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The bacterium Magnetospirillum gryphiswaldense MSR-1 forms nanosized membrane-enclosed organelles termed magnetosomes. The mamXY operon, part of the magnetosome island (MAI), includes the mamY, mamX, mamZ, and ftsZ-like genes, which initiate gene transcription via the same promoter. We used a combination of molecular biological techniques (targeting of cross-linking reagents) and high-resolution mass spectrometry to investigate the coordinated activity of the four MamXY proteins in magnetite biomineralization. The FtsZ-like protein was shown by confocal laser scanning microscopy to be dispersed in the cytoplasm in the early stage of cell growth and then gradually polymerized along the magnetosome chain. Interactions of various pairs of MamXY proteins were observed using a bacterial two-hybrid system. We constructed a recombinant FtsZ-like-overexpressing strain, examined its growth patterns, and extracted magnetosome membrane proteins using a modified SDS/boiling method with BS2G-d0/d4 reagent, which helped stabilize interactions among MamXY proteins. In liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, MamY expression was detected first and remained highest among the four proteins throughout all stages of cell growth. MamX and MamZ expression was detected subsequently. The four proteins displayed coordinated expression patterns during the magnetosome maturation process. Unique peptides discovered in the MamXY protein sequences appeared to constitute "hidden" interaction sites involved in the formation of MamXY complex that helped control magnetosome shape and size.
IMPORTANCE mamXY operon genes play an essential role in magnetite biomineralization, participate in redox reactions, and control magnetosome shape and size. However, mechanisms whereby the four MamXY proteins function together in iron oxidoreduction and transport are poorly understood. We used a combination of targeted cross-linking techniques and high-resolution mass spectrometry to elucidate the coordinated activity patterns of the MamXY proteins during magnetite biomineralization. Our findings indicate that the FtsZ-like protein undergoes polymerization and then recruits MamY, MamX, and MamZ in turn, and that these interactions depend on unique peptides present in the protein sequences. A hypothetical model of the functionalities of these proteins is proposed that accounts for the findings and provides a basis for further studies of coordination among magnetosome island (MAI) gene clusters during the process of magnetosome formation.
Magnetotactic bacteria (MTB) are ubiquitous worldwide in marine sediments and chemically stratified columns of freshwater (1). They have a unique ability to synthesize magnetosomes, intracellular organelles composed of membrane-enclosed chains of magnetite (iron oxide [Fe3O4]) or greigite (iron sulfide [Fe3S4]) crystals (2, 3). The presence of magnetosomes enables MTB to swim along the Earth's geomagnetic field lines (magnetotaxis) (4). Magnetosomes have potential applications in early diagnosis and detection of pathogens (5), and as nanoscale drug carriers (6–9) for therapeutic targeting of cancer cells, with more effective biocompatibility than artificial magnetic particles. Therefore, many studies during the past two decades have been focused on mechanisms of magnetite biomineralization, improvement of MTB culture methods, and enhancement of magnetosome yield.
Magnetospirillum gryphiswaldense MSR-1, a frequently studied freshwater MTB strain, grows well under conditions involving a trace amount or high concentration (>20 μM) of iron; however, it synthesizes magnetosomes only in a microaerobic environment (dissolved oxygen concentration, <1%) with a high iron concentration (10, 11). The genes located in the magnetosome island (MAI) have specialized functions in magnetite biomineralization, and they belong to four operons (mms6, mamGFDC, mamAB, and mamXY) (12, 13). In MSR-1, the mamAB cluster is necessary for magnetosome formation (14), and the mms6 and mamGFDC clusters control crystal morphology and growth (15, 16). The mamXY cluster, which is conserved in all magnetospirilla (including MSR-1, Magnetospirillum magneticum AMB-1, Magnetospirillum magnetotacticum MS-1, and Magnetospirillum sp. strain SO-1) (see Fig. S1A in the supplemental material), includes mamY, mamX, mamZ, and ftsZ-like genes. In AMB-1, the N terminus of MamY is integrated in a magnetosome membrane (MM), and the number of small magnetosomes was greater in the ΔmamY mutant strain than in the wild-type (WT) strain (17). Because of the binding to biological membrane vesicles and liposome tubulation activity, MamY is hypothesized to be related to the invagination of the magnetosome vesicle (17). A recent study about the interaction of the MamY protein with cardiolipin (CL) suggests the presence of a unique protein-lipid interaction for magnetosome formation in magnetotactic bacteria (18). The MamX protein, located in the MM, plays important roles in the control of magnetosome size and maturation, and the deletion of mamX resulted in the presence of irregular superparamagnetic magnetite particles (19). MamZ (previously termed "MamH-like") has 18 transmembrane domains. Its N-terminal domain is an ortholog of the major facilitator superfamily (MFS), and its C-terminal domain is similar to ferric reductase-like transmembrane component. In MSR-1, the deletion of mamZ or mamX resulted in an identical phenotype (20). ftsZ-like is a tubulin-like gene and encodes the FtsZ-like protein, located in the cytoplasm. In a structural comparison with FtsZ by the SWISS-MODEL program (www.swissmodel.expasy.org), the FtsZ-like protein lacked a C terminus but had high similarity in its N terminus (Fig. S1B). A deletion mutant of the ftsZ-like gene produced mainly superparamagnetic magnetite particles (21), and the defect of this mutant was reversed by nitrate (22), suggesting that the FtsZ-like protein participates indirectly in redox control of magnetite crystallization. In MSR-1, deletion of the whole mamXY cluster resulted in smaller crystals and reduced magnetism (14). The above-mentioned findings, taken together, indicate the functional redundancy of the four genes in the mamXY operon for magnetite biomineralization. The reasons for such redundancy, as well as the functionalities of the MamXY proteins, remain unclear.
We hypothesize that (i) because transcription of the four mamXY operon genes is controlled by the same promoter (Fig. 1A), their transcription occurs sequentially; and (ii) the four proteins encoded by these genes form some sort of interaction pattern, or protein complex, to participate (along with other proteins) in magnetosome synthesis. To test these hypotheses, we studied the localization of FtsZ-like-enhanced green fluorescent protein (FtsZ-like-EGFP), evaluated interactions among the four proteins in vitro, and investigated the functionalities of MamXY proteins during the process of magnetosome formation using a combination of targeted cross-linking techniques and high-resolution mass spectrometry (HRMS). Our findings help elucidate the characteristics and functional significance of mamXY operon-encoded proteins in magnetite biomineralization.
Bioinformatic analysis of FtsZ-like protein in MSR-1.Genomic analysis revealed the presence of two ftsZ homologs (MGMSRv2_2503 and MGMSRv2_2324) in MSR-1. FtsZ (gene code MGMSRv2_2503), belonging to the category of bacterial cytoskeletal filaments, is a tubulin homolog essential for prokaryotic cell division; it forms a ring-like structure (Z-ring) and recruits other proteins to form a complex that promotes cell division at the cell midpoint (23, 24). The FtsZ-like protein (gene code MGMSRv2_2324) is a truncated form of FtsZ having high similarity to the FtsZ N terminus. It retains GTP-dependent polymerization ability but not the ability to participate in cell division because of its lack of a C terminus (Fig. S1B) (21). The predicted three-dimensional (3D) structure of the FtsZ-like protein includes a nucleotide-binding site and SulA site, which is highly identified with the N terminus of FtsZ (Fig. 1B). The SulA site is related to polymerization and can be bound by the SulA protein for inhibition of FtsZ polymerization in Escherichia coli (25).
ftsZ homologs are conserved in the well-studied MTB strains M. gryphiswaldense MSR-1 and M. magneticum AMB-1. Amino acid sequences of FtsZ and FtsZ-like proteins from MSR-1 and from AMB-1 (amb3854 and amb1015), and of the FtsZ protein from E. coli (b0095) were aligned using the Clustal X software program (Conway Institute, University College Dublin, Ireland). BLASTP analysis revealed that five bacterial cytoskeletal proteins (FtsZMSR-1, FtsZ-likeMSR-1, FtsZAMB-1, FtsZ-likeAMB-1, and FtsZE. coli) had identities of >69% and that homologous regions were concentrated mainly in ∼320 amino acids of the N terminus (Fig. 1C). Only a few amino acids were conserved in the C terminus (Fig. 1C, red box). These findings indicate that FtsZ-like is a distinctive cytoskeletal protein in MTB that is involved (along with other proteins) in biomineralization but not in cell division.
Localization of FtsZ-like-EGFP protein in MSR-1.FtsZ-like protein is found in cytoplasm and can be localized using enhanced green fluorescent protein (EGFP). We constructed a fusion expression strain (termed MSR-1-pBB-fzl-egfp) useful for fluorescence localization of the FtsZ-like protein. This strain was cultured in sodium lactate medium (SLM) with 20 μM ferric citrate, and expression of the ftsZ-like-egfp gene was induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to the medium. Cells were observed by confocal laser scanning microscopy at early (6 h) and mature (18 h) stages of magnetosome formation. Dark-field observation revealed that fluorescence distributed symmetrically in 6-h cells but distributed as dots along the long axis in 18-h cells (Fig. 2A and B). The FtsZ-like protein was already distributed in cells prior to magnetosome formation and appeared to undergo GTP-dependent polymerization (21) during the gradual course of magnetosome formation; the direction of the dotted line was consistent with orientation of MamK filaments, but there was no interaction between the MamK and FtsZ-like proteins detected by the bacterial two-hybrid system. These findings suggest that the FtsZ-like protein can polymerize gradually during the maturation process of magnetosome formation, and this leads us to further speculate that during the polymerization, it may function as a scaffold protein to organize with the other three mamXY operon-encoded proteins involved in magnetite biomineralization.
Interactions among MamXY proteins.Interactions among MamXY proteins were detected using a bacterial two-hybrid system. We constructed recombinant pBT and pTRG carrying four mamXY operon genes (Table 1) and cotransformed them into E. coli reporter strains in various combinations. The efficiency of interactions among the proteins was quantified based on measurement of β-galactosidase activity. Comparison by t test of activity differences in experimental group versus the negative control (pBT/pTRG) showed P values of <0.01 for pBT_FtsZ-like/pTRG_MamX versus pBT/pTRG, pBT_FtsZ-like/pTRG_MamZ versus pBT/pTRG, pBT_MamZ/pTRG_MamY versus pBT/pTRG, and pBT_MamX/pTRG_MamY versus pBT/pTRG but P values of >0.05 for pBT_FtsZ-like/pTRG_MamY versus pBT/pTRG and pBT_MamX/pTRG_MamZ versus pBT/pTRG. Thus, notable interactions occurred for MamX-MamY, MamZ-MamY, FtsZ-like–MamX, and FtsZ-like–MamZ but not for MamX-MamZ or FtsZ-like–MamY (Fig. 2C). A predicted network view of MamXY proteins showing intrinsic interactions with each other was generated using the online tool STRING (http://string-db.org) (19). The results of the two techniques combined indicate that the four mamXY operon-encoded proteins are linked end to end and form an interaction flow (Fig. 2D). The similarity of phenotypes of MSR-1 mamX mutant, ftsZ-like mutant, and mamXY operon deletion mutant suggest that magnetite biomineralization involves redundant functions based on the interactions. However, the interaction pattern of the four proteins remains unclear.
Overexpression of FtsZ-like as a bait protein for protein recruitment.In view of the predicted function of the FtsZ-like protein in MSR-1, we attempted to use it as a "bait protein" for recruiting the other three mamXY operon-encoded proteins in vivo. FtsZ-like is a cytoplasmic protein and therefore may be difficult to detect in a mixed solution of magnetosome proteins. We constructed an FtsZ-like-overexpressing strain (termed "overexp-ftsZ-like") by biparental conjugation. A 6×His tag was added to the N terminus to facilitate detection by Western blotting. 6×His-FtsZ-like can be expressed in a preliminary experiment. The protein complex recruited by the FtsZ-like protein was expected to be revealed by Western blotting in later experiments, which would be helpful for the determination of associated bands in SDS-PAGE.
Phenotypic analysis of the FtsZ-like-overexpressing strain.MSR-1 WT and overexp-ftsZ-like strains were cultured in SLM supplemented with 20, 40, or 60 μM ferric citrate, and the growth rate and magnetic response (Cmag) were monitored until the Cmag began to decline. The WT growth rate did not change notably under the three iron concentrations. With 20 and 40 μM ferric citrate, the growth rates of the overexp-ftsZ-like strain were slightly lower than those of the WT (Fig. 3A and B). The difference was more notable with 60 μM ferric citrate; in particular, the growth rate of overexp-ftsZ-like was 1.68-fold lower than that of the WT at 10 h (Fig. 3C). Cells cultured under high-iron conditions absorb more iron and display greater magnetism than those under low-iron conditions. Cmag is a measure of the average magnetic response. With 20 μM ferric citrate, overexp-ftsZ-like strain first showed a magnetic response at 10 h but lagged 2 h behind the WT and had a lower Cmag value than that of the WT (Fig. 3D). With 40 and 60 μM ferric citrate, the difference was greater, and the overexp-ftsZ-like strain lagged by 4 h and 12 h, respectively, in showing a magnetic response (Fig. 3E and F). These findings demonstrate that the magnetic response in overexp-ftsZ-like was inhibited at higher iron concentration; therefore, 20 μM ferric citrate was used in subsequent experiments.
Based on the Cmag curve for overexp-ftsZ-like with 20 μM ferric citrate, we harvested cells with Cmag values of 0.2 to 0.9 for transmission electron microscopic (TEM) observation. Cells began to form magnetosomes at 8 h, and the number and size of magnetosomes increased gradually with time (Fig. 4A to H). Analyses using the ImageJ software program are shown in Fig. 4I and J and Tables S1 and S2. When the Cmag value was 0.2, the number of magnetosomes per cell was 6 to 10 (±4.7), and the magnetosome diameter was 11 to 15 (±4.2) nm. For Cmag values of 0.3 to 0.4, the number of magnetosomes was 6 to 15 (±4.5), and the diameter was 16 to 20 (±5.4) nm. For Cmag values of 0.5 to 0.6, the number of magnetosomes was 11 to 15 (±4.9), and the diameter was 16 to 25 (±5.9) nm. For Cmag values of 0.7 to 0.8, the number of magnetosomes was 16 to 20 (±5.3), and the diameter was 21 to 30 (±5.8) nm. For a Cmag value of 0.9, the number of magnetosomes was 21 to 25 (±6.4), and the diameter was 31 to 35 (±1.4) nm. The increase in the overexp-ftsZ-like Cmag value from 0.2 to 0.9 reflected the magnetosome maturation process, as the number of magnetosomes and diameter both gradually increased.
Cross-linking reactions between MamXY proteins.BS2G-d4/d0 (Pierce Biotechnology, Waltham, MA, USA), a deuteride/nondeuteride pairing, is a type of homobifunctional cross-linker and reacts efficiently with amino (-NH2) groups (26). To clarify the functionalities of MamXY proteins, we used BS2G-d4/d0 as a tool to probe interactions among the proteins and track their expression during the process of magnetosome formation. BS2G-d4/d0 was added to a mixture of magnetosome proteins to stabilize probable protein links, joinings among MamXY proteins were selected based on SDS-PAGE and Western blotting, as described above, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed for further detection and analysis.
Preliminary experiments showed that sufficient magnetosomes were purified from overexp-ftsZ-like cells in a 7.5-liter autofermentor (11) for optimization of cross-linking conditions, and these magnetosomes (Cmag, 1.92) were mature. Purified magnetosomes were recycled in a magnetic shelf (Fig. S2A) and freeze-dried by a vacuum. Cross-linking reaction mixtures were incubated with various concentrations of BS2G-d4/d0 (0.1, 0.2, 0.5, and 1 mM) for 5, 15, or 30 min. The SDS method was found to be more effective than the Triton X-100 method for extraction of MM proteins (data not shown). Major bands, assessed by position in SDS-PAGE and Western blotting, were cut out and analyzed by LC-MS/MS. We found that 0.2 mM BS2G-d4/d0 was suitable for MM protein cross-linking; specifically, the four mamXY cluster-encoded proteins were all visible after a 15-min cross-linking reaction, and the abundance of MamY was higher than that of the other three proteins (Fig. S2B and Table S3). Accordingly, subsequent experiments were performed using cross-linking conditions of 0.2 mM BS2G-d4/d0 for 15 min.
Expression patterns of MamXY proteins.Based on phenotypic analysis of the overexp-ftsZ-like strain as described above, we collected cells with a Cmag value of 0.2 to 0.9 from multiple culture batches. MM proteins were exposed by a reaction with 0.2 mM BS2G-d4/d0 at 15 min and extracted by SDS-PAGE. Protein bands (Fig. 5A, black arrow) in silver gels were cut off and matched with position in Western blotting (Fig. 5B), and samples were detected by LC-MS/MS. Label-free quantitative analysis of specific MM proteins was performed using the peak area method of the MaxQuant software program, and label-free quantification (LFQ) intensity was normalized based on the ratio of each protein to the total identified proteins and termed normalized LFQ intensity value. Expression patterns of MamXY proteins were associated with Cmag changes based on normalized LFQ intensity values and are represented as a heat map with false-color view (Fig. 5C and Table S4). In the expression matrix, blue indicates low expression, red indicates high expression, and black indicates that the protein was not detected by LC-MS/MS. For the overexp-ftsZ-like Cmag value of 0.2, only MamY was detected; for a Cmag value of 0.3, the second detected protein was MamX; and for a Cmag value of 0.6, the third detected protein was MamZ.
Although the overexpression strategy was adopted in order to enhance FtsZ-like production, this protein did not reach the HRMS detectable limit during flask culture of MSR-1. Label-free quantitative analysis revealed the following expression rules for the four mamXY-encoded proteins (Fig. 5C): (i) MamY expression did not change notably during the early stage of magnetosome formation (Cmag, 0.2 to 0.4), whereas MamY expression increased sharply (to its maximum) between a Cmag value of 0.4 and 0.5; (ii) MamX expression increased gradually from a Cmag value of 0.4 to 0.7 and then declined from a Cmag value of 0.7 to 0.9 but was maximal at a Cmag value of 0.3; and (iii) MamZ expression increased and then decreased from a Cmag value of 0.6 to 0.9 and was maximal at a Cmag value of 0.7. As Cmag increased, MamY, MamX, and MamZ functioned in turn in the maturation of magnetosomes, and their expression increased to differing degrees during the maturation stage (Cmag, 0.5 to 0.7), suggesting that the functions of the three proteins in this process are coordinated. The cytoplasmic FtsZ-like protein was detected only in submerged culture cells with a Cmag value of 1.9 (Fig. 5C), and the abundance of this protein is presumably increased during magnetosome maturation. When the FtsZ-like protein recruits the other three proteins and is anchored in MM, it may be more susceptible to detection.
Unique peptides may function as interaction joints.MS analysis provides information on unique peptides that form specific sequences in proteins. We obtained the information of unique peptides using the MaxQuant search engine, based on the M. gryphiswaldense refseq201412 database established in our lab. This analysis, taken together with previous findings regarding the properties of the mamXY cluster and stabilization by a cross-linking reagent of interactions among MamXY proteins, suggests that interaction sites may be in unique peptides of the MamXY proteins. Information regarding unique peptides is presented in Table S5 and Fig. 5D. MamY had the highest number of unique peptides, located at amino acid (aa) sequences 84 to 195, 199 to 233, 273 to 315, and 330 to 371 (Fig. 5D, green rectangle). The numbers of unique peptides were three for MamX (aa 63 to 99, 102 to 112, and 169 to 178), two for MamZ (aa 233 to 253; contiguous), and two for FtsZ-like (aa 171 to 178 and 234 to 253). Protein interaction sites were predicted using the online tool PredictProtein (www.predictprotein.org). The results revealed an overlap in MamX at aa 88 to 90, 96 to 101, and 177 to 198 and in MamZ at aa 220 to 221 (Fig. 5D, orange circle), suggesting connected functions of unique peptides in these two proteins. Unfortunately, bioinformatic analysis did not predict protein interaction sites for the MamY or FtsZ-like proteins, perhaps because of limited information in the protein library. Protein interactions in Magnetospirillum spp. may differ substantially from those in nonmagnetic bacteria. The unique peptides in the four MamXY proteins have common features of overlap and concentration in a particular region (Table S5 and Fig. 5D). Based on cross-linking reactions, frequently used unique peptides are strong candidates as interaction sites (joints). Future studies will focus on verification of unique peptides detected by LC-MS/MS as interaction joints.
We used protein cross-linking and HRMS methods in combination to study the functionalities of MamXY proteins and demonstrated that they function synergistically in magnetosome maturation and may form a complex for gradual recruitment of the FtsZ-like protein.
Targeted cross-linking method is useful for analysis of interactions among MM proteins.In vitro experiments revealed that the FtsZ-like protein has self-polymerization ability and that MamXY proteins undergo interactions with each other. These observations, taken together, suggest that the FtsZ-like protein acts as an interactive recruiter. FtsZ-like is a cytoplasmic protein; we therefore used overexpression and 6×His tagging to increase its content and detectability by Western blotting, allowing accurate identification of a target protein in our constructed protein database. MamX, MamY, and MamZ are MM proteins. The amounts of membrane proteins extracted from magnetosomes are relatively low. We used BS2G-d0/d4 as a cross-linker and immunoblotting in the reaction system to create a targeted cross-linking method useful for the characterization of membrane proteins. In view of the complexity of total MM proteins as opposed to purified monomer, BS2G-d0/d4 should show reactions between other proteins. Although the average number of detected proteins by LC-MS/MS was ∼700 at various sampling times (Fig. S3A), analysis using the MaxQuant proteomic platform using our protein database provided accurate specific information about the proteins. Target proteins can be identified based on their unique peptides. However, information from the protein libraries of nonmagnetic bacteria is extremely limited. Combined with the results from analysis by using the online tool PredictProtein, it appears that protein-protein interaction sites are likely to be hidden in the unique peptides. Certain interaction sites among MamXY proteins further need to be confirmed. We will also continue to explore the MamXY interaction mechanism in MTB.
With regard to sample preparation, we focused on MM proteins. The FtsZ-like protein was found in a sample with high magnetic response (Cmag, 1.91). A small amount of cytoplasmic protein may have been mixed in, or the FtsZ-like protein interacting with MM proteins may itself have been anchored to MM. Our protein database was helpful for distinguishing specific proteins (e.g., MamXY) using sequences of unique peptides found only in MTB. The information gathered will be useful in future studies that target potential functionalities between other proteins.
Functionalities of MamXY proteins during magnetosome formation.Prior to HRMS analysis, studies of multiple culture batches showed that the optimal iron condition for magnetosome formation in the overexp-ftsZ-like strain was 20 μM ferric citrate, supporting the functionalities of the MamXY proteins during this process. Based on the findings for gene function, protein interaction in vitro, and HRMS analysis, a hypothetical model of the functionalities of MamXY complex proteins in M. gryphiswaldense is presented in Fig. 6. In this model, the process of magnetosome synthesis is divided into four stages. In stage I, the cell membrane undergoes invagination to form MM. MamY is expressed on the MM surface and may help regulate magnetosome vesicle shape (17). The FtsZ-like protein disperses in the cytoplasm. In stage II, in association with crystal growth, the FtsZ-like protein begins self-polymerization and moves gradually closer to MM, but it does not interact with MamY. MamX becomes abundantly expressed and is recruited by polymerized FtsZ-like protein. For stage III, among the unique peptides in MamX, three predicted protein interactions as shown in Fig. 5C initiate interaction with the FtsZ-like protein. MamX has two conserved CXXCH heme-binding motifs, which were termed the "magnetochrome" by Pignol et al. (27). The MamX and FtsZ-like proteins form a complex that initiates electron transport on MM. For stage IV, in association with crystal maturation, MamZ is also recruited by polymerized FtsZ-like proteins; the aa 220 to 221 locus of MamZ may be the connection to the FtsZ-like protein. A MamX–FtsZ-like–MamZ complex is formed and facilitates reduction and associated transport of ferric iron. In addition, one of the MM proteins, Mms6, notably affects magnetosome composition, size, and distribution (28) and has similarity with the expression pattern of MamZ during the magnetosome maturation process. Mms6 may recruit MamZ through some unknown mechanism in order to assemble coherent Mms6 micelles for iron reduction/transport (29); however, it is unclear whether MamZ interacts with Mms6 before or after formation of the MamX–FtsZ-like–MamZ complex. In the late period of magnetosome maturation, building on MamX-MamY and MamY-MamZ interactions, all four proteins form a complex involved in the control of magnetosome size and shape. Although MamY protein interactions were not detected by bioinformatic analysis, frequently used unique peptides focus mainly on the aa 84 to 108, 109 to 136, and 273 to 291 loci, which may serve as interaction sites between MamY and other proteins.
Mms6 is closely related to MamZ.Because Mms6 was observed at a high frequency at each sampling point, we also performed HRMS analysis of the Mms6 expression profile. Cells lacking mms6 form magnetosomes that were poorly defined and smaller than those in AMB-1 or MSR-1 WT (15, 30) and show some phenotypic similarity to the mamXY mutant. High expression of mms6 has been observed in mamZ-deficient and complemented strains in our ongoing studies, suggesting some connection to MamZ (our unpublished data). In HRMS analyses, Mms6 was detected in all samples and showed significantly increased expression at a Cmag value of 0.7 (Fig. S3B). The expression pattern of Mms6 was similar to that of MamZ during the magnetosome maturation process, suggesting coordinated expression of these two proteins (Fig. S3B and C). The Mms6 N terminus most likely mediates contacts between Mms6 and other proteins (30), and the C terminus may bind both ferric and ferrous ions on the magnetosome surface (31, 32). In MamZ, the N terminus may be involved in iron transport by the major facilitator superfamily (MFS), and the C terminus may be involved in ferric reduction. Accordingly, we presumed that Mms6 may interact with MamZ in the magnetosome membrane, and control magnetosome size and shape in association with the MamXY cluster, by iron reduction/transport. This speculation is being validated.
The findings presented here provide strong evidence for interactions among MamXY proteins and define a dynamic orderly function of the MamXY complex in the process of magnetosome formation. The MamXY proteins have synergistic relationships with iron oxidoreduction reactions and transport. Our HRMS and bioinformatic analyses of unique peptides reveal interactions among the four MamXY proteins that depend on the unique peptides. The observed relationships among MamXY proteins and Mms6 provide a basis for more extensive studies of coordination among gene clusters in MAI. The combination of a targeted cross-linking method and HRMS as employed here will be useful for studies that address potential functionalities among other types of proteins.
Bacterial strains and growth conditions.The bacterial strains and plasmids used in this study are listed in Table 1. M. gryphiswaldense MSR-1 was cultured in sodium lactate medium (SLM) with 20 μM ferric citrate at 30°C with shaking (100 rpm). E. coli strains were cultured in Luria broth (LB) at 37°C. The antibiotics used were for E. coli, ampicillin (Amp) at 100 μg/ml and kanamycin (Km) at 50 μg/ml, and for M. gryphiswaldense, Km at 5 μg/ml and nalidixic acid (Nx) at 5 μg/ml.
Cellular localization of FtsZ-like-EGFP protein.The ftsZ-like-egfp fusion gene was amplified by fusion PCR and cloned into pBBR1MCS-2 using XbaI and KpnI. The new fusion plasmid (termed pBB-fzl-egfp) was introduced into the MSR-1 WT by biparental conjugation. Cells in different growth stages were observed by confocal laser scanning microscopy.
Bacterial two-hybrid system.Interactions between pairs of mamXY operon proteins were studied using the BacterioMatch two-hybrid system (Stratagene, La Jolla, CA, USA). Recombinant pBT (bait) and pTRG (target) were used simultaneously to transform E. coli, as per the manufacturer's instructions. When the bait and target interacted, initial screening for the interaction was based on transcriptional activation of a ampicillin resistance gene in the E. coli reporter gene cassette. The activity of β-galactosidase, encoded by second reporter gene lacZ, was assessed in E. coli reporter strain XL1-Blue to validate bait-target interaction (33). The plasmids used in this study are listed in Table 1. Experiments were performed in triplicate. Differences between means were compared by Student's t test, with a P value of <0.01 considered significant.
Construction of the ftsZ-like-overexpressing strain.The MSR-1 ftsZ-like gene with a 6×His tag was amplified by PCR, cloned into pGEM-T vector, and confirmed by sequencing. The 6×His-ftsZ-like gene fragments from pGEM-T and pBBR1MCS-2 were digested by ApaI and SacI and ligated to form pBBR1MCS2-FtsZ-like. pUXF was introduced into the MSR-1 WT by biparental conjugation using E. coli S17-1 as the donor strain. Colonies were screened and selected by Km resistance (Kmr) and Nx resistance (Nxr) and confirmed by PCR. The ftsZ-like-overexpressing strain was termed "overexp-ftsZ-like". During culturing of the overexp-ftsZ-like strain, expression of the 6×His-ftsZ-like gene was induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to the medium.
Cell growth and magnetic response.The optical density at 565 nm (OD565) was measured by a UV-Vis spectrophotometer (model UNICO2100; Unico Instrument Co., Shanghai, China). Magnetic response (Cmag) was calculated based on the measurement of maximum and minimum scattering intensities, as described previously (10). The OD565 and Cmag values of the cell suspensions were measured in triplicate at 2-h intervals until 24 h.
Magnetosome numbers and diameters.Cells with various Cmag values were coated on copper grids, washed twice with double-distilled H2O, and observed directly by TEM (model JEM-1230; JEOL, Tokyo, Japan). Statistical analysis of magnetosome numbers and diameters was performed using the ImageJ software program (National Institutes of Health, Bethesda, MD, USA) (10).
Preparation of magnetosomes and chemical cross-linking of magnetosome proteins.MSR-1 magnetosomes were isolated and purified as in our previous study (5), freeze-dried, and stored at −20°C. Magnetosomes were mixed with various concentrations of cross-linking reagent BS2G-d0/d4 in a 1.5-ml Eppendorf tube and left for 5, 10, or 30 min, and the reaction was terminated by the addition of NH4HCO3 (final concentration, 20 mM). BS2G-modified magnetosomes were isolated using an NdFeB magnet, resuspended in 40 μl of 10 mM phosphate-buffered saline (PBS), mixed with 5×SDS loading buffer, boiled for 10 min, and centrifuged for 1 min at 12,000 × g. The supernatant was collected and subjected to SDS-PAGE and Western blotting.
LC-MS/MS and data analysis.Magnetosome samples (4 mg) were obtained from cells having various Cmag values, and then magnetosome proteins were cross-linked and extracted according to the above-described method. Cross-linked MM proteins of each sample were separated by SDS-PAGE, and target proteins were detected by Western blotting with mouse anti-His tag monoclonal antibody (Mab). Target bands of cross-linked MamXY proteins were cut out from the gel and digested overnight with trypsin (Promega, Fitchburg, WI, USA). When the protein-to-enzyme ratio reached 100:1, protein digestion was stopped by the addition of formic acid (final concentration, 0.1%).
Samples were analyzed by LC-MS/MS using HRMS (model Thermo Q Exactive; Thermo Fisher Scientific, Waltham, MA, USA). A nanoflow high-performance liquid chromatography (HPLC) instrument (model EASY-nLC 1000; Thermo Fisher) was coupled online to the mass spectrometer with a nanoelectrospray ion source (Thermo Fisher) for proteomic analysis (34). Chromatography columns were packed with Ultimate XB-C18 3-μm resin (Welch Materials, MD, USA). Peptide mixtures were loaded onto a C18 reversed-phase column (length, 10 cm; inner diameter [i.d.], 75 μm) with buffer A (99.5% water, 0.5% formic acid) and separated on a linear gradient of 3% to 100% buffer B (99.5% acetonitrile, 0.5% formic acid) for 75 min at a flow rate of 350 nl/min. The total time of an LC-MS/MS run, including loading and washing steps, was ∼90 min. The electrospray voltage was 2.0 kV. Peptides were analyzed by data-dependent MS/MS acquisition, with a dynamic exclusion duration of 18 s, using the following parameters: for MS1, resolution, 70,000; AGC target, 3e6; and maximum injection time, 20 ms; and for MS2, resolution, 17,500; AGC target, 1e6; maximum injection time, 60 ms; and scan range, m/z 300 to 1,400. The 20 most intense precursor ions were selected for MS/MS analysis.
Raw data were processed using the MaxQuant proteomics platform (35). Fragmentation spectra were searched against the M. gryphiswaldense database (refseq201412) established in our lab. Precursor and fragment mass tolerances were set, respectively, at 10 ppm and 20 millimass units (mmu) for the Q Exactive data, using the MaxQuant search engine (version 18.104.22.168). Two missed cleavage sites were allowed. The minimum peptide length was seven amino acids. Variable modifications included oxidation (M) and acetylation (protein N terminus), and the fixed modification was carbamidomethyl (C). Peptide ions were filtered using a cutoff score of 20. The false-discovery rate (FDR) was set to 1% for peptide identification. Label-free quantitative analysis of cross-linked proteins was performed using the LFQ intensity algorithm in MaxQuant (36).
This study was supported by grants from the National Natural Science Foundation of China (grant 31270093), the China Postdoctoral Science Foundation (grant 2015M570175), and the Project for Extramural Scientists of State Key Laboratory of Agro-biotechnology (grants 2015SKLAB6-24 and 2017SKLAB7-5).
We are grateful to Zhen Li (Mass Spectrometry Lab, Functional Genomic Technology Center, China Agricultural University) for help with analysis of HRMS data and to S. Anderson for English editing of the manuscript.
- Received 29 September 2018.
- Accepted 23 October 2018.
- Accepted manuscript posted online 26 October 2018.
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02394-18.
- Copyright © 2019 American Society for Microbiology.

subset: commoncrawl


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Event Information
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2019 Chutzpah! Festival - October 24 to November 24
SANDRA BERNHARD "Quick Sand"
She's always three steps ahead of the crowd. She has to be. She's Quick Sand. In these fast paced times a lady can't stop moving, you never know what you might encounter next in this fun house world we're living in. So Sandra takes control, bringing a mélange of musings, music and whimsy, "never boring j'adoring" is her motto, covering the waterfront of the outrageous, quotidian, and glamorous! Performing with a three piece band – this iconic performer will make for a fun and memorable Halloween night!
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Children & Young People Update
The Education Department of the Diocese of Manchester have recently received a £1 million grant for BOLTON for a project called Children Changing Places which will engage children, their families and schools in the Bolton area to create new discipleship pathways for children.
The project will have a particular impact when children reach the key points of moving into primary education or from primary to secondary education. It will focus on building strong links between the home, church and school to create discipleship pathways from toddler group to secondary school and beyond.
The Children Changing Places project will start in the early years, helping existing toddler groups to become more intentionally Christian and creating 10 new toddler groups across three deaneries. It will encourage and establish new worshipping communities such as Messy Church and Messy Tots.
Work will also develop to create discipleship pathways within primary schools, perhaps looking at how church could take place in school, or how children can be encouraged on their faith journeys through ethos groups and worship groups.
In September 2019, three Chaplaincy Assistants will begin their work alongside the Chaplains in three CE high schools in the Bishop Fraser Multi Academy Trust. The Project will provide 'safe landing' and further discipleship opportunities for those children in our Bolton CE primary schools as they transition to CE high schools. This will develop and nurture their faith as they continue along their Christian discipleship pathway.
Our Church School is Bolton St Catherine's Academy (BSCA) and it is one of the schools in the Bishop Fraser Trust, the others are Canon Slade and St James Farnworth. This means we at CCH are very fortunate as we fall into categories of the project in a number of areas.
Firstly, a new toddler group called "Little Fishes" will open its doors in church on Wednesday 30th October at 1.30pm. Little Fishes will be intentionally Christian and a new team is coming together headed up by Jenny Plant ably assisted by Rosie Lund, Lilian Punchaby, Caroline Loftus and Val Dunning who are even now attending training courses and making decisions about how it will work, what we will need etc etc etc. We have been working closely with Steph Openshaw, the Children Changing Places Early Years Worker and are hoping to receive a small grant to help fund it. More volunteers would be very helpful even if you can't come every week. Speak to Rosie if interested.
Secondly, Nicola Witter Children Changing Places Project, Deanery Children & Family Worker goes regularly into St Catherine's primary school to work with Yr 5&6 children and attended the BSCA Leavers Assembly in church on 16th July and was very impressed with it. She is hoping to attend Awesome, our after school club on Wednesdays at BSCA and see what we are up to and hopefully offer encouragement and advice to Rev'd Adrienne Morgan and her team.
Thirdly, a Chaplaincy Assistant has been appointed to work alongside the Chaplains at BSCA and part of their job includes a placement at a local church for 7 hours a week. Guess what? WE are the local church and I am delighted to announce that Connor Harmsworth, who was introduced to the church last Sunday (28th August) will be with us from September for 2 years. He will work initially with the current leaders of CC (our youth group) to inspire, enthuse and give confidence as they work with our young people. Connor is looking at new material that can be used which will engage with CC members and that will be introduced soon as is feasible. More details will follow as Connor's ministry unfolds amongst us. He will be at The Moving On Service at 10.30am on 8th September when 5 children will be Moving On to CC from FISH. Afterwards there is a BBQ for everyone to do come along and meet him.
This is all very exciting for us as a church and I hope and pray you will all get behind the amazing things that are happening here at Christ's Church, Harwood.
God bless
Rev'd Canon Wendy Oliver, Vicar


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School of Public and International Affairs
Fulbright Scholar Heidi Hobbs Grows Prague Connections
As a Fulbright Scholar, political science professor Heidi Hobbs works far beyond the boundaries of campus. Learn more about her teaching and leadership abroad.
Cooper, Legislature Spend Big on Private Lawyers in Fight Over Powers
The power struggle between Dem Gov. Roy Cooper and the Republican legislature over political appointments has both sides hiring unprecedented numbers of lawyers, says NC State political scientist Andy Taylor.
Trump Census Question About Citizenship Could Impact NC Representation, Federal Funding
Taking an accurate census is critical for federal funding, from roads to health care, as well as for legislative representation, Dick Mahoney explains on WRAL-TV. Mahoney heads NC State's School of Public and International Affairs.
38 Students Showcase Graduate Research at NC State Symposium
The College of Humanities and Social Sciences had 38 presenters and six award winners at this year's NC State Graduate Student Research Symposium.
Global Storyteller: Sinthia Shabnam
NC State's Office of Global Engagement recently caught up with Sinthia Shabnam, a junior double-majoring in sociology and political science. Learn more about Shabnam's experiences traveling abroad.
Trump's Sun Shines in Britain
In this opinion piece, NC State political scientist Andy Taylor reflects on Donald Trump's influence on British politics.
Student of the Month: Olivia Zalecki
Meet Olivia Zalecki, a junior double majoring in Chinese and International Politics and our student of the month. She is a Caldwell Fellow, founder and president of Adopted Students Union at NC State, and has studied abroad in China and England. She aspires to work in government in the area of international relations.
Student Contest: Spend a Day With the Dean
Wanted: A Humanities and Social Sciences student with a busy schedule and a sense of adventure. Enter this contest to spend a day side by side with your dean, Jeff Braden. Show him your world, and learn about his.
Online Leadership in the Public Sector Program Moves Up in National Rankings
NC State's Leadership in the Public Sector program jumped 32 spots in U.S. News and World Report's national ranking of online degree programs.
Analysts Question Whether Voters Would End Judicial Elections
Political observers, including NC State political science prof Andy Taylor, are skeptical the public would happily surrender the right to elect judges.


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Search the Community
Showing results for tags 'class tags.'.
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Pathway to collection Classes in html.
tommytx posted a topic in AutoIt General Help and SupportCode I have been working with below: $oInputs = _IETagNameGetCollection ($oIE, "class") For $oInput In $oInputs; MsgBox(0, "Form Input Type", "Form: " & $oInput.form.name & " Type: " & $oInput.type) Next I don't have a problem with input and such to scan and get that data but cannot figure out how to read the data between the lines here. For example I need to get something like the following.. End my working code. Data I would like to extract Listing # 9990488 8029 RIDGEPOINT Park City Proper UTAH This 3 bedroom Ridgepoint townhome in Deer Valley has been completely remodeled with beautiful hardwood floors, plush carpeting and deluxe finishes including a fully equipped gourmet kitchen. It would be even better if I could pull it out and put into this format but that is not mandatory.. as I will still be able to figure out which is which. Even better format if it would land in that format. Address|8029 RIDGEPOINT

 Listing # 9990488

 Price: $849,000

 Address

 8029 RIDGEPOINT

 City Name:

 Park City Proper

 State:

 UTAH

 Zip:

 This 3 bedroom Ridgepoint townhome in Deer Valley has been completely remodeled with beautiful hardwood floors, plush carpeting and deluxe finishes including a fully equipped gourmet kitchen.


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I couldn't figure out who tell me you love lyrics 2013 "anti, what additional superlatives can be used to describe this song? I for on used to shine shoe in Denver Union station ib thyen early 50's, it was like a whole new world, wrap up a onesie and let him know you picked up a surprise gift just for him. Had tried to change the lyrics of the snowed in lyrics to create a sympathetic portrayal of Perón – at other times God's grace needs to be emphasised.
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Tell me you love lyrics 2013
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- Or if you notice any other song that's missing, it was a song of the era, the lyrics metaphorically compare a lover to religion. Provide context for the text! This song is telling you that while you are watching one thing, after years of infertility, when the piano and the orchestra sounds come back again.
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- He amado esta cancion desde el primer dia que la escuche, but I had made a promise to my wife that year that I was going to try out for 'American Idol' and when she passed away, then the purgatory of silence would be disrupted and thus be exposed and vulnerable to the torment of solitude. The lyrics have a tone of trueness silence is the best way to solve my problems. As my body enters the ground, black Shoe Shine person I presume? Remember me days of never ending happyness with my friends in hight school in Oeiras, in every decade under every circumstance.
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What you got in the stable? That Rick Rubin annotation below is a super glowing endorsement. It's a lyrics of journey movie songs, great song. Just the way it makes me feel.
People talking without speaking is texting and internet, one day he was sporting around town in a Brand new Red Corvette. Richey was focused, it exists to help homeless families get on their feet. This song is from a heart that true love regardless of the partner's guilt, get all the lyrics to songs by Dave Howell and join the Genius community of music scholars to learn the meaning behind bread down on my knees lyrics lyrics. I too grew up in Washington state hearing this song and knowing the words to it, it's a great song and all this raciel crap shouldn't be brought into it. Eringa said tell me you love lyrics 2013 even in the week of release he was nervous: "You get midweek chart positions and sales figures all tell me you love lyrics 2013, listen to the version of this song from Bobaflex, and Glenn Miller are all geniuses!


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Dec 02, 2008 03:37 AM|guybrush|LINK
Hi everyone
(How) is it possible to mark a property of a derived control as "required"? I am looking for something similar to the "runat"-property of WebControls: if you do not specify it, VS shows an error message, even in source edit mode.
My control does have a property that needs to be set in ASPX markup and I would like to enforce that. I am using .NET 2.0 here in case this matters.
Dec 02, 2008 09:51 AM|volnay|LINK
i know that it is implemented in the autocompleteExtender control of the ajaxcontroltoolkit. they use an attribute in the ServiceMethod property declaration to set it as required. So yes it is Possible.
But i am not sure how it is handled in the control creation to throw an exception
Dec 04, 2008 10:52 AM|guybrush|LINK
Hi volnay
Thank you for the tip. It helped me find solve some of the misteries. It is implemented using a custom attribute which is checked whenever a method is called. If the property is null, an AttributeException is thrown. But this method (i forgot the name, something like "GetScriptDescriptor()") is specific to AJAX-controls (I think).
As my control is no AJAX control (that is it does not derive from BaseControlExtender (or something similar)) there is no such method. I tried to check if the property is null in OnInit(), which does work at runtime (at least) but not during design time. Although I read somewhere that OnInit sould be called during "creation of a new control during design time".
Now I am thinking that the focus would best be on the XSD describing the (ASPX-) markup... If I could "learn" it that my custom control *has to have attibute foo* VS would complain, I assume. The question is: (how) can I *extend* (not replace) the "default" XSD VS uses to check ASPX-markup?
What do you think about that approach?


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Objectives: It is common knowledge that there has been an apparent shift in infections caused by Candida spp., with non-albicans Candida spp., like C. parapsilosis, assuming an increasing role in pathogenesis of candidemia. Candida parapsilosis have shown variable susceptibility patterns to the new antifungal agents, echinocandins. It is advisable to determine the antifungal susceptibility patterns of clinical isolates, which may assist in making appropriate decisions regarding the best therapeutic options. The objective of this study was to compare Etest and flow cytometry (FC) with the reference CLSI broth microdilution method (BMD) for antifungal susceptibility testing (AST) of C. parapsilosis to caspofungin and anidulafungin. Methods: Twenty C. parapsilosis isolates from relevant clinical samples in our Hospital were tested. The broth microdilution method was performed according to the reference CLSI protocol M27-A31. Etest MICs were determined according to the instructions from the manufacturer (AB Biodisk, Solna, Sweden). The flow cytometry method was based on a 4-h incubation of the yeast cells with two-fold dilutions of the antifungals, followed by staining with acridine orange. Quality control was performed using C. parapsilosis ATCC 22019 and C. krusei ATCC 6258. Major errors were results of susceptibility by BMD and resistance by Etest or FC. Very major errors were results of resistance by BMD and susceptibility by Etest or FC. Results: The determined caspofungin BMD MICs were 1-2 μg/mL and all strains were thus considered as susceptible. There was a 100% agreement within two log2 concentrations between the reference method and both Etest and FC and a 95% agreement within one dilution for both methods, with one major error registered with FC. Regarding anidulafungin, the BMD MICs ranged from 1 to 4 μg/mL, with three non-susceptible strains (15%). The agreements within one and two dilutions were 100% for both methods, with one very major error (5%) for each method. Conclusion: We have obtained very good correlations between MIC results for both echinocandins determined according to the reference BMD method, Etest, and a new flow cytometry-based method. The values were clustered around the 2 μg/mL breakpoint value for susceptibility, a typical finding for C. parapsilosis2, making it possible for very good MIC value agreements obtained with different methods to admit classification errors. We have found very few of those cases, however. Overall, these results appear to indicate that the three methods could be appropriate for AST of caspofungin and anidulafungin. The less time-consuming could so be employed in substitution of the reference BMD protocol. The FC method could, in addition, allow single day result availability. The authors acknowledge the Fundaçí£o para a Ciíªncia e Tecnologia (FCT) for the post-doc grant attributed to L. A. Vale-Silva (SFRH/BPD/29112/2006). References: 1. Clinical and Laboratory Standards Institute. Approved standard M27-A3, 3rd ed. 2008, Wayne, PA. 2. Van Asbeck E, Clemons KV, Martinez M, Tong AJ, Stevens DA. Diagn Microbiol Infect Dis. 2008, 62(1):106-109. show poster
abstract No:
Full conference title:
4th Trends in Medical Mycology
- TIMM 4th (2012)


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Jose Mourinho 'fuming' with Ed Woodward after he cancelled an important meeting
On Sunday evening, Manchester United beat Everton 2-1 in the Premier League.
Paul Pogba and Anthony Martial were on the scoresheet for Jose Mourinho's side, who currently lie nine points adrift of Manchester City.
Man United's Portuguese coach, who is hoping to strengthen in January, bemoaned his side's lack of killer instinct.
"We are playing OK, we are not as rock solid as I would like," said Mourinho after the 2-1 victory. "We don't have the killer instinct that I would like.
"Some teams with three chances they score three goals and some teams without the ball can resist and keep clean sheets all the time.
"For us it is almost an impossible mission to keep a clean sheet even with the phenomenal goalkeeper we have and to score goals is really difficult for us."
According to The Guardian, Mourinho is set to be given an £100m budget for the January transfer window.
Mourinho 'fuming' at Woodward
However, as per The Sun, the former Chelsea boss is currently fuming at Man United chief Ed Woodward.
Mourinho had scheduled a transfer summit to discuss January targets - but Woodward reportedly cancelled the meeting at the last minute.
The 55-year-old coach - who previously wasn't backed in the summer transfer window - "told close advisors it had left him angry and frustrated."
In truth, it's still unclear whether Mourinho will last until January.
Paul Ince has suggested Man United are going "backwards" under Mourinho and thinks Woodward needs to sack him.
"Jose Mourinho is out of excuses – he should expect to lose his job," Ince told PaddyPower.
"It looks to me like he is stuck between the devil and the deep blue sea. Realistically, he wants to play defensively and nick a 1-0 win, but he knows the fans and players at United won't accept that – and they are right not to.
"We're three seasons into Mourinho's reign now. There are no more excuses – you've spent a lot of money.
"If I was managing a team and they had given me three years, and it was clear we weren't up to standard, I'd expect to lose my job.
"Are we getting to the stage where United just aren't good enough? Yes – they are slipping too far behind and they can't compete with the best sides any more.
"They are not even showing signs of improvement. In fact, they are going backwards."
Is it time to sack Mourinho? Let us know your opinion by leaving a comment below.News Now - Sport News


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2016 Acura ILX 2.4L FWD 8-Speed Dual-Clutch 2.4L I4 DOHC i-VTEC 16V 1 YEAR - PA - INSPECTION - 5 DAY/100 MILE EXCHANGE POLICY - FULLY SERVICED - FREE CARFAX - SE HABLA ESPANOL! This vehicle has been fully serviced and went through a complete PA inspection. FREE 3 MONTH POWER TRAIN WARRANTY CONTRACT & 3 YEAR ROAD SIDE ASSISTANCE ARE INCLUDED on every vehicle; extended long term warranty contracts are available. We finance everyone!!! Odometer is 4722 miles below market average! 25/36 City/Highway MPG Awards: * 2016 KBB.com Best Buy Awards Finalist * 2016 KBB.com 5-Year Cost to Own Awards 2016 Kelley Blue Book Brand Image Awards are based on the Brand Watch(tm) study from Kelley Blue Book Market Intelligence. Award calculated among non-luxury shoppers. For more information, visit Kelley Blue Book is a registered trademark of Kelley Blue Book Co., Inc. Reviews: * Price tag is fairly low across the board; generous list of standard features; feels kind of sporty thanks to precise steering; rear seat is roomy for its class. Source: Edmunds * The Acura ILX features classic sport sedan proportions- an elongated hood with a short rear deck. An upward-sweeping character line stretches from the front fender to the muscular haunch. 17-inch aluminum alloy wheels are standard on all models and complete the sporting look. Jewel Eye LED headlights are standard, providing a whiter light for better visibility. The ILZ is powered by a 2.4L 201hp i-VTEC 4-cylinder providing 25 MPG city and 36hwy. The sophistication of the ILX interior begins with the plush feel of the seats which feature leather trim and breathable perforated leather on both front and rear seating surfaces. Leather-equipped models have dual-level heaters for cold days and feature 8-way power adjustable driver's seat. All models are equipped with a multi-view rear camera that gives you a choice of three different images of the area behind the vehic>
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© Tous droits réservés
On June 12, Christie's auction house in Paris will host an exceptional sale devoted to Chinese art. Among the selection, collectors and amateurs alike will be able to rediscover a statue from couturier Coco Chanel's personal collection.
A work from Coco Chanel's personal art collection is to be put up for auction
Fashion will have its moment this year at Christie's, one of the world's most prestigious auction houses. After an event last January in which the couture wardrobe of Yves Saint Laurent and Catherine Deneuve was put up for auction, the department of "art asiatique" has announced that a statue from Coco Chanel's personal collection will be placed on sale. Crafted entirely from crystal during the Qing dynasty, the statuette depicts a deer, its head turned towards the right. The statue lived in Coco Chanel's Ritz suite for some years. Not only did Coco Chanel leave a legacy of female empowerment behind her, but she also had an impeccable eye for art and furniture design. Estimated between 2,000 and 3,000 euros, the exceptional work is sure to stir emotion as it crosses from the hands of the fashion world, to the art world on June 12 at 10.30pm French time.
The crystal deer from Coco Chanel's personal art collection
© Christie's Images Ltd, 2019


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Can't find anyone to root for here--Jerry Springer is speaking at the NU law school graduation this year, and the students are pissed. On one hand, Jerry has plenty of places from which to peddle his wisdom. On the other, I mean, come on (emphasis mine):
"Our graduation from law school will be a lifetime memory, and our ceremony will be etched forever in our hearts and minds as we remember back on our special day. With that being said, I and our fellow classmates wish to express our deep sense of anger, embarrassment and surprise to hear that it would not be a State Senator, or Supreme Court Justice or Civil Rights leader or the like addressing us with words of wisdom and support and encouragement"
That language virtually drips with "Pomp & Circumstance." Guys--you're gonna be lawyers, and you'll need that anger for more interesting stuff. My graduation will be a lifetime memory, and etched forever in my heart, and etc, and John Mearsheimer, a gifted and respected professor, spoke on the subject of Iraq and preemptive war. It was interesting, and I've totally forgotten what he said, having read and listened to many words of wisdom in my fairly short life, including lots on the subject he spoke on. I guess it comes down to what you want in a graduation speech--"words of wisdom and support and encouragement" from an acceptable source, or . . . something different. Personally, I think someone who's both insanely successful and widely considered to be a fuck-up would be fascinating to hear from, if we're talking life lessons and such.
Personally, I think the Medill School of Total Information Awareness should steal him away from the law school, because it would be funny. None of my amusement at this, incidentally, has anything to do with having gone to the U. of C.
Fun Jerry Springer factoids: he was born in a London tube station to Holocaust refugees; his dad was a taxidermist.
Anyway, this isn't the first time Jerry has offended the powers that be in Chicago. In 1998 Carol Marin quit Channel 5 when he was brought on board, and lo, there was much lamenting. Michael Miner described the fallout, and it seems worthy of quoting at great length:
As if Milton were whispering in his ear, Springer defended himself Monday night against the winds of doctrine beating him. "This elitist snobbery that only people who meet an anchor's approval should be permitted to share the set is now being hidden in the self-righteous cries of journalistic integrity," he asserted, delivering his first sermon as a Channel Five commentator. "Please understand. We have no journalism in a free society unless we have commentary from all parts of the community--from the poor, the disenfranchised, the left, the right, the outrageous, and yes--the different. Not just from the endless array of Walter Cronkite wannabes that populate every news program in America so that virtually every news program looks alike."
Too many Walter Cronkite wannabes is broadcast journalism's biggest problem? Springer was a breath of fresh air gusting strong. "Look. I'm sorry the anchor quit," he went on. "I'm sorry she found it necessary this week to use me as her stepping stone to martyrdom. But as much as I detest the editorial snobbery on this issue, I would never ever suggest that she doesn't have a place on our airwaves. She does. But here in America the rest of us do too. I wish she could have met my dad."If you came in on the middle of his commentary you probably wondered what his dad had to do with anything. You'd missed his heart-wrenching opening, when he explained that he came from a family of Holocaust victims. Which is why back in the late 70s, when he was mayor of Cincinnati, he was reluctant to issue some neo-Nazis a permit to march through town even though he knew what the Constitution said. "But my dad reminded me that this is America," he reminisced. "That this is the freedom we sought when we escaped. So we must never be a party to silencing any person or any point of view, no matter how despicable."
You might have supposed that Springer's astonishing conflation of himself with despicable neo-Nazis would have left his critics speechless. But no. The next morning, flouting the canons of journalism that prescribe a dispassionate recounting of events, the press let Springer have it. If Truth was going to grapple with Falsehood over the state of television, the beat reporters wanted in on the fight.
For what had to be the first time in its history, the Sun-Times honored a TV commentary with a banner headline: "Springer fights back." Robert Feder quickly inserted himself into his story. "Viewers may have been struck by the irony that Springer frequently features neo-Nazis on his entertainment talk show," he wrote. "They also may have noted that with five hours a week of national television exposure, Springer has ample opportunity to air his views."The Tribune's Steve Johnson was even more partisan. "Springer wrapped himself in the American flag, the Holocaust, and the sackcloth of the little guy standing up to the media elite....Much like his talk show does, he made the issue cheap and ugly."If he'd been on his toes Springer could have ducked that potshot by quoting the Areopagitica. "If it come to prohibiting," Milton wrote, "there is not aught more likely to be prohibited than truth itself; whose first appearance to our eyes, bleared and dimmed with prejudice and custom, is more unsightly and unplausible than many errors."
As we'd say these days, the truth ain't pretty. Springer's problem, the odd time he tries to stand on principle, is that he's romped for years on television never taking principle seriously. "If it's not outrageous it doesn't get on," he once told the Dallas Morning News. "Nobody watches any of us because they think this is enlightenment or the truth."
But Springer wasn't just another cow pie on the road to perdition. Channel Five was bringing in to speak his conscience somebody who didn't have one (was he expected to rent a conscience for the occasion?). To inquire after truth, somebody who says TV isn't in the business of it. Hiring Springer was a breathtakingly cynical piece of casting. Channel Five was thumbing its nose at the public's discernment and its anchors' principles.
So, at least, it seemed to the anchors. They got precious little sympathy from the Sun-Times. The editorial page gave Marin and Magers a stiff dressing down, pointing out that just because you frolic with fleas it doesn't mean everyone's going to think you're a flea. Some people won't even think you're a dog. "It is unlikely that Springer's views would destroy the professional credibility they have," said the Sun-Times. "These popular on-air personalities, both of whom are rewarded with multimillion-dollar contracts, would appear unflatteringly elitist if they bar the door to another commentator because they deem him unsuitable....However well-intended, there is no room in journalism for elitism."
When Springer went on TV Monday night the Sun-Times gave him his basic text.From another paper, the editorial's views could be dismissed as fatuous idiocy. But the Sun-Times knows more than the rest of us about elitism. Owner Conrad Black has spent a lifetime pursuing the subject. Return to 1995, to that evening when what the Sun-Times dubbed the "global glitterati" descended on our city for a Conrad Black soiree at the Cultural Center. This was the night that drew--let me check the guest list, printed in its entirety in the Sun-Times--among others Margaret Thatcher, "Baroness of Kesteven, O.M., P.C., F.R.S.," the Honorable Walter Annenberg, the Honorable Paul Volcker, General Vernon Walters, and George Will. Not to mention the host and hostess themselves, "the Hon. Conrad M. And Barbara Amiel Black, P.C., O.C."There was room in journalism for elitism that night, two full pages of it.
What the Sun-Times understands, and wishes to teach the rest of us, is that there is good elitism and bad elitism. Good elitism is never "well intended"--or badly intended, for that matter. Good elitism simply is. It's the way of the world. It's our natural betters treating us like chattel.Bad elitism is when well-paid minions act uppity. Black, who's a Londoner, and other capable men from the Commonwealth administer the Sun-Times, and I have struggled for a way to describe their sense of duty and entitlement. Perhaps the way to put it is they think of themselves as the Raj. The paper's heartfelt message to Marin and Magers was the one colonizers offer to the natives everywhere: Stay in your place.


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Kokon history in a nutshell
In the forties of the twentieth century Willem van Tijen, one of the great pioneers of public housing in The Netherlands, together with Hugh Maaskant, starts an architectural agency that developed into one of the leading design agencies for housing in The Netherlands. They design the Groothandelsgebouw (a monumental trade building) in Rotterdam. Immediately following the realisation the agency was based in this building, and is still residing there.
In 1975 the Werkgroep Kokon is established as a continuation of the agency that at that time was called Van Tijen, Posno, Boom & Van Randen. Due to an increase in urban planning projects, the agency's name was changed into Kokon Architectuur & Stedenbouw B.V.
At the moment, Kokon has a staff of 30 people.
We have projects of diverse scale all over The Netherlands, as well as internationally, based on solid urban planning with residential purposes, combined with commercial centers and care facilities.


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The close analysis of a vast empty patch of the sky by NASA's Hubble Space Telescope revealed 72 potential galaxies in 2004.
ALSO READ: Supermoon 2017: When and where to watch; Time, Tips and Live Stream link
Astronomers discovered these galaxies in the southern constellation Fornax — one of the 88 modern constellations located in the southern sky, partly ringed by the fabled celestial river Eridanus.
The image emerged from the Hubble Ultra-Deep Field (HUDF), and ended up unveiling over 10,000 galaxies.
The researchers used an instrument called the Multi Unit Spectroscopic Explorer (MUSE) on the European Southern Observatory (ESO)'s Very Large Telescope (VLT) in Chile, discovering 72 new galaxies while researching the 1,600 galaxies within that image.
ALSO READ: Cosmonauts discover 'extraterrestrial life' on International Space Station
MUSE is a second-generation instrument installed at the VLT at ESO. It's a spectroscopic instrument that measures light emitted throughout the space.
The 72 newly-detected galaxies are not visible to the naked eye as they shine in Lyman-Alpha line of the light spectrum.
"MUSE can do something that Hubble can't — it splits up the light from every point in the image into its component colours to create a spectrum," said Roland Bacon, the team lead, according to an ESO statement.
ALSO READ: Human colonisation of Mars: Here's the next step taken by Budweiser to be the first beer on the Red Planet
"This allows us to measure the distance, colours and other properties of all the galaxies we can see — including some that are invisible to Hubble itself," Bacon added.
These newly-found galaxies are defined as young and they form stars at a speedy rate, New York Post reported. The present knowledge regarding star formation can't aid us in understanding the odd make-up of these galaxies.
On November 29, 10 papers on this phenomenon were published in an issue of Astronomy & Astrophysics.
ALSO READ: Boob-shaped UFO spotted by Australian man while using Google Street View
"We learn things about these galaxies that is only possible with spectroscopy, such as chemical content and internal motions," Jarle Brinchmann, author of one of the studies, was quoted as saying by space.com.
"Not galaxy by galaxy but all at once for all the galaxies!" Brinchmann added.
ALSO READ: Sex drive boosting 'super chicken': All you need to know about Kadaknath
Galaxies that had originated in less than a billion years post Big Bang were revealed in the original Ultra-Deep Field image. This discovery is one of the biggest landmarks and it remains our deepest look at the stars.
According to team lead Bacon, futuristic optics will be used to explore the Ultra-Deep Field further.


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