PA2G4 gene. Ebp1 is the human homologue of a previously identified cell cycle-regulated mouse protein p38-2G4. Two transcripts of ebp1 mRNA (1.7 and 2.2 kb) were detected in several normal human organs. The interaction of Ebp1 with ErbB-3 was examined in vitro and in vivo. The first 15 amino acids of the juxtamembrane domain of ErbB-3 were essential for Ebp1 binding in vitro. Treatment of AU565 cells with the ErbB-3 ligand heregulin resulted in dissociation of Ebp1 from ErbB-3. Ebp1 translocated from the cytoplasm into the nucleus following heregulin stimulation. These findings suggest that Ebp1 may be a downstream member of an ErbB-3-regulated signal transduction pathway. © 2000 Cancer Research CampaignThe processes by which ErbB-3, an inactive tyrosine kinase, exerts its biological effects are poorly understood. Using the yeast two-hybrid system, we have isolated an ErbB-3 binding protein (Ebp1) that interacts with the juxtamembrane domain of ErbB-3. This protein is identical to that predicted to be encoded for by the human


Although limited in capacity, visual working memory (VWM) plays an important role in many aspects of visually-guided behavior. Recent experiments have demonstrated an electrophysiological marker of VWM encoding and maintenance, the contralateral delay activity (CDA), which has been shown in multiple tasks that have both explicit and implicit memory demands. Here, we investigate whether the CDA is evident during visual search, a thoroughly-researched task that is a hallmark of visual attention but has no explicit memory requirements.The results demonstrate that the CDA is present during a lateralized search task, and that it is similar in amplitude to the CDA observed in a change-detection task, but peaks slightly later. The changes in CDA amplitude during search were strongly correlated with VWM capacity, as well as with search efficiency. These results were paralleled by behavioral findings showing a strong correlation between VWM capacity and search efficiency.We conclude that the activity observed during visual search was generated by the same neural resources that subserve VWM, and that this activity reflects the maintenance of previously searched distractors. We use visual working memory (VWM) processes to integrate information across events such as eye blinks and eye movements, where sensory input to the visual system is interrupted spatial short-term memory, rather than visual working memory, as tasks which probe spatial short-term memory interfere with this process Numerous studies have explored the role of VWM in visual search, resulting in several different and sometimes competing theories about what effect it may have in the search process number of items currently held in VWM for tasks without any explicit memory requirement, providing insights into the cognitive processes underlying these tasks. More generally speaking, if VWM is indeed a limiting factor in the performance of a given task (as it is for change-detection tasks), performance on these tasks should be constrained by individual VWM capacity, which will be reflected in the CDA amplitude. This means that the CDA can not only be used to predict behavior in typical tests of visual memory but also be used to predict behavior in an attentional task with continuous visual input.Although these behavioral studies indirectly assess the role of VWM under specific conditions, they cannot provide a direct measure of ongoing VWM processing. In contrast, by testing for the presence or absence of the characteristic neural correlate of VWM during search, we can identify whether this cognitive faculty is engaged during a typical visual search task. If the event-related potentials (ERPs) observed in the presence of a continuously presented search display do in fact reflect VWM processing, they should be sensitive to the same manipulations that modulate the CDA during VWM-dependent change detection. That is, the amplitude should be sensitive to the number of items maintained in VWM, and should also reflect individual differences in VWM capacity. Therefore, the CDA may provide a measure of the Overall, the goal of the current study was to determine whether the electrophysiological marker of VWM, the CDA, is present during visual search, and to clarify the relationship between the CDA and visual search performance. The direct comparison between the CDA recorded during a lateralized search task and the CDA recorded during the delay period of a VWM-dependent change-detection task allows for the examination of whether, and to what extent, VWM resources are in fact employed during visual search. The results demonstrate that, for the majority of participants, the visual search and change-detection tasks elicited nearly identical CDAs. Furthermore, VWM capacity predicted both the increase in CDA amplitude during visual search and the behavioral measures of search performance, suggesting that the same neural and cognitive VWM-related resources are used during visual search and change-detection.Eighteen volunteers participated in this experiment for partial course credit and/or for monetary remuneration. All participants provided written and informed consent, and all procedures were approved by the University of Toronto Research Ethics Board. Seven participants were excluded from analyses (see below), resulting in a total of 11 participants . All participants were right-handed and reported normal or corrected-to-normal vision.Both the visual search and VWM tasks were displayed on a 20-inch CRT monitor located 57 cm from the participant. A resolution of 1600 by 1200 was used, with a display refresh rate of 60-Hz. All stimuli were presented on a grey background . Participants were encouraged to maintain central fixation during all trials.At the beginning of each trial, a left or right arrow cue was presented, indicating which side of the display participants should attend. The cue was presented 1° of visual angle above a central fixation cross for 200 ms. Immediately after the arrow cue, search items were presented to the left and right of the fixation cross, randomly arranged within a 3.5°×6.5° invisible grid centred 4° horizontally away from the fixation cross. On each trial, ten search items were presented on both the cued and uncued sides . The seaParticipants always performed the visual search task prior to performing the visual memory task, and were not informed in advance that the second task would test memory performance. Participants were in no way encouraged to utilize a memory strategy while performing the visual search task.The procedure used for the visual working memory task was a change-detection task similar to the one used by Vogel and Machizawa K-estimate formula K) by scaling accuracy, corrected for guessing, by the number of items tested: K = set size * (hits+correct rejections –1).Visual memory capacity was estimated by applying Cowan's The electroencephalogram (EEG) was obtained from 64 active Ag/AgCl electrodes (Biosemi ActiveTwo system), digitally recorded at 512 Hz, mounted on an elastic cap using the International 10/20 system. Digital file conversion was completed using PolyRex software Trials that were contaminated with eye blinks (>80 µV VEOG) or large horizontal eye movements greater than 2.0° (>32 µV HEOG) were excluded from analysis The ERPs were computed by averaging the EEG from 200 ms prior to the onset of the search display or memory sample display and ending 900 ms post onset. The ERPs were baseline-corrected to the 200 ms prior to the search display or memory sample displays onsets. For all ERPs, analysis was restricted to the 10 posterior channels that demonstrate maximal CDA activity in memory tasks Ipsilateral and contralateral waveforms were computed separately for the VWM task, and target present and target absent trials in the search task. Laterality was always defined relative to the attended (cued) side for both tasks. The CDA for each condition was computed by subtracting the ipsilateral waveforms from the contralateral waveforms. Latency and peak amplitude measures were obtained using the jackknife method In addition, to examine the relationship between the increase in CDA amplitude during the search task and VWM capacity, we obtained the change in mean amplitude of the CDA from two 100 ms windows between 300 to 400 ms and 400 to 500 ms in the target absent trials, and correlated these values with VWM capacity obtained in the change-detection task. The comparison of CDA amplitudes between these two early time windows allowed us to assess whether or not VWM capacity was reached quickly during the search task. Specifically, we predicted that participants with low VWM capacity would exhaust their resources with fewer examined search items and as such, their CDA should plateau sooner when compared to high-capacity participants.We also calculated the amplitude of the N2pc in the target absent search condition for each individual, defined as the maximal negative peak  between 200 and 300 ms after the onset of the search display. The N2pc reflects attentional selection process of performing the search, particularly in the first 1,000 ms, electrophysiological analysis was carried out on all trials.Participants correctly reported the presence or absence of the target (upright T) with 61% mean accuracy. The relatively low accuracy of 61% in the search task likely reflects a confluence of factors, including the difficulty of locating targets in the periphery of the display while maintaining eye-fixation on the central fixation cross. In line with previous studies, however, mean reaction times (RTs) in the target present condition  were significantly faster than in the target absent condition , t(10) = 13.5, p<.001. Given that the aim of the current study is to examine the electrophysiological correlates relating to the K formula <1, p = .78.To determine whether the electrophysiological activity recorded during visual search does in fact reflect the use of VWM resources, we compared it to the activity recorded over the same channels during the change-detection task, which is known to depend on VWM resources. Visual inspection of the ERPs recorded during both tasks reveal a striking similarity /B. That adjusted = 3.08, p<.1  and low (mean: 2.1) capacity subjects in r = −.84, p = .001. Similarly, the increase in CSA amplitude from 300–500 ms  was significantly correlated with search RT, r = .7, p = .017 . Our results reveal search-related activity  that is largely indistinguishable from the CDA observed in a four-item, memory-dependent change-detection task. That is, both tasks demonstrate a large, negative, sustained voltage difference between ipsilateral and contralateral channels. Furthermore, the distribution of activity was similar for the two tasks, indicating that the CSA during search and the CDA during a VWM change-detection task likely represent the engagement of the same neural and cognitive resources.In addition to being identified during change-detection, previous studies have observed activity similar to the CDA during a number of tasks that have both implicit and explicit memory requirements. For example, the CDA has been observed while measuring the deployment of attention during a rapid serial visual presentation (RSVP) stream Our results go beyond these previous findings in that they suggest that the neural marker of VWM can be observed even during tasks with continuous, static visual input. Thus, this activity can be used to measure the contribution of VWM to visual search, and potentially other tasks, even when all items remain visible and VWM is not explicitly manipulated. Alternatively, it is possible that the CSA reflects cognitive processing that is unrelated to VWM, such as perceptual or attentional load It remains possible, however, that both the CDA observed in change-detection and the CSA observed during search are mostly driven by attentional selection and not VWM per se. In other words, the more efficient an individual's selection process, the better their VWM performance and the faster their search. In fact, previous studies have shown that high-VWM capacity individuals select relevant items more efficiently, whereas low-capacity individuals show greater interference from irrelevant items As such, our results suggest that VWM resources are employed while performing a visual search task, and that for most individuals, the number of items maintained in VWM during search is similar to that of a change-detection task. Interestingly, our design did not in any way manipulate VWM load during search, and participants were not instructed to use strategies that would elicit VWM activity during the search task; consequently, it would seem that VWM resources were ‘automatically’ recruited to perform the visual search, having potentially important implications for theories of attention.As described earlier, we suggest that VWM supports visual search by remembering what search items have already been processed, allowing them to be subsequently ignored and biasing search towards new items.There are two alternatives to this interpretation. First, the increase in CSA amplitude may be unrelated to the maintenance of search distractors, and instead may reflect other contributions of VWM to search. The relationship between VWM capacity and search RT, however, provides evidence against this alternative. Previous studies have demonstrated how encoding and maintaining distractors in VWM can have a direct effect on search RT spatial short-term memory system that is distinct and independent from VWM Second, numerous behavioral studies have indicated that the mnemonic mechanism that prevents previously searched distractors from being revisited Although our study presents the first demonstration relating the CSA to VWM, previous studies have demonstrated CDA-like activity during visual search In summary, our visual search task elicited activity contralateral to the attended search array that was indistinguishable in mean and peak amplitude from the CDA observed during a four-item change-detection task, despite large differences in the number of items in the display and length of stimulus presentation. The change in amplitude of this CSA over time was strongly correlated with VWM capacity, suggesting that the activity reflected the same resources employed during VWM tasks. Furthermore, behavioral measures of search performance were strongly correlated with electrophysiological measures of VWM processing observed during the search task , as well as with behavioral measures of VWM performance obtained on an independently performed change-detection task, suggesting that VWM plays an integral role in visual search. This occurred despite the absence of explicit VWM requirements. Consequently, the finding that visual search gives rise to the electrophysiological marker of VWM indicates that the CDA may provide a useful tool for identifying the role of VWM in tasks which have continuously presented stimuli and no explicit memory requirement.


A fundamental function of the respiratory system is the supply of oxygen to meet metabolic demand. Morphological constraints on the supply of oxygen, such as the structure of the lung, have previously been studied in birds. Recent research has shown that uncinate processes (UP) are important respiratory structures in birds, facilitating inspiratory and expiratory movements of the ribs and sternum. Uncinate process length (UPL) is important for determining the mechanical advantage for these respiratory movements. Here we report on the relationship between UPL, body size, metabolic demand and locomotor specialisation in birds. UPL was found to scale isometrically with body mass. Process length is greatest in specialist diving birds, shortest in walking birds and intermediate length in all others relative to body size. Examination of the interaction between the length of the UP and metabolic demand indicated that, relative to body size, species with high metabolic rates have corresponding elongated UP. We propose that elongated UP confer an advantage on the supply of oxygen, perhaps by improving the mechanical advantage and reducing the energetic cost of movements of the ribs and sternum. Mm. appendicocostales projects from the proximal edge of the uncinate process, and inserts onto the following vertebral rib Mm. appendicocostales is associated with craniad movement of the ribs and a ventral displacement of the sternum during inspiration M. externus obliquus abdominus, which pull the sternum dorsally during expiration The avian respiratory system consists of a relatively rigid lung coupled with a series (in most birds) of nine compliant air-sacs Mb2/3), i.e. in proportion to body surface area Mb0.72. The projected 0.72 scaling coefficient found by Lasiewski and Dawson Respiration powers locomotion by providing metabolic energy. Resting metabolic rate (RMR) is often used to investigate the relationship between metabolism and body weight The supply of oxygen to gas exchange tissue is critically important for sustaining the metabolic rate of an animal. Numerous studies across a range of phyla have addressed the constraints upon oxygen delivery imposed by body size. For example, an examination of avian respiration by Lasiewski and Calder Mb and RMR in birds adapted to different modes of primary locomotion. An understanding of the relationship between the UPL and metabolic rate will shed further light on the evolution of morphological variation in these processes.UP are now confirmed to be important respiratory structures in birds involved in inspiration and expiration Measurements of UPL were taken from the left-hand side of skeletons from 112 species using a Mayr digital calliper . The length of process 4 was used in all statistical analyses. This process occurs in all specimens and has a relatively stable morphology Mb. This procedure is appropriate for analysis of morphological characters because the variation in both x and y variables is taken into account 10 transformed data were independently generated in each locomotor category. ANCOVA was used to test for significant differences between regression lines.Reduced major axis (RMA) regression 10 transformed Mb and UPL data, with the CRUNCH facility in the Comparative Analysis of Independent Contrasts (CAIC) software, version 2.6.9 Mb. RMA regressions of independent contrasts were performed through the origin The potentially confounding effects of the birds having a shared phylogenetic ancestry were controlled by calculating independent contrasts 10 transformed, and only data corresponding to true RMRs, as defined by them, were used. To ensure that the regression equations describing the relationship between UPL and Mb in the species subset did not differ significantly from those in the larger dataset, RMA analyses were repeated. This procedure is important for validating the assumption that an association between process length and RMR can be extrapolated to the larger dataset. RMA regression was used to investigate the linear relationship between Mb and RMR. Finally, the scaling of RMR with UPL was explored using RMA regression.The relationship between RMR and UPL was studied in a subset of 35 species, taken from the previous dataset. These species were selected because corresponding RMR values are available Mb on RMR. By accounting for the variation attributable to Mb and group structure, the GLM provides an estimate of the association between RMR and process length independent of Mb. In the first analysis, locomotor category  was designated as a group factor, while Mb and UPL were the covariates (RMR = locomotor group+UPL+Mb). A second GLM analysis repeated the prior method with the group factor comprising diving and non-specialist species. Lastly, the influence of UPL and Mb on RMR, in the absence of a grouping variable, was examined (RMR = UPL+Mb).General linear models (GLM) were used to assess the influence of locomotor mode, UPL and Mb on RMR whilst controlling for phylogeny we employed the following method: first the linear relationship between log10Mb and log10 UPL was determined (UPL = 0.2137 Mb+0.4336). Residual UPL was then calculated by subtracting the length predicted from the log10Mb/log10 UPL relationship from actual UPL for each species. The relationship between log10 RMR and residual uncinate process length was then calculated for each of the three locomotor groups using CAIC. RMA regressions were calculated to assess the linear relationship between the independent contrasts generated for RMR and residual UPL within each locomotor mode.A phylogenetically controlled analysis in the form of the GLM (above) was not possible. Therefore, to assess the influence of locomotor mode, UPL and GLM analyses were conducted in SPSS  and ANCOVAs, and RMA regressions in MATLAB® 2007b . Ninety five percent confidence limits are displayed in parentheses immediately after the scaling exponent.Mb in diving birds, intermediate in non-specialists and shortest in walking birds . Greater variation around the regression line was evident for the walking category, as indicated by the lower correlation coefficient, r  for all locomotor groups  UPL scaled isometrically with Mb in the diving and non-specialist birds . The group and Mb interaction term was included to ensure that the slope of RMR against Mb for the three groups was not significantly different. The regression line exponents for RMR regressed on Mb did not match the expected (1.0) for geometric similarity . The scaling exponent determined for RMR against UPL was significantly less than 2 for both diving and non-specialist groups  and RMR, and the group differences determined above, UPL is significantly positively correlated (coefficient = 6.77) with RMR . Given the non-significant association between RMR and UPL for walking birds . Therefore, the group factor was removed from the GLM, leaving bM and UPL as covariates. As found for the GLM including all three groups, after accounting for the variation in RMR attributable to bM, the length of the UP was again significantly positively correlated (coefficient = 3.21) with RMR .A GLM suggested that after controlling for the well-established relationship between size  may explain why there is no association between uncinate process length, body size, and RMR in these birds. The GLM analysis indicates that when we control for body size, increases in the length of the UP correspond to an increase in RMR. Therefore, birds with higher RMR for a given body mass have proportionately longer UP. Consequently, relatively longer UP appear to be associated with an elevated metabolic rate. Perhaps elongation of the UP facilitates an increase in metabolic demand by improving ventilation via the action of the UP in moving the ribs and sternum. Improving the work involved in ventilation may also explain the long UP found in diving species such as the penguins Of course, if it is advantageous, the question of why all birds don't have proportionally longer UP arises. Presumably, process elongation comes at a cost, possibly in terms of changing the shape of the associated musculature. Furthermore, UP act as levers for rib and therefore sternal movement and longer levers will not always provide a mechanical advantage depending on the overall body shape of the birds. Crucially, the rib cage, sternal morphology and associated musculature, appear to be driven by adaptations to different forms of locomotion.


Chromodomain-containing Gypsy LTR retrotransposons or chromoviruses are widely distributed among eukaryotes and have been found in plants, fungi and vertebrates. The previous comprehensive survey of chromoviruses from mosses (Bryophyta) suggested that genomes of non-seed plants contain the clade which is closely related to the retrotransposons from fungi. The origin, distribution and evolutionary history of this clade remained unclear mainly due to the absence of information concerning the diversity and distribution of LTR retrotransposons in other groups of non-seed plants as well as in fungal genomes.in silico analysis of chromodomain-containing LTR retrotransposons in 25 diverse fungi and a number of plant species including spikemoss Selaginella moellendorffii (Lycopodiophyta) coupled with an experimental survey of chromodomain-containing Gypsy LTR retrotransposons from diverse non-seed vascular plants . Our mining of Gypsy LTR retrotransposons in genomic sequences allowed identification of numerous families which have not been described previously in fungi. Two new well-supported clades, Galahad and Mordred, as well as several other previously unknown lineages of chromodomain-containing Gypsy LTR retrotransposons were described based on the results of PCR-mediated survey of LTR retrotransposon fragments from ferns, horsetails and lycophytes. It appeared that one of the clades, namely Tcn1 clade, was present in basidiomycetes and non-seed plants including mosses (Bryophyta) and lycophytes (genus Selaginella).In present study we preformed The interkingdom distribution is not typical for chromodomain-containing LTR retrotransposons clades which are usually very specific for a particular taxonomic group. Tcn1-like LTR retrotransposons from fungi and non-seed plants demonstrated high similarity to each other which can be explained by strong selective constraints and the 'retained' genes theory or by horizontal transmission. Retrotransposons are a class of mobile genetic elements, which use reverse transcription in their transposition. Five orders of retrotransposons are recognized: those having long terminal repeats (LTRs) (LTR retrotransposons); those lacking LTRs (non-LTR retrotransposons); DIRS retrotransposons; Penelope-like retrotransposable elements; and short interspersed nuclear elements (SINEs). According to the modern classification, LTR retrotransposons are divided into several superfamilies: Copia (Pseudoviridae), Gypsy (Metaviridae), Bel-Pao, Retrovirus (Retroviridae), and ERV .Chromodomain-containing LTR retrotransposons or chromoviruses are the most widespread lineage of Gypsy LTR retrotransposons and are present in genomes of fungi as well as in plants and vertebrates ,3. The cOur previous comprehensive survey of chromoviruses from mosses (Bryophyta) suggested that the diversity of CHD-containing Gypsy LTR retrotransposons in plant genomes is underestimated ,9. Therein silico analysis of Gypsy LTR retrotransposons in 25 species of fungi, genomes of which available in public databases, along with survey of related LTR retrotransposons from whole genome sequences (WGS) and expressed sequence tags (ESTs) of diverse plants including spikemoss Selaginella moellendorffii (Lycopodiophyta) and PCR-based screening of ferns, horsetails and lycophytes showed that a common clade of CHD-containing Gypsy LTR retrotransposons can be found in mosses, lycophytes from genus Selaginella, and basidiomycetes. According to the classification of CHD-containing Gypsy LTR retrotransposons proposed by Gorinsek et al. (2004) [The questions addressed in current investigation are as follows: (1) what kind of Gypsy LTR retrotransposons from fungi are closely related to the LTR retrotransposons detected in mosses; (2) how widely those clades which were previously identified in mosses are distributed among other non-seed plants including lycophytes, ferns and horsetails; and 3) what is the origin of the clade which is common for non-seed plants and fungi. The  what is http://ugene.unipro.ru/. The transposable elements thus identified were then classified into families based on RT and Int domains sequence similarity. Members of the same family shared high amino acid identity (90-100%) but had very little similarity to elements from other families. Our survey has identified more than 150 novel Gypsy LTR retrotransposon families which have not been described previously  . The surntity 90-00% but hLaccaria bicolor and CopConTy3-14 from Coprinus cinereus) formed their own lineage closely related to Ylt1-like LTR retrotransposons. This lineage cannot be attributed to Ylt1 because of low bootstrap support  [Candida albicans (Tca3 element) [Cryptococcus neoformans [The newly identified Gypsy LTR retrotransposons fall into two major, distinct lineages according to phylogenetic analysis based on RT and partial Int domains: chromoviruses (or CHD-containing Gypsy LTR retrotransposons) and Ylt1-like LTR retrotransposons. Two retrotransposons (LacBicTy3-15 from nsposon) , Candidaelement) , and basoformans . In the Batrachochytrium dendrobatidis JEL423)  were found in Gag for some of the identified Gypsy LTR retrotransposons  and carboxyl-terminus of Pol (PosPlaTy3-4) or between PR and RT domains (PosPlaTy3-5)  has been found in several LTR retrotransposons from the basidiomycete nosporum ,15. The nosporum . It was nosporum .Physcomitrella patens formed a common branch with Tcn1-like LTR retrotransposons from fungi . A Tcn1-like LTR retrotransposon search was implemented with BLAST (blastp and blastx). Amino acid sequences of RT and Int domains of known Tcn1-like  and newly identified retrotransposons (SpoRosTy3-4 and BatDenTy3-1) were used as the queries. The Tcn1-like LTR retrotransposons were identified only in the whole genomic sequence of Selaginella moellendorffii  encodes a 323 amino acid (aa) protein with strong similarity to retroviral Gag proteins (pfam03732). ORF2 or pol (3714 bp) encodes a 1238 aa polyprotein, with characteristic retroviral aspartyl protease (PR), RT, Int, and CHD domains  using SM-Tcn1 putative Pol protein as a query yielded more than 500 hits. The size of S. moellendorffii genome is only ~100 Mbp [The whole sequence of SM-Tcn1 LTR retrotransposon was obtained from WGS. SM-Tcn1 is 5704 bp in length and carries two putative ORFs. ORF1 or ~100 Mbp , thus SMP. patens and S. moellendorffii, did not provide a satisfactory answer to the question concerning distribution of this clade among non-seed vascular plants. Therefore, we used PCR with degenerate primers to investigate the distribution of CHD-containing Gypsy LTR retrotransposons in 26 ferns and horsetails (monilophytes) belonging to three classes: Psilotopsida, Polypodiopsida and Equisetopsida, and in 10 lycophytes from two classes: Isoetopsida and Lycopodiopsida , two ophioglossoid ferns  and three horsetails  were included [Isoetes and Huperzia species (Isoetaceae) as well as two Selaginella species (Selaginellaceae), which belong to the class Isoetopsida, and seven diverse species from Lycopodiaceae .The estimated diversity of monilophytes (= Infradivision Moniliformopses) is about 9000 species and includes horsetails, whisk ferns, and all eusporangiate and leptosporangiate ferns . Most ofincluded . Lycophyincluded . Among lThe presence of CHD-containing Gypsy LTR retroelements among the listed plants was tested by amplifying genomic DNA with previously developed degenerate oligonucleotide primers ,24. ConsDrosophila melanogaster were used as an outgroup. The newlyidentified LTR retrotransposon grouped into four large clusters on the phylogenetic tree. The group of clones from diverse monilophytes and one LTR retrotransposon from lycophytes Lycopodium alpinum (LycAlpTy3-1 clone) form a common group with previously described retroelements from mosses Tetraphis pellucida and Vesicularia dubyana [The phylogenetic relationships among obtained clones and known CHD-containing Gypsy LTR retrotransposons, extracted from databases, were reconstructed using neighbor-joining (NJ) analysis based on the multiple alignment of nucleotide sequences of RT fragment Figure . The Gyp dubyana . Althoug dubyana .Lycopodium clavatum (LycClavGty3 clones), L. japonicum (LycJapGty3-1 clone), and Diphasiastrum complanatum (DiphComGty3 clones). Another lineage seems to have had a long-term association with fern genomes, since it appears to be widely distributed among leptosporangiate ferns and can be found in Dennstaedtiaceae (Pteridium aquilinum), Pteridaceae (Adiantum pedatum), Aspleniaceae (Asplenium viride), Woodsiaceae (Athyrium distentifolium and Cystopteris fragilis), and Dryopteridaceae (Dryopteris crassirhizoma and Polystichum tripteron) , Aspleniaceae (A. viride) and Dryopteridaceae . The second cluster has a bootstrap support of 77% and is represented by clones isolated from Lycopodium annotinum (Lycopodiaceae) and Woodsia polystichoides (Woodsiaceae). The last monophyletic cluster, clade Mordred, is the largest, well-supported clade which is widely distributed among representatives of all investigated classes except Isoetopsida. It was found to be present in fern genomes from families Ophioglossaceae (Ophioglossum vulgatum and Botrychium multifidum), Pteridaceae (Adiantum pedatum), Woodsiaceae , Onocleaceae (Onoclea sensibilis), and Dryopteridaceae (Dryopteris filix-mas and D. crassirhizoma); in horsetails Equisetum hiemale and E. fluviatile (Equisetaceae); and lycophyte Lycopodium magellanicum (Lycopodiaceae).The largest group is represented by 37 LTR retrotransposons. Three clearly separated clusters can be found inside this group  and S. pulvinata (SelPulGty3 clones) in addition to the previously described LTR retrotransposons from mosses and SM-Tcn1 LTR retrotransposon from Selaginella moellendorffii . Such a wide distribution makes the Tcn1 clade unique among the CHD-containing Gypsy LTR retrotransposons. The interkingdom distribution of Tcn1 clade could be the result of horizontal transmission (HT) of LTR retrotransposons among fungi and plants; otherwise, Tcn1-like LTR retrotransposons could have been 'retained' by mosses and lycophytes from the most recent common ancestor of plants and Fungi/Metazoa lineage of eukaryotes [As a rule chromoviruses clades are specific for a particular group of eukaryotic organisms such as Ascomycota fungi , Basidiomycota fungi , or plants  . In the karyotes ,28.Physcomitrella contained a fraction of transcripts derived from putative genes ('retained' genes), which are not present in seed plants but can be found in other kingdoms including fungi. It was proposed that such retained genes along with Physcomitrella-specific (or moss-specific) genes encode functions that make mosses unique in terms of physiology and metabolism [Physcomitrella, which showed a high similarity with functional genes from fungi: uric acid-xanthine permease  and inorganic phosphate transporter .The hypothesis of 'retained' genes is based on the observation that EST data of tabolism . We usedCoprinus cinereus Okayama7#130, Phanerochaete chrysosporium and Cryptococcus neoformans revealed 51.3% to 64.9% similarity whereas only 23.0% identical amino acid residues was found on average in pairwise comparisons between fungal proteins and putative Pho88 from Physcomitrella (Table P. patens was found in Schizosaccharomyces pombe (26.8% of similarity). The similarity between uapA from Physcomitrella and Cryptococcus (36.9%) was almost the same as between proteins from Cryptococcus and Coprinus (39.6%) or Cryptococcus and Phanerochaete (43.2%). More then 67% of amino acid residues are identical in permeases from Coprinus and Phanerochaete. Predicted uapA from Ashbya gossypii and Physcomitrella share 40.7% of amino acid residues.The pairwise comparisons between hypothetical Pho88 proteins from basidiomycetes It seems to be that the hypothesis of 'retained' genes cannot be implemented as explanation for Tcn1 clade distribution since investigated RT-Int fragments of Tcn1-like LTR retrotransposons from fungi and plants have higher similarity to each other than functional proteins which were proposed to be 'retained' . RT-Int Despite a number of whole genome sequence studies, the distribution and diversity of CHD-containing Gypsy LTR retrotransposons is still poorly understood. Current knowledge of distribution and evolution of this group of mobile elements has been mainly obtained from diverse model organisms . The quiSelaginella)  and lycophytes (genus ) Figure . Such anosophila  and has osophila ,24. Recea [Oryza  and Routa [Oryza .Several criteria can be used for HT event recognition. The first criterion is inconsistencies between the phylogenies of transposable elements (TEs) and host species ,34. TherPhyscomitrella represented an attractive alternative to horizontal transmission as an explanation of the phylogenetic inconsistencies as well as the existence of a number of functional genes in Physcomitrella genome, which seem to have non-plant origin and can be found in bacteria, fungi and protozoa but not in higher plants [Selaginella since only representatives of this genus carry this group of retrotransposons among all investigated lycophytes . PyrTriTy3-2 belongs to Pyggy clade and appeared to be more closely related to LTR retrotransposons from Sordariomycetes (NecHaemTy3-4 from Nectria haematococca MPVI and ChaGloTy3-8 from Chaetomium globosum CBS 148.51) than to the elements from other Dothideomycetes such as AltBraTy3-2 from Alternaria brassicicola ATCC 96866, REAL from Alternaria alternata (AB025309) [Pyrenophora graminea (AF533704) [Another putative case of HT based on the results of present survey of LTR retrotransposons from fungal species was found for PyrTriTy3-2 LTR retrotransposon from B025309) , and PYGF533704)   to Gnetum (gymnosperms) [While extremely rare, horizontal transfer seems to be quite common and recurrent in eukaryotes. An incomplete list of putative HT events includes: HT as a key event in the evolution of several fungal genes -48; HT fase gene ; numerouase gene -38; as wiew, see ); and a osperms) .Amborella trichopoda, it has been demonstrated that direct plant-to-plant transfer can take a place [The actual mechanisms of horizontal transfer for eukaryotic genes and transposable elements are still unknown since it is not possible to show experimentally how HT can occur. Parasites, symbionts, bacteria, or viruses all could be suggested as potential vectors for horizontal transfer. Moreover, based on an example of massive HT from a land plant donor to the basal angiosperm  a place . The ass a place -55. Both a place ).Selaginella. Such interkingdom distribution is not typical for chromodomain-containing LTR retrotransposons clades which are usually very specific for a particular taxonomic group and can be explained by strong selective constraints and the 'retained' genes theory or by horizontal transmission. The close examination and comparison of evolutionary rates for LTR retrotransposons including representatives of Tcn1 and two other clades of LTR retrotransposons, and evolutionary rates estimated for putatively 'retained' genes from mosses and fungi suggests that a horizontal transmission of Tcn1-like LTR retrotransposons took place among fungi and mosses/lycophytes. However the evidence is not strong enough to discard other explanations; such as selective pressure coupled with vertical transmission of retrotransposons in genomes of non-seed plants and loss of these elements by other plants.Tcn1-like LTR retrotransposons were found in basidiomycota fungi and non-seed plants, including all investigated mosses and lycophytes from genus Fungal genomic sequences are available at: Fungal Genome Initiative ; The DOEWe used UniPro uGENE software  for LTR The whole nucleotide sequences of the transposable elements, if possible, were also extracted with the assistance of UniPro uGENE software. After localization of amino acid sequences obtained during HMMER search in the initial genomes in its nucleotide representation, the sequences were expanded up to 15 Kb and used for long terminal repeats (LTRs) search. The algorithm for repeats search, 'Repeat Find', is included to the UniPro uGENE as well as the visualization feature and 'ORF Find' option which were used to identify the putatively intact copies of LTR retrotransposons. Structural features of newly identified LTR retrotransposons can be found in Additional file Selaginella moellendorffii (Lycopodiophyta) is located in scaffold_0 (1426925-1421008) of genomic sequence version 1.0 which is available at The U.S. Department of Energy Joint Genome Institute web-site [Porphyra yezoensis at Kazusa DNA Research Institute [Cyanidioschyzon merolae Genome Project [Tcn1-like LTR retrotransposon search was carried out using BLAST (blastp and blastx). BLAST analysis was performed using sequence databases accessible from the National Center for Biotechnology Information (NCBI) server , The U.Sweb-site . The whoweb-site , NCBI coweb-site , ESTs frnstitute , Cyanidi Project , The Pla Project , The Ins Project , CassavaAll multiple DNA alignments were performed by ClustalW  and editTable 4)2SO4, 4 mM MgCl2, 200 μM each of four dNTPs, 0.5 μM primers, and 2.5 units of Taq polymerase. After an initial denaturation step for 3 min at 94°C, the PCR reactions were subjected to 30 cycles of amplification consisting of 30 sec denaturation at 94°C, 42 sec annealing at 50°C, and 1 min extension at 72°C. PCR products were separated by agarose gel electrophoresis. The resulting PCR products were directly ligated into a pGEM vector using a pGEM-T-Easy cloning kit (Promega) for sequence determination.Previously designed degenerate PCR primers for chromodomain-containing Gypsy LTR retrotransposons were used in present study: GyRT1 = 5'-MRNATGTGYGTNGAYTAYMG-3'  and ty3-GQ443314-GQ443445 and AY959294-AY959313.Clones were amplified by PCR with M13 primers, and 40 ng of the product was used in a 10 μl cycle sequencing reaction with the ABI BigDye Terminator Kit on an ABI 310 Genetic Analyser (Applied Biosystems); or sequencing reactions were performed with Dye Terminator Cycle Sequencing Kit (Beckman Coulter) and analyzed on CEQ 8000 Genetic Analysis System. Sequences were deposited to GenBank under Acc. Numbers LTR: long terminal repeat; SINE: short interspersed nuclear element; CHD: chromodomain; RT: reverse transcriptase; Int: integrase; ORF: open reading frame; PR: proteinase; dUTPase: deoxyuridine triphosphatase domain; PPT: polypurine tract; PBS: primer-binding site; HT: horizontal transfer or horizontal transmission; TE: transposable element; LCA: last common ancestor; AM: arbuscular mycorrhiza; HMM: hidden Markov models; Myr: million years; Mya: million years ago.ON participated in the design of the study, carried out the analysis, participated in the sequence analysis and drafted the manuscript. GS contributed to data acquisition, participated in analysis and interpretation. AB participated in the design of the study, performed coordination and has given final approval of the submitted version. All authors read and approved the final manuscript.Structure of novel LTR retrotransposons from Fungi. Table contained list of novel LTR retrotransposons from Fungi detected in present study, their copy numbers and putative structure including predicted enzymatic domains.Click here for filePhylogenetic analysis of dUTPase. Neighbor-Joining phylogenetic tree reconstructed based on dUTPase amino acid sequences from eukaryotes, viruses, and dUTPase domains from CHD-containing Gypsy LTR retrotransposons.Click here for fileSM-Tcn1 CHD-containing Gypsy LTR retrotransposon. Sequence of SM-Tcn1 CHD-containing Gypsy LTR retrotransposon from spikemoss Selaginella moellendorffii (Lycopodiophyta) with annotations in GenBank format.Click here for filePhylogenetic analysis of Athila-like LTR retrotransposons. Neighbor-joining (NJ) phylogenetic tree based on RT nucleotide sequences of Athila-like LTR retrotransposons including newly described elements.Click here for fileList of fungal species, genomes of which were analyzed. Table contained the list of fungal species, genomes of which were analyzed in silico in the present study and the sources of genomic sequences.Click here for fileNovel Gypsy LTR retrotransposons from Fungi. Table contained the list of novel Gypsy LTR retrotransposons from Fungi detected in present study and their accession numbers.Click here for file


EGF-R was expressed in 68% of the HCC examined and showed correlation with the proliferating activity, stage, intrahepatic metastasis and carcinoma differentiation. c-erbB-2 was expressed in only 21% of the cases and showed no relationships with the clinicopathological parameters. c-erbB-3 protein was observed in 84% of the HCC and 38.1% of the noncancerous lesions. Its expression in HCC was equal to or greater than noncancerous lesions in 90.5% of the cases, and was related to the stage, portal invasion, cell proliferating activity, tumour size, intrahepatic metastasis and carcinoma differentiation. c-erbB-4 protein was expressed in 61.0% of HCC and in as much as 86.1% of the noncancerous lesions. Unlike the expression of c-erbB-3, that of c-erbB-4 in HCC was less than that of the adjacent noncancerous lesions in 51.2% of the cases. No statistical significance could be established between this protein expression in HCC and clinicopathological features. EGF-R and c-erbB-3 affected disease-free survival, but were not recognized as independent prognostic factors by multivariate analysis. The present study suggests that, of the four receptors, EGF-R and c-erbB-3 play important roles in the progression of HCC. © 2001 Cancer Research Campaign


In April 1999, isolation of avian influenza A (H9N2) viruses from humans was confirmed for the first time. H9N2 viruses were isolated from nasopharyngeal aspirate specimens collected from two children who were hospitalized with uncomplicated, febrile, upper respiratory tract illnesses in Hong Kong during March 1999. Novel influenza viruses have the potential to initiate global pandemics if they are sufficiently transmissible among humans. We conducted four retrospective cohort studies of persons exposed to these two H9N2 patients to assess whether human-to-human transmission of avian H9N2 viruses had occurred. No serologic evidence of H9N2 infection was found in family members or health-care workers who had close contact with the H9N2-infected children, suggesting that these H9N2 viruses were not easily transmitted from person to person. In April 1999, two World Health Organization reference laboratories independently confirmed the isolation of avian influenza A (H9N2) viruses for the first time in humans H9N2 viruses have been prevalent in domestic poultry  throughout Asia since the early 1990s and were also isolated from swine in Hong Kong in 1998 The first and only documented human outbreak of highly pathogenic avian influenza A (H5N1) virus infections resulted in 18 hospitalizations and six deaths among Hong Kong residents during 1997  hemagglutinin (HA) subtypes, including H5 and H9 viruses The identification of two children who had acute infection with novel H9N2 virus strains provided the first opportunity to investigate their transmissibility and pandemic potential among humans. We report the results of four retrospective cohort studies designed to detect serologic evidence of H9N2 virus infection among family members and health-care workers (HCWs) exposed to the two H9N2 patients, as well as unexposed controls.The target populations included HCWs at the two hospitals where the H9N2-infected patients received care, as well as family and household members of the patients. The infectious period for an H9N2 patient was defined as a 15-day period beginning from the day before illness onset to the 14th day after illness onset . The infectious period was defined conservatively to reflect the potential for prolonged viral shedding, especially since children can shed influenza viruses for longer periods than adults. Close contact was defined as coming within 3 m of an H9N2-infected patient. Participants were defined as exposed if they had close contact with an H9N2 patient during the infectious period. An unexposed person was defined as having had no contact with the H9N2 patients during the infectious periods. Unexposed subjects included family members and relatives who did not live in the same household as and had no contact with an H9N2 patient, and HCWs who worked on hospital units different from those where the H9N2 patients were located and who denied exposure to the H9N2 patients.We conducted four retrospective cohort studies of either HCWs or family and household members of the H9N2 patients. During face-to-face interviews conducted in either English or Cantonese, staff from the Hong Kong Department of Health administered a detailed questionnaire to a group of household members, family members, and relatives of each H9N2-infected child. The questionnaire assessed the level of exposure and contact with the H9N2-infected patient during the infectious period, along with other suspected risk factors for H9N2 infection, such as recent contact with poultry and swine. A similar questionnaire administered to HCWs asked about contact with each H9N2-infected patient during the patient’s hospitalizations , and recent exposure to poultry and swine. All participants provided written, signed informed consent. Approximately 10 cc of blood was provided by each participant approximately 5 to 6 weeks (except where indicated) after the onset of the H9N2 patients’ illnesses to test for antibody to H9N2.Serum samples from all study participants and the two H9N2 patients were tested for antibody to FLUAV H9N2 by a microneutralization assay at both the Centers for Disease Control and Prevention (CDC), Atlanta, and the Hong Kong Department of Health Government Virus Unit Laboratory, as described At CDC, a Western blot assay with bromelain-purified or baculovirus-expressed recombinant hemagglutinin  from HK/1073 virus was used to confirm each positive microneutralization result, as described At the Government Virus Unit in Hong Kong, microneutralization-positive sera were confirmed by a single radial hemolysis assay for H9N2 antibodies, based on a modified, previously described protocol A490 value greater than the mean A490 plus 3 standard deviations of six to seven negative age-matched controls at an equivalent dilution of sera. A titer ≥1,600 was considered positive.Sera from the two H9N2-infected children were also tested by enzyme-linked immunosorbent assay (ELISA) to detect immunoglobulin (Ig) G and IgM antibodies to H9 as described Univariate analysis of associations between exposure variables and antibodies to H9N2 virus results were done by SAS 6.12 .Patient 1 was positive for H9 antibodies to H9 by all serologic assays and had substantial titers of H9 HA-specific IgG and IgM antibodies . Low titThe demographic characteristics of the study participants are shown in Fourteen of 15 eligible persons were enrolled in the family cohort study (3 exposed immediate family members and 11 unexposed relatives) of H9N2 patient 1. One exposed participant reported respiratory symptoms within the 2 weeks after onset of illness in the patient. This participant’s serum was obtained 3 weeks after H9N2 patient’s illness onset and was seronegative for H9N2 antibodies. No other participant reported respiratory illness. All 14 study participants tested seronegative for H9N2 antibodies .All seven family and household members eligible for the family cohort study of H9N2 patient 2 were enrolled (six exposed and one unexposed family and household members). Two exposed participants reported respiratory symptoms within 2 weeks after onset of illness in H9N2 patient 2. The unexposed participant reported no respiratory illness. All seven study participants tested seronegative for H9N2 antibodies .The HCW study population for H9N2 patient 1 consisted of 30 exposed HCWs from 4 hospital units and 75 unexposed HCWs from 14 hospital units. Three exposed and three unexposed HCWs reported respiratory symptoms  during H9N2 patient 1’s hospitalization or within 5 days of the date of hospital discharge. All 30 exposed study participants were seronegative for H9N2 antibodies. One of the 75 unexposed HCWs was seropositive . The HCWThe HCW study population for H9N2 patient 2 was 15 exposed and 23 unexposed HCWs from four hospital units. One exposed HCW declined to participate. Four exposed HCWs reported respiratory symptoms beginning 2 to 5 weeks after contact with the patient. All 38 study participants tested seronegative for H9N2 antibodies.These cohort studies suggest that influenza A (H9N2) viruses were not transmitted from the two H9N2-infected children to family and household members or HCWs who were exposed to the H9N2 patients during their acute illness infectious periods. As described for the avian influenza A (H5N1) viruses (Evidence for influenza A (H9N2) infection as the cause of acute illness in the two patients includes the direct isolation of H9N2 viruses from nasopharyngeal aspirate specimens during the acute phase of illness The apparent lack of human-to-human transmission of avian H9N2 viruses and the low transmissibility of avian H5N1 viruses among humans have several possible explanations . The N2 NA of the H9N2 viruses isolated from patients is antigenically distinct from that of recent H3N2 human viruses, although some cross-reactivity with human H2N2 and early H3N2 viruses has been reported Because only two H9N2 cases were identified, we did not conduct a case-control study to identify risk factors for H9N2 infection. Thus, the sources and modes of acquisition of H9N2 for the two infected children are unknown. The Hong Kong Department of Health found that one H9N2 patient had very brief exposure to live chickens 11 days before onset of illness but did not directly touch the birds. No other contacts with live poultry, swine, or other animals for either H9N2 patient were found. There was no known contact or common exposure between the two H9N2 patients.During the 1997 FLUAV (H5N1) outbreak in Hong Kong, a case-control study found that visiting a poultry stall or market with live poultry during the week preceding illness was the main risk factor for H5N1 infection We were able to obtain only one convalescent-phase blood specimen from study participants, which limited our ability to document seroconversion. However, none of the exposed persons were seropositive for H9N2. Currently, there are no seroprevalence data on rates of H9N2 infection in children or the general population. One study of a cohort of poultry workers in Hong Kong found that approximately 30% were seropositive for antibodies to H9N2 In addition to H9N2, other avian influenza viruses have been isolated from specimens collected from Hong Kong poultry since 1997, including H6, H4, and H11 viruses These limited studies suggest that avian influenza A (H9N2) viruses were not transmitted from the two infected children to exposed household members, relatives, or HCWs in Hong Kong. However, H9N2 viruses are widely distributed in avian populations, can infect humans, and could evolve or undergo genetic reassortment with potential for increased pathogenicity and transmissibility in humans. The recent emergence of human infections with avian influenza A (H9N2) and (H5N1) viruses highlights the need to improve surveillance for influenza viruses in poultry, swine, and humans, especially in Asia. Further studies to assess the health risks posed by H9N2 and other avian influenza viruses are warranted.


Vibrio species are several that have been reclassified as novel members of the Vibrionaceae. The description of two putative new Vibrio species, Vibrio sp. RC341 and Vibrio sp. RC586 for which we propose the names V. metecus and V. parilis, respectively, previously characterized as non-toxigenic environmental variants of V. cholerae is presented in this study.In recent years genome sequencing has been used to characterize new bacterial species, a method of analysis available as a result of improved methodology and reduced cost. Included in a constantly expanding list of rpoB similarity, MLSA, and phylogenetic analysis, the new species are concluded to be phylogenetically closely related to V. cholerae and V. mimicus. Vibrio sp. RC341 and Vibrio sp. RC586 demonstrate features characteristic of V. cholerae and V. mimicus, respectively, on differential and selective media, but their genomes show a 12 to 15% divergence (88 to 85% ANI and 92 to 91% AAI) compared to the sequences of V. cholerae and V. mimicus genomes (ANI <95% and AAI <96% indicative of separate species). Vibrio sp. RC341 and Vibrio sp. RC586 share 2104 ORFs (59%) and 2058 ORFs (56%) with the published core genome of V. cholerae and 2956 (82%) and 3048 ORFs (84%) with V. mimicus MB-451, respectively. The novel species share 2926 ORFs with each other (81% Vibrio sp. RC341 and 81% Vibrio sp. RC586). Virulence-associated factors and genomic islands of V. cholerae and V. mimicus, including VSP-I and II, were found in these environmental Vibrio spp.Based on results of whole-genome average nucleotide identity (ANI), average amino acid identity (AAI), V. cholerae strains, are new species which have evolved from ancestral lineages of the V. cholerae and V. mimicus clade. The presence of conserved integration loci for genomic islands as well as evidence of horizontal gene transfer between these two new species, V. cholerae, and V. mimicus suggests genomic islands and virulence factors are transferred between these species.Results of this analysis demonstrate these two environmental vibrios, previously characterized as variant  Vibrio comprises a diverse group of gamma-proteobacteria autochthonous to the marine, estuarine, and freshwater environment. These bacteria play a role in nutrient cycling, degrade hydrocarbons, and can be devastating pathogens for fish, shellfish, and mammals as well as humans . However, the variant encoded in Vibrio sp. RC341 is, to date, unique.Vibrio sp. RC341 encodes V. cholerae GI-33, a ca. 2615 bp region, (VCJ_001870 to VCJ_001874) similar to RS1Φ-like phage in Vibrio sp. RC586, V. cholerae strains VL426, SCE264, TMA21, TM11079-80, and 623-39, showing 93 to 96% nucleotide sequence similarity across 67 to 79% of the phage  and 82% (GI-62) ANI , and two with and 85%   received a VSP-I similar to that of Vibrio sp. RC341 and Vibrio sp. RC586 via horizontal gene transfer. We also found evidence of horizontal transfer of V. cholerae GI-2 from V. cholerae to Vibrio sp. RC586, as well as Vibrio sp. RC341 Islet-3 and V. cholerae GI-4 from Vibrio sp. RC341 to V. cholerae strains.Phylogenetic analysis using homologous ORFs of the genomic islands yielded evidence of recent lateral transfer of VSP-I, and GIs-2, 41, and 61 among V. cholerae genome and in Vibrio sp. RC341, showed no evidence of horizontal gene transfer. Most likely there are many undescribed variants of these elements, in both structure and nucleotide sequence, yet to be found in the natural environment, with certain variants more frequently transferred among strains of the same species. Coevolution of the island and host genome over time no doubt occurs. In any case, based on the data reported here V. cholerae is not alone in propagating these elements. They surely cycle among different but closely related species in the environment.VSP-II, islets-2, -4, -5, and GIs-1, -2, -3, -9, -10, all present in at least one Vibrio sp. RC586 putatively encodes five unique genomic islands and islets not yet reported for V. cholerae , with high similarity to V. parahaemolyticus and V. harveyi zot (VOA_001598-VOA_001600). This phage-like element is inserted at the homologous locus for V. cholerae O1 Classical CTXΦ insertion (VCA0569-VCA0570). Vibrio sp. RC586 GI-4 encodes sequences homologous to the Tn7 transposition tnsABCDE, a transposon known to integrate into phylogenetically diverse organisms and form genomic islands [Vibrio sp. RC586 GIs-1, -3, -4, and islets-1 through 6 all share homologous insertion loci with previously described V. cholerae GIs , ca. 4962 bp region of hypothetical proteins and transposases, is inserted at the homologous locus for V. cholerae O1 Classical CTXΦ, a locus shown to harbor a variety of GIs and phages [d phages   from each other and from V. cholerae and V. mimicus genomes, supporting the conclusion that both represent unique species not described before. Moreover, genes conserved among V. cholerae, V. mimicus, and the two new species varied sufficiently to suggest ancient speciation via genetic drift of the ancestral core genomic backbone. Furthermore, results of our analyses suggest Vibrio sp. RC341 to have evolved from a progenitor of V. cholerae and V. mimicus, whereas Vibrio sp. RC586 is concluded to have evolved from an early V. mimicus clade. Although the ANI of all genomes analyzed in this study demonstrates divergence, putative genomic islands were found to cross species boundaries, often at an higher ANI than the conserved backbone. These data, coupled with phylogenetic analyses, point to lateral transfer of the islands and phages among V. cholerae, V. mimicus, Vibrio sp. RC341, and Vibrio sp. RC586 in the natural environment. Furthermore, homologous GI insertion loci were present in both new species and in the case of V. cholerae, these insertion loci were not GI-specific. The pool of DNA laterally transferred between and among members of the Vibrionaceae strongly suggests that near-neighbors of V. cholerae act as reservoirs of transferable genetic elements and virulence in the environment and that V. cholerae is not alone in propagating these elements therein. Results of this study also demonstrate a widespread allelic variation in these elements and evidence of evolution of mobile genetic elements, including pathogenicity islands, through a multistep mosaic recombination with other elements, including phage. The ability of vibrios to incorporate exogenous DNA at several loci that encode a large combination of GIs, thereby, allows optimization of the genome for success in a specific niche or wider ecology in the natural environment.The genomes of two new NZ_ACZT00000000 and NZ_ADBD00000000).Draft sequences were obtained from a blend of Sanger and 454 sequences and involved paired end Sanger sequencing on 8 kb plasmid libraries to 5× coverage, 20× coverage of 454 data, and optional paired end Sanger sequencing on 35 kb fosmid libraries to 1-2× coverage (depending on repeat complexity). To finish the genomes, a collection of custom software and targeted reaction types were used. In addition to targeted sequencing strategies, Solexa data in an untargeted strategy were used to improve low quality regions and to assist gap closure. Repeat resolution was performed using in house custom software . TargeteGenome to genome comparison was performed using three approaches, since completeness and quality of nucleotide sequences varied from strain to strain in the set examined in this study. Firstly, nucleotide sequences, as whole contigs were directly aligned using the MUMmer program . SecondlVibrionaceae [To determine average nucleotide (ANI) and average amino acid identities (AAI) for the purpose of assigning genetic distances between strains and strains to species groups, a recripocal best match BLASTN analysis was performed for each genome. The average similarity between genomes was measured as the average nucleotide identity (ANI) and average amino acid identity (AAI) of all conserved protein-coding genes, following the methods of Konstantinidis and Tiedje . By thisionaceae . Data foPutative genomic islands (GIs) were defined as a continuous array of five or more ORFs discontinuously distributed among genomes of test strains following the methods of Chun et al . CorrectV. cholerae N16961 was obtained for different sets of strains, and individually aligned using the CLUSTALW2 program [A set of orthologues for each ORF of  program . The res program . The evo program . The MEG program .J.C., A.H. and R.R.C. designed research; T.S.B., D.C.B., J.C.D., C.S.H., N.A.H. performed research; J.C., C.J.G., N.A.H., B.J.H., and S.Y.C. analyzed data; B.J.H. and R.R.C. wrote the paper. All authors have read and approved the manuscript.Vibrio strains used in the comparative genomics utilized in this study. Species, strain ID, serogroup/serotype and biotype (where available), geographical location and source of isolation and year of isolation are listed in this table. NCBI Genbank accession numbers are listed in the right column.Click here for fileVibrio sp. RC586 as query and V. cholerae N16961 as referenceMUMmer plot of . Vibrio sp. RC586 contigs are on Y-axis and V. cholerae N16961 chromosomes are on X-axis. V. cholerae N16961 chromosome I begins at XY-intercept and chromosome II is located on the right section of the X-axis.Click here for fileVibrio sp. RC341 as query and V. cholerae N16961 as referenceMUMmer plot of . Vibrio sp. RC341 contigs are on Y-axis and V. cholerae N16961 chromosomes are on X-axis. V. cholerae N16961 chromosome I begins at XY-intercept and chromosome II is located on the right section of the X-axis.Click here for fileVibrio sp. RC341Average nucleotide identity analysis of . Average nucleotide identity (ANI%) between Vibrio sp. RC341 and Vibrio genomes used in this study.Click here for fileVibrio sp. RC586Average nucleotide identity analysis of . Average nucleotide identity (ANI%) between Vibrio sp. RC586 and Vibrio genomes used in this study.Click here for fileBLAST atlas key. BLAST atlas key for Additional files Click here for fileVibrio sp. RC341Evolutionary distance analysis of . Evolutionary distance of strains used in this study from Vibrio sp. RC341 as determined by ANI between Vibrio sp. RC341 and all strains used in this study.Click here for fileVibrio sp. RC586Evolutionary distance analysis of . Evolutionary distance of strains used in this study from Vibrio sp. RC586 as determined by ANI between Vibrio sp. RC586 and all strains used in this study.Click here for fileV. mimicus MB451Evolutionary distance analysis of . Evolutionary distance of Vibrio sp. RC586 and Vibrio sp. RC341 from V. mimicus MB451 as determined by ANI between V. mimicus MB451 and all strains used in this study.Click here for fileV. cholerae BX 330286Evolutionary distance analysis of . Evolutionary distance of Vibrio sp. RC586 and Vibrio sp. RC341 from strains V. cholerae BX 330286 as determined by ANI between V. cholerae BX 330286 and all strains used in this study.Click here for fileVibrio sp. RC341Putative genomic islands of . Putative genomic islands of Vibrio sp. RC341, showing insertion loci, homologous flanking loci in V. cholerae N16961, %GC, other carrier strains used in this study, ANI with homologous islands, δ*, direction of transfer, islands sharing same insertion loci, and annotation.Click here for fileVibrio sp. RC586Putative genomic islands of . Putative genomic islands of Vibrio sp. RC586, showing insertion loci, homologous flanking loci in V. cholerae N16961, %GC, other carrier strains used in this study, ANI with homologous islands, δ*, direction of transfer, islands sharing same insertion loci, and annotation.Click here for fileStrain legend. Legend for Additional files Click here for filePhylogeny of the genomic island GI-2. Phylogeny of the genomic island GI-2 as determined by reconstructing a neighbor-joining tree using the Kimura-2 parameter as a nucleotide substitution model.Click here for filePhylogeny of the genomic island GI-41. Phylogeny of the genomic island GI-41 as determined by reconstructing a neighbor-joining tree using the Kimura-2 parameter as a nucleotide substitution model.Click here for filePhylogeny of the genomic island GI-4. Phylogeny of the genomic island GI-4 as determined by reconstructing a neighbor-joining tree using the Kimura-2 parameter as a nucleotide substitution model.Click here for filePhylogeny of VSP-I. Phylogeny of the genomic island VSP-I as determined by reconstructing a neighbor-joining tree using the Kimura-2 parameter as a nucleotide substitution model.Click here for filePhylogeny of the genomic island GI-61. Phylogeny of the genomic island GI-61 as determined by reconstructing a neighbor-joining tree using the Kimura-2 parameter as a nucleotide substitution model.Click here for fileVibrio sp. RC341 Islet-3Phylogeny of . Phylogeny of Vibrio sp. RC341 Islet-3 as determined by reconstructing a neighbor-joining tree using the Kimura-2 parameter as a nucleotide substitution model.Click here for file


Avian influenza virus H9N2 isolates cause a mild influenza-like illness in humans. However, the pathogenesis of the H9N2 subtypes in human remains to be investigated. Using a human alveolar epithelial cell line A549 as host, we found that A/Quail/Hong Kong/G1/97 (H9N2/G1), which shares 6 viral "internal genes" with the lethal A/Hong Kong/156/97 (H5N1/97) virus, replicates efficiently whereas other H9N2 viruses, A/Duck/Hong Kong/Y280/97 (H9N2/Y280) and A/Chicken/Hong Kong/G9/97 (H9N2/G9), replicate poorly. Interestingly, we found that there is a difference in the translation of viral protein but not in the infectivity or transcription of viral genes of these H9N2 viruses in the infected cells. This difference may possibly be explained by H9N2/G1 being more efficient on viral protein production in specific cell types. These findings suggest that the H9N2/G1 virus like its counterpart H5N1/97 may be better adapted to the human host and replicates efficiently in human alveolar epithelial cells. Genetic characterization and phylogenetic analysis revealed that there are multiple lineages of H9N2 viruses isolated from various types of poultry including chickens, ducks, quail and pigeons. The H9N2 virus lineages found to be the most prevalent in poultry in southern China include the H9N2/G1-like lineage represented by A/Quail/Hong Kong/G1/97 (H9N2/G1) and the H9N2/Y280-like lineage represented by A/Duck/Hong Kong/Y280/97 (H9N2/Y280) and A/Chicken/Hong Kong/G9/97 (H9N2/G9) since 1997 .H9N2 viruses have repeatedly infected humans albeit causing a mild disease -5. The l50) assays using Madin-Darby canine kidney (MDCK) cells. Their replication efficiencies were compared with the human influenza A/Hong Kong/54/98 virus (H1N1). As shown in Fig. 50 titre of H9N2/G1 viruses at 24 h and 48 h post-infection were 103.6 and 104.5 per 0.1 ml, respectively. Similar viral titres were observed in H1N1-infected cells at the corresponding time points. In contrast, both H9N2/Y280 and H9N2/G9 viruses exhibited poor replication competence in A549 cells  of 0.01 -12. The ells Fig . Thus ouNext we examine the host-specific effects on the replication of H9N2 viruses, the leghorn male hepatoma chicken liver (LMH) cells were infected by H9N2 subtypes or H1N1 virus. We also infected embryonated chicken eggs with these viruses and their viral titres were determined by a hemagglutination assay. As shown in Fig. To delineate the mechanisms underlying the differences in replication of H9N2 subtypes, we investigated the infectivity, transcription and translation of different viruses in A549 cells. A549 cells were first infected by H9N2/G1, H9N2/Y280, H9N2/G9 or H1N1 at an m.o.i. of 2 and the expression of viral nucleoprotein (NP) and matrix protein (M1) in the infected cells was examined by using immunofluorescent staining and Western analysis, respectively. Positive staining of the NP protein was found in both H9N2-infected and H1N1-infected A549 cells at 24 h post-infection Fig. . InteresSince 1999, the H9N2 viruses have been intermittently isolated from patients manifesting influenza-like symptoms ,4 and thIn this report, we found that all three H9N2 lineages tested can infect different cell types including A549 cells, MDCK and LMH as evident by the detection of H9N2 viral protein NP in the infected cells and embryonated chicken eggs. Interestingly, only H9N2/G1 virus could produce a high titre of virus particles in A549 cells comparable to the H1N1 infection. These results suggested that the disease severity of H9N2/G1-infected patients may be associated with the replication ability of the virus in specific cell types. From our results, we showed that the viral growth efficiency may not be due to the difference of infectivity of the virus subtypes. Instead, we found that the translation of M1 protein is impaired in H9N2/Y280 and H9N2/G9. Previous reports have shown that M1 forms the major structural component of the virion and plays an important role in virus budding and assembly ,17. HoweThe genetic background of different H9N2 subtypes Table  may contSince both of the H9N2 human isolates from 1999 and 2003 caused mild respiratory symptoms in infected patients, we postulate that there may be some common factors that lead to effective infection among those viruses. The HA, NA, NP genes of H9N2 viruses that have infected humans belonged to either H9N2/G1 or H9N2 Y280-like sub-lineages. Interestingly however, irrespective of the derivation of the HA and NA genes, the PB2, PB1 and M genes of these human isolates all belong to the H9N2/G1 sublineage. The PA genes of these viruses belong to either the H9N2/G1 or the contemporary Z genotype H5N1 viruses . It is nInfection of patients with avian influenza virus subtypes including H4N8, H6N1, and H10N7 is inefficient and this may be due to the inefficient virus replication competence in human cells . Here, wThe authors declare that they have no competing interests.DCWL participated in experimental design, data analysis and manuscript preparation, CMKP performed experiments, data analysis and draft the manuscript, AHYL carried out experiments and data analysis, MP participated in data analysis and manuscript preparation, ASYL participated in project design, data analysis and manuscript preparation. All authors read and approved the final manuscript.


P = 0.003;n = 77). Highest specific activities and cancer/normal ratios (C/N) for activity were measured in Dukes' B and C stage carcinomas, cancers involved in local spread and invasion to lymph nodes. In contrast, cathepsin B and L activities analysed in the same paired extracts had been shown to be most frequently elevated in earlier stage carcinomas (Dukes' A and B), confirming that cathepsin H demonstrates a distinct pattern of expression during colorectal cancer progression. Although cathepsin H activities were most commonly elevated in Dukes' C cancers at all colon sites, both specific activity and C/N ratios were significantly higher for cancers of the left colon compared to other colon locations. A subset of 43 paired extracts analysed on Western blots also revealed consistent changes in cathepsin H protein forms in cancers. Normal mucosa typically showed a strong protein doublet at 31 and 29 kDa while cancers demonstrated decreased expression or total loss of the 31 kDa protein (90% of cases), equal or increased expression of the 29-kDa protein (67% of cases) and the new appearance or up-regulation of a cathepsin H band at 22 kDa (78% of cases). C/N ratios for cathepsin H enzyme activity correlated significantly with C/N ratios for the 29 kDa mature single-chain protein form (P< 0.001), with increased activity most commonly associated with elevated expression of 29-kDa cathepsin H but also with up-regulation of the 22-kDa band, suggesting a shift to more fully processed, mature active cathepsin H protein forms in cancers. Changes in cathepsin H expression were also detected by immunohistochemistry as elevated cathepsin H staining in tumour epithelial cells. © 2000 Cancer Research CampaignOur analyses of cathepsin H activity levels and protein forms in human colorectal cancers compared to matched control mucosa support the concept that altered proteinase expression patterns may reflect both cancer stage and site. Cathepsin H-specific activity was significantly increased in colorectal cancers compared to control mucosa (


Levels of all eicosanoids decreased rapidly after a peak which was seen as early as 5 min. Enzyme markers of cellular contents of neutrophils and mononuclear cells, MPO and NAG respectively, decreased rapidly after ionophore injection; this was followed by increases after 60 min. Indomethacin, a selective cyclooxygenase inhibitor, and zileuton and ICI D-2138, two selective 5-lipoxygenase inhibitors attenuated prostaglandin and leukotriene pathways respectively. Oral administration of zileuton  inhibited LTB4 biosynthesis for up to 6 h suggesting a long duration of pharmacological activity in the rats consistent with its longer half-life. The rapid onset and the magnitude of increases in levels of eicosanoids render the ionophore induced peritoneal eicosanoid biosynthesis a useful model to evaluate pharmacological profiles of inhibitors of eicosanoid pathways in vivo.The present investigation characterizes calcium ionophore (A-23187) induced peritoneal eicosanoid biosynthesis in the rat. Intraperitoneal injection of A-23187 (20 μg/rat) stimulated marked biosynthesis of 6-keto-PGF


C. elegans results in dopaminergic neuron degeneration, the effects of α-synuclein on dopamine homeostasis and its contribution to dopaminergic neuron degeneration in C. elegans have not been reported. Here, we examined the effects of α-synuclein overexpression on worm dopamine homeostasis. We found that α-synuclein expression results in upregulation of dopamine synthesis and content, and redistribution of dopaminergic synaptic vesicles, which significantly contribute to dopaminergic neuron degeneration. These results provide in vivo evidence supporting a critical role for dopamine homeostasis in supporting dopaminergic neuron integrity.Disruption of dopamine homeostasis may lead to dopaminergic neuron degeneration, a proposed explanation for the specific vulnerability of dopaminergic neurons in Parkinson's disease. While expression of human α-synuclein in  Drosophila embryos induces cellular degeneration vesicular monoamine transporter (VMAT) loss-of-function mice show nigrostriatal neurogdegeneration Abnormal dopamine (DA) metabolism, which produces reactive oxygen species (ROS), may lead to dopaminergic (DAergic) neuron degeneration and has been proposed to be related to the pathogenesis of Parkinson's Disease (PD) in vitro or ex vivo evidence also suggests a connection between dopamine homeostasis and α-synuclein, the central player of PD pathology in vitroSome Drosophila and C. elegans are valuable surrogates for the study of certain aspects of neurodegenerative diseases, including investigations of α-synuclein toxicity Genetic model organisms such as yeast, C. elegans results in their degeneration C. elegans lines to examine the toxic effects of hαSyn on dopamine homeostasis and its contribution to hαSyn-mediated DAergic neuron degeneration.Expression of human α-synuclein (hαSyn) in DAergic neurons of dat-1 promoter-driven hαSyn by using immunohistochemistry and confocal microscopy. Positive hαSyn immunostaining was found exclusively in DAergic neurons, marked with dat-1 promoter-driven DsRed, demonstrating the specificity of hαSyn expression in our transgenic lines . Dopamine deamination also yields 3,4-dihydroxyphenylacetic acid (DOPAC), a non-toxic metabolite that can be used to monitor dopamine deamination-specific oxidative stress in vivo. This quinone can be oxidized to several other species We found that hαSyn-expressing worms displayed an age-related accumulation of DOPAC leading to a significantly higher DOPAC content than control worms , therebyDopamine is loaded into synaptic vesicles by a VMAT and pathogenic α-synuclein impairs dopamine storage in mammalian cell lines Also consistent with previous reports cat-2 mutant background  abnormalities ckground , indicatin vivo overexpression of VMAT/CAT-1 would ameliorate hαSyn mediated DAergic neuron degeneration. Indeed, we found that VMAT/CAT-1 overexpression Dopamine is toxic in the cytosol but not in synaptic vesicles cat-2 mutant background  also plays a critical role in DAergic neuron loss in mammals dat-1 was cloned and linked to a full-length cDNA encoding hαSyn, DsRed or GFP according to a previous description dat-1 (encoding dopamine transporter) promoter sequence cat-1 and cat-2 mutants used were e1111 and e1112, respectively. N2 was used as the wild type.The promoter of Worms were fixed with formaldehyde and stained with goat anti-hαSyn antibody according to published protocols with slight modifications ex = 543nm and λem = 580–630nm) and GFP(λex = 488nm and λem = 510–530nm). To count fluorescent DAergic neuron numbers, living worms were immobilized with 30 mM sodium azide on 5% agarose pads and examined with a Leica DMI3000 microscope or a Leica TCS SP2 confocal microscope according to a published method with modifications EM 885 EMCCD camera and SimImaging  (when Lecica DMI3000 microscope was used) or a Leica TCS SP2 confocal microscope. All images were processed and analyzed with National Instruments Vision Assistant 7.1.All confocal experiments were conducted with a Leica TCS SP2 confocal microscope. The spectra used were: DsRed with a protocol described previously Worm basal/enhanced slowing responses with and without dopamine pretreatment were obtained as previously described Samples were prepared as described Statistical significance was analyzed by using Statistica . T-tests, ANOVA with Bonferroni corrections or Dunnet's post-hoc analyses were used for their appropriate applications.Figure S1C. elegans. A–D, Confocal images of a formaldehyde-fixed day 2 adult worm with DAergic neuron specific expression of hαSyn and DsRed. A, Bright field (BF). B, DsRed. C, hαSyn immunostaining (green). D. Merged image of B and C.Immunohistochemical analysis of hαSyn expression in transgenic (0.80 MB TIF)Click here for additional data file.Figure S2C. elegans. A–B, Confocal images of formaldehyde-fixed day 0 (A) or day 10 (B) worm with DAergic neuron specific expression of hαSyn and DsRed. Left, TH immuostaining; Middle, DsRed; Right, Merged image of TH staining and DsRed; C, Quantification of DAergic neuron degeneration by using TH staining (green) or DsRed (Red). Data represents mean ± S.E.M., n = 10. 1 represents 6.8 and 6.3 DAergic neurons in DsRed and TH staining experiments, representatively.Immunohistochemical analysis of TH expression in transgenic (0.69 MB TIF)Click here for additional data file.Figure S3Correlation of DAergic neuron degeneration with CAT-2::GFP and DsRed. A–B, Confocal images of living day 0 (A) or day 10 worms (B) with DAergic neuron specific expression of CAT-2::GFP, DsRed and hαSyn. Left, CAT-2::GFP; Middle, DsRed; Right, Merged image of CAT-2::GFP and DsRed. (C) Quantification of DAergic neuron degeneration by using CAT-2::GFP (green) or DsRed (red) in hαSyn-expressing (diamonds) and control (circles) lines. Data represent mean ± S.E.M., n = 30. Error bars may hide in symbols. ***, p<0.005 (Two-way ANOVA to compare hαSyn expressing and control line) . 1 represents 7.9 and 7.7 in DsRed and CAT-2/TH::GFP experiments, respectively.(1.44 MB TIF)Click here for additional data file.Figure S4hαSyn expression does not affect serotonin neurotransmission. Enhanced slowing responses of day 2 adult worms. GFP indicates a wild type worm line expressing GFP in DAergic neurons. Food response experiments were conducted with (grey bars) or without (black bars) food.(0.23 MB TIF)Click here for additional data file.Figure S5hαSyn expression disrupts dopamine synaptic vesicle distribution in TH/CAT-2 knockout background. A–D, Typical bright field (A) or confocal laser scanning VMAT/CAT-1::GFP (B–D) images of living L2 worms expressing both VMAT/CAT-1::GFP and hαSyn in a TH/CAT-2 knockout background. C and D are magnified areas of B that show DAergic and serotonergic somas (C) or DAergic dendrites of CEPs (D), respectively. E, Quantification of CAT-1::GFP redistribution in CEPs of L2 worms expressing both VMAT/CAT-1::GFP and hαSyn in wild type (n = 5) or a TH/CAT-2 knockout background (n = 5). Error bar:SEM.(0.98 MB TIF)Click here for additional data file.


The c-erbB-2 product is thought to be a unique and useful target for antibody therapy of cancers overexpressing the c-erbB-2 gene. In vitro and in vivo anti-tumour effects of a humanised antibody against the extracellular domain of the c-erbB-2 gene product, rhu4D5, were examined. Rhu4D5 was less effective than its murine counterpart, mu4D5, for the direct antiproliferative activity against the c-erbB-2-overexpressing SK-BR-3 cell line. In vivo treatment of severe combined immunodeficient (SCID) mice carrying the c-erbB-2-overexpressing 4-1ST human gastric carcinoma xenograft with 4hu4D5 revealed that the recombinant protein had potent anti-tumour activity. Furthermore, cytotoxicity of human peripheral blood mononuclear cells against 4-1ST was significantly augmented with rhu4D5, but not with mu4D5. These results indicate that rhu4D5 might perform better in patients than predicted from preclinical studies.


This study aimed to find novel biomarkers for colorectal cancer.Fluorescent mRNA differential display PCR (DD-PCR) was used to screen the genes differentially expressed in colorectal cancer tissues and their adjacent tissues. The differentially expressed genes were confirmed by real-time PCR and then their clinical relevance (such as association with tumor location and lymph gland metastasis) was further investigated.DHX32, which showed higher expression in colorectal cancer tissues than their adjacent tissues, and this result was confirmed by real time RT-PCR. In addition, we found that the level of DHX32 gene expression in colorectal cancer was significantly associated with cancer location, lymph gland metastasis, cancer nodal status, differentiation grade, and Dukes, stage.We identified by DD-PCR a novel RNA helicase, DHX32 may play an important role in the development of colorectal cancer and could serve as a novel biomarker for colorectal cancer after additional investigation. Colorectal cancer (CRC) is one of the most common causes of cancer death throughout the world. Multistage development of the disease has been associated with remarkable genetic events, mainly at the level of oncogenes and oncosuppressor genes, most notably the adenomatous polyposis coli gene (APC) , ras 2,,2,3, andSeveral approaches have been taken to identify and compare gene expression in normal and disease states -11. The DHX32, a novel RNA helicase, was significantly up-regulated in colorectal cancer compared to its adjacent normal tissue using a combination of DD-PCR and real-time PCR methods. Our results suggested that the level of DHX32 gene expression in colorectal cancer was significantly associated with cancer location, lymph gland metastasis, cancer nodal status, differentiation grade and Dukes' stage.In this study, we found that 34 pairs of specimens  and 14 tumor tissues were obtained from patients with colorectal cancer who underwent surgical resection at the Xiamen Zhongshan Hospital, Xiamen University in Xiamen during 2006 and 2007. The detail clinical and pathological characteristics of these 48 cases of samples were listed in a table Total RNA was prepared using Trizol reagent  according to the manufacturer's instructions. RNA was treated with DNase?  in the presence of 50 μM T7(dT12)AP2, T7(dT12)AP7 primer in 20 μl RT buffer , at 25°C for 5 minutes, followed by 42°C for 10 minutes and 50°C for 60 minutes. Reverse transcriptase was inactivated at 70°C for 15 minutes..Differential display was performed using Hieroglyph mRNA Profile kit . Briefly, PCR amplification was done using 1.5 μl of the cDNA, primed with arbitrary P primer and anchored T primer. Amplification at (95°C 2 minutes) 1 cycle,  4 cycles,  25 cycles, followed by a final extension at 72°C for 7 minutes on a GeneAmp PCR system 9600 . Following amplification of randomly primed mRNAs by RT-PCR, the cDNA products were heated at 94°C for 2 minutes and separated on a denaturing 5.6% polyacrylamide gel using a Genomyx LR DNA Sequencer . Bands exclusively present in either of two samples were considered as candidates of differentially expressed transcripts, which were excised, eluted, re-amplified, and subcloned into the pGEM-T easy vector . The sequence reactions were performed by Invitrogen Corp . Sequence homology to published database was analyzed with the BLAST program at the internet site of NCBI  DHX32 expression in 48 tumor samples by real-time quantitative RT-PCR using TaqMan methodology in an ABI PRISM 7500 Sequence Detection System. The real-time RT-PCR allows, by means of fluorescence emission, the identification of the cycling point when PCR product is detectable. The Ct value inversely correlates with the starting quantity of target mRNA. Measurements were performed in duplicate and the controls were included in which the reaction mixture contained no cDNA. The amount of target mRNA after normalized to the endogenous reference β-actin was calculated by the Ct method as described by Liu W [We measured by Liu W .β-actin and DHX32 mRNAs were chosen using the Primer Express 2.0 software . The primers, placed in different exons, were designed to ensure that genomic DNA would not be amplified. Primer and probe nucleotide sequences for DHX32 (GenBank accession number NM_018180) were: DHX32-Fw 5'-GTCTTTCCATCCACTACCAGCAC-3', DHX32-Rev 5'-ATGATGACCCCATAGCT ACCCAA-3', and TaqMan probe 5'-(FAM) CGTGATATGCACACAGGTCCACAAG C (TAMRA)-3'. Primers and probe for β-actin mRNA were: β-actin-Fw 5'-TCACCCACACTGTGCCCATCTACGA-3', β-actin-Rev 5'-CAGCGGAACCGCTCATTGCCAATGG-3', and TaqMan probe 5'-(FAM)ATGCCC-X(TAMRA)-CCCCCATGCCATCCTGCGT-3'. Probes were purchased from Invitrogen Corp . The thermal cycling conditions were: 5 minutes at 95°C, followed by 60 cycles of 30 seconds at 95°C and 1 minute at 60°C.Primers and probes for DHX32 gene expression between tumor tissue and its adjacent normal tissue in each patient, respectively. The Mann-Whitney U test was used for comparing the gene expression of DHX32 between the different groups according to various clinical and pathological variables. All of the statistical analyses were performed with SPSS 14.0 . A P value of less than 0.05 was considered to be statistically significant.The Levene's test was performed to determine the homogeneity of variance for all the data, and then the paired Chi-Square test or 2-related samples Wilcoxon nonparametric test was performed to compare the positive rates and the levels of DHX32 in the GenBank database.The modified DD-PCR method was used to identify genes uniquely and/or highly expressed in human CRC tissues by comparing with those in adjacent normal tissues. One cDNA band (size ranging between 700 bp and 800 bp) was found to be highly expressed in colorectal cancer tissues while barely expressed in matched adjacent normal tissues. The identified band was recovered, re-amplified, subcloned and sequenced. BLAST analysis of this nucleotide sequence revealed 99% homology to the gene DHX32 gene expression between colorectal tumors and their adjacent normal tissues. We found that the positive rate of DHX32 gene expression was significantly higher in the colorectal tumors (76.5%) than that in the adjacent normal tissues (26.4%) , we compared the positive rate and the levels of DHX32 gene expression between the different groups according to various clinical and pathological variables. Although we did not observe significant differences of the positive rate of DHX32 gene expression between the groups according to each parameter (data not shown), our results suggested that the level of DHX32 gene expression in colorectal carcinoma was significantly associated with tumor location, lymph gland metastasis, tumor nodal status, differentiation grade and Dukes' stage (P < 0.05) (Figure DHX32 gene expression between the different groups classified by age, gender, and Polypi.In order to determine the relationships between DHX32, a novel RNA helicase, was up-regulated in colorectal cancer compared to its adjacent normal tissues. Furthermore, our results suggested that the level of DHX32 gene expression in colorectal carcinoma was significantly associated with tumor location, lymph gland metastasis, tumor nodal status, differentiation grade, and Dukes' stage.The study of the molecular biology of colorectal cancer has progressed rapidly, but the survival of patients with this neoplasm has improved rather modestly . ConsequDHX32 was originally identified as a novel RNA helicase with unique structure in the helicase domain, but with overall similarity to the DHX family of helicases [DHX32 is composed of 12 exons spanning a 60-kb region at human chromosome 10q26 and encodes for a 743 amino acid protein with a predicted molecular weight of 84.4 kDa. DHX32 has a widespread tissue distribution and also has cross-species counterparts, such as 84 and 80% amino acid identity with mouse and rat counterparts, respectively. The high level of similarity between human and murine DHX32 and the widespread expression of DHX32 message suggest that it is an evolutionally conserved and functionally important gene.elicases . RNA helelicases ,20. The elicases . DHX32 iDHX32, are not very well characterized. In our study, we found that DHX32 was overexpressed in colorectal cancer compared with the adjacent normal tissues, suggesting that abnormal expression of DHX32 is associated with the development of colorectal cancer. The involvement of DHX32 in other cancer development was previously demonstrated by other groups. For example, the expression of DHX32 was dysregulated in several lymphoid malignancies [With a few notable exceptions, the biochemical activities and biological roles of RNA helicases, including gnancies ,22. DHX3gnancies . The ovegnancies .DHX32 involves in cancer development, but the exact biochemical activities and biological functions of DHX32 are still elusive. DHX32 contains sequences which are highly conserved between a subfamily of DEAH RNA helicases, including the yeast pre-mRNA splicing factor Prp43 [DHX32 to RNA helicases involved in mRNA splicing suggests a role in pre-mRNA splicing. It is possible that the dysregulation of the normal function of RNA helicases can potentially result in abnormal RNA processing with deleterious effects on the expression/function of key proteins in normal cell cycles and contribute to cancer development and/or progression. There are several examples of dyregulation of RNA helicases in cancer [DDX5 [DDX6 [So far, several groups have attempted to reveal the underlying mechanisms by which or Prp43 , and itsn cancer , mostly er [DDX5  and DDX6X5 [DDX6  which weDHX32 in colorectal carcinoma was significantly associated with tumor location, lymph gland metastasis, tumor nodal status, differentiation grade, and Dukes' stage. These results not only further confirmed the possible critical role of DHX32 in human colorectal development, but also suggested that additional studies may help develop DHX32 as a potential biomarker to judge the prognosis of colorectal cancer patients: the patients with higher gene expression of DHX32 may have worse prognosis.Our results indicated that the differential expression of DHX32 in human CRC than that of the adjacent normal tissue, indicating that overexpression of DHX32 may play a pivotal role in the multistage carcinogenesis of human CRC. It still remains to be further investigated for the functions of DHX32 during the progression of colorectal cancer. DHX32 may also serve as a bio-marker for judging the levels of malignancy of colorectal cancer, which may guide the development of anticancer therapy regime after additional studies.In conclusion, to our knowledge, we are the first to report the more frequent and significant overexpression of human DHX32 may play an important role in the development of colorectal cancer and additional studies may help use DHX32 as a novel biomarker for colorectal cancer.The authors declare that they have no financial competing interests.ZZ conceived of the study and guided the biochemical experiments.CH performed DD-PCR and drafted the manuscript.XL performed real-time PCR, analyzed data, collected tissue specimens and clinical records, and helped write the manuscript.RH conceived of the idea and provided helpful comments.All authors read and approved the final manuscript.