoms, postoperative outcomes, malignant transformation, and follow-up reexaminations, including imaging, laboratory, and tumor marker tests. Results. Seven of the 196 patients initially treated at our hospital developed postoperative biliary malignancy, and the surgical approaches were extrahepatic bile duct cyst resection combined with hilar cholangioplasty and Roux-en-Y cystojejunostomy (n = 5), and intra- and extrahepatic bile duct cyst resection and Roux-en-Y hepaticojejunostomy (n = 2). The overall malignancy rate was 3.6% (7/196). Forty-eight patients initially treated at other hospitals developed malignancy postoperatively: 15 (31.2%) remained untreated and 33 (68.8%) had undergone incomplete resection procedures. Because Todani type IV cysts were seen in 268 patients, the postoperative malignancy rate of this group of patients was 12.3% (33/268). Conclusions. Radical resection of both intra- and extrahepatic cysts combined with hepatic resection and Roux-en-Y hepaticojejunostomy is associated with a reduced risk of subsequent cancer development. Procedures in which radical cyst excision is not performed are associated with a greater risk of subsequent malignancy. Congenital cystic dilatation of the bile duct, also known as choledochal cyst, is highly prevalent in Asia, especially in China , 2. CholMost surgical interventions aimed at removing a choledochal cyst are successful in preventing the postoperative development of CCC . HoweverThe aim of this study was to explore the relationship between the postoperative malignancy rate of Todani type IV bile duct cysts and the type of surgical method used to remove the cyst and to stress the importance of both intra- and extrahepatic cysts combined with hepatic resection and Roux-en-Y hepaticojejunostomy.The ethics committee of our hospital approved this retrospective study. Every patient who received surgery signed an informed consent form detailing the surgery and procedures carried out during the operation. For patients who received surgery at other hospitals, ethics approval from other hospitals was not required as only the recorded information of past history of medical and surgical treatment was analyzed.We reviewed all patients with a definite diagnosis of a choledochal cyst from January 1994 to June 2013. Data were collected using patients' medical records and operative reports. Patients' demographic information, presenting symptoms, surgical approaches for removal of choledochal cysts, postoperative outcomes, malignant transformation, and follow-up information were collected.During postoperative follow-up, the patients were asked to come to our center for reexamination. For the first two years, reexamination occurred every three months and the examination items included computed tomography (CT) and magnetic resonance imaging (MRI) as well as biochemistry blood testing and tumor marker testing, such as CEA and CA19-9. Two years after surgery, for the patients from outside Beijing, reexamination occurred at the local hospitals instead of our center. If any abnormalities were observed in these patients, they would come to our center for reassessment. Our center keeps track of these nonlocal patients, and the reassessment items are consistent with those taken at the local hospitals. For the patients who remained asymptomatic for a certain period after surgery, reexamination could occur every six to twelve months.The demographic and operative data were presented using descriptive analysis. For continuous variables, the mean ± standard deviation (SD) was calculated, and categorical data were presented as the number with or without percentage.As shown in According to the data collected at our center over the past 20 years, the overall malignancy rate of Todani type IV bile duct cysts was 17.9% (48/268), and the postoperative malignancy rate was 12.3% (33/268).Of the 196 patients with follow-up data, 117 underwent extrahepatic bile duct cyst resection combined with hilar cholangioplasty and Roux-en-Y cystojejunostomy. The other 79 patients had intra- and extrahepatic bile duct cyst resection and Roux-en-Y hepaticojejunostomy with partial liver resection. The median follow-up duration of the 196 patients was 100 months . As shown in The results of this study indicate that cancer development after surgical treatment of Todani type IV bile duct cysts is related to the surgical approach. Radical resection of both intra- and extrahepatic cysts combined with hepatic resection and Roux-en-Y hepaticojejunostomy is associated with a reduced risk of subsequent cancer development. Procedures in which radical cyst excision is not performed are associated with a greater risk of subsequent cancer development.To resolve the finding of a higher cancer rate after surgery in Todani type IV duct cysts, we analyzed the 33 patients who developed cancer after surgery at other hospitals. The results showed that simple internal drainage was the most commonly used surgical method, accounting for 54.6%, followed by extrahepatic bile duct cyst resection plus cholangioenterostomy, accounting for 30.3%, simple external drainage, accounting for 12.1%, and extrahepatic bile duct cyst excision and Roux-en-Y hepaticojejunostomy with partial hepatic resection, accounting for 3%. Of note is that the above 33 patients who developed cancer after surgery for Todani type IV cysts did not have radical removal of the bile duct cysts at the time of their initial surgery nor did they have adequate biliary drainage. We also found that all of the 33 patients had cholangitis, and this may be related to the subsequent malignancy because it is well known that recurrent cholangitis is a risk factor of cholangiocarcinoma . These fExtrahepatic bile duct cyst resection plus Roux-en-Y hepaticojejunostomy has become a relatively common surgical treatment for Todani type IV bile duct cysts –22. SurgThe ideal surgical method for treatment of Todani type IV bile duct cysts should (1) achieve diversion of the bile and pancreatic juice and (2) enable adequate biliary drainage. Based on these two criteria, radical resection of the involved bile ducts and restoration of normal physiological function of the bile ducts are necessary. Thus, radical resection of intra- and extrahepatic bile duct cysts combined with partial hepatic resection and Roux-en-Y hepaticojejunostomy represents the ideal surgical method for the treatment of Todani type IV bile duct cysts.If the intrahepatic bile duct lesions are confined to a segment or a lobe of the liver, radical resection of intra- and extrahepatic bile duct cysts combined with partial hepatic resection and Roux-en-Y hepaticojejunostomy can achieve curative resection of the bile duct lesions. Patients who received radical resection of Todani type IV bile duct cysts had a zero rate of cancer (data not shown). However, in actual clinical practice, not all Todani type IV patients have bile duct lesions confined to a hepatic segment or lobe. Rather, a certain proportion of patients have widely distributed intrahepatic bile duct lesions, and because of the limitations of liver resection a partial hepatectomy cannot achieve radical resection of all bile duct lesions. The only surgical method that satisfies all the above conditions is liver transplantation, which is unrealistic for most Todani type IV cyst patients. Therefore, there are only two surgical options: extrahepatic bile duct cyst excision plus hilar cholangioplasty and Roux-en-Y hepaticojejunostomy or partial hepatectomy plus intra- and extrahepatic bile duct cyst resection and Roux-en-Y hepaticojejunostomy. Both can achieve diversion of bile and pancreatic juice and improve biliary drainage.Our data showed there were seven cases of postoperative cancer in Todani type IV bile duct cysts patients, with an overall cancer rate of 3.6%. Thus, although a certain percentage of patients developed postoperative cancer after treatment using these two surgical methods, the cancer rate was greatly reduced. Of particular note, due to widely distributed intrahepatic bile duct lesions, complete resection was not achieved in these seven patients although the surgical techniques were the same as those in the remaining 189 patients. This is the fundamental reason for cancer developing following surgery for Todani type IV bile duct cysts and confirms the conclusion that radical resection of intra- and extrahepatic bile duct cysts combined with partial hepatic resection and Roux-en-Y hepaticojejunostomy represents the ideal surgical method for surgical treatment of Todani type IV cysts.This study has several limitations including its retrospective nature, which precludes the data collection of the total number of patients receiving operation at other hospitals. In addition, the follow-up length of several patients was relatively short, which may have affected the results. Future research findings with longer follow-up times are, therefore, needed to confirm our findings.In summary, postoperative malignancy in Todani type IV bile duct cysts appears closely related to the surgical approach used. Radical resection of both intra-and extrahepatic cysts, combined with hepatic resection and Roux-en-Y hepaticojejunostomy, is associated with a reduced rate of subsequent malignancy and thus may constitute the ideal surgical method for the treatment of Todani type IV bile duct cysts. When radical resection of the cysts cannot be achieved, the rate of cancer development after surgery can be reduced as long as adequate bile drainage is provided during surgery.