GFAP-Cre to ablate β-catenin in neuronal cells of the cerebellum after embryonic day 12.5, and found an unexpected role of β-catenin in determination of the foliation pattern. In the mutant mice, the positions of fissure formation were changed, and the meninges were improperly incorporated into fissures. At later stages, some lobules were formed by Purkinje cells remaining in deep regions of the cerebellum and the laminar structure was dramatically altered. Our results suggest that β-catenin is critical for cerebellar foliation and lamination. We also found a non cell-autonomous role of β-catenin in some developmental properties of major cerebellar cell types during specific stages.The cerebellum has a conserved foliation pattern and a well-organized layered structure. The process of foliation and lamination begins around birth. β-catenin is a downstream molecule of Wnt signaling pathway, which plays a critical role in tissue organization. Lack of β-catenin at early embryonic stages leads to either prenatal or neonatal death, therefore it has been difficult to resolve its role in cerebellar foliation and lamination. Here we used  In addition to its well-known role in coordinating motor processing, recent studies indicate that the cerebellum is also important for some higher order brain functions, including cognition, emotion, and language processing The cerebellum also exhibits an ordered laminar organization. Defined cell layers are formed by a series of well-orchestrated cell migration events. Purkinje cells, Bergmann glia and interneurons migrate radially from the primary germinal zone toward the cerebellar surface. Granule cell precursors (GCP) first move tangentially from the rhombic lip across the surface of the developing cerebellum. These cells then form a secondary germinal zone, the external granule cell layer (EGL), which lies between the meninges and the Purkinje cell layer (PCL). Granule cells that are generated in the EGL subsequently migrate radially along Bergmann glial fibers through the PCL to form the internal granule cell layer (IGL) β-catenin at early stage of embryogenesis using the Wnt1-Cre promoter leads to agenesis of the cerebellum β-catenin at midgestation using the nestin-Cre promoter causes premature neural precursor cell fate commitment β-catenin is a downstream signaling component of Wnts GFAP-Cre to ablate β-catenin in neural cells. The hGFAP-Cre promoter activates at a later time point and in a narrower distribution than the nestin-Cre promoter In the present study, we used hGFAP-Cre line T2hGFAP-CreER line L7-Cre line β-catenin line cre gene were crossed to fl/flβ-catenin or fl/wtβ-catenin mice. F2-generation mice carrying cre and two floxed β-catenin alleles were used as the mutant type. All the other littermates were used as the control type. In some cases the ROSA26 reporter allele (R26R) R26R line, in which the lacZ reporter construct is integrated into the ROSA locus, is common to detect Cre-mediated recombination. In the absence of Cre expression, the lacZ-encoding protein β-galactosidase  is not expressed due to a floxed stop signal. Cre expression results in the removal of the stop signal and expression of β-Gal, which could cleave 5-bromo-4-chloro-indolyl-β-D-galactopyranoside  and form an intensely blue product. The results obtained from the control or mutant mice were consistent with or without the R26R allele, so we did not distinguish them in the descriptions below. In experiments relating to timed pregnancies, the plug date was defined as embryonic day E0.5.All the animal protocols were approved by the Animal Committee of Institute of Neuroscience. To generate conditional knockout mice, the 2HPO4, 1.9 NaH2PO4, 154 NaCl, 2 MgCl2, 5 K3[Fe(CN)6], and 5 K4[Fe(CN)6]), 0.5% Triton X-100 and 1% Xgal stock solution .Mice were deeply anesthetized with Pentobarbital sodium salt (Sigma) and perfused with 0.9% NaCl, followed by 3% or 4% paraformaldehyde/Phosphate buffered saline (PBS). Brains were removed and postfixed for 2–5 h for Xgal staining, or overnight for immunohistochemical staining. After cryoprotection in 30% sucrose and subsequent freezing in Optimal Cutting Temperature compound (OCT), sections were cut 30–40 μm in thickness on a cryostat and either placed in PBS or allowed to air-dry on slides. Sections were stained with hematoxylin and eosin following the manufacturer's instructions (Beyotime Institute of Biotechnology). For Xgal histochemistry, sections were stained for 2 h to overnight at 37°C in Xgal staining solution which consisted of the staining buffer , mouse anti-GFAP , mouse anti β-catenin , rabbit anti-calbindin-D28K , mouse anti-NeuN , rabbit anti-BLBP , mouse anti-nestin , 4D7 anti-TAG1 , and rabbit anti-laminin . For immunohistochemistry using the rabbit anti-phospho histone H3 antibody , slides were subjected to antigen retrieval by heating in a microwave at 30% power for 7 min in 10 mM sodium citrate, pH 6.0. Then the slides were left to cool at room temperature for 45 min before washing in PBS and the permeabilization step. After washing to remove excess primary antibodies, the sections were incubated for 2 h at room temperature with Alexa fluro 488, cy3, or cy5, or TRITC-conjugated secondary antibodies.In some experiments, staining was performed using the HRP/DAB-based staining system  according to the manufacturer's specifications.Images were collected with the laser confocal microscope (Olympus IX71) driven by Fluoview 500 software, or the NIKON E600FN microscope equipped with an automatic stage driven by the Neurolucida software.5′-ACATAATGAGGACCTACAC-3′). An siRNA that does not target any known mammalian gene (5′-TTCTC CGAACGTGTCACGT-3′) was cloned into pSUPER as a control Astrocytes were prepared from the cortex and hippocampus of C57/BL6 mice on P0, and cultured in minimal essential medium (MEM) with 10% FBS (Gibco). After 7–10 days when cells reached confluence, they were detached with trypsin and transfected with plasmid DNA by nucleofection (Amaxa). Vector-based siRNA against β-catenin was encoded by pSUPER vectors, with the described sequence (in vivo electroporation was conducted as described previously Cerebellar 9 pfu/ml), which expresses EGFP driven by the mCMV promoter, was purchased from Vector Gene Technology Co. Ltd; AdCreM2 (5×109 pfu/ml), which expresses Cre driven by the mCMV promoter, was purchased from Microbix Biosystems Inc.Ad5-GFP  at E13.5 .We also examined β-catenin protein expression in the nd E18.5 . Meningend E18.5 . Neithernd E18.5 . Heterozfl/flhGFAP-Cre, β-catenin mice were ataxic , which could label radial glia and astroglia. At E14.5, radial glial fibers spanned the cerebellar cortex in both control and mutant mice, with no apparent difference in alignment and density . At E17.The astroglia in the interior of the mutant cerebellum expressed BLBP and was rather compact, compared with the scattered distribution of astroglia in the EGL. They had foliation pattern similar to Purkinje cells . The borfl/flhGFAP-Cre, β-catenin mice.In control cerebellum, white-matter glia , a highlfl/flhGFAP-Cre, β-catenin mice, there were defects in the arrangement of Purkinje cells after P3 . The fl/flL7-Cre, β-catenin mice lived to adulthood and showed no apparent motor defects. They had inflammation in the cornea and tissues near the eyes. Inflammation was not found in fl/wtL7-Cre, β-catenin mice (data not shown). By immunostaining with anti-calbindin antibody and DAPI, we found that the morphology and lamination of Purkinje cells were both normal in the fl/flL7-Cre, β-catenin mice . First we examined the effect of AdCreM2 in cultured astrocytes. The astrocytes from fl/flβ-cat mouse were first transfected with a plasmid containing a floxp-flanked stop sequence between a ubiquitous promoter and the EGFP gene. In these cells AdCreM2 efficiently excised the floxp-flanked stop sequence and initiate EGFP expression. The expression of β-catenin was apparently downregulated (fl/wtβ-cat or fl/flβ-cat cerebellum with AdCreM2 and another adenovirus carrying a GFP gene (AdGFP) at E13.5. Many choroid plexus cells were infected by both viruses. In the fl/flβ-cat cerebellum these cells showed clear down regulation of β-catenin at P0 , and rarely in the EGL , granule cells (after E13.5 or P4) or Bergmann glia (after E14.5 or P0).We inactivated the β-catenin gene selectively in precursors of neurons and glia by Cre/Lox-mediated gene inactivation and found that the mutant mice developed severe cerebellar abnormalities. The defects included changes in the pattern of foliation, fusion of adjacent lobules, and abnormal laminar organization. The differentiation and distribution of granule cells and astroglia were also dramatically changed. However, at the stages we examined, β-catenin did not play a cell-autonomous role in the lamination or the general differentiation of major cerebellar cell types.In this study, we focused on the role of β-catenin in late embryonic and early postnatal cerebellar development. Our data provide evidence that Purkinje cells formed folia-like structures with unseparated fissures, but these fissures appeared at positions different from those of wild-type mice, and different patterns formed in different mutant mice.fl/flhGFAP-Cre, β-catenin mouse. However, the positions of ectopic fissures in the fl/flhGFAP-Cre, β-catenin mouse was different from those in the mutant mice mentioned above The proliferative kinetics of granule cells is an important factor for cerebellar foliation fl/flhGFAP-Cre, β-catenin cerebella did not resemble any of these reported mutants. In addition, after P3 in the rostral part of the mutant cerebellum, some lobules remained in deep zones; while in the caudal part, lobules were still near the meninges. Some genes are differentially distributed in rostral and caudal parts of the cerebellum Several molecules have been found to function in the patterning of cerebellar folia. Removal of the transcription factor Gbx2 after E9 results in lobules reduced in size to varying degrees in the vermis fl/flhGFAP-Cre, β-catenin cerebella found in the present study. It is possible that in addition to Wnt1, other members of the wnt family may be involved in mice with β-catenin mutation. Alternatively, as a partner of cadherin β-catenin is a critical molecule in the canonical Wnt signaling pathway. It acts downstream of Wnt, which is a morphogen regulating tissue patterning fl/flhGFAP-Cre, β-catenin mice, the deposition of meningeal basement membrane at cerebellar fissures was defective. The morphology of Bergmann glia has been proposed to be critical for the remodeling of meninges and the formation of fissures fl/flhGFAP-Cre, β-catenin mice, radial fibers attaching to the meninges were decreased from E17.5, and replaced by ectopic cell bodies in the EGL. This may account for the defects in the deposition of meninges. However, we cannot exclude the involvement of granule cells, which might draw the meninges into fissures together with Bergmann glia, or secrete factors to attract meningeal components into fissures. Our results also indicate that when only part of the granule cells or glial cells were depleted of β-catenin, the change in adhesive force or concentration of secreted factors was not enough to generate obvious defects in meningeal deposition.Interestingly, although β-catenin was only inactivated in neurons and glia in fl/flNestin-Cre, β-catenin mice leads to premature neural precursor cell fate commitment in the ventricular zone. The EGL was also disrupted at E14.5 fl/flhGFAP-Cre, β-catenin mice was maintained with normal morphology until P3. It is likely that β-catenin was important in formation of the EGL from the RL, which initiates at about E13 fl/flhGFAP-Cre, β-catenin mice, the IGL formed where a relatively normal PCL formed near the pial surface, suggesting that β-catenin-deficient granule cells were still able to migrate. Accumulation of differentiated granule cells was found beneath the meninges in some fl/flhGFAP-Cre, β-catenin mice, suggesting premature differentiation. Our loss of function studies, by electroporating wild-type granule cells with β-catenin siRNA or infecting fl/flR26R, β-catenin granule cells with Cre-expressing adenovirus, demonstrated a non-cell-autonomous role of β-catenin in granule cell differentiation and distribution. Several factors may contribute to the retention of granule cells in ectopic places of the fl/flhGFAP-Cre, β-catenin mice. The gross changes of the cerebellar structure  may be unpermissive for granule cell migration. Alternatively, disordered interactions of granule cells with neighboring β-catenin-deficient cells, either by adhesive force or secreted factors, may not allow granule cells to migrate.β-catenin has been reported to play a cell-autonomous role in the neuronal proliferation and differentiation of cerebral cortical precursor cells hGFAP promoter was active in some VZ and cortical cells at E14.5 and E16.5, possibly including pre-migrating or migrating Purkinje cell and glial cells. However, apart from the fissures, Purkinje cells of the fl/flhGFAP-Cre, β-catenin mice had a well-organized laminar structure until P3. This may be due to the delay of β-catenin down regulation compared to Xgal expression, or because β-catenin did not play a role in Purkinje cell migration at this stage. After P3, the laminar structure of Purkinje cells was gradually disordered. The PCL was not tightly arranged, but rather interrupted by gaps. Some Purkinje cells delaminated from the PCL. Such defects were not seen in the fl/flL7-Cre, β-catenin mice, which expressed Cre after E17.5. The defects may be caused by changed interaction with other β-catenin-deficient cells (e.g. changed adhesive force or secreted factors), or by the disrupted glial scaffold, which became defective at as early as E17.5. The defect in astroglia at either prenatal or postnatal stage may be caused by synergistic interactions between astroglia and other cells, rather than cell-autonomous effects, as demonstrated by infection of AdCreM2 at E14.5 and induction of the T2, R26R, β-cateninfl/flhGFAP-CreER mice at P0.Purkinje cells are produced in VZ at E11-13, with a peak production at E11/12. They migrated radially from the VZ towards the cortical surface between E13 and E17. Glial cells also originate from VZ. Their cell bodies migrate between E15 and P7 to form a compacted BGL. It was suggested that the arrangement of radial glia and expression of adhesion molecules may be involved in the control and guidance of Purkinje cell migration Movie S1Ataxic movement of the fl/flhGFAP-Cre, β-catenin mouse. A P18 mutant mouse and its control littermate are shown. The mutant mouse was unable to keep balance during walking.(AVI)Click here for additional data file.Figure S1Recombination in Purkinje cells driven by the hGFAP-Cre promoter. (A) Example cerebella slice stained with Xgal from a P22 hGFAP-Cre, R26R mouse. It was the same slice shown in fl/flhGFAP-Cre, R26R, β-catenin mouse stained with Xgal and anti-Calbindin antibody (DAB staining). The right panels are enlargement of corresponding squares labeled with d-h in the left panels. In all the figures Xgal-positive Purkinje cells tended to form clusters (red arrows). Such clusters scattered throughout the cerebellum and mixed up with Xgal-negative Purkinje cells (white arrows). Scale bars: 250 μm.(TIF)Click here for additional data file.Figure S2Variable foliation patterns of the fl/flhGFAP-Cre, β-catenin mice. Midsagittal sections were analyzed by the hematoxylin and eosin method. The white dotted line indicates the position of the pr, or the deepest fissure in the anterior lobe (pcu in the control mice and dp in the mutant mice). In the mutant mice recognizable fissures were designated by an asterisk after the corresponding name. Other fissures were assigned from rostral to caudal as ‘a1, a2...an’. (A) Example sections of three E18.5 mutant mice. Mutant 1# had two fissures (a1 and a2) anterior to the dp. In the control mouse there was only one fissure (pct) anterior to the deepest fissure (pcu). Mutant 2# and 3# had one ectopic fissure between the dp and pr. The right panels are enlargement of the squared areas in the left panels (a and b). (B) Sections of six P3 or P4 mutant mice. They were different from each other. Anterior to the dp, there were two (mutant 2# and 5#) or three  fissures. In the control mouse there was only one (pct). Between the pr and dp, mutant 1#, 2# and 3# had no ectopic fissure, whereas mutant 4#, 5# and 6# had one ectopic fissure . Scale bars: 100 μm.(TIF)Click here for additional data file.Figure S3Distribution and differentiation of granule cells in the fl/flhGFAP-Cre, β-catenin mouse. (A) Parts of the P7 control and mutant cerebellum in (TIF)Click here for additional data file.Figure S4Defects in Bergmann glia of the fl/flhGFAP-Cre, β-catenin mouse. (A) Example sections showing radial fibers spanned the EGL in the rostral part of E17.5 control and mutant cerebellum. (B) Example sections showing ectopic cell bodies appeared in the EGL of the E18.5 mutant cerebellum. (C) Neighbouring sections from a P4 mutant mouse were stained with antibodies against Calbindin or BLBP, revealing similar foliation pattern. White arrows indicate separated fissures. Yellow arrows indicate fused fissures. The green arrow indicates a fissure fused at the base. (D) Midsagittal sections from a P8 mutant mouse were stained with antibodies against Calbindin or BLBP and counterstained with DAPI. The squared region in the left panel were enlarged and shown in the three right panels. The dotted lines indicate the border of inner egl, which was corresponding to the border of dendritic terminal of Purkinje cells (a) or compact astroglia in the interior of cerebellar cortex (b). The arrowheads indicate the scattered astroglia beneath the pial surface or in the egl. Scale bars: 20 μm in (A), 100 μm in (B), (C) and (D).(TIF)Click here for additional data file.Figure S5Inhibition of β-catenin expression by siRNA or Cre-expressing virus. (A) Cultured cortical astrocytes were electroporated with pSuper vectors carrying either control siRNA or β-catenin siRNA. After 3 days, cells were fixed and immunostained with anti-β-catenin antibody. The expression level (mean fluorescence density) of β-catenin within individual GFP-positive cells was quantified. The percentages of cells within each range of expression are shown in the histograms. Differences were statistically significant (p<0.001) as determined by the Mann-Whitney two-sample rank sum test. (B) Astrocytes from fl/flβ-cat mouse were cultured and transfected with a plasmid containing a floxp-flanked stop sequence between a ubiquitous promoter and the EGFP gene (flstopfl-GFP). The cells were fixed and immunostained with anti-β-catenin antibody 6 days after infection with or without AdCreM2. GFP expression was initiated and β-catenin expression was inhibited in the AdCreM2-infected cells. Note that AdCreM2 affected more cells than the flstopfl-GFP plasmid, as judged by the down-regulated β-catenin expression in some GFP-negative cells. (C) E13.5 fl/wtβ-catenin (upper panels) or fl/flβ-catenin (lower panels) mice injected with a viral mixture of AdCreM2 plus Ad5-GFP. The mice were killed and the choroid plexus was stained with anti-β-catenin antibody at P0. GFP-positive cells of the fl/flβ-catenin mouse showed an apparent decrease in the expression level of β-catenin (arrows). (D) The cerebella from the same mice as in (C) were stained with anti-β-catenin antibody. Note an apparent down-regulation of β-catenin in the EGL of fl/flβ-catenin mouse, although no GFP-positive cell was detected in the region, possibly due to higher infection efficiency of AdCreM2 than Ad5-GFP in the cerebellum. Scale bars: 100 μm in (A) and (B), 20 μm in (C) and (D).(TIF)Click here for additional data file.Figure S6Infection of AdCreM2 in the cerebellum. (A and B) The pregnant dams of fl/wtR26R, β-catenin or fl/flR26R, β-catenin mice were injected at E13.5 with AdCreM2. Sections were first stained with Xgal and then with neutral red to reveal layers. (A) Example sections from P0 mouse showing many infected cells were located in the EGL. (B) Example sections from P16 mouse showing that most Xgal-positive cells had migrated into the IGL. (C and D) The pregnant dams of fl/wtR26R, β-cat  mice or fl/flR26R, β-cat  mice were injected at E14.5 with AdCreM2, and the offspring were analyzed at P21. (C) Example sections stained with Xgal. Some cells located in the BGL showed Bergmann glia-like morphology (arrows). (D) The sections were first stained with Xgal and then with anti-BLBP antibody using the DAB method. A few cells located in the BGL were double-positive for Xgal and BLBP (red arrows) and extended radial fibers to the pial surface. (E) A  fl/flR26R, β-cat mouse was injected with a viral mixture of AdCreM2 plus Ad5-GFP at P0, and analyzed at P19. The section was first stained with Xgal, and then with anti-GFP antibody using the DAB method. Although Xgal-positive cells were located mostly in the IGL with no GFP expression, some were located in the BGL and positive for GFP (arrows). GFP staining showed that they extended normal radial processes to the pial surface. Left panel: staining with Xgal alone. Right panel: enlargement of the square in the middle panel. Cells showing colocalization were recognized by comparison of the left panel with the middle or right panel. Abbreviations: egl, external granule cell layer; igl, internal granule cell layer. Scale bars: 100 μm in (A), (C) and (E), 500 μm in (B), and 20 μm in (D).(TIF)Click here for additional data file.


AbstractStaphylinini rove beetle genus Euryporus Erichson from the subtribe Quediina is restricted to include only three species from the Western Palearctic region: Euryporus picipes , Euryporus aeneiventris , and Euryporus princeps Wollaston, 1864. Euryporus argentatus Fauvel, 1881, Euryporus warisensis Last, 1987 and Euryporus multicavus Last, 1980, which do not even belong to the subtribe Quediina, are excluded fromthe genus. Of these, two were transferred to different genera: Tympanophorus argentatus , comb. nov., from Sumatra;and Hesperus warisensis , comb. nov.,from New Guinea. “Euryporus” multicavus could not be placed to any of the described genera of Staphylinini and is left as incertae sedis pending a broader study of the relevant fauna of this tribe in New Guinea and adjacent regions. The taxonomic history of Euryporus is reviewed, and an updated diagnosis of this genus is provided.The  Staphylinini  , and Euryporus princeps Wollaston, 1864), and three poorly known “exotic” species: Euryporus argentatus Fauvel, 1881 from Sumatra  , Eurypor Sumatra , as wellst, 1987  and Euryst, 1980  from NewEuryporus immediately obvious. But while the correct identity of Euryporus argentatus and Euryporus warisensis as members ofthe genera Tympanophorus Nordmann, 1837 and Hesperus Fauvel, 1874, respectively, also became clear, proper classification of Euryporus multicavus faced a problem of poor generic limits in the subtribes Philonthina and Anisolinina, and even a problem of blurred limit between these subtribes BPBM Hungarian Natural History Museum, Budapest (G. Makranczy)HNHM Manchester Museum, the University of Manchester (D. Logunov)MMUE Netherlands Centre for Biodiversity Naturalis, the Netherlands (M.E. Gassó Miracle and A. van Assen)NCBNLabels of the examined types are quoted verbatim; data from each label are separated by a slash [/].PageBreak Pro . The photo in Photos in http://isodp.hof-university.de/fuzzyg/query/ ).Correspondence of the locality names from old collection labels to modern toponyms was checked with the on-line resource .Euryporus Erichson, 1839 was described by Pelecyphorus to include one European species Euryporus picipes   was a preoccupied name, Euryporus and described the second species in the genus, Euryporus puncticollis from North America  for his own species Euryporus flavipes. Finally, Euryporus: Euryporus multicavus Last, 1980 and Euryporus warisensis Last, 1987, both from Papua New Guinea.The rove beetle genus l, 1800) . Since P America . Soon, Ere added . Later Fre added , 1884 deEuryporus included six species before this study  widely distributed in Europe , n Europe ; the WesQuediina, Euryporus can be distinguished by the following combination of characters: fully developed infraorbital ridges; mandibles elongate with broad basal part but narrow and sharp apical portion; last segment of maxillary palps fusiform, slightly setose; last segment of labial palps enlarged, apically obliquely truncated, densely setose; first antennal segment elongate, as long as second and third antennal segments together; anterior tarsi narrow, not enlarged in both sexes; apical margin of abdominal sternite VIII in both sexes concave, in male without median incision. Other recent descriptions and synopses of the genus can be found in Among other genera of the subtribe PageBreakFor phylogenetic purposes adult comb. n.http://species-id.net/wiki/Tympanophorus_argentatusIndonesia: Holotype, female, “Euryporus argentatus Fvl. [in Fauvel's handwriting] / Suon Exp. Moeara Laboe 11/77 [circle label]/ Museum Leiden Euryporus argentatus det. Fauv. / argentatus Fauvel n. sp. [handwritten label]/ Holotype Euryporus argentatus Fauv. revised by A. Solodovnikov 2012 [red label]/Tympanophorus argentatus (Fauvel) A. Solodovnikov det. 2012” (NCBN).Comments. In the original description of Euryporus argentatus, 1°29'0"S, 101°3'0"E]. Based on the habitus ”, apparently meaning a synonymy of his species with Tympanophorus rugosus . This so vaguely annotated transfer of Euryporus argentatus to Tympanophorus was overlooked by later authors. For example Euryporus argentatus Fauvel, 1881 as a valid species and “Tympanophorus argentatus Fauvel”, erroneously, as nomen nudum. Synonymy of Tympanophorus argentatus  and T. rugosus  remains to be verified.It is noteworthy that long after the description of comb. n.http://species-id.net/wiki/Hesperus_warisensisPapua New Guinea: Holotype, female, “Holotype [red circular label]/ New Guinea Neth. Waris, S. of Hollandia, 450–500 m, VIII-16-23-1959/ T.C. Maa collector Bishop/ Euryporus warisensis sp. n. H.R. Last det., Holotype [H.R. Last's label]/ Hesperus warisensis (Last) A. Solodovnikov det. 2012” (BPBM).Euryporus warisensis is strikingly different from the Palearctic Euryporus , the enlarged apical labial palpomeres of Euryporus warisensis easily distinguish this species from any other known species of Hesperus.Because of its rather elongate mandibles and maxillary palps , as wellanalysis , this geHesperus warisensis displays a “fake” superior line extended towards anterior angles of pronotum, while the right side has no such structure ; paratype, male, “New Guinea SE Kiunga, 23.VII-2.VIII.1969/ No. NGK-B.3. leg. Dr. Ballogh/ Euryporus multicavus sp. n. H.R. Last det., Paratype [H.R. Last's label]/ Staphylinini genus nov.? A. Solodovnikov det. 2012” (MMUE).Comments. As in the above described case, Euryporus multicavus is strikingly different from the Palearctic Euryporus in habitus (cf. Euryporus multicavus is not congeneric with Euryporus and even does not belong to the subtribe Quediina. On the other hand, the combination of characters of that species does not allow its unambiguous placement in any of the currently recognized subtribes of Staphylinini.itus cf.  and 8, bitus cf. ; presentTympanophorus, I assume that “Euryporus” multicavus is phylogenetically close to the Tympanophorus-lineage of the subtribe Anisolinina (as defined in PageBreakPageBreakmere of the aedeagus (Euryporus” multicavus with several species from New Guinea described in the genera Philonthus and Hesperus. But, except Hesperus warisensis moved to that genus here, none of those species have robust and dilated labial palpi, and all of them differ from “Euryporus” multicavus in other details. It is possible that “Euryporus” multicavus represents a new genus whose description must be postponed until a more inclusive phylogenetic study of relevant lineages is performed. Such study should be based not only on an extensive taxonomic revision of the hitherto poorly described relevant species but also include additional material from the collections of Staphylinini from New Guinea and adjacent regions, which I am aware of and which have remained largely untouched by modern workers.Because of the short and stout labial palps with dilated last segment, shape of the mandibles , stronglaedeagus , cast do


Prescription errors that occur due to the process of pill splitting are a common medication problem; however, available prescription information involving inappropriate pill splitting and its associated factors is lacking.We retrospectively evaluated a cohort of ambulatory prescriptions involving extended-release or enteric-coated formulations in a Taiwan medical center during a 5-month period in 2010. For this study, those pill splitting prescriptions involving special oral formulations were defined as inappropriate prescriptions. Information obtained included patient demographics, prescriber specialty and prescription details, which were assessed to identify factors associated with inappropriate pill splitting.There were 1,252 inappropriate prescriptions identified in this cohort study, representing a prescription frequency for inappropriate pill splitting of 1.0% among 124,300 prescriptions with special oral formulations. Among 35 drugs with special oral formulations in our study, 20 different drugs  had ever been prescribed to split. Anti-diabetic agents, cardiovascular agents and central nervous system agents were the most common drug classes involved in inappropriate splitting. The rate of inappropriate pill splitting was higher in older (over 65 years of age) patients . Eighty-seven percent (1089/1252) of inappropriate prescriptions were prescribed by internists. The rate of inappropriate pill splitting was highest from endocrinologists , nephrologists  and cardiologists . Multivariate logistic regression analysis revealed that the strongest factor associated with individual specific drug of inappropriate splitting was particular physician specialties.This study provides important insights into the inappropriate prescription of special oral formulation related to pill splitting, and helps to aggregate information that can assist medical professionals in creating processes for reducing inappropriate pill splitting in the future. Pill splitting is common in oral drug therapy in clinical practice Medication errors may occur at any stage of medication usage, including: prescription, dispensation, and administration. However, prescription is often noted as the stage where most errors occur The primary purpose of this study was to assess the frequency, and identify the risk factors, associated with inappropriate pill splitting.st to May 31, 2010. The study variables which were analyzed included: patient gender and age, diagnosis codes , specialty of prescribing physician, prescribed drugs, dose, frequency and route of administration. Patients filled their prescriptions from the same hospital pharmacy.This study was conducted in a tertiary care medical center in Taiwan which has more than 2.5 million outpatient visits per year. More than 25,000 ambulatory prescriptions were delivered daily. Data analysis was based on a computerized ambulatory prescription database from January 1This study was approved by the institutional review board of Taipei Veterans General Hospital. Since the research posed no more than minimal risk to the participants and involved no procedures, the review board approved that written consent from patients was not required.Drugs were recognized as “special oral formulations” if they were ER or EC formulations that were not originally intended to be split. Drug formulation and the appropriateness of splitting were noted on the instruction leaflets accompanying the medicines. Whenever the prescribed drug was fragmented despite being a special oral formulation, we defined that the drug as split, and regarded it as an inappropriate prescription of pill splitting. The drugs with special oral formulations were supplied consistently and stably over the whole study period.p value <0.05 was considered to indicate statistical significance.All data were linked by the SQL server 2008  and analyzed by SPSS software version 19.0 . Descriptive statistics was used to summarize prescription characteristics. For each specific drug with special oral formulation, multivariate logistic regression analysis was used to examine the potential association of independent variables  with inappropriate pill splitting. The dependent variable was defined as a single prescription with inappropriate pill splitting. We assume that the prescriptions are independent of each other. Patients aged less than 18 years were excluded due to the sample size being too small to yield statistically reliable results of logistic regression analysis. Results are presented as odds ratios and 95% confidence intervals (CIs). A Thirty-five different drugs with special oral formulations in this study are shown in The rate of inappropriate pill splitting was higher in older (>65 years) patients . Eighty-seven percent of prescriptions (1089/1252) with inappropriate splitting were from internists. The rate of inappropriate pill splitting was highest from endocrinologists , nephrologists  and cardiologists .Among drugs with special oral formulations, 20 kinds of drugs  had ever been prescribed to split. For half of these drugs , the prescribed splitting was unnecessary since they had a corresponding lower strength dose available in the study hospital. The detailed information of prevalence of inappropriate pill splitting for adults is shown in Multivariate logistic regression analysis revealed that the strongest factor associated with individual specific drug of inappropriate splitting was particular physician specialties. The risk of the top 6 prescribed inappropriate splitting drugs and physician specialties are shown in This study revealed the frequency and characteristics of inappropriate prescriptions related to pill splitting. Among all prescribed medications during ambulatory visits, 5.7% of all medications were drugs with special oral formulations. Inappropriate splitting occurred in 1% of drugs with special oral formulations. Fifty-seven percent of drugs with special oral formulations had ever been prescribed to be split. For half of these drugs, the prescribed splitting was unnecessary since they had a corresponding lower strength dose available in the study hospital. Unexpectedly, internists delivered the greatest number of prescriptions with inappropriate pill splitting. The rate of inappropriate pill splitting was highest from endocrinologists (3.4%), to nephrologists (1.3%) and cardiologists (1.3%). This study provides insights into the inappropriate prescriptions with pill splitting. The results demonstrate that efforts to reduce inappropriate tablet splitting are necessary and pressing.Lewis et al. reported an overall total prescribing error rate of about 7% in a recent systemic review Anti-diabetic agents, cardiovascular agents and central nervous system agents were the most common drug classes involved in inappropriate splitting. This finding may indicate that physicians are not fully aware of critical splitting restrictions, which may be due to the deficiency of knowledge regarding special oral formulations. For example, we identified surgery as significantly associated with inappropriate splitting for centain drugs . Surgeons rarely initiate drug therapy outside of their specialty service, and knowledge about these drugs may be poor. In view of these facts, two strategies can be suggested. First, printed hospital formularies should provide detailed prescribing information on tablet splitting, thereby reducing inappropriate splitting of drugs with special oral formulations. Second, a computerized clinical decision-support function within the computerized physician order entry system needs to be implemented, which could be a powerful intervention tool to help avoid inappropriate prescriptions for pill splitting The reason for internists associated with inappropriate pill splitting may be due to their service specialties. Our data showed that endocrinologists, nephrologists and cardiologists were more likely to be associated with inappropriate splitting among all medical specialties. The probable reason may be that the majority of endocrinologists and nephrologists’ clients are patients with renal insufficiency, such that dose adjustment may be indicated, and pill splitting prescribed Old age was associated with a high risk of inappropriate pill splitting. The changing pathophysiology occurring with the aging process results in complex alterations to the pharmacokinetics and pharmacodynamics of medications This study has provided possible factors associated with inappropriate pill splitting for drugs with special oral formulations. The risk factors identified in this study may imply how to develop strategies for preventing medication errors for drugs with special oral formulations. The findings of this study are informative for the assessment and development of medication prescription policy in hospitals. In addition, identification and acknowledgement of risk factors regarding inappropriate pill splitting are needed to incorporate guidelines for medication safety and the educational curriculum of health professionals.This study has some limitations. First, the study was conducted in a single hospital over a short period of time, which limits the generalizability of our findings. Second, we only assessed the frequency of inappropriate pill splitting by drug formulation without analyzing associated clinical outcomes. Other characteristics of drugs such as score marking, size or shape of pills, and multiple ingredients were not taken into consideration In conclusion, medical prescriptions involving inappropriate pill splitting are not rare in clinical practice. This phenomenon may be due to the lack of knowledge of special oral formulations which cannot be split. This study provides fundamental information about inappropriate pill splitting. However, our study may imply that healthcare providers should take further steps to employ enhanced safety modalities, to prevent or reduce the occurrence of inappropriate prescription pill splitting.Table S1Number of prescriptions with special oral formulation by the specific drug and physician specialty.(DOCX)Click here for additional data file.


Three different Wnt signaling pathways function to restrict the anterior neuroectoderm state to the anterior end of the sea urchin embryo, a mechanism of anterior fate restriction that could be conserved among deuterostomes. Patterning the neuroectoderm along the anterior–posterior (AP) axis is a critical event in the early development of deuterostome embryos. However, the mechanisms that regulate the specification and patterning of the neuroectoderm are incompletely understood. Remarkably, the anterior neuroectoderm (ANE) of the deuterostome sea urchin embryo expresses many of the same transcription factors and secreted modulators of Wnt signaling, as does the early vertebrate ANE (forebrain/eye field). Moreover, as is the case in vertebrate embryos, confining the ANE to the anterior end of the embryo requires a Wnt/β-catenin-dependent signaling mechanism. Here we use morpholino- or dominant negative-mediated interference to demonstrate that the early sea urchin embryo integrates information not only from Wnt/β-catenin but also from Wnt/Fzl5/8-JNK and Fzl1/2/7-PKC pathways to provide precise spatiotemporal control of neuroectoderm patterning along its AP axis. Together, through the Wnt1 and Wnt8 ligands, they orchestrate a progressive posterior-to-anterior wave of re-specification that restricts the initial, ubiquitous, maternally specified, ANE regulatory state to the most anterior blastomeres. There, the Wnt receptor antagonist, Dkk1, protects this state through a negative feedback mechanism. Because these different Wnt pathways converge on the same cell fate specification process, our data suggest they may function as integrated components of an interactive Wnt signaling network. Our findings provide strong support for the idea that the sea urchin ANE regulatory state and the mechanisms that position and define its borders represent an ancient regulatory patterning system that was present in the common echinoderm/vertebrate ancestor. The initial regulatory state of most cells in many deuterostome embryos, including those of vertebrates and sea urchins, supports anterior neural fate specification. It is important to restrict this neurogenic potential to the anterior end of the embryo during early embryogenesis, but the molecular mechanisms by which this re-specification of posterior fate occurs are incompletely understood in any embryo. The sea urchin embryo is ideally suited to study this process because, in contrast to vertebrates, anterior–posterior neuroectoderm patterning occurs independently of dorsal-ventral axis patterning and takes place before the complex cell movements of gastrulation. In this study, we show that a linked, three-step process involving at least three different Wnt signaling pathways provides precise spatiotemporal restriction of the anterior neuroectoderm regulatory state to the anterior end of the sea urchin embryo. Because these three pathways impinge on the same developmental process, they could be functioning as an integrated Wnt signaling network. Moreover, striking parallels among gene expression patterns and functional studies suggest that this mechanism of anterior fate restriction could be highly conserved among deuterostomes. Wnt signaling pathways play fundamental roles in many developmental processes. One of the earliest and most crucial of these roles is the activation of gene regulatory programs that specify different cell fates along the embryo's primary anterior–posterior (AP) axis. Recent comparative analyses suggest that Wnt/β-catenin signaling is an ancient AP patterning mechanism that establishes posterior identity in most metazoan embryos Tribolium castaneumSaccoglossus kowalevskiiWnt/β-catenin signaling also transforms the initial regulatory state that specifies anterior neuroectoderm (ANE) development in those deuterostome embryos in which it has been examined six3Remarkably, Six3 activates a large cohort of genes in the sea urchin ANE that are orthologs of genes expressed in the vertebrate ANE (forebrain/eye field) , raisingXenopus and zebrafish, the initial widespread regulatory state promotes neuroectoderm specification throughout most of the embryo in the absence of β-catenin, which blocks dorsal organizer formation, as well as BMP2, BMP4, and BMP7. Expression of neuroectoderm markers is radialized around the AP axis in these embryos, but remarkably they retain normal AP neuroectoderm patterning, and the ANE expands posteriorly when both maternal and zygotic Wnt/β-catenin function is blocked Recent studies suggest that early AP neuroectoderm patterning in vertebrate embryos is independent of information from the dorsal organizer. In both Here we show that the Wnt-dependent restriction of neuroectoderm to the anterior pole involves not only Wnt/β-catenin but also a series of linked steps mediated by Wnt/JNK signaling through Wnt1, Wnt8, and Fzl5/8, the homolog of vertebrate Fzl8. Coordinated progression of signaling through these Wnt pathways and activation of the secreted Wnt antagonist Dkk1 in anterior-most blastomeres establish the definitive ANE around the anterior pole. Signaling through a second Wnt receptor, Fzl1/2/7, and its activation of PKC suppress Wnt/β-catenin and Wnt/JNK ANE restriction activities to coordinate the correct temporal progression of ANE restriction. Collectively, signaling through three different Wnt signaling pathways provides precise spatiotemporal control of neuroectoderm AP patterning along the AP axis.foxq2 expression at the 32-cell stage , ΔFzl5/8-injected embryos expressed foxq2 ectopically throughout the anterior half of the embryo, indicating that the second phase of its restriction to the anterior pole requires Fzl5/8 function  that acts as a dominant negative by competing for Wnt ligands age cf., , suggestion cf., . Expressced cf., ,E. We usignaling . Furtherignaling  were sigA itself , indicat neurons . In cont neurons . These o neurons .fzl5/8 at mesenchyme blastula stage  (wnt1 expression was first detected midway through the 60-cell stage (9 hpf) in the micromeres, and it also depended on nβ-catenin (wnt8 expression first moved into the next most anterior tier of blastomeres (veg1) and then, during late blastula stages (18 and 24 hpf), into both veg1 and overlying posterior ectoderm cells (wnt1 expression continued in the micromeres until midblastula stages (15 hpf) after which it, too, progressively moved to more anterior blastomeres until it reached the endoderm/ectoderm boundary during later blastula stages (24 hpf) , Wnt8 expression was normal in Wnt1 morphants  in posterior blastomeres and that also depend on Wnt/β-catenin activity. We confirmed the previously reported expression profile of r (veg2) . Similar-catenin . As deverm cells . wnt1 ex(24 hpf)  and S4A.orphants . The con on Wnt8 . We concfoxq2 expression in the ANE    . These r n = 126) and, to  n = 83) . These dfzl1/2/7, suggests that Fzl1/2/7 signaling also could affect neuroectoderm restriction  2+-dependent PKC isoforms by competing for the ATP binding site foxq2 expression was never initiated in embryos treated with the inhibitor continuously from fertilization to mesenchyme blastula stage (24 hpf)  . Moreovexpressed , and in initiating expression of upstream ANE regulatory factors was entirely unexpected because at early stages, Wnt signaling is thought to antagonize this process. We hypothesized that Wnt signaling through this receptor is necessary either for the expression of regulatory genes that specify the ANE or for antagonizing the ANE restriction mechanism from the very earliest stages. To distinguish between these alternatives, we first asked whether Fzl1/2/7 signaling is part of the maternal mechanism that can drive ubiquitous expression of ANE regulatory genes in the absence of Wnt/β-catenin signaling. Within each of three batches of embryos, we injected one set of fertilized eggs with Axin mRNA, a second set with Fzl1/2/7 morpholino, and a third with both Fzl1/2/7 morpholino and Axin mRNA  of embryos lacking Fzl1/2/7. However, it was expressed at high levels throughout all Fzl1/2/7-deficient embryos (47/47) when Wnt/β-catenin signaling was also blocked. These results indicate that maternal factors are still capable of activating foxq2 in embryos lacking Fzl1/2/7 and that the loss of foxq2/ANE fate in Fzl1/2/7 morphants requires a functional Wnt/β-catenin pathway. Thus, Fzl1/2/7 signaling is not a positive regulator of the initial maternal regulatory state that supports ANE specification, but rather it inhibits the Wnt/β-catenin-dependent ANE restriction mechanism.Our findings that a Wnt signaling branch utilizing Fzl1/2/7 and PKC activity is necessary for xin mRNA . As showfoxq2 in 99% (n = 72) or 93% (n = 70), respectively, of embryos also lacking Fzl1/2/7. These results suggest that Fzl1/2/7 antagonizes Fzl5/8-JNK-mediated ANE restriction. In the final set of experiments, we tested whether PKC signaling also antagonizes Fzl5/8-JNK-mediated ANE restriction , demonstrating that, like Fzl1/2/7, PKC antagonizes the ANE restriction mechanism by antagonizing Fzl5/8 signaling. Collectively, these results support the idea that the Fzl1/2/7-dependent suppression of Fzl5/8-mediated ANE restriction works through PKC  during the early stages of ANE restriction using the TCF-luciferase reporter plasmid, TopFlash foxq2 expression patterns , restriction of the ANE is complete and it constitutes a separate regulatory domain at the anterior end of the embryo with well-defined borders. Expression of s domain , raising by qPCR , and incrulation , inset. ignaling , raisingfoxq2 expression    . Togethe n = 64) , suggestern cf., , and 6Dbern cf.,  and 2Ag.ern cf., .fzl5/8 itself. Clearly, Fzl5/8 plays a pivotal role in the ANE restriction process because it is necessary not only for the second phase of ANE restriction but also to stop that process in the third phase of ANE patterning when Fzl5/8 signaling leads to the expression of the Wnt receptor antagonist Dkk1 at the anterior pole. Thus, the coordination between the timing of auto-repression of fzl5/8 transcription and activation of Dkk1 by Fzl5/8 ensures that this negative feedback loop reproducibly defines the ANE at the anterior pole of the embryo by mesenchyme blastula stage (24 hpf). The relative timing of Dkk1 production in the anterior ectoderm and ANE restriction in the rest of the embryo is critical and carefully controlled by a third Wnt pathway working through Fzl1/2/7 and PKC activities that limit Wnt/β-catenin and Wnt/JNK functions during the first two phases of ANE clearance.The data presented here show that patterning the neuroectoderm along the AP axis of the early sea urchin embryo depends on an elegant spatiotemporal coordination and integration of the activities of three different Wnt signaling pathways. Throughout this process, a balance is achieved between the initial regulatory mechanisms that can specify the ANE ubiquitously, those that subsequently suppress it in posterior regions, and those that limit ANE suppression. The consequence is that ANE tissue is stably positioned only at the anterior pole of the embryo by the mesenchyme blastula stage. To summarize our current model , the firwnt8 expression moves into posterior ectoderm cells as ANE factors move out. Alternatively, Wnt1 and Wnt8 may act indirectly by reinforcing the nβ-catenin gradient in the anterior-most cells of the posterior half of the embryo , activating production of an unidentified intermediate Wnt ligand that is secreted from even more anterior cells and that activates Fzl5/8 signaling.Because all of these Wnt pathways affect the same developmental process , they may function as components of an interactive Wnt signaling network rather than as separate pathways with different roles. Yet it appears that posterior Wnt/β-catenin and anterior Wnt/JNK signaling define two adjacent early regulatory domains in the sea urchin embryo. While our data suggest that these two signaling pathways activate different downstream regulatory programs in order to down-regulate ANE factors, both pathways are linked spatially and temporally by the activities of at least two common signaling components, Wnt1 and Wnt8 six3 and foxq2, are not expressed in Fzl1/2/7 knockdowns. This unexpected phenotype is the exact opposite of the ANE expansion produced by interference with Wnt/β-catenin, Fzl5/8, and JNK signaling. The function of Fzl1/2/7 begins as early as the 32-cell stage, around the time that nβ-catenin is first detectable in posterior blastomeres We found that the cardinal ANE regulatory genes, 2+ pathway. This mechanism may be similar to the situation in several other systems where Wnt/Ca2+ signaling affects early development either through the intracellular messengers CamKI, Calcineurin, and the transcription factor, NF-AT, or through PKC 2+ pathway has been shown to antagonize Wnt/β-catenin signaling during vertebrate D/V axis specification 2+ signaling in the sea urchin embryo, it could affect several different downstream parallel pathways, any or all of which are necessary to prevent premature and complete elimination of the ANE regulatory state. Moreover, the involvement of Wnt/Ca2+ signaling in AP neuroectoderm patterning would be a first.Since Fzl1/2/7 does not appear to signal through either the Wnt/JNK or the Wnt/β-catenin pathways during ANE restriction, we propose that it transduces signals through the Wnt/CaXenopus embryo +2) that operates throughout the embryo. The balance between these pathways may favor Wnt/β-catenin signaling in the posterior half of the cleavage stage embryo because of localized Wnt/β-catenin pathway-specific co-factors in that part of the embryo These considerations suggest that the function of Fzl1/2/7 in the early embryo is context-dependent, and we propose that the balance of information sent by this receptor through different Wnt signaling pathways is essential for correct specification and patterning. Recent data from several laboratories suggest that the same Fzl receptors can activate different Wnt signaling pathways, even in the same cells all of the cells in the sea urchin embryo supports development of ANE from the very beginning of its specification. These data indicate that an initial ubiquitous maternal regulatory state activates ANE specification and that one of the most important roles of posterior Wnt/β-catenin signaling is to break the symmetry of this neural-promoting state. Similarly, in vertebrate embryos, an initial regulatory state is capable of activating ANE markers throughout the embryo in the absence of Wnt, Nodal, and BMP signaling Striking parallels are emerging in the regulatory mechanisms that sea urchin and vertebrate embryos use to establish neural regulatory states at the anterior pole. Both embryos require Six3 for anterior neural development and share many homologous factors 2+ signaling. At present these are completely novel findings, but the fact that orthologs of several Wnt signaling components that function in these additional pathways in sea urchins   and those specifying endomesoderm (wnt1 and wnt8) between the invertebrate chordate amphioxus and the sea urchin embryo   also are8, Dkk1)  [17],[31n embryo . For exaStrongylocentrotus purpuratus sea urchins were obtained from Point Loma Marine Invertebrate Lab, Lakeside, CA; The Cultured Abalone, Goleta, CA; or Marinus, Garden Grove, CA. Embryos were cultured in artificial seawater at 15°C. For drug treatments, eggs attached to a protamine sulfate-coated plate were fertilized in the presence of 2 mM 4-Aminobenzoic acid (PABA), and fertilization envelopes were removed by shear force. Treatments with the cell-permeable JNK Inhibitor 1, (L)-form,  and the PKC inhibitor, Bisindolylmaleimide 1 , were performed by diluting the stock solution to 50 µM or 3 µM, respectively. JNK Inhibitor 1, (L)-form is a specific inhibitor that blocks interactions between JNK and its transcriptional substrates, such as c-Jun and c-Fos, resulting in a knockout phenotype dkk1, frizzled5/8, frizzled1/2/7, wnt1, and a partial clone of jnk by PCR. The following primers were based on the sea urchin genome sequence: Sp-dkk1 Forward ATGGCGGCTCCTTCTGC-3′5′-AGA, Reverse 5′-TCATAATACAGTTAACTGGC-3′; Sp-frizzled5/8 Forward ATGGCTGCCTTCAGTGGAAC-3′5′-AGA, Reverse 5′-TCACACCTGTACATTTGGTA-3′; Sp-frizzled1/2/7 Forward ATGGGTTGGTTGGTGAGA-3′5′-AGA, Reverse 5′-TCATACATTGGCTGGTGCAC-3′; Sp-wnt1 Forward ATGAAACTTGAGTGGTTTG-3′5′-AGA, Reverse 5′-CTACAAGCATCTGTGCACG-3′; Sp-jnk Forward 5′-GAATGTGACGCATGCCAAGC-3′, Reverse 5′-GATCACCGCCGTCGTCTATTG-3′.The 24-h blastula total cDNA was used to obtain full-length clones for dkk1 and wnt1 cDNA sequences were inserted into pCS2+ vector for mis-expression studies. ΔFzl5/8-pCS2 and Wnt8-pCS2 were obtained from Jenifer Croce  and Christine Byrum , respectively. pCS2 constructs were linearized with Not1 and mRNA was synthesized with mMessage Machine kit (Ambion), purified by LiCl precipitation and ∼20 pl injected at the following concentrations: Fzl1/2/7 mRNA = 1.0–1.5 µg/µL; Fzl5/8 mRNA = 2.0 µg/µL; ΔFzl5/8 mRNA = 2.0 µg/µL; Wnt1 mRNA = 0.01–0.1 µg/µL; Wnt8 mRNA = 0.5–1.0 µg/µL; Dkk1 mRNA = 3.0 µg/µL; Axin mRNA = 1.0 µg/µL; Tcf-Eng mRNA = 0.5–1.0 µg/µL.Full-length S. purpuratus EST sequences for wnt1, fzl1/2/7, and fzl5/8 as well as sequence information from 5′ RACE on dkk1 were used to generate translation-blocking morpholino oligonucleotides. A splice-blocking morpholino oligonucleotide was designed for the second exon–intron boundary of wnt8, which produces transcripts encoding a protein lacking sequence from the second exon, which was verified by PCR (5′-ACGCTACAAACCACTCAAGTTTCAT-3′ (1.8 mM); Wnt1 MO2: 5′-ATCCTCATCAAAACTAACTCCAAGA-3′ (0.4 mM); Wnt8 splice MO: 5′-GTAAAGTGTTTTTCTTACCTTGGAT-3′ (0.7 mM); Wnt8 MO2: 5′-GTACACTCCAATAAAAGAAATCAAA-3′ (0.6 mM) 5′-CATCTTCTAACCGTATATCTTCTGC-3′ (1.3 mM); Fzl1/2/7 MO2: 5′-ACAGATCTCCTTTAACAAGGGTAGA-3′ (2.2 mM); Fzl5/8 MO1: 5′-GGATTGTTCCACTGAAGGCAGCCAT-3′ (2.25 mM); Fzl5/8 MO2: 5′-ATGTTTATGGTCTGATGGCAATCGC-3′ (0.6 mM); Dkk1 MO1: 5′-GCGTCTAAATCCTAAATTCCTTCCT-3′ (1.5–1.6 mM); Dkk1 MO2: 5′-ATCGTTGGTAGTTGCAGAAATTCGT-3′ (0.7–0.85 mM); and JNK splice MO: 5′-CCTCATCGTTCTAGACTCACCGTTC-3′ (1.0–1.25 mM).d by PCR . The morEmbryos were injected immediately after fertilization with solutions containing FITC, 20% glycerol, and mRNA and/or morpholino oligonucleotides. All injected embryos were cultured at 15°C. Microinjection experiments were performed using at least three different batches of embryos, and each experiment consisted of 50–150 embryos unless otherwise stated. Experiments were scored only if a change in phenotype or marker expression was seen in at least 85%–90% of the manipulated embryos.qPCR was performed as described previously foxq2 was labeled with fluorescein and detected with Cy5-TSA, wnt1 was labeled with DNP and detected with Cy3-TSA, and wnt8 was labeled with DIG and detected with fluorescein-TSA.The probes for each gene analyzed correspond to the full-length cDNA sequence. Alkaline phosphatase and three-color fluorescent in situ hybridization were carried out as previously described Embryos were fixed in 2%–4% paraformaldehyde in artificial seawater at RT for 20 min and washed 5 times in phosphate-buffered saline containing 0.1% Tween-20. Embryos were incubated with primary antibodies at 4°C overnight at a dilution of 1∶1,000 for serotonin  and synaptotagminB/1e11 S. purpuratus PKC isoforms. Membranes were washed 3–5 times in room temperature PBST and probed for 1 h at room temperature in PBST+3% Bovine Serum Albumin with an enhanced chemiluminescent anti-rabbit IgG horseradish peroxidase secondary antibody . After 3–5 more washes in PBST, the membranes were developed and imaged.Protein extracts were prepared by adding 30 µL of lysis buffer  to a pellet of 300 injected embryos. Embryos were crushed 4–5 times with a pestle, immediately spun at 16,000 RCF for 15 min at 4°C, and the supernatant was stored at −80°C until use. Samples were thawed on ice and 4× NuPage Running Buffer containing 4% SDS and 10% 2-ME was added. Samples were heated at 80°C for 3–5 min and 20 µL of each sample was run on 4%–12% NuPage Bis-Tris gradient gel , transferred to nitrocellulose. Membranes were probed overnight at 4°C in Phosphate Buffered Saline+0.1% Tween-20 (PBST)+3% BSA with a poly-clonal Phospho-PKC(pan) (β11 Ser660) antibody  (1∶250) that recognizes a region that includes serine 660 and detects endogenous levels of phosphorylated PKC α, β1, β11, δ, ε, η, and θ. The recognition sequence is conserved in Promega Dual Luciferase Reporter System (Promega) was used to perform dual luciferase assays. Embryos (350–400) were injected with linearized TopFlash-Firefly Luciferase (REF) and Endo16-Renilla Luciferase plasmids at concentrations of 20 ng/µL and 10 ng/µL, respectively, along with 10 ng/µL of linearized genomic DNA carrier. The Firefly and Renilla luciferase signals were recorded with a plate style luminometer using Promega's suggested protocol. The level of luciferase activity was normalized to the level of Renilla activity for each experiment. All experiments were repeated three times using separate batches of embryos.Figure S1foxq2 expression at early cleavage and blastula stages, related to blimp1b, hox11/13b, and gcm.  Axin injected embryos develop into dauer blastulae.TCF-Eng and Axin mRNA overexpression block Wnt/β-catenin-dependent specification of endomesoderm and patterning of the ANE.  TCF-Eng mRNA overexpression prevents restriction of (TIF)Click here for additional data file.Figure S2fzl5/8, fzl1/2/7, fzl9/10, and fzl4 during early development of the sea urchin embryo, related to fzl5/8, fzl1/2/7, fzl9/10, and fzl4 transcripts per embryo at 0 hpf, 60-cell, 120-cell, hatched blastula, and mesenchyme blastula stages. The y-axis shows the approximate number of transcripts per embryo based on the Ct value of z12 transcripts, whose absolute concentrations are known at each stage  fzl1/2/7, fzl5/8, and foxq2 mRNAs during ANE restriction. (Ba–d) fzl1/2/7 expression. (Be–h) fzl5/8 expression. (Bi–l) foxq2 expression. All samples were examined at the stages indicated above each column. (C) Whole mount in situ hybridization showing that JNK is ubiquitously expressed during the ANE restriction process (60-cell to mesenchyme blastula stage).Expression of (TIF)Click here for additional data file.Figure S3wnt8 = 357 bp; expected Wnt8-MO1 PCR product size for wnt8 = 315 bp; expected control PCR product size for jnk = 690 bp; expected JNK splice MO PCR product size for jnk = 530 bp. Diagrams of the intron-exon organization of wnt8 (Jb) and jnk (Kb) pre-mRNAs. Primers used to characterize the mRNA products in (Ia) and (Ja) (arrows). Position of the target sequence for the morpholino (red bar). JNK catalytic domains is in the deleted exon (blue bar) (Kb). MO, morpholino.Additional morpholino and inhibitor phenotypes, related to (TIF)Click here for additional data file.Figure S4wnt1 during early development and effects of perturbing Wnt signaling via Dkk1, Axin mis-expression, and a Wnt1 morpholino, related to wnt1 during ANE restriction. (B) Overexpression of Dkk1 blocks the expression of the endomesoderm marker z13; (C) overexpression of Axin blocks expression of wnt1; (D) wnt8 expression is not regulated by Wnt1 signaling; (E) wnt1 expression does not depend on Wnt8 signaling.Expression of (TIF)Click here for additional data file.Figure S5foxq2 expression , related to foxq2 expression in more than half the embryos . (B) Fzl5/8 is necessary for and Dkk1 antagonizes posteriorization by Wnt1 signaling; related to wnt1 have a severe vegetalized/posteriorized phenotype. (c) Embryos misexpressing both wnt1 and dkk1 have a normal phenotype. (d) Embryos misexpressing wnt1 and Δfzl5/8 have the Δfzl5/8 phenotype.Phenotypes produced are severely vegetalized/posteriorized by Wnt1 misexpression, require pJNK and Fz5/8, and can be antagonized by Dkk1. (A) pJNK is necessary for full down-regulation of the ANE by Wnt1. Wnt1 mRNA overexpression eliminates (TIF)Click here for additional data file.Figure S6The expression level of Fzl receptors is not a rate-limiting step that influences the balance of Wnt signaling in embryos during ANE restriction, related to (TIF)Click here for additional data file.Table S1qPCR primer pairs used for expression analysis.(DOCX)Click here for additional data file.


The mechanisms that underlie the critical dendritic cell (DC) function inmaintainance of peripheral immune tolerance are incompletely understood,although the β-catenin signaling pathway is critical for this role. Themolecular details by which β-catenin signaling is regulated in DCs areunknown. Mechanical disruption of murine bone marrow-derived DC (BMDC) clustersactivates DCs while maintaining their tolerogenic potential and this activationis associated with β-catenin signaling, providing a useful model with whichto explore tolerance-associated β-catenin signaling in DCs. In this report,we demonstrate novel molecular features of the signaling events that control DCactivation in response to mechanical stimulation. Non-canonical β-cateninsignaling is an essential component of this tolerogenic activation and ismodulated by adhesion molecules, including integrins. This uniqueβ-catenin-dependent signaling pathway is constitutively active at lowlevels, suggesting that mechanical stimulation is not necessarily required forinduction of this unique activation program. We additionally find that theimmunomodulatory cytokine TGF-β antagonizes β-catenin in DCs, therebyselectively suppressing signaling associated with tolerogenic DC activationwhile having no impact on LPS-induced, β-catenin-independent immunogenicactivation. These findings provide new molecular insight into the regulation ofa critical signaling pathway for DC function in peripheral immune tolerance. TGF-β is a cytokine whose signaling is strongly associatedwith immune suppression and tolerance -/-mice were generously provided by T. Mayadas . Animalstudies were approved by the Brigham and Womens' Hospital/Harvard MedicalSchool institutional review and ethics committee . Mice were maintained in specific pathogen-free conditions in accordancewith institutional guidelines.WT C57BL/6 mice were purchased from the Jackson Laboratories. CD11b6 cells/ml in RPMI medium with 10ng/ml murine GM-CSF (Peprotech) and various concentrations of CHO-derived humanTGF-β1 (Peprotech). Media was refreshed every other day.Bone marrow-derived dendritic cells were cultured as previously described Inflammatory stimulation: Day 5 BMDCs were stimulated for 24 hours with 100 ng/mlLPS (Sigma-Aldrich). Tolerogenic stimulation: loosely adherent day 5 BMDCs werecollected by repeated pipetting and resuspended in fresh media for 24 hours. Insome cases, CD11c magnetic beads (Miltenyi Biotec) were used according tomanufacturer's instructions to simultaneously purify CD11c+ cells andintroduce mechanical stimulation. Integrin-mediated stimulation: LEAF-purifiedantibodies (Biolegend) were added to cultured cells to 1.5 µg/ml finalconcentration for 24 hours. Following stimulation, loosely adherent cells werecollected by pipetting, for analysis by flow cytometry.hi/CD86+/CCR7+ DCs in thestimulated condition after subtracting the percentage in the unstimulatedcondition. Fold-induced maturation is calculated as the ratio of inducedmaturation in the experimental group to the induced maturation in the controlgroup.To calculate the impact of TGF-β on DC responsiveness to stimulation requiredthat we first correct for the effect of TGF-β on unstimulated levels ofspontaneously mature DCs. Percent induced maturation is therefore the percentageof MHC-IIpLKO vector-encoded lentivirus was produced as previously described Immunoprecipitations were completed using anti-E-Cadherin  withlysates prepared from CD11c-purified BMDCs using the Pierce Classic IP Kitaccording to manufacturer's instructions. Protein fractions were run on4–20% polyacrylamide gels (Biorad) and transferred to PVFDmembranes. After blocking, membranes were stained either with unconjugated wholerabbit anti-serum against β-catenin (Sigma) or unconjugated anti-tubulin. HRP-conjugated goat anti-rabbit  was used as asecondary stain.Day 2 control or TGF-β BMDCs were infected with lentiviral reporterconstructs  following the protocol outlined above withMOI 2. Infected cells were maintained in control or TGF-β medium untilanalysis on D5. BMDC lysates were prepared and analyzed using a luciferase assaykit (Agilent Technologies) according to manufacturers instructions.BMDCs were cultured for 5 days. CD11c+ cells were purified by magnetic beads(Miltenyi) and plated onto glass coverslips. Cells were allowed to adhere for 24hours before fixation with 4% paraformaldehyde. Cells were thenpermeabilized and stained with antibodies or phalloidin (actin), were washed,and mounted with DAPI-containing medium (Invitrogen).We cultured immature bone marrow-derived dendritic cells (BMDCs) to studymechanisms that may orchestrate tolerogenic DC maturation. We began our studiesby reproducing aspects of this model reported previously . DC matuhi immunophenotype) . As previously reported,we also find that maturation is robustly induced by mechanical disruption ofBMDC clusters . Weconfirmed by flow cytometry that both types of maturation stimuli activated the“core” aspects of the DC maturation response  . We findresponse .ex vivo with cognate antigen. We find that whileboth LPS- and mechanically-stimulated BMDCs prime the recall response equallywell ,the cyAs an initial step to understanding tolerogenic function in BMDCs, we sought tobetter understand the molecular mechanisms by which mechanical stimulationinduces maturation. Previous studies with mechanical stimulation have utilizedrelatively poorly defined stimuli to introduce mechanical stimulation, such assimple pipetting -/- BMDCs  was reduced with respect to maturation achieved on standard culturesubstrates . Cytoskeletal activation is associated with DCmaturation Selective suppression of mechanically-stimulated DC maturation by TGF-βindicates that activation by this stimulus is uniquely dependent on aTGF-β-sensitive signaling molecule. We first investigated the impact ofTGF-β on DC morphology during culture. We found no effect of TGF-β onBMDC cluster formation . However+ cells from TGF-β+ orTGF-β- BMDC cultures, a process that in itself providesmechanical stimulation To distinguish these two possibilities, we used magnetic beads to purifyCD11cThree observations suggested to us that in addition to the disruption of thecytoskeleton and adhesion molecules, TGF-β additionally disrupts downstreamβ-catenin signaling. First, although TGF-β reduced the surface levels ofCD11b, a substantial amount did remain , yet wasTo address this possibility, we examined the effects of TGF-β onβ-catenin function. Given the morphological changes we observed afterTGF-β treatment, we first examined the structural function of β-catenin.To investigate the possibility that TGF-β disrupts β-catenin/E-Cadherinassociations, we immunoprecipitated (IP) E-Cadherin from TGF-β-cultured DClysates. Western blots demonstrate that TGF-β did not inhibitco-precipitation of β-catenin with E-Cadherin, suggesting that thisparticular association is not disrupted . InteresIn addition to its structural role, β-catenin serves as a transcriptionfactor. To evaluate the impact of TGF-β on β-catenin transcriptionalactivity, we infected BMDCs with a lentiviral TCF/LEF-luciferase reporter. Wefound that TGF-β suppresses β-catenin transcriptional activity . Thus, wWe set out to better define the molecular mechanisms by which mechanical stimulationinduces the unique tolerogenic activation program in BMDCs and to investigate thefunction and regulation of β-catenin signaling in this response. By thisapproach, we describe several novel features of β-catenin signaling in DCs.Although it has been proposed that disruption of E-Cadherin-mediated cell-cellinitiates the tolerogenic program invivoinvivo suggesting the possibility of autocrine activation in the absenceof external signals in vitro model, wepropose that β-catenin-depdendent tolerogenic DC function invivo may not be driven by mechanical stimulation perse, but rather by a constitutively or stochastically active signalmodulated by adhesion molecules and intensified by mechanical stimulation. Furtherstudies will be required to determine the contributions of canonical wnt signalingand alternative signaling mediated by adhesion molecules to steady state tolerogenicDC function in vivo.β-catenin signaling is constitutively active in tolerogenic DC subsets in vivo. TGF-β is awell-established regulator of DC development, chemoattraction and function,particularly for DC subsets in peripheral tissues such as skin, the lungs, and thegut β is strongly associated with immune suppression, it isperhaps counterintuitive that TGF'β antagonizes a signaling pathwayassociated with tolerogenic DC function. However, peripheral tissues arecontinuously subject to mechanical stress that would be predicted to initiatewide-spread activation of DCs. We speculate that TGF-β may act directly tosuppress DC activation by opposing β-catenin-dependent signaling. Consistentwith this model, mice in which DCs are deficient in TGF-β signaling displayenhanced spontaneous DC maturation and migration in vitro model,we continued to observe low levels of spontaneous maturation. Therefore, even in aTGF-β-rich environment our model predicts infrequent but measurable maturationto occur, consistent with in vivo observations per se.Our data provide insight into mechanisms that may regulate signaling pathwaysassociated with tolerogenic DC function in vivo might beselectively targeted, in order that one might “steer” DC functiontowards a desired functional outcome and thereby shift the balance of immunity andtolerance in human disease. Further research will be required to explore thevalidity of this concept.The unique role of β-catenin in tolerogenic DC function, and the finding thatthis molecule may be regulated independently of immunogenic DC signaling, isparticularly intriguing with respect to therapeutic manipulation of antigen-specificimmune responses. It suggests that signaling pathway components uniquely associatedwith immunogenic or tolerogenic DC function Figure S1Disruption of cell-cell contacts is not essential for BMDC response tomechanical stimulation. Day 5 CD11c+ BMDCs werepurified by magnetic bead separation (Miltenyi). TGF-β was included ininitial culture to prevent maturation during purification. Purified DCs werereplated at sufficiently low density to minimize cell-cell contactformation. 24 hours after replating, DCs were either left untreated ormechanically-stimulated by repeated pipetting and replated. 48 hours afterinitial purification, DCs were assessed for maturation by flow cytometry.Plots are representative of 3 experiments.(TIF)Click here for additional data file.Figure S2Integrins and DC activation. A) CD11b expression on Day 5 BMDCswas assessed by flow cytometry using either the M170 or 5C6 clones. Greyindicates isotype control staining. Both clones show similar levels ofbinding. B) BMDCs were either left untreated (Cont) or pretreated withunconjugated 5C6 before staining with PE-conjugated M170. Pretreatment with5C6 did not block M170 binding. C) CD11a and CD11b expression levels wereanalyzed by flow cytometry on Day 5 conventional (Cont) orTGF-β-cultured BMDCs.(TIF)Click here for additional data file.Figure S3Lentiviral shRNA knockdowns in BMDCs. A) shRNA-infected BMDCsdisplay normal flow cytometry scatter profile after puromycin selection. B)BMDCs infected with control shRNA were analyzed for maturation before andafter stimulation by flow cytometry. Stimulated DCs display over 20-foldincrease in levels of DCs bearing markers of maturation afterstimulation.(TIF)Click here for additional data file.


Burkholderia mallei isolates from equines in Punjab, Pakistan from 1999 through 2007. We show that recent outbreaks are genetically distinct from available whole genome sequences and that these genotypes are persistent and ubiquitous in Punjab, probably due to human-mediated movement of equines.We collected epidemiologic and molecular data from  Burkholderia mallei, an obligate pathogen of horses, donkeys, and mules (Equidae), with occasional infections in felines, canines, and humans  of B. mallei . Furthermore, B. mallei can remain viable in contaminated stables for up to 6 weeks  the industrial nature of Faisalabad or 2) from high endemism of B. mallei disseminates throughout a region. For example, epidemiologic data suggests that 2 horses from a farm in the district of Sargodha (PRL3 and PRL4) contracted glanders while at a polo club in the Lahore district. This is supported by VNTR data, as these 2 isolates clustered phylogenetically with one of the samples obtained from an outbreak that occurred at the same polo club 3 months prior  are not viable options. Therefore, other solutions to curbing the spread of glanders need to be identified. We suggest that a focus on finding methods to improve the sanitary conditions of communal water troughs and stables may lead to a practical solution for disease reduction and containment. Finally, our study demonstrates the utility of VNTRs paired with extensive epidemiologic data for analyzing the distribution of B. mallei genotypes throughout endemic regions.Our study suggests that numerous lineages of Epidemiologic and 23-locus VNTR data for 15 isolates of Burkholderia mallei from the Punjab province, Pakistan, and 9 Burkholderia mallei whole genome sequences*


Drosophila males and increases the transcription level of X-linked genes by approximately twofold. Male-specific lethal 2 (MSL2) together with MSL1 mediates the initial recruitment of the DCC to high-affinity sites in the X chromosome. MSL2 contains a DNA-binding cysteine-rich CXC domain that is important for X targeting. In this study, we determined the solution structure of MSL2 CXC domain by NMR spectroscopy. We identified three zinc ions in the CXC domain and determined the metal-to-cysteine connectivities from 1H-113Cd correlation experiments. The structure reveals an unusual zinc-cysteine cluster composed of three zinc ions coordinated by six terminal and three bridging cysteines. The CXC domain exhibits unexpected structural homology to pre-SET motifs of histone lysine methyltransferases, expanding the distribution and structural diversity of the CXC domain superfamily. Our findings provide novel structural insight into the evolution and function of CXC domains.The dosage compensation complex (DCC) binds to single X chromosomes in  Drosophila melanogaster, the transcriptional level of most genes in the single male X chromosome is increased by approximately twofold to match that from two female X chromosomes  or chromatin entry sites (CES) in the X chromosome and then spreads Drosophila cells MSL2 is a core component of the DCC MSL2 is composed of an N-terminal RING domain, a cysteine-rich CXC domain and a C-terminal region rich in proline and basic residues (Pro/Bas patch). The RING domain binds MSL1 3Cys9 cluster. Interestingly, the CXC structure with a Zn3Cys9 cluster shows strong similarity to pre-SET motifs of histone lysine methyltransferases, suggesting that the CXC and pre-SET domains share a common evolutionary origin.The CXC domain is remarkable by having 9 invariant Cys within about 50 residue region. In this study, we have determined the first structure of MSL2 CXC domain by NMR spectroscopy. The structure reveals a compact fold that encages an unusual ZnD. melanogaster MSL2 CXC domain was conducted mainly on two constructs. One construct containing residues 517–572 and the C560G mutation (CXC-2) was used in early experiments, and another slightly shorter construct containing residues 520–570 and the C560G mutation (CXC-3) was used in the majority of experiments. The two fragments display identical NMR spectra in folded regions and thus should have the same structure. In both constructs, the nonconserved residue Cys560 was replaced by glycine, an amino acid that is frequently found at the corresponding position in MLS2 homologs, to avoid formation of intermolecular disulfide bonds. The 1H-15N HSQC spectrum of the MSL2 CXC domain displays a single set of well-dispersed peaks, indicating that the fragment is autonomously folded and amenable for further structural characterization . Analytical ultracentrifuge sedimentation velocity assay further showed that the CXC domain is a monomer in solution (data not shown). These biophysical results indicate that the CXC domain folds into a monomeric structure with three bound zinc ions.The CXC domain is characterized by nine invariant cysteine residues and conserved spacing between them, suggesting that these cysteine residues may coordinate metal ions. Indeed, inductively coupled plasma mass spectrometry of recombinant CXC protein revealed a significant enrichment of Zn compared with other metals Fe, Mg, Ca, Mn, Ni and Cu (data not shown). To assess the Zn binding stoichiometry, the CXC-3 protein was analyzed with electrospray mass spectroscopy under conditions that preserve the native protein structure . The mon2+ ions are most likely coordinated by nine invariant Cys residues in the MLS2 CXC domain. The CXC domain has two histidines at positions 557 and 565, but they are not conserved and are unlikely to bind structurally important Zn2+ ions. As each Zn2+ ion needs to be coordinated to four ligands, the unusual Zn to Cys ratio of 3∶9 suggests that these Zn2+ ions are bound in an unconventional way. To assign the zinc ligands, we replaced Zn with 113Cd, which has a similar coordination pattern to Zn but has more favorable NMR properties 113Cd-loaded protein was prepared during protein expression by substituting Zn2+ with 113Cd2+ in minimal M9 medium.Three Zn1H-15N HSQC spectrum of 113Cd/15N-labeled CXC protein displays a single set of peaks, indicating complete 113Cd substitution  split these resonances into triplets.The 46.6 ppm , in agre1H-113Cd HSQC spectra and an HMQC-TOCSY spectrum to correlate 113Cd ions to Hβ and Hα protons of coordinating Cys residues . Inspection of structures calculated without the Zn-C restraints suggested that Cys539 and Cys553 are the remaining two ligands for Zn-C that could not be identified with 113Cd NMR. Other assignments of Zn-C ligands were not consistent with the existing structural restraints. The numbers and types of restraints used in the final structure calculation and the statistics for the 20 lowest energy structures are given in The structure of MSL2 CXC domain was initially calculated in CYANA based solely on autoassigned NOE cross-peaks without incorporation of zinc ions. The resulting structures converged and showed a compact fold with Cys clustering at the center. The structure was further refined in CNS with additional TALOS-derived dihedral restraints, protein-zinc restraints deduced from 1H-15N heteronuclear NOE values (The NMR structures are well defined with an average of 27 restraints per residue. The root mean square deviation (RMSD) values of the 20 best structures to the mean structure are 0.38 Å and 0.87 Å for backbone and heavy atoms in the structure core (residues 523–529 and 538–565), respectively . A few tE values .3Cys9 cluster in a right-handed manner. The N-terminal loop (residues 521–550) including a short α-helix (residues 546–550) harbors five Cys residues, whereas the C-terminal loop (residues 551–567) contains four Cys residues.The CXC domain adopts a small globular fold that encapsulates three triangularly arranged zinc ions . Among n3Cys9 cluster appears to be important for the structure and function of CXC domains. The Ala double substitution of Cys544 and Cys546 of MSL2 was previously shown to delay development and reduce male fly viability D. melanogaster cells An intact ZnBesides invariant Cys ligands, residues Arg526, Gly528, Thr538, Arg543, Try547 and Asn563 are also highly conserved among MSL2 homologs . These r2H2O and monitored with 1H-15N HSQC spectra for disappearance of amide proton. Seventeen amide protons were observed in the first recorded spectrum, ten persisted after 2 h and four remained after 24 h  and the SUV39 family of SET domain histone lysine methyltransferases (HKMTs). MTs are ubiquitous Cys-rich small proteins (6–7 kDa) with a high content of divalent metal ions such as Zn2+, Cd2+ and Cu1+ and are involved in metal homeostasis, detoxification and protection against reactive oxygen 3Cys9 metal-thiolate cluster and an α-domain that binds an M4Cys11 cluster 3Cys9 clusters are also present in other families of MTs Similar ZnSurprisingly, the CXC domain structure shows remarkable resemblance with structures of pre-SET motifs in the SUV39 family of HKMTs Structures of SUV39 family HKMTs show that the pre-SET domain coordinates three zinc ions with nine invariant Cys residues 2Cys7 cluster . The mutation C560G was introduced by QuikChange site-directed mutagenesis (Stratagene), yielding the GST-fused CXC-2 construct. The coding sequence consisting of residues 520–570 and the C560G mutation was amplified from the CXC-2 plasmid and subcloned into an engineered pET28a vector, yielding the CXC-2 construct. The CXC-3 protein was expressed as fusion to an N-terminal HisEscherichia coli Rosetta(DE3) cells containing CXC expression vectors were grown at 37°C in LB broth. When the OD600 reached 0.8, the growth temperature was lowered to 16°C, and the culture media was supplemented with 40 µM ZnSO4 and 0.2 mM isopropyl-β-D-thiogalactopyranoside to induce protein expression. After additional growth for 16 h, the cells were harvested, suspended in buffer A consisting of 50 mM Tris-HCl (pH 8.0) and 250 mM NaCl, lysed by sonication and centrifuged.The clarified lysate of GST-fused CXC-2 was loaded onto a GSTrap column. The GST-tag was cleaved on column by PreScission protease overnight. The released CXC-2 protein was washed with buffer A, diluted 3-fold with 25 mM HEPES-K (pH 7.6), loaded onto a heparin column  equilibrated in 25 mM HEPES-K (pH 7.6) and 80 mM KCl and eluted using a linear gradient of KCl. Proteins eluting at less than 300 mM KCl were pooled, concentrated with 3-kDa cutoff ultrafiltration devices (Amicon) and further purified with a Superdex 75 column in buffer 50 mM phosphate (pH 6.0).6-SMT3-tagged CXC-3 protein was purified with a HisTrap column  and eluted with 500 mM imidazole in buffer A. The pooled factions were incubated with ULP1 for 1 h on ice to cleave the His6-SMT3 tag. After a threefold dilution in 25 mM HEPES-K (pH 7.6), the protein was loaded onto a Q column. The flow-through containing CXC-3 was further purified by heparin and gel filtration chromatography following the same procedure as for CXC-2. Protein concentrations were determined spectrophotometrically with a calculated molar extinction coefficient of 4470 M−1 cm−1 at 280 nm for both CXC-2 and CXC-3.The His15N- or 15N/13C- labeled CXC proteins were prepared in M9 minimal medium with 1 g/L of (15NH4)2SO4 and, if needed, 2 g/L of 13C-glucose as the sole nitrogen and carbon sources, respectively (Cambridge Isotope Laboratories). For 113Cd labeling, ZnCl2 in M9 media was substituted with 10 nM 113Cd acetate. E. coli growth and protein yields were normal in 113Cd-containing media.2+ ion was calculated as molecular mass of 64Zn (63.929) minus 2, to compensate for two positive charges of Zn2+ ion.The CXC-3 protein was exchanged into 200 mM ammonium acetate and analyzed by electrospray ionization mass spectrometry with a Q-Star mass spectrometer (Applied Biosystems). To calculate the monoisotopic mass of a species, the mass-to-charge (m/z) ratio of its monoisotopic peak was multiplied by the charge, and the charge was then subtracted. The monoisotopic mass of CXC-3, which contains residues 520–570, mutation C560G and an extra N-terminal Ser from the vector, is 5638.48 Da. The exact mass of Zn2H2O. NMR data were recorded at 298 K on a Bruker DMX600 spectrometer equipped with a triple resonance cryoprobe, unless indicated otherwise. Three-dimensional (3D) CBCA(CO)NH, HNCACB and HNCO spectra were collected to obtain sequence-specific backbone resonance assignments m 12 ms), HCCH-TOCSY (τm 12 ms), 1H-15N TOCSY-HSQC (τm 60 ms) and 2D 1H-13C HSQC spectra were collected for side-chain assignments. All spectra were processed with Felix (Accelrys Inc.) and analyzed with NMRViewJ 1H chemical shifts were referenced to internal DSS, and the 15N and 13C chemical shifts were referenced indirectly. The 1H-15N steady-state heteronuclear NOE values were calculated from the ratios of peak intensities in a 1H-15N HSQC spectrum collected with a 3 s period of initial proton saturation to those in an unsaturated spectrum. The error of peak intensity was estimated from spectrum background noise and propagated into the error of 1H-15N heteronuclear NOE.The NMR samples contained 0.5–1.5 mM CXC-2 or CXC-3 proteins, 50 mM potassium phosphate (pH 6.0), 0.01% (w/v) sodium 2,2-dimethylsilapentane-5-sulfonate (DSS) and 10% (v/v)  2H2O. A series of 1H-15N HSQC spectra were recorded 0 min, 30 min, 75 min, 120 min, 160 min, 390 min and 24 h after sample preparation.In hydrogen-deuterium exchange experiments, the CXC-3 protein originally prepared in 500 µl of 50 mM potassium phosphate (pH 6.0) was lyophilized and redissolved in 500 µl113Cd, 2D 1H-113Cd HSQC and 2D 1H-113Cd HMQC-TOCSY spectra were recorded on a Bruker 400 MHz NMR spectrometer equipped with a broadband double resonance probe. The delays for magnetization transfer (1/2JH-Cd) were set to 10, 16.7 and 25 ms. The resonances of 113Cd-loaded CXC were reassigned according to the assignments of Zn-loaded CXC and 3D 1H-15N TOCSY-HSQC and 1H-15N NOESY-HSQC spectra collected on 113Cd/15N-labeled CXC protein. The 113Cd chemical shifts are reported relative to external 1 M Cd(CH3COO)2, which serves as the 0 ppm reference.1D 1H-15N NOESY-HSQC (τm 200 ms), 3D aliphatic 1H-13C NOESY-HSQC (τm 200 ms) and 3D aromatic 1H-13C NOESY-HSQC (τm 200 ms) spectra. Inter-proton distances were obtained with NMRViewJ using an exponential calibration from peak volumes and the upper limits of restraints were set to 2.2–6.0 Å. The CXC structure was initially calculated in CYANA solely from autoassigned NOE peaks NOE-based distance restraints were derived from 3D 113Cd NMR experiments were incorporated first. Zn-C was assigned by 113Cd NMR to be ligated to Cys558 and Cys561. Inspection of the resulting structures suggested that Cys539 and Cys553 are the remaining two ligands for Zn-C. Incorporation of these two Zn-C restrains caused no NOE violations during the structure calculation, whereas coordination of Zn-C with different ligands caused violations around the Zn-cysteine cluster, indicating the correctness of the assignment.The CYANA-generated model was further refined in CNS 100 structures were calculated, and the 50 lowest energy structures were further refined with electrostatic potentials and explicit water using CNS and RECOORDScript The NMR resonance assignments for CXC-3 have been deposited in the BioMagResBank with accession number 18514. The atomic coordinates and experimental NMR restraints for the MSL2 CXC domain have been deposited in the Protein Data Bank with accession code 2LUA.