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Targeting the Wnt/β-catenin pathway in endometriosis: a potentially effective approach for treatment and prevention

Endometriosis is a chronic, estrogen-dependent disease associated with infertility and pelvic pain. Endometriosis is defined by the presence of extra-uterine endometrial tissue. It affects approximately 10% of reproductive-aged women. However, the underlying etiology, pathogenesis and pathophysiology remain to be fully elucidated. Knowledge of these factors is indispensable for the development of targeted therapies for prevention and treatment of endometriosis. Several studies, including those from our laboratory, have suggested that aberrant activation of the Wnt/β-catenin pathway may be involved in the pathophysiology of endometriosis. This is a review of the literature focused on the aberrant activation of the Wnt/β-catenin pathway in patients with endometriosis, and on how targeting the Wnt/targeting pathway may be a potentially effective approach for treating and/or preventing endometriosis.

Background
Endometriosis is a chronic, estrogen-dependent disease associated with infertility and pelvic pain. Endometriosis is defined by the presence of extra-uterine endometrial tissue. It affects approximately 10% of reproductive-aged women [1]. However, the underlying etiology, pathogenesis and pathophysiology remain to be fully elucidated. Knowledge of these factors is indispensable for the development of targeted therapies for prevention and treatment of endometriosis.
Several studies, including those from our laboratory, have suggested that aberrant activation of the Wnt/β-catenin pathway may be involved in the pathophysiology of endometriosis [2][3][4][5][6][7][8][9]. The Wnt/β-catenin pathway has crucial roles in embryonic development, tissue self-renewal, and various diseases [10][11][12][13]. In the absence of Wnt ligands ("off" state), β-catenin is degraded by the APC/Axin/GSK-3ß complex [10][11][12][13] (Figure 1A). Binding of Wnt ligands to the Frizzled transmembrane receptors and their LRP co-receptors ("on" state) leads to the inactivation of GSK-3ß and accumulation of β-catenin in the cytoplasm. Then, the elevated cytosolic β-catenin can translocate to the nucleus, where it interacts with the Tcf/LEF transcription factors, leading to transcriptional activation of Wnt-responsive genes ( Figure 1B). Many Wnt-responsive genes have crucial roles in cell proliferation, migration, and invasion [10][11][12][13]. These processes are also common in endometriosis [1]. This is a review of the literature focused on the aberrant activation of the Wnt/β-catenin pathway in patients with endometriosis, and on how targeting the Wnt/targeting pathway may be a potentially effective approach for treating and/or preventing endometriosis.

Review
Aberrant activation of the Wnt/β-catenin pathway in endometrium of patients with endometriosis Menstrual endometrium of patients with endometriosis Pathogenesis of endometriosis remains unclear. However, the implantation theory is the most widely accepted [1]. Endometriosis originates from retrograde menstruation of endometrial tissue that passes through patent Fallopian tubes into the peritoneal cavity. As retrograde menstruation is a common physiological event, it remains unknown why endometriosis only occurs in about 10% of women during their reproductive years. One possible explanation is that the eutopic endometrial cells of women with endometriosis may be functionally and biochemically different from those of women without endometriosis [1].
Our previous study showed significantly higher total and active forms of MMP-9 in the menstrual epithelial and stromal cells of patients with endometriosis compared to those of patients without endometriosis [4]. MMP-9 is one of the Tcf/β-catenin target genes (www.stanford.edu/group/ nusselab/cgi-bin/wnt/target_genes). Treatment with PKF 115-584, a small-molecule antagonist of the Tcf/β-catenin complex, decreased the amount of total MMP-9 approximately 75% in epithelial cells and 85% in stromal cells in patients with endometriosis [4]. Furthermore, treatment with PKF 115-584 decreased the amount of active MMP-9 to undetectable levels in both epithelial and stromal cells of patients with endometriosis [4]. MMP-9 activity is known to be involved in cell invasion [14][15][16][17]. In addition, recent studies clearly demonstrated that a latent form of MMP-9 may play an important role in cell migration [18,19]. Our previous study demonstrated that the inhibitory effects of cell migration and invasion of menstrual endometrial epithelial and stromal cells of endometriosis patients by treatment with PKF 115-584 were much higher than those of patients without endometriosis [4]. These findings are consistent with those of a previous study that demonstrated that MMP-9 secretion, as assessed by zymography and enzyme-linked immunosorbent assay (ELISA), was increased in women with endometriosis compared to healthy women [20]. According to the implantation theory, two processes appear to be critical for the establishment of endometriosis: migration and invasion [1,21]. These findings suggested that aberrant activation of the Wnt/β-catenin pathway may result in increased migration and invasion of menstrual endometrial cells of patients with endometriosis.
Furthermore, a recent study showed that human endometrial basal glandular epithelial cells expressed nuclear SOX9, a Wnt target gene, and contained a rare subpopulation of cells with nuclear β-catenin [22]. These findings suggested that the Wnt pathway was activated in the basal endometrium. They also showed that the embryonic stem cell-surface marker, SSEA-1, marked the human endometrial basal glandular epithelial cells. Interestingly, cells in ectopic endometriosis lesions also expressed SSEA-1 and nuclear SOX9, and SOX9 and SSEA-1 expression patterns were similar to those in the matched eutopic basalis epithelia [22]. These investigators speculated that the ectopic lesions might be basalis in origin following retrograde menstruation [22]. A growing body of evidence suggests that endometriosis may arise from stem cells [23][24][25][26][27]. The Wnt signaling pathway plays an essential role in stem cell regulation [13]. Although further studies are required to examine the role of the Wnt/β-catenin signaling in SOX9-and SSEA-1-expressing endometrial glandular epithelial cells, these findings suggested that manipulation of Wnt signaling for stem cell regulation might be a novel therapeutic strategy for prevention and treatment of endometriosis.

Mid-secretory endometrium of infertile patients with endometriosis
Endometriosis affects approximately 25%-50% of all women with infertility [1]. However, the underlying molecular mechanisms of endometriosis-associated infertility remain to be elucidated. One potential cause may be endometrial molecular defects during the implantation window [1]. Previous findings, including those from our laboratory, suggested that the Wnt/beta-catenin signaling pathway might be aberrantly activated in infertile patients during the mid-secretory phase [2,6,7]. Our previous study demonstrated punctate membranous expression of dephosphorylated beta-catenin in endometrial epithelial cells of infertile patients during the mid-secretory phase [2]. Expression of the N-terminally dephosphorylated form of beta-catenin has been shown to be well correlated with Wnt activity [28]. Our previous study showed significantly higher basal cell proliferation of endometrial epithelial and stromal cells of patients with endometriosis compared to patients without endometriosis in the mid-secretory phase [4]. In addition, mRNA expression of Cyclin D1, a Tcf/β-catenin target gene, was significantly higher in endometrial epithelial cells of patients with endometriosis compared to patients without endometriosis in the mid-secretory phase [4]. Expression levels of Cyclin D1 tended to be higher in the secretory phase stromal cells of patients with endometriosis compared to patients without endometriosis, which is in agreement with with the results of a previous study [29]. Cell proliferation and Cyclin D1 mRNA expression in epithelial and stromal cells of patients with endometriosis were effectively decreased by treatment with PKF 115-584, a small-molecule antagonist of the Tcf/β-catenin complex.
A study showed that increased E2 levels activate Wnt/βcatenin signaling to promote endometrial cell proliferation during the proliferative phase of menstrual cycle, whereas during the secretory phase, progesterone levels inhibit Wnt/β-catenin signaling, resulting in counterbalancing E2-induced proliferation and enhancing differentiation [30]. In addition, a mouse study demonstrated that stabilization of beta-catenin in the uterus resulted in endometrial glandular hyperplasia and lack of a decidual response [31]. In these mice, estrogen receptor (ER)-alpha expression was increased in the epithelium [31]. A growing body of evidence suggests that endometrium of patients with endometriosis has an altered response to progesterone and persistence of the proliferative phenotype [7,29,32]. Studies have demonstrated impaired decidualization in endometrial stromal cells and elevated endometrial ER-alpha expression during the mid-secretory phase in patients with endometriosis [33]. Furthermore, regulation of cyclin D1, one of the target genes of the Wnt pathway via activation of β-catenin, is impaired in endometrial stromal cells of patients with endometriosis [7]. These findings suggest that progesterone resistance might fail to inhibit activation of Wnt/β-catenin signaling, resulting in the persistence of the proliferative phenotype and impaired decidualization in the endometrium of infertile patients with endometriosis during the window of implantation.
Aberrant activation of the Wnt/β-catenin pathway in endometriosis Cell proliferation, migration and invasion of endometriotic cells The Wnt/beta-catenin pathway is involved in cell proliferation, migration, and invasion [34], which are also involved in the pathophysiology of endometriosis [1]. Our previous study showed that PKF 115-584, a smallmolecule antagonist of the Tcf/β-catenin complex, could significantly decrease cell proliferation, migration and invasion of endometrial and endometriotic epithelial and stromal cells [4]. However, our previous findings suggested that the Wnt/β-catenin signaling pathway might not be essential for cell proliferation of endometriotic cells [4]. The inhibitory effect of treatment with PKF 115-584 on cell proliferation in ovarian endometriotic tissue was significantly lower than that of matched eutopic endometrium of the same patients [4]. The cell proliferation inhibitory effect of deep infiltrating endometriotic tissue and superficial peritoneal endometriotic tissue tended to be lower than that of matched eutopic endometrium of the same patients [4]. Furthermore, we showed that in either non-treated or treated cells with PKF 115-584, no significant difference in the number of migrated epithelial and stromal cells was observed between endometriotic tissue and matched eutopic endometrium of the same patients [4]. In addition, we observed that no significant difference in percent inhibition of cell migration by treatment with PKF 115-584 in either epithelial or stromal cells was noted between endometriotic tissue and eutopic endometrium of the same patients [4]. Activation of the Wnt/β-catenin pathway may not be as involved in the cell proliferation and migration of diseased cells-endometriotic cells-than their normal cell counterparts-endometrial cells.
In contrast, our previous study demonstrated that endometriotic epithelial cells and stromal cells were more invasive than those of matched eutopic endometrium of the same patients [4]. These findings are in agreement with the results of previous in vitro experiments studies that showed that endometriotic cells have invasive and metastatic phenotypes similar to metastatic carcinoma cells [35,36]. The numbers of invasive endometriotic epithelial and stromal cells were effectively decreased by treatment with PKF 115-584 [4]. Levels of active MMP-2 in endometriotic epithelial cells and total and active MMP-9 in endometriotic stromal cells were significantly decreased compared to those of matched eutopic endometrium following treatment with PKF 115-584 [4]. Both MMP-9 and MMP-2 are Tcf/β-catenin target genes [4]. These findings suggested that inhibition of active MMP2 and MMP9 by treatment with PKF 115-584 decreased the numbers of invasive endometriotic epithelial cells and stromal cells. Aberrant activation of the Wnt/β-catenin signaling pathway may be involved in the invasive phenotype of endometriotic cells.
Furthermore, a recent study demonstrated that the activated TNFα-MMP-9-SRC-1 axis protects the ectopic endometrium from proinflammatory cytokinemediated apoptosis [37]. Endometriotic cells exhibit abnormal apoptotic regulation [38]. Aberrant activation of the Wnt/β-catenin signaling pathway might also be involved in resistance of endometriotic stromal cells to apoptosis through the TNFα-MMP-9 axis.
Recent studies demonstrated the presence of ectopic endometrium in the rectovaginal septum, in the Douglas pouch, in the rectum of human female fetuses [39,40]. They hypothesized that ectopic endometrium might be misplaced outside the uterine cavity during the organogenesis [39,40]. The Wnt/beta-catenin signaling pathway is essential for organogenesis [41]. Endometriosis in some patients may arise from müllerian duct remnants. The Wnt/beta-catenin signaling pathway may possibly be activated by hormonal inputs in the ectopic endometrium after puberty starts [30,42]. However, it is unlikely that all endometriosis could arise from müllerian duct remnants, because the distribution of pelvic endometriosis differs from that of embryonic duct remnants.

Fibrosis in endometriosis
Endometriosis is histologically characterized by dense fibrous tissue mainly composed of collagen type I [1,43]. Excess fibrosis may cause severe clinical symptoms, such as pelvic pain, severe dysmenorrhea, and deep dyspareunia in patients with endometriosis [44,45], Endometriosis is an estrogen-dependent disease. However, hormonal suppressive therapy is not usually effective for deep infiltrating endometriosis [45]. Complete surgical removal of the deep endometriotic lesions results in the best long-term results and symptomatic relief [45]. However, in addition to the dense fibrosis, deep infiltrating endometriosis frequently invades vital pelvic organs [45]. Surgical treatment of deep infiltrating endometriosis should be performed by laparoscopic surgeons who are highly skilled and competent in performing bowel, bladder, and ureteral surgery [45].
Despite of its clinical importance, only a few studies have been conducted to evaluate new therapies for fibrosis in endometriosis. The cellular and molecular mechanisms underlying fibrosis in endometriosis remain to be fully elucidated.
Recent studies have demonstrated the involvement of activated Wnt/β-catenin signaling in fibrosis in several organs [46][47][48][49][50]. Our previous study showed that Wnt3a treatment in the endometrial stromal cells of patients without endometriosis significantly increased cell proliferation and migration, cell-mediated contraction of collagen gels, and expression of fibrotic marker genes (alpha-smooth muscle actin, type I collagen, connective tissue growth factor, and fibronectin) [5]. We showed a significantly lower cell-mediated collagen gel contraction in stromal cells from patients without endometriosis compared to patients with endometriosis [5]. Cell-mediated contraction of collagen gel in stromal cells of patients without endometriosis was increased by treatment with Wnt3a to a level comparable with that of patients with endometriosis [5]. Treatment with Wnt3a induced clearly visible αSMA-positive stress fibers, the hallmark of activated myofibroblasts, in endometrial stromal cells of patients without endometriosis. These findings suggested the involvement of the aberrant activation of the Wnt/β-catenin pathway in the molecular and cellular mechanisms underlying fibrogenesis of endometriosis [5]. Further studies are required whether overexpression of Wnt3a is one of the underlying mechanisms for the development of fibrosis in endometriosis.
Furthermore our previous study demonstrated that mRNA expression of fibrotic marker genes was significantly decreased by treatment with PKF 115-584 and CGP049090, small-molecule antagonists of the Tcf/β-catenin complex [5]. Treatment with PKF 115-584 and CGP049090 significantly decreased stromal cellmediated contraction of collagen gels in endometrium and endometriosis [5]. Our previous study also showed that cell proliferation and migration of endometrial and endometriotic stromal cells were significantly decreased by treatment with PKF 115-584 [4]. Fibroblast migration, proliferation, and collagen contraction are main hallmarks of fibrogenesis [51]. Small-molecule antagonists of the Tcf/β-catenin complex may inhibit fibrogenesis in endometriosis [4,5]. Furthermore, we showed that treatment with CGP049090 prevented the progression of fibrosis in a xenograft model of endometriosis in nude mice [5]. More importantly, we observed that treatment with CGP049090 revresed established fibrosis in our mouse model of endometriosis [5]. A study demonstrated that treatment with ICG-001, a selective inhibitor of Wnt/β-catenin-CBP-dependent transcription, prevented and reversed fibrosis in a mouse model of bleomycin-induced pulmonary fibrosis [49]. These findings and our findings support that aberrant activation of the Wnt/β-catenin signaling pathway play an important role in the pathogenesis of fibrosis.
Chronic pelvic pain in endometriosis: potential involvement of aberrant activation of the Wnt/β-catenin pathway Pain is a major clinical problem in patients with endometriosis [1,[52][53][54][55]. However, the underlying mechanisms are not yet very well understood. The group of Berkley demonstrated in endometriosis is associated with central sensitization that underlies pain hypersensitivity [54]. In addition, the same group recently proposed that painful endometriosis can be classified as a mixed inflammatory/neuropathic pain condition [55]. Thus, therapeutic strategies for neuropathic pain might be applied to endometriosis [53,55]. Central sensitizationassociated synaptic plasticity in the spinal cord dorsal horn (SCDH) critically contributes to the development of chronic pain [52,53]. A recent study showed that both Wnt3a and β-catenin are up-regulated in the SCDH of various mouse pain models: the capsaicin pain model, the HIV-gp120 pain model, and the neuropathic pain model [56]. These results suggested that Wnt signaling pathways are regulated by nociceptive input [56]. The activation of Wnt signaling may contribute to the spinal cord central  sensitization [56]. In addition, a recent animal experiment showed that nerve injury caused rapid-onset and longlasting expression of Wnts and activation of Wnt/Frizzled/ β-catenin signaling in primary sensory neurons, SCDH neurons, and astrocytes [57]. Furthermore, Wnt signaling activation stimulated production of the proinflammatory cytokines IL-18 and TNF-α, which play an important role in the generation of neuropathic pain, through the β-catenin pathway in the spinal cord [57]. Blocking Wnt/β-catenin signaling may provide a strategy for treating neuropathic pain through IL-18 and TNF-α inhibition [56]. Although to date no studies have investigated the activation of Wnt signaling in the SCDH in animal models of endometriosis pain, previous animal experiments suggested the potential involvement of Wnt signaling activation in the molecular mechanisms that underlie chronic pelvic pain in endometriosis.

Conclusions
Studies, including those from our laboratory, suggested that the aberrant activation of the Wnt/β-catenin pathway may be involved in the pathophysiology of endometriosis. The aberrant activation of the Wnt/β-catenin pathway in menstrual endometrium may facilitate development of endometriosis through increased cell migration and invasion. In addition, the aberrant activation of the Wnt/β-catenin pathway may facilitate growth of endometriosis through an increased invasive phenotype and resistance to apoptosis of endometriotic cells. Furthermore, the aberrant activation of the Wnt/β-catenin pathway in mid-secretory endometrium may result in the persistence of the proliferative phenotype and impaired decidualization in infertile patients with endometriosis. The findings from our laboratory also showed the involvement of the Wnt/β-catenin signaling pathway in the cellular and molecular mechanisms underlying fibrosis in endometriosis. A small-molecule antagonist of the Tcf/β-catenin complex prevented the progression of fibrosis and, more importantly, reversed established fibrosis in a xenograft model of endometriosis in immunodeficient nude mice. These findings suggested that targeting the Wnt signaling pathway may be a potentially effective approach for treating and/or preventing endometriosis (Table 1).
However, one major concern in targeting the Wnt/βcatenin pathway is the potential for side effects on stem cell maintenance and tissue homeostasis [58]. Close attention should be paid to potential side effects of in vivo use of the Wnt/β-catenin pathway inhibitors in patients with endometriosis in further studies.


extfieldsofstudy: Biology Medicine

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The implications of a Silurian and other thylacocephalan crustaceans for the functional morphology and systematic affinities of the group

Background Thylacocephala is a group of enigmatic extinct arthropods. Here we provide a full description of the oldest unequivocal thylacocephalan, a new genus and species Thylacares brandonensis, which is present in the Silurian Waukesha fauna from Wisconsin, USA. We also present details of younger, Jurassic specimens, from the Solnhofen lithographic limestones, which are crucial to our interpretation of the systematic position of Thylacocephala. In the past, Thylacocephala has been interpreted as a crustacean ingroup and as closely related to various groups such as cirripeds, decapods or remipeds. Results The Waukesha thylacocephalan, Thylacares brandonensis n. gen. n. sp., bears compound eyes and raptorial appendages that are relatively small compared to those of other representatives of the group. As in other thylacocephalans the large bivalved shield encloses much of the entire body. The shield lacks a marked optical notch. The eyes, which project just beyond the shield margin, appear to be stalked. Head appendages, which may represent antennulae, antennae and mandibles, appear to be present. The trunk is comprised of up to 22 segments. New details observed on thylacocephalans from the Jurassic Solnhofen lithographic limestones include antennulae and antennae of Mayrocaris bucculata, and endites on the raptorial appendages and an elongate last trunk appendage in Clausocaris lithographica. Preserved features of the internal morphology in C. lithographica include the muscles of the raptorial appendage and trunk. Conclusions Our results indicate that some ‘typical’ thylacocephalan characters are unique to the group; these autapomorphies contribute to the difficulty of determining thylacocephalan affinities. While the new features reported here are consistent with a eucrustacean affinity, most previous hypotheses for the position of Thylacocephala within Eucrustacea (as Stomatopoda, Thecostraca or Decapoda) are shown to be unlikely. A sister group relationship to Remipedia appears compatible with the observed features of Thylacocephala but more fossil evidence is required to test this assertion. The raptorial appendages of Thylacocephala most likely projected 45 degrees abaxially instead of directly forward as previously reconstructed. The overall morphology of thylacocephalans supports a predatory mode of life.

Background
The monophyletic group Thylacocephala is known to range from at least 435 million years ago (Silurian) [1,2] to 84 million years ago (Cretaceous) [3]. Vannier et al. [4] described a possible Cambrian species, Zhenghecaris shankouensis, from the lower Cambrian Chengjiang fauna of China and discussed whether other Cambrian arthropod species (of Isoxys and Tuzoia) might represent thylacocephalans (see also [5]). These arthropods, however, do not preserve the characteristic raptorial appendages [4,6]. The enigmatic Ainiktozoon loganense, from the Lower Silurian of Lesmahagow, Scotland, which has compound eyes and possible spiny limbs, has also been interpreted as a thylacocephalan [7]. Its complex morphology [8], however, is not easy to reconcile with that of thylacocephalans. Thylacocephalans are characterised by a large bivalved shield often termed 'carapace' that encloses almost the entire body. Many representatives are known only from their valves. Where other aspects of the 'soft part' morphology are preserved thylacocephalans typically show a pair of large anterior compound eyes and three pairs of large sub-chelate raptorial appendages. It is not clear to which body segments these raptorial appendages belong. Posterior of them the trunk consists of a series of homonomous segments that bear relatively simple appendages.
The interpretation of the morphology and systematic position of Thylacocephala has been controversial since their discovery [4,6,[9][10][11][12]. The first specimens of Thylacocephala described were isolated shields of Concavicaris sinuata interpreted at that time (1868) as phyllocarids [13]. In the 1880s thylacocephalans from the Cretaceous of Sahel Alma, Lebanon, were interpreted as larvae of stomatopod crustaceans [14,15], see [3]. A century later species from the Jurassic of Osteno, Italy, were interpreted as relatives of thecostracan crustaceans, i.e. barnacles and their parasitic relatives [16]. Other thylacocephalan species have been compared to non-stomatopod malacostracan crustaceans, particularly decapods [4,12,17]. Thus, while there is some agreement that thylacocephalans are representatives of Eucrustacea, their placement within this higher taxon is uncertain.
The best preserved specimens are from Jurassic Lagerstätten. Details of the internal morphology are known from the famous La Voulte Lagerstätte in south-eastern France [12,18]. Specimens from the Jurassic of southern Germany preserve possible appendages anterior of the raptorial limbs [19]. Even these details, however, have not resulted in a more satisfactory systematic assignment, possibly because these relatively young species are derived representatives of the group. The evidence of these Jurassic fossils has led to a consensus on the mode of life of Thylacocephala, which are thought to have been mobile predators or ambush predators [4,20].
The oldest well preserved material of unequivocal thylacocephalans is from the Silurian Waukesha biota of Wisconsin, USA. It is to these Paleozoic fossils that we look for evidence of the more plesiomorphic morphology of the group, and possible insights into the systematic affinities of Thylacocephala. New details of Jurassic species from the Solnhofen lithographic limestones also provide evidence of the possible systematic affinity and ecology of Thylacocephala.

Material
The Silurian specimens UWGM 1748-1750, 1767-1769 (all with part and counterpart) are held by the Geology Museum, Department of Geology and Geophysics, University of Wisconsin, Madison, U.S.A.. These six specimens are from the Brandon Bridge Formation (late Telychian) at Waukesha, near Milwaukee, Wisconsin (see [21][22][23] for details of the setting). The enclosing lithology is finely laminated organic-rich argillaceous mudstone and dolomudstone that occurs in the lowest 2 m of the Brandon Bridge strata. Fine scale lamination with limited bioturbation suggests deposition in an anoxic, possibly brackish, environment. Arthropods, dominantly represented by exuviae, are the major component of the fauna and include trilobites, phyllocarids and ostracods, and a number of undescribed arthropods and worm-like animals of uncertain affinity [1,2]. Shelly fossils are rare and usually decalcified. The exceptionally preserved assemblage clearly represents an unusual environmental setting related to restricted circulation associated with initial flooding at the beginning of a sequence [24].
The Solnhofen Limestone is a pure laminated micritic limestone interpreted as a result of deposition in a restricted lagoon [25]. Limited circulation led to salinitystratified water and benthic anoxia. The diverse fauna includes species of Archaeopteryx and Compsognathus, pterosaurs, fishes, shrimps and other arthropods, molluscs, echinoderms and rarer insects and plants.

Documentation methods
The Silurian specimens were photographed with a Canon Rebel T3i and a MPE-65 mm macrolens. Cross-polarised light was provided by Canon Macro Twin Flash MT 24. Several image details were stitched to generate a complete image of the specimens with Adobe Photoshop CS3. Resulting images were color-inverted and their histograms optimised. Prominent structures were traced by hand and color marked. Documentation of the Jurassic specimens followed the principles of fluorescence composite imaging and macro-fluorescence imaging (see [26,27]). 3D-models were produced with Blender.
Diagnosis: Thylacocephalan with a large bivalved shield enveloping the entire body, only eyes and distal extremities of raptorial appendages projecting beyond it. Shield in lateral view with straight dorsal margin; anterior, ventral and posterior margin of shield continuous, rounded. Optical notch very weak. Eyes relatively small, stalked. Raptorial appendages robust and stout. Trunk with up to 22 segments.

Description of the specimens General appearance
Arthropod (Figures 1, 2, 3, 4, 5 and 6), length from the anterior extremity of the eyes to the distal extremity of the trunk appendages from 44 (UWGM 1767, Figure 5A) to 74 mm (UWGM 1769) (UWGM 1748 is 69 mm long, Figures 1A, 2A). The entire body is enclosed by a large bivalved shield; only the extremities of the three pairs of raptorial appendages extend beyond it ( Figures 1A, 2A). The raptorial appendages appear to be borne by the head although the insertion of the third pair is close to the boundary with the trunk.

Shield
The attachment of the shield to the body is not clearly evident: it appears to be restricted to the anterior region of the body, i.e. the head (Figures 1A, 2A). The entire outline of the valves is smoothly rounded; the optical notch is weakly developed (Figures 1A, 2A, 3A, B). In lateral view, the posterior margin of the valves is more rounded, the anterior is slightly less so ( Figure 4A).

Sensory structures
A pair of circular structures, presumably compound eyes, projects anteriorly from under the shield ( Figures 1B, 2B, 3B, 4B, 5D-F). The eyes appear to arise from elongate stalks ( Figure 6A-D). The insertion of these stalks is unknown due to preservation. Posterior of the eyes there appear to be elongate structures lying superimposed on the body, which may represent appendages (Figures 2A, 6E). Based on their anterior position, these may represent remains of the antennulae (first antennae) and/or (second) antennae, but that is unclear due to preservation. Other areas of relief in the head region might also represent appendages; the preservation is inadequate and the number of specimens too few to be sure.

Possible mouth parts
A pair of triangular structures lies posterior to the possible antennae ( Figure 2A). Based on their position and apparently stronger sclerotisation these may represent the mandibles. Although this specimens affords a lateral view, the left and right eyes are offset suggesting that both left and right mandible appendages might both be evident, particularly if they are strongly sclerotised and likely to preserved in relief (see examples in [31,32]. Alternatively, these structures might also represent bundles of phosphatised muscles. Further ventral to the presumed mandibles (or muscle bundles) lies the first of three pairs of raptorial appendages. The proximal part of these appendages is obscured.

Raptorial appendages
The first of the series of three raptorial appendages is the smallest (Figures 1C, 2C) and is probably oval-shaped in cross section. Two elements can be differentiated. The proximal element is shorter than wide and bears spines along its inner margin. The exact nature of the proximal region is unclear, as the spines are broken off. A cluster of three spines originating together is apparent distally on the proximal element. The distal element is small, providing an attachment for at least four stronger spines. They are arranged along the median edge; the proximal spines point medially, the more distal ones progressively more distally.
The second raptorial appendage is significantly larger than the first, and appears to be composed of at least five elements ( Figures 1D, 2D, 4D, E); the appendage is presumably oval-shaped in cross section. The most proximal preserved element is short, about as long as wide, and lacks armature. The second element is the most elongate of the five, about 1.6 times as long as wide. The distal margin of the element is oriented oblique to the appendage axis (the outer edge of the element is about 1.6 times as long as the inner edge). The third element is only slightly shorter than the second, spinose, and angled about 45°inward. Due to the oblique joint between elements two and three, and the attitude of element three, this joint allows the appendage to function like a sub-chela. Three spines arise from the inner margin of element three, and are about three times as long as their diameter at the base. The third spine arises from the distal-median edge of the element. The two more proximal spines are about half as long as the width of the element; the third one is about twice as long but only slightly wider at its base. All three spines curve slightly inward. The position of the fourth element is evidenced only by a set of three spines, resembling those of the third element, but about 20% smaller. The fifth element is likewise only revealed by a triplet of spines, which are smaller than those of the fourth element.
The third raptorial appendage is the largest of the series, being more than 20% longer than the second one, and is presumably oval-shaped in cross section ( Figures 1D, 2D, 4D, E). Four elements are known. The most proximal one is the longest and appears to correspond to the second element (or the first two elements) of the second raptorial appendage. This first element is about 2.5 times as long as wide. The distal margin is oblique; the outer edge of the element is about 1.2 times longer than the inner edge. The second element is about 70% the length of the first, spinose and angled about 45°inward. Due to the oblique joint between elements one and two, and the attitude of element two, this joint forms a functional sub-chela like that of the second raptorial appendage. A single spine arises distomedially from element two. The spine is about as long as the width of the element, and curves slightly inward. The more distal elements are evidenced only by their spines. Element three corresponds to element four in the second raptorial appendage and strongly resembles it, differing only in its slightly larger dimensions. Element four bears three spines of which the second is the largest; it is about 1.5 times longer than the spine on element two. Proximally there is a smaller spine which is only about half the length of the second spine. Distally, part of an even smaller spine is preserved, presumably indicating the extremity of the appendage.

Trunk
A series of twenty-two short (in anterior-posterior dimension) bands, decreasing in height (dorsal-ventral) towards the posterior, is evident in UWGM 1748 and interpreted as the trunk (Figures 1A, 2A). The bands are probably defined mainly by internal rather than external structures, most likely the muscles within each somite ( Figures 1E,  2E, 4C). The number of divisions may vary. There appear to be twenty-two in UWGM 1769 but UWGM 1767 ( Figure 5A-C) preserves evidence of~20 (based on a combination of boundaries and limbs), a lower number that may reflect the smaller size of this specimen or be a result of preservation. A narrow bundle of muscles is preserved running the length of the trunk along its dorsal margin in UWGM 1767 ( Figure 5B). It is not clear whether one or both longitudinal muscles (overlapping) are preserved. Remnants of this longitudinal muscle may also be represented in UWGM 1748 (Figure 2A) running along the dorsal margin of the anterior part of the trunk; there is no evidence of a ventral longitudinal muscle. A pair of simple paddle-shaped appendages arises ventrally from each segment ( Figure 3A). The position of the ventral margin of the trunk is unclear but is constrained by the trace of the gut or musculature surrounding it (which must lie within the trunk!). The position of the gut is evident in UWGM 1748 (Figure 2A), 1749 and 1767 ( Figure 5B). The height (dorsalventral dimension) of the segment is at least 4 times the length of the appendages, which decrease in length toward the posterior. The appendages are 1.5 to 2 times as long as their preserved width. Posterior to the last segment a stout structure appears to bear the anus and is thus interpreted as the telson (Figures 2A, 3A).

Clausocaris lithographica Amended description
An extensive description of Clausocaris lithographica was provided by Polz [33]. Here we report new morphological details ( Figures 7A-G, 8, 9) and confirm most of his observations, for example the serration of the postero-dorsal area of the shield ( Figure 8D). New evidence shows that the raptorial appendages bear significantly more setae than previously observed ( Figure 7B, C). The proximal region (most likely the basipod) bears enditic protrusions: at least three are present on raptorial appendage 2 ( Figure 7D) and at least five on raptorial appendage 3 ( Figure 7F). Each endite is equipped with at least one row of at least 8 long setae ( Figure 7G). The appendages preserve long muscles ( Figures 7A, 8A, B, 9A, E) with fan-like attachment areas ( Figure 7E). Discrete muscle bundles are also apparent in the trunk region ( Figures 8C, 9D) consisting of a bundle of shorter muscles followed by a bundle of longer ones. This pattern allows the arrangement and number of trunk segments to be determined. Displacement of the muscle bundles ( Figure 9D, furthest left bundles) does not disturb this pattern. Eleven segments are evident posterior of the last raptorial appendages. Each one bears a pair of paddleshaped appendages equipped with setae along the lateral edge ( Figure 9B, F-G). The most posterior appendages are similar in structure to the preceding ones, but slightly longer ( Figure 9D). Dorsally at the anterior of the trunk a pair of leaf-like structures is apparent. These may represent gills.

Mayrocaris bucculata Amended description
An extensive description of Mayrocaris bucculata was provided by Polz [34]. We have new evidence of two pairs of appendages lying between the two large compound eyes (preserved compressed through the eyes) ( Figure 7H-J). The more anterior appendage lies slightly dorsal of the other, due to the strongly convex shape of the body in the anteriormost region. A more proximal and a distal part can be differentiated. The proximal part of the appendage consists of at least three similar elements, apparently tubeshaped and longer than wide. The distal part of the appendage appears flagellate and is comprised of at least nine elements. These also appear tube-shaped, but are significantly smaller than those of the proximal part; each element of the distal part is slightly longer than wide, the elements decreasing in width distally. It remains unclear whether additional distal elements might have been present but are not preserved. It is also uncertain whether the most proximal preserved element is the originally most proximal one, although this seems plausible. This appendage, with its anterior position, is interpreted as the antennula. The second appendage comprises five elements. These elements appear more robust than those of the first appendage, and vary in length. The two most proximal ones are about twice as long as wide. The third element is only one third of the length of the preceding one. The fourth one is longer again, about twice as long as element 3. Element 5 appears to be similar in length to element 3, but may be incomplete. It remains unclear whether further distal elements were present but are not preserved. This appendage is interpreted as the antenna.
Further posterior under the shield dorso-ventral bands are apparent. Such structures have been interpreted as gills [34]. Yet, here these structures seem more likely to represent the more anterior trunk segments.

Preservation of details
The relatively common preservation of muscles in specimens of Clausocaris lithographica ( Figure 10D) is remarkable. The long muscles ( Figures 7A, 8A-B, 9A, D), and their fan-shaped attachment ( Figure 7D), are unusual. Even more so are the tightly arranged muscles in the trunk, which also preserve a distinct pattern ( Figure 8C). These structures were observed by Polz [33], who interpreted them as parts of the trunk appendages. The distinction between trunk and appendages is evidenced by the setae on the latter (Figure 9C, F, G). The appendages are represented mainly by muscle and setae indicating that their cuticle was rather weakly sclerotised and lost through decay. Muscles are also preserved in each trunk segment of Thylacares brandonensis (Figures 2E, 4C) and help to define their boundaries and identify the segment count.

Assignment of Thylacares brandonensis to Thylacocephala
Thylacares brandonensis shows many characters typical of thylacocephalans, but also some features that are unusual for the group ( Figure 10A). The large bivalved shield lacks the characteristic well developed optical notch ( Figure 3A, B), a feature that was used to argue that some Cambrian taxa should be assigned to Thylacocephala [4]. Yet, other thylacocephalans appear to lack a pronounced optical notch [6]. This observation emphasizes that the shield morphology of bivalved arthropods is not a reliable guide to their affinity (e.g. [35]).
The eyes of T. brandonensis are also unusual in their small size and apparent stalked nature ( Figure 6A-D). The large compound eyes of other species of Thylacocephala are generally regarded as sessile [11]. Yet the eyes of many other thylacocephalans are unknown and may also have been small and stalked.
T. brandonensis bears three pairs of sub-chelate appendages (Figure 2A) that resemble those of other thylacocephalans in general structure and size, i.e. the first one is the smallest, the third the largest. Yet they are significantly shorter than those described in most other species (compare Figure 10B and Figure 10E).
The trunk of T. brandonensis is very similar to that of other representatives of Thylacocephala, although it is comprised of more segments.
The differences between this Silurian form and other thylacocephalans can be interpreted as plesiomorphic. A marked optical notch in some later thylacocephalans may be a derived feature, yet shield structures are highly likely to be convergent, and the plesiomorphic condition is difficult to infer. From a functional point of view the evolution of a notch is likely coupled to the reduction of the eye stalks and enlargement of the eyes in some forms. The long raptorial appendages of some later thylacocephalans, which contrast with the short limbs in the Silurian form, also appear to be a derived character, as other younger forms retain relatively short raptorial appendages [6].

The absence of gills
The presence of eight gills has been advocated as an important character of Thylacocephala (e.g. [12,17]). While it is difficult to judge whether a structure in a fossil is a gill [36], the exquisite preservation in some of the fossils from La Voulte allows virtually no other interpretation [12,17].
We have observed no features in T. brandonensis that resemble gills. T. brandonensis may represent a sister species to all other thylacocephalans, in which case the absence of gills might be plesiomorphic; alternatively gills may have been lost through decay.
The features interpreted as gills in M. bucculata [34] may simply represent the upper preserved part of the anterior trunk segments; they are in a similar position to these segments in T. brandonensis. Eight poorly preserved gills have also been reported in C. lithographica [33], but they are not evident in the specimens we investigated. Thus, while some thylacocephalans appear to have eight sets of (supposed) gills, it is not clear whether this is a diagnostic character of the group.

Assignment to Eucrustacea
Most authors have considered Thylacocephala as an ingroup of Eucrustacea, yet unequivocal evidence for this assignment has been lacking. Lange et al. [37] noted that the presence of two pairs of antennae in Thylacocephalus cymolopos from the Upper Cretaceous of Lebanon supports the assignment of thylacocephalans to crustaceans. There is also evidence of two pairs of antennae in Thylocaris brandonensis. Although the second appendage is called an 'antenna' in Eucrustacea, it is antenniform only in Eumalacostraca. In other eucrustaceans it is used mainly for locomotion, and sometimes resembles the mandible but never the antennula (see discussion in [38]). Thus the morphology of the second antenna varies in different eucrustaceans and this character must be used with caution in determining affinity.
The morphology of the appendages of Thylacocephala appears to be highly derived, which makes comparison with other arthropods difficult. One new observation reported here supports a eucrustacean affinity. The proximal region of the raptorial appendages of Clausocaris lithographica bears up to five enditic projections with rows of setae. Median enditic armature is widespread among Euarthropoda. Slight elevations in early chelicerates bear just a single strong spine accompanied by two smaller spines. Similar arrangements are found in early crustaceans. In labrophoran crustaceans the endites are more strongly pronounced and bear more complex armature (see e.g. [38]). Several strong endites with setose armature are developed in entomostracan eucrustaceans and in at least some appendages of malacostracan eucrustaceans. Hence the presence of up to five pronounced endites with numerous setae on the raptorial appendages of Thylacocephala supports a eucrustacean affinity.

Systematic position: earlier ideas
Since their recognition as a separate group [16,30] thylacocephalans have been assigned to a range of eucrustacean groups including stomatopods, decapods and cirripedes, and a superficial resemblance to Remipedia has also been noted [3,39].
The comparison with stomatopods (see discussion in [3] and [40] was based on the shape of the shield of some Cretaceous species, which in lateral aspect resembles that of certain stomatopod larvae. The appendage morphology, however, is incompatible with stomatopods. Although the raptorial appendages of stomatopods are described as subchelate they differ strongly from those of thylacocephalans in overall structure. Most important of these differences is the double flexure that results in a Z-shape in stomatopods, whereas the raptorial appendages of thylacocephalans are only "folded" once, and do not close fully. The distal movable finger in stomatopods is formed by a single element, while it comprises four or five in thylacocephalans. There are five pairs of sub-chelate appendages in stomatopods, the second of the series being the largest (at least in extant forms), while in Thylacocephala the last of three pairs is the largest.
The arrangement of tagmata in thylacocephalans, and especially in Thylacares brandonensis, argues against a malacostracan affinity, including stomatopods. The trunk of up to 22 undifferentiated segments strongly differs from that of Malacostraca, which is consistently differentiated into a thorax of eight segments and a pleon of six (sometimes five in Eumalacostraca) or seven (Phyllocarida). The arrangement of tagmata in Thylacocephala also rules out a decapod affinity, a suggestion prompted by the similarity of the gills in certain thylacocephalan species to those in decapods [17]; the nature of the gills in fossil arthropods is difficult to infer without evidence of the ultrastructure of the surface epithelium [36].
The long trunk does not support a close affinity between Thylacocephala and Cirripedia (as pointed out by [41,42]); the trunk of cirripedes includes only six segments. Like cirripedes, thylacocephalans possess pits on their shield [42], which may represent a dorsal organ. While the special arrangement of these pits in the so-called lattice organ has been argued to be an autapomorphy of Euthecostraca (which also includes cirripedes: [43]) such pits are widespread among crustaceans and even other euarthropods [44]. Abbreviations: a = antennula; a2 = antenna; ce = compound eye; ed = endites; gs = presumed gill structure; m = muscles; ma = muscle attachment areas; ra = raptorial appendage; tr = trunk.

Systematic affinities: sistergroup to Remipedia?
The new details reported here prompt a reconsideration of a possible affinity of thylacocephalans to remipedes supporting suggestions by Schram [39]). Remipedes possess three pairs of sub-chelate appendages ( Figure 10C): the posterior two head appendages (maxillula and maxilla) and the first trunk appendage (maxilliped).
Three pairs of limbs are present anterior to the raptorial appendages in Clausocaris lithographica, namely antennula, antenna and mandibles [19]. Our observations reveal at least two pairs of anterior appendages in Mayrocaris bucculata (most likely representing antennula and antenna, similar to strucures observed in Thylacocephalus cymolopos [37]) and possibly three in Thylacares brandonensis (antennula, antenna and mandibles). Thylacocephala in general appear to bear at least three pairs of appendages anterior to the first raptorial one.
The segmental affiliation of the three raptorial appendages in Thylacocephala is uncertain. They have been interpreted as belonging to the anterior trunk segments (see summary in [19]), but their position in the specimens described here makes this unlikely. The raptorial appendages are broad proximally and apparently too robust to attach to the short trunk segments, except perhaps the most anterior ones. Both their position and size indicates that at least some of the raptorial appendages belong to the posterior divisions of the head.
Thus the three pairs of raptorial appendages in Thylacocephala could represent maxillula, maxilla and trunk limb one (maxilliped), or maxilla and trunk limbs one and two. Additional material is required to resolve this question but a positional homology (homotopy) between the raptorial appendages of Thylacocephala and Remipedia is at least plausible.
Morphological similarities between the raptorial appendages in the two groups strengthen this assumption. The proximal part of the appendages (probably a basipod) bears setose endites in both. More importantly, three or more distal elements form the functional finger of the subchela in thylacocephalans as well as in remipedes ( Figure 10B, C, E). This is an unusual character state, as the functional finger of other subchelae in crustaceans comprises only the most distal element (e.g. in mantis shrimps and gammarids), or the distalmost two (e.g. in slipper lobsters).
The multisegmented and relatively undifferentiated trunk in Thylacares brandonensis, which bears more than twenty appendages, is unusual among eucrustaceans. Apart from the modern branchiopod ingroups Polyartemia and Phyllopoda, which, unlike thylacocephalans, possess phyllopodous appendages (i.e. limbs differentiated into a basipod with median endites, a reduced endopod, a paddle-shaped exopod and lateral epipods; e.g. [45]), only Remipedia show such a high number of trunk segments. The eucrustacean trunk is usually differentiated into at least two tagmata: thorax and pleon in malacostracans and thorax and abdomen in entomostracans [46], in contrast to the trunk in thylacocephalans and Remipedia, where there is only one tagma. The specialisation of the posterior head appendages and anteriormost trunk appendage as sub-chelate raptorial appendages with setose endites and a finger made up of the three or more distal elements represents a potential synapomorphy of Thylacocephala and Remipedia. This, together with the multisegmented trunk, suggests a sister-group relationship.
Additional material is necessary to determine whether the three raptorial appendages in Thylacocephala are homologous in position with maxillula, maxilla and maxilliped and to test the possibility of a sister group relationship with Remipedia. Such evidence is a prerequisite for a rigorous phylogenetic analysis.

Functional morphology and 3D modelling of Thylacocephala
Previous authors (e.g., [4,20]) have considered the thylacocephalans to be nectic or necto-benthic predators. The new evidence presented here allows us to refine these interpretations.
Thylacocephalans are usually reconstructed with their raptorial appendages pointing forward, i.e., more or less in the axial plane of the body. However, our new reconstruction ( Figure 10A, D) shows that such an arrangement is unlikely. Given the narrow ventral gape, which has been reconstructed in different species [4], and the relatively large size of the appendages, only certain positions are possible. The proximal podomeres with their endites and Close up of trunk region; note the distinct arrangement of muscles: in each segment a short muscle is followed by a long muscle which appears to extend into the appendages. E-G. SMNS 70193/4. E. Overview. F. Close up of paddle-shaped appendage, setae color-marked. G. Same as in F, without color-markings. Abbreviations: ce = compound eye; lm = long muscles; ma = muscle attachment areas; pa = paddle-shaped appendages; ra = raptorial appendage; sh = shield; sm = short muscles; so = shield outline; sr = serrations; st = setae; tr = trunk.
armature must have interacted with the opposing appendage of the pair and therefore cannot face directly forward, but at most antero-medially. When the valves are closed, the largest (third) appendages would occupy almost the entire width of the ventral gape; there is no space for the two other pairs of raptorial appendages to lie inside or outside the valves. This problem is overcome by rotating the proximal podomeres about 45°abaxially to accommodate all three appendages within the narrow ventral gape in an anterior-posterior sequence.
In remipedes the appendages are also held in an abaxial orientation, at an even higher angle than in thylacocephalans. Mantis shrimps, although not closely related, provide a further functional comparison. They accommodate four pairs of sub-chelate appendages which are also rotated away from the axis. The arrangement of the raptorial appendages in mantis shrimps, near parallel to the axial plane, is achieved by the presence of an additional joint, resulting in a z-shape.
The attitude of thylacocephalan raptorial appendages reconstructed here is supported by several specimens that preserve them directed posteriorly ( Figures 7E, F, 8A). Preservation of appendages projecting forwards in some specimens and backwards in others is less likely if the appendages are held in an orientation parallel to the axial plane of the body.
The appendages are reconstructed here forming a kind of basket. Such an arrangement would have facilitated predation. In the absence of a second, strongly flexed joint, such as that in mantis shrimps and some greatappendage arthropods [47], the raptorial appendages of thylacocephalans could not extend forward. More likely, the crustacean swam forward using the trunk appendages and trapped prey or at least grabbed food items with the raptorial appendages.
The paddle-shaped trunk appendages identified here in Clausocaris lithographica are compatible with a swimming function. They are equipped with setae enlarging the surface, and powered by well-developed muscles. The last pair of trunk appendages, which are more elongate than those lying anterior to them, may have functioned as a steering device, in a manner similar to the uropods of eumalacostracans and the furca of phyllocarid and entomostracan eucrustaceans.

Conclusions
1. The Silurian thylacocephalan Thylacares brandonensis, described here for the first time, appears less derived than many of the later representatives of the group. 2. Features "typical" for many thylacocephalans, such as a marked optical notch on the shield, sessile hypertrophied eyes and extremely elongate raptorial appendages, as well as a relatively short trunk, evolved after the early Paleozoic.


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RUNX3 regulates vimentin expression via miR-30a during epithelial–mesenchymal transition in gastric cancer cells

Runt-related transcription factor 3 (RUNX3) is a putative tumour suppressor via regulating the expression of a series of target genes. Clinical studies demonstrated that loss of RUNX3 expression is associated with gastric cancer progression and poor prognosis, but the underlying mechanism is not entirely clear. Accumulating evidence shows that the epithelial–mesenchymal transition (EMT) plays an important role in cancer relapse and metastasis. Therefore, we addressed whether RUNX3 has a role in the EMT in gastric cancer. Knockdown of RUNX3 promoted cell invasion and increased the protein expression of the mesenchymal marker vimentin in human gastric cancer cells. Overexpression of RUNX3 suppressed cell invasion and decreased the protein expression of vimentin in the cells and inhibited gastric cancer cells colonization in nude mice. Furthermore, overexpression of RUNX3 increased the expression of microRNA-30a (miR-30a), and miR-30a directly targeted the 3′ untranslated region of vimentin and decreased its protein level. miR-30a inhibitor abrogated RUNX3-mediated inhibition of cell invasion and downregulation of vimentin. Thus, RUNX3 suppressed gastric cancer cell invasion and vimentin expression by activating miR-30a. In gastric cancer patients, levels of RUNX3 were positively correlated with miR-30a and negatively associated with the levels of vimentin. Collectively, our data suggest a novel molecular mechanism for the tumour suppressor activity of RUNX3. Effective therapy targeting the RUNX3 pathway may help control gastric cancer cell invasion and metastasis by inhibiting the EMT.

Introduction
Gastric cancer is one of the most common malignant tumours worldwide and ranks second in terms of global cancer-related death [1,2]. The most common cause of the death from gastric cancer is invasion and metastasis of the tumour [3]. Malignant tumour invasion and metastasis is a complex biological process, including the isolation of cancer cells from the primary lesion, invasion into the surrounding tissues, penetration of the basement membrane into blood vessels and lymphatic vessels, oozing from blood and lymphatic vessels, and reformation of metastatic nodules and then proliferation to new metastases [3][4][5]. In the metastatic cascade, local invasion is considered an initial and essential step leading to distant metastasis [5,6]. In this process, the epithelial-mesenchymal transition (EMT) was thought to play a critical role.
The EMT is a complex biological process whereby immotile, polarized epithelial cells are converted to cells with a motile mesenchymal cell phenotype, which enhances migratory capacity, invasiveness and resistance to apoptosis, thus contributing to cancer progression [7][8][9][10]. The EMT programme is regulated by some transcriptional factors, including the zinc finger proteins Snail1, Slug and the basic helix-loop-helix factors Twist1/2 and Six, and the TGF-b and Wnt/ b-catenin signalling pathways. These transcriptional factors or signalling pathways repress the expression of the epithelial marker Ecadherin and induce the expression of the mesenchymal marker vimentin [11][12][13][14][15][16].
Human runt-related transcription factor (RUNX) genes are homologues to Drosophila Runt [17] and Lozenge [18] genes and contain highly conserved sequences encoding the 'runt domain', which mediates the binding of RUNX protein to specific DNA sequences and causes transcriptional activation or repression of the downstream target genes [19,20]. The human RUNX family has three members: RUNX1/AML1, RUNX2/CBFA1 and RUNX3/ PEBP2aC/AML2. All three members share similar structures, but have different functions [21][22][23]. Runt-related transcription factor 3 (RUNX3) is involved in the development of the gastrointestinal tract and neurogenesis during embryonic development. Recently, inactivation or loss of RUNX3 was found closely associated with tumourigenesis and tumour progression [24]. RUNX3 is involved in transforming growth factor b (TGF-b)-induced growth inhibition and apoptosis. On TGF-b stimulation, RUNX3 cooperates with SMADs to directly up-regulate P21 and Bim, which inhibits cell growth and increases cell apoptosis respectively [25][26][27]. RUNX3 deficiency caused a precancerous state in the gastric epithelia of mice [28]. Recently, Lin et al. [20] showed that loss of RUNX3 expression enhanced the Akt1-mediated signalling pathway and increased tumourigenesis in human gastric cancer. Besides, RUNX3 expression was significantly reduced in clinical tissue samples of peritoneal metastasis in gastric cancer as compared with normal gastric mucosa or primary tumours [29]. In addition, RUNX3 overexpression inhibited gastric cancer angiogenesis by decreasing the expression of VEGF and suppressed gastric cancer metastasis [30]. Chang et al. [31] showed that RUNX3 could directly bind to the promoter of Claudin-1 and inhibit tumourigenesis and metastasis in gastric epithelial cells. As well, RUNX3 suppressed gastric cancer metastasis by inactivating MMP9 via up-regulating TIMP-1 [32].
Here, we investigated whether RUNX3 regulates the EMT in gastric cancer cells. We examined the effect of increased or decreased RUNX3 expression on the invasion potential of human gastric cancer cells and the expression of the EMT molecules vimentin and E-cadherin. Our data provide a novel mechanism for RUNX3-mediated suppression of gastric cancer invasion and metastasis.

Patients
We obtained tumour specimens and surrounding normal tissue from 55 patients with primary gastric cancer who underwent gastrectomy at the Cancer Hospital of Shandong Province in 2012-2013. Samples were stored at À80°C. We collected data on patient age, sex and tumour histology, differentiation status, size (diameter), invasiveness, and regional and distant metastases at the time of surgery (pathologic tumour-node-metastasis classification). Detailed patient and disease characteristics are documented in Table 1. The study was approved by the ethics committee of School of Medicine, Shandong University.

Reporter vector construction and luciferase assay
Luciferase reporter vector pMIR-REPORT (Ambion, Austin, TX, USA) was used to generate luciferase reporter constructs. The 366-bp miR-30a binding sequence at the 3′ untranslated region (3′ UTR) of human vimentin gene (Vim) was amplified and cloned into the SpeI/HindIII sites of a luciferase gene in the pMIR-REPORT luciferase vector (pMIR-Vim/wt). Two miR-30a complementary sites with the sequence GTTTAC in the 3′ UTR were mutated to remove complementarity with miR-30a by use of a QuikChange site-directed mutagenesis kit with pMIR-Vim/ wt as the template. All the primer sequences were listed in Table 2. The mutants were named pMIR-Vim/mut1 and pMIR-Vim/mut2. Gastric cancer cells were seeded in 24-well plates and transiently transfected with appropriate reporter plasmid and miRNA by use of Lipofectamine 2000. The cells were harvested and lysed after 48 hrs. Luciferase activity was measured by use of the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Renilla luciferase was used for normalization. For each plasmid construct, transfection experiments were performed in triplicate.

RNA extraction and real-time PCR
Total RNA in cells and tissue was extracted by use of TRIzol reagent (Invitrogen). Primers for miR-30a, miR-30e and U6 were from RiBo-Bio. Primers for vimentin, E-cadherin, Snail, Slug, Twist and b2-M were listed in Table 3. The first-strand cDNA was synthesized with random primers (N6) or miRNA-specific primers and M-MLV reverse transcriptase (Ferments). Real-time PCR involved the use of the ABI7700 sequence detector (Applied Biosystems, Foster City, CA) with the SYBR Green kit (TakaRa, Dalian, China). Calculation of target mRNA levels was based on the CT method and normalization to human b2-M or U6 expression. All reactions were run in triplicate. ‡Semi-quantitative analyses of Runx3 and vimentin protein levels in cancer specimens using western blot and relative abundances of each protein were calculated based on band density values and normalized with b-actin.-: undetectable; RUNX3, runt-related transcription factor 3.

Microarray analysis
Total RNA was harvested from cells by use of TRIzol (Invitrogen) and the miRNeasy mini kit (Qiagen, Valencia, CA, USA). Samples were labelled by use of the miRCURY LNA microRNA Hy3/Hy5 Power labelling kit and hybridized on the miRCURY LNA Array (v.16.0). After washing steps, the slides were scanned by use of the Axon GenePix 4000B microarray scanner (Molecular Devices, Inc., Sunnyvale, CA, USA). Scanned images were imported into GenePix Pro 6.0 (Axon) for grid alignment and data extraction.

Matrigel invasion assay
AGS, BGC-823 and SGC-7901 cells transfected with pcDNA3.1 or the RUNX3 overexpression plasmid RUNX3/pcDNA3.1 were harvested and re-suspended in serum-free RPMI-1640 medium, and 1 9 10 5 cells were seeded into the upper 24-well chambers coated with 150 mg Matrigel in a cell invasion system (BD Biosciences). Medium with 10% FBS was added to the lower chambers as a chemoattractant. After 36 hrs, cells remaining on the upper surface of the membrane were removed with the use of a cotton swab, and cells that had invaded the membrane filter were fixed with 100% methanol, stained in a dye solution containing 0.05% crystal violet and photographed under a microscope. The number of invading cells was counted from three independent experiments.

Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation assay was as previously described [7]. Briefly, AGS cells (no endogenous RUNX3 expression) transfected with pcDNA3.1 or RUNX3/pcDNA3.1 plasmid were cross-linked by incubation in 1% (v/v) formaldehyde-containing medium for 10 min. at 37°C, then sonicated to form soluble chromatin. Antibodies against RUNX3 (Abcam) were used to precipitate DNA fragments bound by their corresponding elements. The protein-DNA complex was collected with the use of protein A Sepharose beads (Millipore), eluted and reverse crosslinked. After treatment with protease K (Qiagen), samples were extracted with phenol/chloroform and precipitated with ethanol. The recovered DNA was re-suspended in TE buffer and amplified by PCR with primers for the miR-30a promoter listed in Table 3.

Mouse model
Female athymic BALB/c nude mice (Shanghai Slac Laboratory Animal Co., China) were used to analyse metastatic potency in vivo. BGC-823 cells were transfected with pcDNA3.1 or RUNX3/pc3.1, then screened with G418 to obtain stably transfected cells. Gastric cancer cells (6 9 10 6 per mouse) were injected intravenously into the mouse tail vein. Animals were killed after 4 weeks, lungs were excised and tumours on the lung surface were counted. Lungs were fixed in formaldehyde solution and stained with haematoxylin and eosin to examine metastatic nodules.

Statistical analysis
Data are presented as mean AE SD and compared by two-sided unpaired t test. Correlation analyses of RUNX3, miR-30a and vimentin in GC samples were made using linear regression. All experiments were repeated three times. Data analysis involved the use of SigmaStat3.1 (Systat Software, Inc., Richmond, CA, USA). P < 0.05 was considered statistically significant. 5′-CAACATAATCTATCGCGACGAAAAATCT-3′ 5′-AGATTTTTCGTCGCGATAGATTATGTTG-3′ Table 3 Primer sequences used in the study

RUNX3 inhibition induced a mesenchymal phenotype in gastric cancer cells and stimulated tumour cell invasion
First, we investigated the expression of RUNX3 in gastric cell lines. The human immortalized gastric epithelial cell line GES-1 showed the highest expression of RUNX3; AGS and KATOIII lines show no expression of RUNX3; BGC-823 and SGC-7901 lines showed a moderate expression of RUNX3 (Fig. 1A). The result was consistent with the Guo et al. results [33]. Therefore, we chose BGC-823 and SGC-7901 cells for RUNX3 siRNA and expression vector transfection and AGS cells for RUNX3 expression vector transfection.
Runt-related transcription factor 3-specific siRNA transfected into BGC-823 and SGC-7901 cells to knock down RUNX3 expression produced an elongated fibroblastoid-like morphologic alteration in transfected cells (Fig. 1B). Therefore, we investigated the expression of the epithelial and mesenchymal markers E-cadherin and vimentin, respectively, in gastric cancer cells transfected with control siRNA and RUNX3 siRNA. Gastric cancer cells transfected with RUNX3 siRNA showed decreased RUNX3 protein expression (Fig. 1C). The expression of E-cadherin was not changed significantly, but that of vimentin was increased in cells with RUNX3 knockdown (Fig. 1C). Because the EMT programme activation contributes to cancer cell invasion, we determined the invasive potential of cells transfected with control siRNA or RUNX3 siRNA. Invasion of BGC-823 and SGC-7901 cells was greater with RUNX3 siRNA than control siRNA ( Fig. 1D and E).

RUNX3 inhibited tumourigenesis and colonization of gastric cancer cells in vivo
We then examined whether RUNX3 overexpression inhibited the metastasis of gastric cancer cells in vivo. We injected BGC-823 cells transfected with pcDNA3.1 or RUNX3/pc3.1 into the tail vein of nude mice and examined their lungs for tumour seeding. Metastatic nodules were fewer with RUNX3/pc3.1 than pcDNA3.1 transfection (Fig. 2D-F).

RUNX3 inhibition or overexpression had no effect on vimentin mRNA level
To further test whether RUNX3 affects the transcription of genes regulating the EMT, we examined the effect of RUNX3 on the mRNA level of vimentin, E-cadherin, Slug, Twist and Snail in BGC-823 and SGC-7901 cells. Neither RUNX3 inhibition nor overexpression had an effect on the mRNA levels of these genes ( Fig. 3A and B). Therefore, RUNX3 did not regulate vimentin at the transcriptional level.
We next determined whether the RUNX3-mediated decrease in vimentin resulted from proteasomal degradation. We treated cells transfected with RUNX3 expression vector with the proteasome inhibitor MG132 and detected the protein level of vimentin by western blot analysis. The addition of MG132 did not abolish the ability of ectopically expressed RUNX3 to decrease vimentin protein level in gastric cancer cells (Fig. 4A and B).

RUNX3 activated miR-30a expression
Because miRNAs can suppress gene expression by binding to the 3′ UTR of target mRNAs and these interactions may inhibit translation of the targeted mRNAs or their degradation, we next determined whether RUNX3 activated an miRNA, then whether the miRNA targets to degrade vimentin. We first used miRNA array to screen miRNAs regulated by RUNX3 and found 15 miRNAs activated by RUNX3. We searched miRNA databases to analyse these differentially expressed miRNAs and found that among the 15 miRNAs, only miR-30a and miR-30e were partly complementary to the conserved site within the 3′ UTR of vimentin (Fig. 5A). Therefore, we overexpressed or knocked down RUNX3 in BGC-823 and SGC-7901 cells and used qRT-PCR to  detect the miR-30a and miR-30e mRNA levels in these cells. We found that RUNX3 overexpression up-regulated and inhibition downregulated the expression of miR-30a, but had no effect on miR-30e level ( Fig. 5B and C).
To further determine whether RUNX3 regulate the expression of miR-30a by directly binding to the corresponding genomic sequences, we used Genomatrix to analyse the upstream region of mature miR-30a and found a consensus binding sequences of RUNX3 'TGTGGT' (RBS) between the nucleotides -3854 to -3861. As AGS cell has no engeneous RUNX3 expression, we transfected RUNX3/ pcDNA3.1 into the cells and used ChIP assay to confirm the direct binding of RUNX3 to the RBS sites in RUNX3-overexpressing AGS cells using anti-Runx3 antibody (Fig. 5D).

miR-30a targeted the vimentin 3′ UTR directly and downregulated vimentin protein expression
Next, we investigated whether miR-30a affected the expression of vimentin. We transfected the inhibitor or mimics of miR-30a into SGC-7901 cells and used qRT-PCR and Western blot analyses to detect the expression of vimentin. miR-30a mimics inhibited and miR-30a inhibitor induced vimentin protein level, but had no effect on its mRNA level (Fig. 6A and B). To further determine whether miR-30a directly targets vimentin mRNA in gastric cancer cells, we inserted the full-length 3′ UTR of vimentin into the pMIR-REPORT luciferase vector (pMIR-Vim; Fig. 6C) and investigated the effect of miR-30a on the luciferase activity of pMIR-Vim. miR-30a significantly reduced the activity of the luciferase reporter of pMIR-Vim (Fig. 6D). Because two conserved sites within the 3′ UTR of vimentin were identified to be complementary to miR-30a, we generated two 3′ UTR mutants, pMIR-Vim/mut1 and pMIR-Vim/mut2 (Fig. 6C), and investigated the effect of miR-30a on their luciferase activity. The luciferase activity was significantly reduced in the presence of miR-30a with pMIR-Vim/ mut1 but not pMIR-Vim/mut2 (Fig. 6D). Therefore, the second region of the 3′ UTR of vimentin is important for miR-30a binding.

mir-30a was critical for RUNX3-mediated cell invasion inhibition and vimentin downregulation
We next ascertained whether RUNX3 regulates EMT and vimentin expression via an miR-30a-dependent mechanism. We knocked down miR-30a with an inhibitor of miR-30a in RUNX3-overexpres-sed gastric cancer cells and detected cell invasion and the expression of vimentin. The miR-30a inhibitor abrogated RUNX3-mediated inhibition of cell invasion (Fig. 7A and B). The RUNX3-mediated downregulation of vimentin protein level was also abrogated with the miR-30a inhibitor (Fig. 7C, Figure S2). Therefore, RUNX3-mediated cell invasion inhibition and vimentin downregulation depended on miR-30a.
RUNX3 expression was associated with miR-30a and vimentin in primary gastric cancer analysis showed that the expression of RUNX3 in tumour tissue was significantly lower than that in surrounding normal mucosa (Fig. 8A and B). A highly significant negative correlation between RUNX3 and vimentin protein was observed in these gastric cancer samples (P < 0.0001; Fig. 8C). The expression of miR-30a was reduced in 43/ 55 (78.2%) of the gastric cancer samples as compared with the surrounding normal mucosa (Fig. 8D). In all, 29/55 samples showed the same trend of decreased RUNX3 and miR-30a expression in tumour rather than normal tissue. Statistical analysis showed that RUNX3 and miR-30a levels were highly correlated in gastric cancer samples (P < 0.0001; Fig. 8E).

Discussion
Runt-related transcription factor 3 is closely associated with the development and progression of gastric cancer. Clinical studies showed that a decrease or loss of RUNX3 expression was inversely related to survival and was an independent predictor of poor patient outcome. As well, restoration of RUNX3 expression greatly suppresses tumour growth and metastasis [34]. However, how RUNX3 is involved in cancer progression remained incompletely understood. In this study, we addressed whether RUNX3 has a role in the EMT, related to cancer relapse and metastasis, in gastric cancer. Knockdown of RUNX3 promoted the EMT and increased the protein expression of the mesenchymal marker vimentin in human gastric cancer cells. Overexpression of RUNX3 suppressed cell invasion and decreased the protein expression of vimentin in gastric cancer cells and inhibited gastric cancer cell colonization in nude mice. Furthermore, overexpression of RUNX3 increased the expression of miR-30a, which directly targeted the 3′ UTR of vimentin and decreased its protein level. An miR-30a inhibitor abrogated RUNX3mediated inhibition of cell invasion and downregulated vimentin. In gastric cancer patients, levels of RUNX3 were positively correlated with miR-30a and negatively associated with vimentin. Thus, RUNX3 suppressed gastric cancer cell invasion and vimentin expression by activating miR-30a. We provide evidence that RUNX3 plays a significant role in regulating the EMT, thereby contributing to cancer progression. In this study, RUNX3 inhibition and overexpression increased and decreased human gastric cancer cell invasion respectively. In vivo, RUNX3 inhibits human gastric cancer cells metastasis. Tanaka et al. [35] also found that RUNX3 reversed the EMT in hepatocellular carcinoma, but the mechanism underlying RUNX3 action is different in gastric cancer cells. Tanaka et al. [35] showed that ectopic RUNX3 protein expression induced E-cadherin expression and suppressed vimentin expression in hepatocellular carcinoma cells with low EMT. However, in our study, RUNX3 did not induce E-cadherin in human gastric cancer cells. Different tumour cell types may have different mechanisms in regulating the EMT.
We found that RUNX3 greatly inhibited vimentin protein expression, but had no effect on vimentin transcription. Because the tran- scription factors Snail, Slug/Snail2 and Twist play an important role in regulating the EMT [36], we determined whether RUNX3 regulates the expression of these transcription factors. Our results showed that RUNX3 did not affect the mRNA level of these factors, so RUNX3 does not regulate the EMT at the transcription level. We further investigated whether RUNX3 regulates vimentin protein expression through a ubiquitin-proteasome degradation pathway. The addition of the proteasome inhibitor MG132 had no effect on RUNX3-mediated downregulation of vimentin protein, so the ubiquitin-proteasome pathway was not involved.
MiRNAs can interact with complementary sequences in the 3′ UTR of the target mRNA to induce translational repression or target degradation [37]. Recently, many studies have suggested that miR-NAs contribute to the invasion and metastasis of various types of human cancers by regulating the EMT [37][38][39][40][41][42]. Vimentin is one of the targets that can be regulated by miRNAs. Yamasaki et al. showed that miR-138 targeted vimentin and inhibited cell migration and invasion in renal cell carcinoma. Cheng et al. [38] showed that miR-30a could directly bind to the 3′ UTR of vimentin and inhibit its translation, then reduce the protein level of vimentin in breast cancer. Therefore, we considered that RUNX3 regulates the expression of some miRNAs, which contributes to the downregulation of vimentin protein and the inhibition of invasion and metastasis of gastric cancer. First, we used miRNA array to detect the miRNAs that could be regulated by RUNX3 and detected 15 miRNAs activated by RUNX3. Among these miRNAs, miR-30a and miR-30e were predicted to target to vimentin. We overexpressed or knocked down RUNX3 in gastric cancer and detected the mRNA expression of miR-30a and miR-30e. Only miR-30a was significantly activated by RUNX3. To investigate whether miR-30a regulates the expression of vimentin, we transfected gastric cancer cells with miR-30a mimics and detected the protein and mRNA levels of vimentin. Overexpression of miR-30a decreased the protein level of vimentin in human gastric cancer cells, but had no effect on the mRNA level. In contrast, inhibition of miR-30a increased the protein level of vimentin. As well, miR-30a could directly bind to the 3′ UTR of vimentin, which is consistent with the results of Cheng et al. [38] in breast cancer. To further detect whether RUNX3 downregulated vimentin expression through miR-30a, we added the miR-30a inhibitor into gastric cancer cells transfected with the RUNX3 expression vector and found that the miR-30a inhibitor abolished the RUNX3-mediated downregulation of vimentin and the inhibition of gastric cancer cell invasion. Therefore, RUNX3 downregulated vimentin and inhibited gastric cancer cell invasion through an miR-30adependent mechanism. Collectively, our data suggest a novel molecular mechanism for the tumour suppressor activity of RUNX3. Targeting the RUNX3 path-way may help to control gastric cancer invasion and metastasis by inhibiting the EMT.


extfieldsofstudy: Medicine Biology

text:
IMP-1 encoded by a novel Tn402-like class 1 integron in clinical Achromobacter xylosoxidans, China

Achromobacter xylosoxidans strain A22732 is isolated from a pneumonia patient in China and produces carbapenemases OXA-114e and IMP-1, which are encoded by chromosome and plasmid, respectively, and confer resistance to multiple ß-lactam antibiotics including carbapenems. The blaIMP-1 gene together with aacA7 and orfE is captured by a novel Tn402-like class 1 integron in a conjugative IncP-1ß plasmid. In addition to the intrinsic integron promoter PcW, there is still a blaIMP-1 gene cassette-specific promoter. This is the first report of carbapenemase-encoding IncP-1ß plasmid in clinical bacterial isolate.

negative E. coli transconjugant, designated A22732-IMP-EC600, was obtained by conjugal transfer, indicating that A22732 harbored a conjugative IMP-encoding plasmid, which was designated pA22732-IMP. As determined by a modified CarbaNP test 6 , strain 22732-IMP-EC600 had class B carbapenemase activity, while A22732 probably expressed class B/D carbapenemases (Fig S1), being consistent with the above PCR/sequencing results.
The minimum inhibitory concentration (MIC) values (Table 1) were determined for A22732, 22732-IMP-EC600, and EC600. A22732 and 22732-IMP-EC600 show almost identical drug resistance profiles. These two strains are highly resistant to penicillins, aztreonam, and ephalosporins tested, but they remain susceptible to the fluoroquinolone, furane, aminoglycoside, and sulfanilamide drugs tested. Both A22732 and 22732-IMP-EC600 are resistant to imipenem and meropenem, but the MIC values of imipenem and meropenem (both .516) against A22732 are much higher than those (4 and 2, respectively) against 22732-IMP-EC600, which is consistent with the fact that A22732 expresses two different arbapenemase IMP-1 and OXA-114e while 22732-IMP-EC600 produces only a single one IMP-1.
The entire pA22732-IMP sequence had .70% query cover and .80% identity with the above IncP-1ß plasmids. pA22732-IMP was aligned with four representative plasmids R751 (accession number U67194) 8 , pB10 9 (AJ564903), pB8 (AJ863570) 10 , and pAKD31 (JQ436721) 11 through pairwise whole plasmid sequence comparison (Fig 2). R751 is from a clinical Enterobacter aerogenes isolate and represents the prototype IncP-1b drug-resistance plasmid, pB10 is isolated from a bacterial community residing in activated sludge compartment of a waste-water treatment plant, and pB8 and pAKD31 are from agricultural soil bacteria. The above IncP-1ß plasmids were selected for further analyses, because they harbored different forms of Tn501-like or Tn402-like elements (see below).
The backbone regions are highly conserved in pA22732-IMP, R751, pB10, pB8, and pAKD31. All the former four plasmids harbor two accessory modules, namely Tn501-like element and Tn402-like integron, each of which has different gene organizations among these four plasmids (Fig 2). The above two accessory modules are inserted into downstream of trfA and that of traC2, respectively, which are the targeting locations typically interrupted by mobile elements in IncP-1b plasmids. By contrast, only Tn501-like element rather than Tn402-like integron is found in the corresponding region of pAKD31 (Fig 2).
Tn501-like loci can be identified in all the above five plasmids (Fig 3). The Tn501-like element of pB10 is flanked by a 5-bp target site (direct repeats of TGCCT), but those of all the other four plasmids leave no trace of insertion.
pB10 harbors an greatly extended Tn501-like mer locus, because tnpA is interrupted by IS1071 (which is further interrupted by Tn1721-like tetracycline-resistance locus) plus a Tn5393c-like streptomycin resistance locus, thereby leading to partial deletion of tnpA. Loss of IRi and tnpAR as well as 3'-terminus deletion of orf2, because of insertion of IS1071 plus IS21 into orf2, is observed for the Tn501like mer locus of pAKD31. The Tn501-like mer locus of pA22732-IMP has undergone loss of IRi, tnpAR, and orf2 as well as an inversion event of the whole locus.
Both R751 and pB8 carry a 'cryptic' Tn501-like element, which is composed of IRi, orf1, orf2, a merR remnant and IRt in the absence of all the other features identified for the ancestral Tn501-like mer locus. In addition, R751 has acquired two copies of IS4321 (IS4321L and IS4321R), which flank the merR remnant and orf2, respectively. By contrast, evolution of pB8 involves insertion of a Tn501-like quaternary ammonium compound resistance (qacF) locus into orf2, thereby disrupting this gene.
The pB8 and pB10 integrons, each of which contain 59-CS, drug resistance gene cassettes, 39-CS, and completely or partially truncated tni module, appear to undergone all the above three steps of evolution. By contrast, the evolution step II (generation of 39-CS) is mostly likely omitted for the pA22732-IMP integron, while the R751 integron might represent a primitive Tn402-like integron due to absence of evolution steps II and III (truncation of tni module).
The Tn402-like integron of pA22732-IMP is inserted into the traC-parA intergenic region, leaving parA and its downstream gene upf31.0 intact. The parA upstream or around region represents a hot spot for Tn402 targeting, most likely due to it contains the multimer resolution site II for recognition by Tn402 transposase 20,21 . Interestingly, the insertion of Tn402-like integrons into the hotspot target region leads to further deletion of parA from pB8 and R751, and that of parA/upf31.0 from pB10.
The pA22732-IMP, R751 and pB8 integrons contains IRi and IRt of Tn402, but only IRi rather than IRt is identified for pB10. The lack of IRt in pB10 might due to the above-mentioned deletion removing of parA/upf31.0. The 5-bp target site (direct repeats of AGCAT) is still intact to flank the pA22732-IMP integron, but all the other three integrons do not leave traces of insertion.
Integrase IntI1 recognizes two different types of recombination site attI1 (integron attachment site) and attC (recognition site for integrase), and it catalyzes integration or excision of gene cassettes through site-specific recombination commonly between one attI1 site and one or more attC sites 22,23 . One attI1 site and three attC sites, upstream of aacA7, orfE, bla IMP-1 and tniA, respectively, are indentified in the pA22732-IMP integron (Fig 4a). These sites are long inverted-repeat-containing sequences of variable length and sequence, and each inverted repeat begins with a core sequence RYYYAAC and ends with an inverted core sequence GTTRRRY as described previously 24,25 , which would form imperfect cruciform structures and be required for capture of aacA7, orfE, and bla IMP-1 .
Expression of integron gene cassettes. The pA22732-IMP integron gene cassettes aacA7, orfE and bla IMP-1 , but not intI1 and tniA, are organized in the same transcriptional direction (Fig 5a). PCR generates an amplicon ranged from 59-untranslated region (59-UTR) of aacA7 to 59-terminal of the bla IMP-1 coding region, when using A22732 genomic DNA as template (Fig 5a). The positive RT-PCR amplification with the same primer pair, using cDNA sample generated from A22732 total RNA as template (Fig 5b), indicates that aacA7, orfE, and bla IMP-1 are transcribed into a single RNA transcript and thereby, these three genes constitutes a single operon aacA7-orfE-bla IMP-1 (Fig 5a). two broken-line arrows represent primary RNA transcripts transcribed for the aacA7-orfE-bla IMP-1 operon and the bla IMP-1 gene, respectively. Line with filled circles at both termini indicates location of primer pair plus expected PCR amplicon. b) PCR and RT-PCR. cDNAs generated from total RNA of strain A22732, and genomic DNA of A22732 were used as templates for RT-PCR and PCR, respectively. c) Primer extension. Primer extension assay of the RNA transcript of aacA7 or bla IMP-1 was done for A22732 cultured with addition of increasing amounts of imipenem. Lanes C, T, A and G represent Sanger sequencing reactions. The transcription start of aacA7 or bla IMP-1 is indicated by the arrow with nucleotide T or G, respectively, and the minus number under arrow indicate the nucleotide position upstream of the aacA7 or bla IMP-1 start codon. Representative data from at least two independent biological replicates are shown. Integrons act as natural gene expression platforms due to the presence of an intrinsic promoter (Pc) that is recognized by RNA polymerase to drive transcription of inserted gene cassettes that generally do not have their own promoters. At least eight distinct types of Pc promoter, PcS (strong, TTGACA-N 17 -TAAACT), PcW (weak, TGGACA-N 17 -TAAGCT), PcH1 (hybrid 1, TGGACA-N 17 -TAAACT), PcH2 (Hybrid 2, TTGACA-N 17 -TAAGCT), PcSS (super-strong, TTGATA-N 17 -TAAACT), PcIn42 (TTGGCA-N 17 -TAAACT), PcIn116 (TTGACA-N 17 -TGAACT), and PcPUO (TCGACA-N 17 -TAAACT) have been described for class 1 integrons 26 . As detected by primer extension (Fig 5c), a transcription start site (nucleotide C) is located at 227-bp upstream of aacA7 start codon (i.e. a 227-bp 59-UTR of aacA7), validating presence of the PcW promoter to drive aacA7-orfE-bla IMP-1 transcription. The PcW promoter can be found for all the pA22732-IMP, pB8, R751, and pB8 integrons.
Remarkably, the primer extension assay detects another transcription start site (nucleotide G) which is located at 155-bp upstream of bla IMP-1 start codon. This assay discloses presence of an internal promoter (TTGCCA-N 16 -TATCAT) driving transcription of the bla IMP-1 gene cassette (Fig 5c). Being very rare, the internal gene cassette-specific promoter is an extra element in evolution of antimicrobial resistance phenotype and act independent of Pc promoter 27 .
In addition, the primer extension assay shows that addition of increasing amounts of imipenem during bacterial cultivation has no effect on promoter activity of either aacA7-orfE-bla IMP-1 or bla IMP-1 (Fig 5c), validating constitutive expression of the aacA7-orfE-bla IMP-1 operon and the bla IMP-1 gene cassette. Notably, constitutive transcription of integron gene cassettes has been suggested previously 23,28,29 .
Concluding remarks. We present the first complete sequence of IMP-encoding plasmid from A. xylosoxidans, and this is also the first report of identification of a carbapenemase-encoding IncP-1ß plasmid from a clinical bacterial isolate. The detected bla IMP-1 gene is captured by a novel class 1 integron with a novel gene cassette array in the IncP-1ß plasmid pA22732-IMP. The class 1 integron is embedded in a Tn402-like transposon and inserted into pA22732-IMP by transposition. Most of the characterized IncP-1ß plasmids are isolated from bacteria in agricultural soils or waters and frequently associated with mercury resistance 7,[9][10][11]13 . Only a few of them, e.g. pA22732-IMP and R75, are of clinical origins and harbor an array structure of multiple resistance gene cassettes. The IncP-1ß plasmids thus could represent important vehicles for spreading clinically relevant resistance determinants across a number of bacterial species. The transfer of bla IMP to A. xylosoxidans, which already carried bla OXA-114 intrinsically, would lead to more severe drug resistance, making high difficulty in timely choosing sensitive antibiotics for treatment. The IMP-producing A. xylosoxidans should be taken seriously as the surveillance target especially in East Asia countries such as China and Japan.

Methods
Bacteria isolation and identification. Fresh sputum specimens were sampled from the indicated patient and inoculated onto Mueller-Hinton agar for bacterial isolation. The use of human specimens and all related experimental protocols were approved by the Committee on Human Research of Chinese People's Liberation Army General Hospital and carried out in accordance with the approved guidelines, and moreover the informed consent was obtained from the indicated patient. Single colony of each bacterial strain tested was subjective for species identification by VITEK 2 (BioMérieux), Bruker MALDI Biotyper, and 16s rRNA gene sequencing. For determination of16S rRNA gene sequence, the almost complete coding region of 16S rRNA gene was amplified by PCR with the universal primers 27f (AGAGTTTGATCCTGGCTCAG) and 1492r (TACCTTGTTACGACTT) 30 and then sequenced on ABI 3730 Sequencer.
PCR detection of bla genes. All known carbapenemase and ESBL genes as listed in Table S1 were subjected to PCR detection. Primer pair GTCCAAGACCGGCAACTC/CACCAGCAGGATCGACAG was designed from whole-genome shotgun sequences of strain A22732 (data not shown) for amplifying the DNA fragments containing the whole coding region of bla OXA-114 , because all the available primers gave negative amplification. All amplicons were sequenced on ABI 3730 Sequencer with the same primers for PCR.
Conjugal transfer. Plasmid conjugal transfer experiments were carried out with rifampin-resistant E. coli EC600 being used as recipient and bla IMP -positive A. xylosoxidans as donor. 3 ml of overnight cultures of each of donor and recipient bacteria were mixed together, harvested and resuspended in 80 ml of Brain Heart Infusion (BHI) medium. The mixture was spotted on a 1 cm 2 filter membrane that was placed on the BHI agar plate, and then incubated for mating at 37uC for 12-18 h. Bacteria were washed from the filter membrane and spotted on the Muller-Hinton agar plate containing 1500 mg/ml rifampin and 100 mg/ml ampicillin for selection of the bla IMP -positive E. coli transconjugant.

Determination of minimum inhibitory concentration (MIC).
The MIC values of indicated bacterial strains were tested by using VITEK 2 according to manufacturer's instructions, and antimicrobial susceptibility was judged by Clinical and Laboratory Standards Institute (CLSI) standard.
Determination of plasmid DNA sequence. The chromosome DNA-free plasmid DNA was isolated from the cell cultures of the bla IMP -positive E. coli transconjugant using a Qiagen large construct kit, and then sequenced by using whole-genome shotgun strategy in combination with Illumina HiSeq 2500 sequencing technology. The contigs were assembled with Velvet, and the gaps were filled through combinatorial PCR and Sanger Sequencing on ABI 3730 Sequencer. The genes were predicted with GeneMarkS and further annotated by BLASTP against Uniport and NR databases.
RNA isolation and reverse transcription (RT)-PCR. Bacteria were cultured overnight in Mueller-Hinton broth (BD) with or without addition of 2 mg/ml imipenem (Sigma). Total RNAs were extracted from harvested bacterial cells using TRIzol Reagent (Life Technologies). RNA quality was monitored by agarose gel electrophoresis, and RNA quantity was determined by spectrophotometry. The contaminated DNA in the total RNA samples was removed by using Amibion's DNA-free TM Kit. cDNAs were generated by using 5 mg of RNA and 3 mg of random hexamer primers in a 40 ml reaction mixture. The cDNA samples were generated by RT from total RNAs. Genomic DNA and cDNA were used as the templates for PCR and RT-PCR, respectively, with the primer pair TGTTTGATGTTATGGAGCAG/ AGCCGTAAATGGAGTGTC. To ensure that no contamination of genomic DNA in the RT reactions would occur, RT-PCR of negative controls was performed using the 'cDNA' sample generated without reverse transcriptase as template. Reactions containing primer pairs without templates were also included as blank controls.
Primer extension assay. The [c-32 P] ATP end-labeled primer CAGTCATAACAAGCCAT or CATACTTTTCCTTTTCTAACGG, which was complementary to aacA7 or bla IMP-1 transcript, respectively, was annealed with total RNA sample of strain A22732 for primer extension assay as described previously 31 . For different cell cultures (lanes) in a single experiment, equal amounts of total RNA were used as starting materials. The corresponding end-labeled primer was also used for sequencing the PCR amplicon generated by the primer pair TGACGATGCGTGGAGACC/CAGTCATAACAAGCCAT or TGTTTGATGTTATGGAGCAG/AGCCGTAAATGGAGTGTC. DNA sequencing was carried out using the AccuPower & Top DNA Sequencing Kit (Bioneer). Primer extension products and sequencing materials were analyzed on an 8 M urea-6% polyacrylamide gel electrophoresis. Radioactive species were detected by autoradiography.
Nucleotide sequence accession number. The complete sequence (File S1) of plasmid pA22732-IMP was submitted to the GenBank nucleotide sequence database under accession number KJ588780. www.nature.com/scientificreports