Saccharomyces cerevisiae has recently been described as an emerging opportunistic fungal pathogen. Fungal cell wall mannoproteins have been demonstrated to be involved in adhesion to inert surfaces and might be engaged in virulence. In this study, we observed four clinical isolates of S. cerevisiae with relatively hydrophobic cell surfaces. Yeast cell wall subproteome was evaluated quantitatively by liquid chromatography/tandem mass spectrometry. We identified totally 25 cell wall proteins (CWPs) from log-phase cells, within which 15 CWPs were quantified. The abundance of Scw10p, Pst1p, and Hsp150p/Pir2p were at least 2 folds higher in the clinical isolates than in S288c lab strain. Hsp150p is one of the members in Pir family conserved in pathogenic fungi Candida glabrata and Candida albicans. Overexpression of Hsp150p in lab strain increased cell wall integrity and potentially enhanced the virulence of yeast. Altogether, these results demonstrated that quantitative cell wall subproteome was analyzed in clinical isolates of S. cerevisiae, and several CWPs, especially Hsp150p, were found to be expressed at higher levels which presumably contribute to strain virulence and fungal pathogenicity.The budding yeast  Saccharomyces cerevisiae was considered to be a harmless microorganism; however, occasional reports of uncommon infections that were attributed to S. cerevisiae were revealed since 1980 [S. cerevisiae were first genetically characterized as pathogens in 1994 [S. cerevisiae has been regarded as one of emerging opportunistic yeast infections [S. cerevisiae, such as thermotolerance and pseudohyphal formation [Yeast nce 1980 . The cli in 1994 . Some clfections –14. Seveormation ; howeverS. cerevisiae cell wall, β-glucan and chitin, are served as scaffold and recognized by specialized innate immune receptors [Candida albicans and Candida glabrata [S. cerevisiae may highlight new determinants of fungal virulence.Yeast cell walls are essential for the survival of fungal cells. Two primary components of the inner part of eceptors , 16. It eceptors . In addieceptors –23. The eceptors , 25. Muteceptors . It has eceptors , 27.In aglabrata , 29. TheS. cerevisiae proteome. SILAC (stable isotope labeling by amino acids in cell culture) labeling of yeast was applied to generate one-to-one pairs of peptide signals which facilitates a highly reliable measurement of relative and absolute protein amounts [HSP150 were more potent to elicit proinflammatory cytokine TNF-α from macrophages, suggesting that alteration of cell wall composition and architecture causes yeast hypervirulent.Mass spectrometry-based proteomics has provided a powerful tool for systematic elucidation of yeast  amounts –32. Here600 = 0.8–1.2. YYC1-3 were isolated and obtained from National Taiwan University Hospital, Taipei, Taiwan. YJM309 (YYC38) was the gift from John McCusker of Duke University Medical Center, North Carolina [his3Δ1/ his3Δ1 leu2Δ0/ leu2Δ0 LYS2/lys2Δ0 MET15/met15Δ0 ura3Δ0/ ura3Δ0) is the diploid strain in S288c background generated by mating BY4741 and BY4742, the parental strains of Yeast Deletion Project obtained from Wen-Hsiung Li at Academia Sinica, Taipei, Taiwan. YYC370 (MAT a/α ADE2/ade2Δ::hisG HIS3/his3Δ200 leu2Δ0/leu2Δ0 lys2Δ0/lys2Δ0 MET15/met15Δ0 TRP1/trp1Δ63 URA3/ ura3Δ0) is also the diploid strain in S288c background generated by mating BY4704 and BY4739 obtained from Wen-Hsiung Li at Academia Sinica, Taipei, Taiwan. The hsp150 deletion strain in BY4741 background (MAT a his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 hsp150Δ::KanMX4) was obtained from Wen-Hsiung Li at Academia Sinica, Taipei, Taiwan. For Calcofluor white (CFW) or Congo Red (CR) sensitivity assay, overnight cultures were diluted and spotted to YPD plates with 50 μg/mL CFW or 100μg/mL CR and incubated at 30°C for 3 days.Strains were grown in YPD (1% (w/v) yeast extract, 2% (w/v) bactopeptone, and 2% (w/v) glucose) media and harvested at ACarolina . Σ1278b HSP150-173 and HSP150-288 alleles were amplified by PCR from YYC173 and S288c strains respectively with a Myc tag inserted into the end of the ORFs. The PCR products were digested with restriction enzymes BamHI and SalI and then subcloned into the vector pRS415-GAL1, which is the gift from Tien-Hsien Chang at Academia Sinica, Taipei, Taiwan. The constructs were transformed into hsp150Δ deletion strain, and the expression of HSP150 alleles were driven by GAL1 promoter by growing in SC-galactose media (0.67% (w/v) yeast nitrogen base and 2% (w/v) galactose). For CFW or CR sensitivity assay, overnight cultures were diluted and spotted to SC-galactose plates with 500 μg/mL CFW or 150 μg/mL CR and incubated at 30°C for 3 days.YYC173 is the segregant of YYC1. 600 of the suspension (A0) was 1.0. 80 μL o-xylene was added to a 800 μL suspension, vortexed at top speed for 1 min, and allowed to settle for 10 min. The OD600 of the aqueous phase (At) was measured. The hydrophobicity was calculated by the percentage of cells adhered to o-xylene: Adhesion to xylene (%) = (1- At /A0) x 100. The strains were considered more hydrophobic if the percentage of adhesion is higher. Statistical significance was measured by Student’s t test.Determination of cell surface hydrophobicity using MATH  test was performed as described before , with soS. cerevisiae to polystyrene surface was performed as describe before [600 in YPD media with 0.1% glucose. 100 μL of cell suspension was transferred into wells of a 96-well polystyrene plate  and incubated at 30°C for 1 hr. Cell suspension was then removed and cells adhere to polystyrene were stained with 100 μL 1% (w/v) crystal violet for 15 min [t test.Adherence of e before , 35. Str600 = 0.8–1.2. Equal amounts of each light and heavy cells (as determined by A600 measurement) were then mixed 1:1, harvested and washed for cell wall preparation. The mixture of equal amounts of light and heavy YYC370 was served as controls of our quantification later.SILAC labeling was performed as described before  with modCell walls were isolated as described , 37. BriPeptides for LC-MS/MS analysis were obtained from the proteolytic digestion directly from isolated cell wall fractions. 5 biological replicates of proteomics analyses were performed. The SDS-extracted cell walls were reduced by dithiothreitol (10 mM) at 55°C for 30 min followed by alkylation by iodoacetamide (50 mM) at room temperature for 30 min in the dark. Cell wall fractions were digested overnight by sequencing grade trypsin. After enzymatic reaction, peptides in the supernatants were collected and dried under vacuum.6 ions, 1000 ms and 5 x 104 ions, 200 ms respectively. Ions were fragmented by use of CID (collision induced dissociation) with the following parameters: the normalized collision energy to 35%, the activation Q to 0.3, and the activation time to 30 ms.Nano-LC-MS/MS experiments were performed on a LTQ-FT mass spectrometer  with a nano-electrospray ion source  in positive ion mode. The liquid chromatographic separation was using Agilent 1100 Series binary high-performance liquid chromatography system  with Famos autosampler . Peptide samples were injected onto a self-packed pre-column  followed by a self-packed reversed phase C18 nano-column  for chromatographic separation. A linear gradient of 5 to 40% mobile phase B (0.1% formic acid in 80% acetonitrile) was applied for 100 min at a flow rate of 300 nL/min. The positive ion mode was used in MS analysis with electrospray voltage of 2 kV and capillary temperature at 200°C. A MS scan cycle was initiated with a survey MS spectrum (m/z 300–2000) performed on the FT-ICR mass spectrometer with resolution of 100,000 at 400 Da. Ten most abundant ions detected in this scan were subjected to a MS/MS experiment performed in the LTQ mass spectrometer. Ion accumulation (Auto Gain Control target number) and maximal ion accumulation time for survey MS and MS/MS were set at 1 x 10http://www.massmatrix.net [For data analysis, all MS/MS spectra were converted to mzXML format from experiment RAW files by MM File Conversion Tools (trix.net ), and thtrix.net  for MS/Mt test.Murine RAW 264.7 macrophage cell line was a gift from Betty Wu-Hsieh at National Taiwan University, Taipei, Taiwan, originally obtained from the American Type Culture Collection, Rockville, Md., and cultured in DMEM-10  with 10% heat-inactivated FCS , 1x of Hyclone non-essential amino acids , 100 unit/mL penicillin, 0.1 mg/mL streptomycin, and 250 ng/mL amphotericin B) , 41. YeaS. cerevisiae by the MATH test, in which the partition of cells in o-xylene represents a quantitative measurement of hydrophobicity [o-xylene, the clinical isolates YYC1-3 all showed 13–18% of adherence, about increased up to 1.5 to 2 folds comparing to lab strain S288c  except Cwp1p, Mkc7p, and Pry3p. Bgl2p is characterized as a beta-1,3-glucanase which introduces intrachain linkages and is involved in cell wall structure maintenance [To investigate the variation of fungal cell wall proteomics between the lab strain and the clinical isolates, we established analyses of nd Tdh3p , which antenance , 45. Ssantenance . Tdh3p intenance .To further quantitate the covalently linked CWPs between the clinical and lab strains, SILAC labeling followed by LC-MS/MS was used as an easy and accurate approach for CWP relative quantitation. The experimental scheme was illustrated in HSP150 in S288c and clinical isolates and found that HSP150 transcripts in clinical isolates were surprisingly slightly reduced (data not shown). We suspected that the increased Hsp150p protein abundance was more likely due to post-transcriptional regulation. Scw10p, one of ASL-CWPs with potential glucanase activity, has been suggested to play a role in conjugation during mating [C. albicans ortholog of Pst1p is not found, but Ecm33p is required for its virulence [S. cerevisiae might be correlated to strain pathogenicity. The role of Pst1p up-regulation in clinical isolates of S. cerevisiae still remains to be further investigated. However, there is no direct evidence showing that Scw10p or Pst1p is related to fungal pathogenicity to our knowledge.Within 15 CWPs analyzed, cell wall proteins Cis3p, Crh1p, Hsp150p, and Scw10p were all expressed significantly higher in all 4 clinical isolates than in S288c lab strain, and higher levels of Ecm33p, Pir1p, Pst1p, and Scw4p were observed in 3 out of 4 clinical isolates . Scw10p,g mating . Howeverg mating , 50. Nevirulence . We therS. cerevisiae, apparently attach to cell wall via 1,3-glucan through alkali-sensitive linkages. An N-terminal signal peptide, a Kex2 site, multiple PIR repeat-containing regions, and a carboxyl-terminal region with four cysteine residues were highly conserved in Pir family. Hsp150p/Pir2p is one of the members in Pir family which are regulated by cell integrity pathway and required for cell wall stability [S. cerevisiae, despite the fact that Pir5p is still a putative protein of unknown function, and Pir3p was unable to be quantified in our analyses, Pir1p, Hsp150p/Pir2p, and Cis3p/Pir4p levels were all considerably increased in clinical isolates analyzed  or CFW (500 μg/ml). Although hsp150Δ was still more sensitive than WT at low concentrations of CR (100 μg/ml) or CFW (50 μg/ml), our data revealed that at higher concentrations of CR or CFW, strains with highly expressed Hsp150p were much more resistant to cell wall-perturbing agents, while the WT and hsp150Δ were very sensitive  have been recognized as a virulence-associated trait [C. albicans is closely related to its ability of biofilm formation [Aspergillus fumigatus, a protein phosphatase 2A SitA was identified as a possible modulator for adhesion, cell wall integrity, biofilm formation, and virulence [S. cerevisiae such as thermotolerance and pseudohyphal formation [S. cerevisiae is related to its hydrophobicity, adhesion, cell wall integrity, and virulence.Fungal virulence was considered multi-factorial. Several key virulence factors have been addressed in ed trait , 57. Theormation , 59. In irulence . Severalormation . AlthougS. cerevisiae by SILAC labeling followed by LC-MS/MS analysis. Our results indicated that clinical isolates of S. cerevisiae showed distinct characteristics of cell wall, such as higher hydrophobicity, higher ability of adherence, and higher levels of certain CWPs, including Hsp150p, indicating the discrepancy of the cell wall structure or composition between clinical isolates and lab strain S288c. In addition, we also showed that overexpression of Hsp150p enhances the virulence of yeast. Our findings in this study will lead to a better understanding of CWPs as well as the pathogenicity of clinical isolates of S. cerevisiae.The yeast cell wall is a dynamic structure with a layer of glucan and chitin molecules as scaffold for the outer layer of glycoproteins, and we reported here the first study of cell wall quantitative proteomics in clinical isolates of S1 Fig(A) MS spectrum shows a 2 m/z difference for doubly charged peptides; and (B) MS/MS spectrum shows no different for b11, b12, and b13 ions but with a 4 m/z shift for y11 and y12 ions. (C) Manual quantification of peaks for peptides with both Lys-D0 and Lys-D4 labeling from LC chromatogram.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Dickeya contains bacteria causing soft rot of economically important crops and ornamental plants. Here, we report the draft genome sequences of two Dickeya sp. isolates from rotted leaves of Phalaenopsis orchids.The genus  Dickeya genus of the family Enterobacteriaceae cause diseases on many plant species  and S1 (NIB Z 2099), belonging to the undefined lineages UDL-3 and UDL-4  and concentrated to an approximate concentration of 500 ng/µl , as determined with the Qubit double-stranded DNA (dsDNA) HR assay . Two single-read libraries were constructed with the Ion Xpress Plus fragment library kit (Life Technologies), according to the manufacturer’s protocol , with mode novo assembler . Both assemblies were processed using standard program settings with minor modifications in the GS de novo assembler: (i) the all-contig threshold was set to 200 bp, and (ii) the long-contig threshold was changed to 1,000 bp. Assembled contigs were integrated using CISA from the Orione  portal (http://orione.crs4.it/). Contigs with average coverage of <10×, contigs <1,000 bp, and all contaminates identified with BLASTn were discarded. The draft genomes comprise 61 and 59 contigs, with a total size of 4,885,657 bp and 4,892,969 bp and an N50 of 167,592 bp and 160,162 bp for strains B16 and S1, respectively.Sequences in FASTQ format were quality checked, and adapters were trimmed using CLC Genomics Workbench version 7.5 , with the following modifications: (i) sequence quality score was set at 0.1, (ii) eight nucleotides were trimmed off every read at the 5′ terminal end, and (iii) reads of <15 nucleotides were discarded. Trimmed reads were independently assembled with CLC Genomics Workbench version 7.5 and the GS Dickeya strains are available in GenBank under the accession numbers JXBN00000000 (BioProject PRJNA268269) and JXBO00000000 (BioProject PRJNA268270) for B16 and S1, respectively. The versions described in this paper are the first versions, JXBN01000000 and JXBO01000000.The draft sequences of these two


Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase.Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an  Data are presented as average mΔψ±s.e.m. Cell-by-cell mΔψ change rates for some time points were evaluated and are presented as distribution histograms.rCCA were labeled with 600 ng/ml JC-1 for 15 min at 37°C. The medium was replaced and time-lapse recordings were made with a 10X 0.4 N.A. objective and the same microscope set up described above with a Hammamatsu Orca R2 CCD and homemade white LED illumination. Time-lapse recordings were acquired at 1 f/min. JC-1 orange-red fluorescence was recorded with a 562/35 bandpass excitation filter, a 600 longpass dichroic mirror and a 610 longpass emission filter. JC-1 green fluorescence was recorded with a 485/30 bandpass excitation filter, a 505 longpass dichroic mirror and a 510/50 bandpass emission filter. After obtaining basal readings in 3 frames  distribution using Graphpad Prism 5.01 (Graphpad Software Inc.). Significance is claimed with p≤0.01. For mΔψ experiments, statistical differences at each time point of the time-lapse were determined by the student’s t-test with Excel (Microsoft). Significance is claimed with p≤0.05 or p≤0.01. Cell-by-cell change rates for mΔψ are presented as average ± s.e.m.Statistical differences in We initially characterized the purity of our rCCA by GFAP staining and found that >95% of cells were positive . Then weIn addition, rCCA were labeled by Abs against all NMDAR subunits: a polyclonal Ab against GluN2A EC domain; a monoclonal Ab against the GluN2B EC domain; a polyclonal Ab against the GluN2C EC domain; a monoclonal Ab against the GluN2D EC domain; a polyclonal Ab against the GluN3A EC domain; and a polyclonal Ab against the GluN3B EC domain . PhenotyWe next investigated NMDAR subunit mRNA expression by qRT-PCR and found mRNA expression of all NMDAR subunits . These dr), was recognized by Abs against both the C- and N- terminal domains  (0) plus twice its s.d.  value above the RTV after perfusion with NMDA than with vehicle alone  that displayed a modest iCa2+ increase just above the RTV, and another ≈18% of cells distributed in additional peaks, indicating heterogeneous, larger responses. These experiments strongly suggested that rCCA possess functional NMDARs.We then tested for NMDAR function by measuring response ; Fig 4A,response . In contresponse ; Fig 4B.onse (0) . In addile alone . This isle alone . These hiCa2+ response of rCCA to NMDA (0) analysis showed that the APV blockade was significant and reached almost 70% with only 19.3% of cells above the RTV (iCa2+ response of rCCA to NMDA (0) examination showed that KYNA significantly reduced by 80% the rCCA response to NMDA with only 8.9% of cells above the RTV  site in GluN1 subunit. Our experiments showed that APV (100 μM) blocked the averaged  to NMDA ; Fig 4C. the RTV ; Fig 4F. to NMDA ; Fig 4D. the RTV ; Fig 4F.iCa2+ increase, and they rule out unspecific effects associated with the NMDA concentration employed.Taken together these results demonstrated that rCCA express functional NMDARs that, after ligand binding, mediate 0 = 0.34, but with a larger decrease with time (0) analysis showed that this reduction was statistically significant and reached 76% when compared with siC transfected cells and only 12.7% of cells had a response above the RTV (0) analysis showed that, although the maximal averaged response of siC-transfected cells was similar to that of untransfected cells, siC transfection significantly decreased ∫(ΔF/F0) by 46% when compared with untransfected cells  knock down. Initially, we determined that the highest knock down efficiency (>55%) and cell viability were obtained with 2 μl/ml Lipofectamine and 166 nM siRNA  and the ith time ; Fig 5A.ith time ; Fig 5B. the RTV . These eed cells . This wa the RTV , Fig 5C. the RTV .0) examination showed no significant difference with NMDA perfused cells  analysis showed no difference with cells treated with NMDA in the presence of extracellular Ca2+  did not block the averaged response to NMDA ; Fig 6A,ed cells . MK-801 ed cells ; Table 1e groups –43 , an inhibitor of the Inositol trisphosphate (IP3) receptor (IP3R) in the ER that mediates Ca2+ exit. XesC (100 nM) blocked the averaged iCa2+ response of rCCA to NMDA (0) analysis showed that this inhibition was significant . Ry (50 μM) reduced the averaged iCa2+ response of rCCA to NMDA (0) analysis showed that this reduction was significant although to a lower extent  (10 μM), a tyrosine kinase inhibitor, doubled the maximal averaged response to NMDA, eliciting a transient peak that reached ≈0.8 ΔF/F0 and did not returned to basal levels by the end of the recording (0) examination showed that this increase was significant  analysis showed that the response to ATP was significantly different from that to NMDA alone (0) analysis showed that the ATP response was non-significantly different from the GX-NMDA response  and ∫(ΔF/F0) values of the ATP and GX-NMDA responses, their population dynamics were different. Here, it is important to note that analysis of the ∫(ΔF/F0) distribution histograms from averaged responses that were not significantly different showed meaningful differences; thus these analyses may be a useful tool for examining population-based studies.These comparisons demonstrated that NMDA and the well-known gliotransmitter ATP both evoke an iCa2+ buffer. We found that NMDA treatment (1 mM) significantly reduced mΔψ  in comparison with vehicle-treated cells  and three other time points: at t2 (t2mΔψ) during basal conditions; at t9 (t9mΔψ) after NMDA application; and at t12 (t12mΔψ) after CCCP treatment. These change rates were plotted on distribution histograms for vehicle , whereas cells within this range were considered non-responsive , vehicle- and NMDA-treated cells showed similar distributions (t0mΔψ/t9mΔψ rate (post NMDA) increased in NMDA-treated cells , and that of NMDA-treated cells was larger than that of vehicle-treated cells. These results confirmed functional NMDAR expression by rCCA and demonstrated that its activation regulates mitochondrial function.We next analyzed the NMDA effect on mΔψ at the individual cell level, because responses were heterogeneous, as reported previously , 47. For vehicle  and NMDA vehicle , and datsponsive . For theibutions . In conted cells , but noted cells ; Table 2iCa2+ rise and/or evoked currents in human astrocytes in response to NMDA, although these effects were not sensitive to APV and only partially sensitive to extracellular Ca2+ depletion [iCa2+ rise and cell death induced by Glu were sensitive to memantine or MK-801, evidence of canonical NMDAR function [iCa2+ rise was induced by NMDA [iCa2+ rise was found and non-canonical NMDAR function was suggested [2+ block, argued against functional NMDARs in astrocytes.Previous studies have reported functional, canonical ionotropic NMDARs in astrocytes using brain slices or acute tissue disaggregation , 22–25, epletion , 30. In function . Also, i by NMDA . Additio by NMDA . Recentluggested . These ain situ hybridization experiments for GluN1 were non-conclusive [In the present work, we detected all NMDAR subunits by IF that exhibited a phenotype characteristic of transmembrane molecules Figs . Intrigunclusive , 29, 51.r in astrocytes. A previous study reported efforts using eGFP-tagged astrocytes acutely isolated from mouse brain, but the amounts harvested were insufficient for WB analysis [Since non-functional NMDAR may result from incorrect GluN1 assembly and transport –21, we aanalysis . Thus, ranalysis , 7. We aanalysis , 17, 21.iCa2+ rise in rCCA, although the heterogeneous cell responses suggest that NMDAR activity varies in rCCA. This could be related to: a) different expression levels of NMDAR subunits, and/or b) cell differences in NMDAR regulation and/or assembly. Intriguingly, we observed that iCa2+ increases not only in the cytoplasm but apparently also within the nucleus, however more experiments are needed to confirm this possibility  distribution analysis, may provide additional insight into the cellular and molecular mechanisms and enable a more integrative view of these complex phenomena.We also found that tyrosine kinases regulate NMDAR. In this regard, it is well known that the GluN1 and GluN2A-B subunits are subject to tyrosine phosphorylation that regulates NMDAR activity . HoweveriCa2+ metabolism [iCa2+ rise and mΔψ depletion depends on different cellular mechanisms. First, NMDARs induce the iCa2+ rise that reaches maximal levels within seconds; then, mitochondria initiate iCa2+ uptake at the expense of mΔψ. Finally, mΔψ takes time to recover, because it requires the activation of mitochondrial electron transport and metabolic pathways. We assume that mΔψ eventually recovers, because the rCCA were alive for weeks after NMDA challenge. Here, it is critical to clarify that our mΔψ measurements comprise populations and not single mitochondria; therefore, temporal scales are different from those described by Keil et al. [iCa2+ rise. NMDA also elicited heterogeneous mΔψ responses that could be associated with the diversity of iCa2+responses, or alternatively, with other cell conditions that may include: NMDAR status, redox conditions, substrate availability, enzyme activity and/or availability and mitochondrial metabolism. These cellular conditions could also help to explain the different number of cells that rise iCa2+ of those that depleted mΔψ.We measured mΔψ considering the role of mitochondria in tabolism , 34, 58,tabolism , 59, usitabolism , 59–61. l et al. . On the 2+, enabling its activation at physiological resting potentials, but with lower Ca2+ permeability than neuronal receptors [Importantly, the NMDA concentration used in our experiments is within the Glu physiological levels reported within the synaptic cleft , where aeceptors . Such NMiCa2+ rise, that occurs in astrocytes in an oscillatory manner, that it may in turn regulate Ca2+ dependent secretion [in vivo. Moreover, considering previous in situ and in vitro studies with astrocytes, rCCA seem to exacerbate the metabotropic-like function of the NMDAR, a fact that could represent an advantage to study this NMDAR function. These findings also open the question about how NMDAR function, their mΔψ depolarization and their metabolic outcomes in astrocytes are involved in neuronal death, survival and communication, considering the close interaction between these two cell types in health and disease in the CNS.This work employed different approaches to demonstrate that rCCA express non-canonical functional NMDARs with metabotropic-like signaling. This supports that astrocytes are active participants in brain function as proposed by the tripartite synapse hypothesis, in this case through NMDAR as a putative player in ecretion , 58, 64.S1 Fig(A) GFAP labeling of rCCA. (B) High magnification of representative cells showing GFAP phenotype. (C) Glutamine synthase (Gln Syn) labeling of rCCA. (D) High magnification of representative cell showing Gln Syn phenotye. (E) Texas red hydrazide (Tex Red H)  vital labeling of rCCA. (F) MAP2 labeling of rCCA; as expected no cells were positive since neurons do not proliferate nor do they survive trypsinization. All images are representative of each staining. For GFAP, Gln Syn and Tex Red H <95% of cells were stained. Bar = 50μm for A, C, E and F; for B and D = 10μm.(TIF)Click here for additional data file.S2 FigAmplification curves for the indicated gene products with cDNA obtained from rat brain. One representative experiment for each probe is shown.(TIF)Click here for additional data file.S3 FigiCa2+ response. One representative experiment is shown.rCCA were labeled with Fluo-4 AM, recorded as described in the materials and methods section and perfused with 100 μM NMDA. As observed, this treatment did not modify the averaged (TIF)Click here for additional data file.S4 FigrCCA seeded into 24 well plates were transfected with 2 μl/ml Lipofectamine 2000 and the indicated nM concentration of siRNA (siC or siGrin1). 24 h after cells were fixed, stained against extracellular NMDAR subunit GluN1, photographed  and analysed. Data represent average fluorescence per cell ± s.e.m. from 35–60 cells evaluated with background subtracted obtained from control cells without primary Ab. Higher amounts of Lipofectamine 2000 or siRNA caused substantial cell death and detachment.(TIF)Click here for additional data file.S5 FigrCCA were labeled with JC-1, recorded as described in the materials and methods section and incubated with 100 μM NMDA. As observed, this treatment did not change mΔψ. One representative experiment is shown.(TIF)Click here for additional data file.S6 FigrCCa lysates were prepared and WB was performed with an Ab against the EC domain of GluN1 subunit as described in the materials and methods section. As observed, a conspicuous band was detected below the full-length GluN1 (115 kDa). One representative experiment is shown.(TIF)Click here for additional data file.S7 FigiCa2+ response with a time course similar that of NMDA alone (see text). One representative experiment is shown.rCCA were labelled with Fluo-4 AM, recorded as described in the materials and methods section and then perfused with 1mM NMDA+100 μM Glycine (Gly). As observed, this treatment increased the averaged (TIF)Click here for additional data file.S8 FigFor each experimental condition discussed in the text (rows) a representative cell was chosen and three frames were extracted from the recording representing basal (left column), peak (middle column) and end (right column) conditions.(TIF)Click here for additional data file.S1 Video(MPEG)Click here for additional data file.


Mechanical stimuli play crucial roles in bone remodeling and resorption. Osteopontin (OPN), a marker for osteoblasts, is important in cell communication and matrix mineralization, and is known to function during mechanotransduction. Hypergravity is a convenient approach to forge mechanical stimuli on cells. It has positive effects on certain markers of osteoblast maturation, making it a possible strategy for bone tissue engineering. We investigated the effects of hypergravity on OPN expression and cell signaling in osteoblasts. Hypergravity treatment at 20 g for 24 hours upregulated OPN expression in MC3T3-E1 cells at the protein as well as mRNA level. Hypergravity promoted OPN expression by facilitating focal adhesion assembly, strengthening actin bundles, and increasing Runx2 expression. In the hypergravity-triggered OPN expression pathway, focal adhesion assembly-associated FAK phosphorylation was upstream of actin bundle assembly. This is largely attributed to the lack of mechanical stimuli during cell culture. Hypergravity, or centrifugation, is a convenient approach to place mechanical stress on cultured cells, and is known to have an impact on osteogenesis [g), mid-level (10–100 ×g), and high-level (>100 ×g) hypergravity conditions were all observed to have significant effects on the morphology, proliferation, gene transcription, extracellular matrix protein synthesis, and alkaline phosphatase activity of osteoblasts. These findings suggested that hypergravity is a possible approach for bone tissue engineering [Mechanical stimuli play important roles in bone remodeling. Bone cells differentiate, proliferate, and mature under mechanical stress cal cues –5. Enginogenesis . In previneering –10. To ein vivo on earth [During bone regeneration and development, OPN, a marker for osteoblasts, plays an important role in cell communication and matrix mineralization –16. Receon earth –28. HoweIn the present study, we aimed to understand the mechanism by which hypergravity affects OPN expression in osteoblasts. Using a custom-designed centrifuge, we cultured cells under 20-g hypergravity conditions, and found that OPN expression and secretion were upregulated in MC3T3-E1 osteoblast cells. Runx2, a major regulator of osteogenic differentiation, was required for hypergravity-induced OPN expression. A multi-index quantification showed that the cytoskeleton and focal adhesions of MC3T3-E1 cells were altered by hypergravity, and they also contributed to hypergravity-induced OPN expression. Taken together, our findings showed that hypergravity may affect OPN expression via changes in focal adhesions, the cytoskeleton, and Runx2.g was applied for 24 h in an incubator at 37°C. The control cells were placed on the platform of the centrifuge, as shown in A custom-made low-speed centrifuge was used to apply hypergravity to the cells in this study. Cells in culture flasks (or dishes) were placed on the horizontal rotors in the centrifuge, and 20 ×2. Cells were sub-cultured (1:3), every 3 days, using 0.25% trypsin with 1 mM EDTA. 15mM Hepes was added to adjust the pH in culture medium. Unless otherwise specified, 105 cells per flask were exposed to 20 ×g hypergravity for 24 h, after seeding and spreading for 2 h. The control cells were subjected to the same conditions as the hypergravity-exposed cells in terms of timing, incubation media, and other procedures. The MC3T3-E1 cells used in this study were from passages 25 to passages 30. We immediately collected n = 6 1 ×g controls and n = 6 20 ×g hypergravity treated samples for western blotting analyses as well as n = 6 1 ×g controls and n = 6 20 ×g hypergravity treated samples for real-time PCR, respectively.MC3T3-E1 osteoblastic cells  were purchased at the passage number of 20, and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, and 1% penicillin–streptomycin at 37°C in 5% COg and 20 ×g treatments; and samples were collected at 25 h, 26 h, and 27 h. We collected n = 6 1 ×g controls and n = 6 20 ×g hypergravity treated samples from each class. In experiments with varies cell densities, 103, 104, 105, or 106 cells were exposed to 20 ×g hypergravity for 24 h. In experiments with altered preparatory adherent period, cells were preparatory adhered over a range of 6 h, 12 h, and 24 h and then exposed to 20 ×g hypergravity for 24 h. We collected n = 6 1 ×g controls and n = 6 20 ×g hypergravity treated samples from each class. In addition, cells were exposed to 20 ×g hypergravity for a longer period of 48 h or 72 h. We then collected n = 4 1 ×g controls and n = 4 20 ×g hypergravity treated samples from each condition for western blotting analyses.In some experiments, groups of cells were placed in incubator for a period of time after 1 ×g hypergravity stimulation (n = 6). In FAK inhibitor  experiments, cells were treated with 10 mM PF573228 for 24 h during 20 ×g hypergravity stimulation. We collected n = 6 1 ×g controls and n = 6 20 ×g hypergravity treated samples for western blotting analyses as well as n = 4 1 ×g controls and n = 4 20 ×g hypergravity treated samples for real-time PCR, respectively. To measure the secreted OPN in the culture medium, MC3T3-E1 cells were first seeded on culture flasks with complete media. After adhering and spreading, cells were cultured in serum-free media and then exposed to 20 ×g hypergravity for 24 h. The culture media were collected and concentrated using Millipore’s Amicon Ultra 15 mL centrifugal filter device, according to the manufacturer’s protocol. We collected 1 ×g controls and 20 ×g hypergravity treated samples without drug (n = 9), with blebbistatin (n = 6) or with PF573228 (n = 5) for western blotting analyses.In blebbistatin  treatment experiments, MC3T3-E1 cells were treated with 50 μM blebbistatin for 24 h during 20 ×2. The osteoblasts from passages 2 to passages 6 were used in this study. We collected n = 6 1 ×g controls and n = 6 20 ×g hypergravity treated samples for western blotting analyses.The primary osteoblasts were obtained from 3-day-old mouse calvarias following the published method , 30. In g) and hypergravity-exposed (20 ×g) cells were prepared with RIPA lysis buffer. Protein content was determined using a Protein Assay Kit (Thermo). Equal amounts of protein samples were resolved by reducing 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked for 2 h in 5% nonfat milk and incubated with primary antibodies against OPN , Runx2 , Paxillin , p-FAK , FAK , and GAPDH  overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG  or anti-rabbit IgG  for 2 h at room temperature. Lastly, the membranes were developed with enhanced chemiluminescence reagents (Millipore) and exposed to an X-ray film .Whole cell lysates from control , according to the manufacturer’s instructions. The Runx2 siRNA sequences  were used in a previous study . After 8Total RNA was extracted from cell samples using TRIzol , and cDNA was generated using a reverse transcription kit (Tiangen), according to the manufacturer’s instructions. SYBR Green Realtime PCR Master Mix (TOYOBO) was used for the real-time PCR analysis. The reactions were performed in a Mastercycler ep realplex . The following primers were designed using the Primer 3 website and were synthesized by Invitrogen:OPN ,Runx2, ,GAPDH, .A comparative CT method was used for quantification. The housekeeping gene GAPDH was used for internal normalization.Cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized in 0.1% Triton X-100 buffer for 5 min. After blocking in 5% BSA for 30 min at room temperature, the cells were incubated with primary antibodies against OPN , Runx2 , and paxillin  diluted in 5% BSA at 4°C overnight. Cells were then incubated with Anti-mouse IgG conjugated to TRITC , anti-rabbit IgG conjugated to FITC , and rhodamine phalloidin  for 2 h at room temperature. Lastly, cell nuclei were visualized by DAPI (Sigma) and viewed under a Leica TCS SP5 confocal microscope system. The fluorescence images were selected randomly for quantification. The quantification of the fluorescence signal was done using the image processing software Image-Pro plus  and the ImageJ software. To measure the thickness of actin bundles, we drew a line across each cell, and the bundle width at the cross line section was quantified. To avoid background noise, only regions with a fluorescence intensity higher than 40 were considered as actin bundles and thus counted.P-value < 0.05 was considered significant.All experiments in this study were performed at least quadruplicate. The Wilcoxon matched pairs test and Kruskal–Wallis test were carried out to compare the differences between the experimental groups in realtime-PCR and western blotting. Unpaired t-test was used to compare the differences between the two experimental groups in immunofluorescence staining. A g centrifugal force for 24 h. After exposure to hypergravity, cells were assayed for the mRNA and protein levels of OPN and Runx2 by real-time PCR, immunofluorescence staining, and western blotting, respectively   . These rg hypergravity-treated cells had higher actin staining intensity and thicker actin filaments. The average thickness of actin bundles was 1.39 μm in unloaded cells and 1.6 μm in the centrifuged cells. In addition, 36% of all visualized fibers in the loaded cells were thicker than 1.5 μm, compared with those in unloaded cells   are known to be the mechanosensing elements in several mechanotransduction pathways –38. Prev fibers) .g hypergravity. The number of observable FAs in the hypergravity-treated cells and the fluorescence intensity of each FA were higher than those in the unloaded cells (2) and post-hypergravity (2.3 μm2) samples  that can undergo tension-dependent conformational changes to affect kinase activity, phosphorylation site availability, intracellular localization, and/or ligand affinity. Actin bundles, FAs, and their interactions play pivotal roles in several mechanosensory pathways –42. Therg- and 1 ×g-treated cells disappeared after the blebbistatin treatment , 24 h continuous hypergravity stimulation upregulated OPN expression, and the hypergravity-increased OPN expression can last for at least 3 h after hypergravity deprivation. After 48 h hypergravity treatment, the expression of OPN and Runx2 returned to the same levels of the control group. Independent group has reported that OPN plays a role in the early the initial early molecular events during the course of bone and tendon remodeling [OPN expression is reported to be sensitive to altered gravity in space flight . Our resmodeling , 46. TheSome transcription factors such as AP-1 and ERK1 are known to bind the OPN promoter and affect OPN expression in certain cell types under hypergravity conditions . In thisFAs and the cytoskeletal system, including actin bundles, microtubules, and intermediate filaments, have long been known to respond to hypergravity and microgravity , 47,48. g hypergravity treatments [Since actin bundles are crucial to the cell’s reaction to hypergravity, the signaling pathways interacting with actin bundles, such as Rho/ROCK, ERK/ERK1, and ERK-c-Fos, are worth investigating ,44. In aeatments . Here, wIn conclusion, our findings show that a 24-h medium hypergravity treatment can upregulate OPN expression and secretion in osteoblasts. Furthermore, FAK phosphorylation, actin bundle contractility, and Runx2 are closely involved in the hypergravity sensing and signal transduction process. These results suggest that hypergravity conditions induced by centrifugation may be a convenient approach in bone tissue engineering.


Head trauma affects millions of Americans each year and has significant morbidity and economic costs to society. The objective of this study is to describe the epidemiology of head traumas presenting to emergency departments in the United States.The National Electronic Injury Surveillance System-All Injury Program was queried to conduct a retrospective analysis of head traumas treated in U.S. emergency departments. 207,159 cases of nonfatal head trauma from January 1, 2007 to December 31, 2011 were included in this study.An estimated 10,746,629  head traumas were treated in U.S. emergency departments (EDs) during the study period, averaging 2,149,326 cases annually. The annual injury rate per 10,000 population increased from 55.2 in 2007 to 85.4 in 2011, with the largest increases seen in children ≤11 years of age and in adults >65 years of age. Traffic-related head trauma accounted for an estimated 1,819,824 visits to U.S. EDs over the study period and was associated with a 1.74 times greater risk of a hospital admission compared to injuries due to non-traffic-related causes. Assaults (95.9 %) were the most common reason for head trauma in cases where injury intent was documented, and 16.9 % of assault-related head trauma occurred in children 0-17 years of age. When analyzed separately from other head traumas, concussions increased by 37.5 % over the study period, and nearly a third (29.9 %) of all concussions were sports-related.The increase in the number and rate of head traumas treated in U.S. EDs warrants continued injury prevention efforts and improvements in injury nomenclature and surveillance. Head trauma encompasses a wide variety of injuries with differing severities, ranging from trivial head wounds to traumatic brain injuries. In the United States, a large emphasis has been placed on traumatic brain injuries (TBIs); more than 1.7 million TBIs are estimated to occur each year in the U.S., 75 % of which are concussions or other mild traumatic brain injuries . A diverThe epidemiology of head trauma, which encompasses both TBIs as well as more minor head injuries, has not been well represented in the literature. Since patients with head trauma often utilize healthcare resources, understanding head trauma injury patterns is valuable in helping shape public policy and providing information to inform injury prevention efforts. Given that most patients who present with head trauma are often triaged and treated in an emergency department (ED) setting , a natioInternational Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnosis codes in its classification of injury diagnosis [The use of the NEISS in head trauma surveillance was identified in a report to Congress in 2003 by the Centers for Disease Control and Prevention (CDC) as a priority recommendation . Its useiagnosis , 14. Reciagnosis –17. The This study was a retrospective cohort study utilizing the National Electronic Injury Surveillance System-All Injury Program, a nationally representative database maintained by the U.S. Consumer Product Safety Commission (CPSC). This study was exempt from review by the University of Pennsylvania Institutional Review Board.The NEISS-AIP is a collaborative database maintained jointly by the CDC and the CPSC . ComposeAll nonfatal head traumas reported through NEISS-AIP from January 1, 2007 to December 31, 2011 were retrospectively analyzed. Cases of head trauma were identified using the following NEISS diagnosis and body part codes in concordance with previous studies –17: concAll study variables were coded by NEISS coders using a standardized data dictionary, and these variables were regrouped for further analysis in this study. The NEISS variable for age was regrouped into 6 age groups: (1) 0–11, (2) 12–17, (3) 18–24, (4) 25–44, (5) 45–64 and (6) 65+. Disposition from the ED was categorized as (1) treated and released, (2) admitted/hospitalized , (3) left against medical advice, or (4) not documented. The precipitating cause of injury was regrouped into (1) motor vehicle—occupant, (2) motorcyclist, (3) pedal cyclist, (4) pedestrian, (5) other transport, (6) fall, (7) struck by/against, (8) other specified, or (9) unspecified/unknown.Perpetrator relationship in assault cases were categorized as (1) multiple, (2) friend/acquaintance, (3) spouse/partner, (4) other relative, (5) stranger, (6) other specified, and (7) unspecified/unknown. The reason for assault was regrouped as (1) altercation, (2) robbery/burglary, (3) other specified, or (4) unspecified/unknown. All analyses involving mechanism of injury utilized the precipitating cause of injury variable (the cause of injury that started the chain of events that leads to an injury) rather than the direct cause of injury, as the precipitating cause is more relevant to injury prevention efforts. The 39 individual sports categories provided by NEISS-AIP were examined, and the top 7 head trauma associated organized sports and the top 12 head trauma associated individual sports were isolated based on prevalence for further analysis.χ2 analysis to assess differences between years for secular trends and calculation of relative risk (RR) with 95 % confidence intervals (CIs).Data were analyzed using SPSS version 21.0  statistical software. National injury estimates were calculated using statistical weights provided by the CPSC; each sample weight represents the inverse probability of the selection of a logged case. Analyses that involved fewer than 20 unweighted NEISS-AIP cases, 1200 weighted cases, or a coefficient of variation >30.0 % were considered unstable and were excluded from the results. All data reported in this article are national estimates unless otherwise noted. U.S. Census Bureau intercensal and postcensal population estimates from 2007 to 2011 were used to calculate injury rates for the study period . StatistBetween 2007 and 2011, an estimated 10,746,629  patients were treated for head trauma in United States EDs, averaging 2,149,326  or 70.1 (95 % CI: 54.6-85.6) injuries per 10,000 population annually. Nearly a quarter (23.3 %) of head traumas was documented in children ≤11 years of age, and males (54.6 %) were injured more often than females  over the study period, from 1,662,593  visits in 2007 to 2,659,942  visits in 2011  head trauma visits to U.S. EDs during the study period were documented traffic or motor vehicle crashes on highways and roadways. Nearly a third of traffic-related head traumas (31.2 %) involved 25–44 year-olds, followed by 18–24 year-olds (23.8 %). The majority of traffic-related head traumas occurred in males (55.6 %). When occupant status in the vehicle was documented, 42.8 % of traffic-related head traumas involved the driver and 23.9 % involved the passenger. An estimated 444,842  hospital admissions were associated with traffic-related head trauma, and explicitly documented traffic-related head traumas were 1.74 times (95 % CI: 1.45-2.08) more likely to lead to a hospital admission compared to other causes.In cases of traffic-related head trauma where the precipitating cause of injury was documented, 78.0 % of all injuries occurred when the patient was in a motor vehicle Table . A great1,714,388  sports-related head traumas were treated in U.S. emergency departments from 2007 to 2011. Nearly two-fifths of all sports-related head traumas (37.9 %) occurred in children 12–17 years of age, and over three-quarters (78.6 %) of all sports-related head traumas occurred in patients ≤24 years of age. Males (68.9 %) accounted for the majority of sports-related head traumas. When head traumas were stratified by the most commonly associated individual and organized sports, many have a clear male or female predominance (Table Explicitly documented sports-related head traumas were associated with a 0.60 times (95 % CI: 0.51–0.71) risk of hospitalization compared to other causes. An estimated 24,978 patients , or 4.0 % of all patients who participated in a top 7 organized sport, were hospitalized while 95.3 % were treated and released. In contrast, 127,837  patients (14.5 %) injured while participating in one of the 12 individual sport or recreational activities were admitted to a hospital. Bicycling (37.6 %), ATV use (22.1 %), and horseback riding (12.3 %) accounted for nearly three quarters of all individual sports-related hospital admissions.RR = 16.77, 95 % CI: 9.90-28.39) to be admitted to a hospital compared to other intents of injury.Among cases where intent of injury was documented, assaults were the most common reason associated with head traumas (95.9 %), followed by legal intervention (1.1 %) Table . Multiplχ2 = 18.21, p = 0.001). The majority of the increase in concussions was attributed to sports-related injuries  concussions occurred over the study period, averaging 401,295  concussions or 13.1 (95 % CI: 10.4-15.8) concussions per 10,000 population annually. The number of concussions increased from 344,001  in 2007 to 472,881  in 2011, an increase of 37.5 %  of all head traumas. This figure was in concordance with previously published studies, with another large nationally representative database study documenting motor vehicle traffic accidents as a mechanism of injury in approximately 16 % of TBIs in its sample in 2010 . MoreoveNearly a fifth of both assault-related head traumas (17 %) and assault-related concussions (19 %) occurred in children <18 years of age. These proportions suggest that a number of children may be presenting to U.S. EDs with assault-related head traumas, some of which are clinically mild. The mechanisms of assault-related injury are diverse and include blows to the head or other body parts, dropping, throwing, or shaking . Most ofNearly a third (30 %) of concussions was associated with a sport or recreational activity. Clear guidelines currently exist with respect to sports concussion management and opportunities for injury prevention exist through rule and culture changes in many sports . DespiteAs the NEISS is designed specifically for injury surveillance purposes , 14, it This study has several limitations. Injuries treated in urgent care centers, physician offices, or on athletic fields that do not present to an emergency department are not captured by the NEISS database. The number of head traumas is likely to be underestimated, particularly minor head traumas, of which a substantial portion are treated in an outpatient setting . TherefoIn summary, the increase in the number and rate of diagnosed head traumas treated in U.S. EDs warrants continued injury prevention efforts and public awareness. Currently, major injury prevention efforts have focused on improving motor vehicle safety and safe play in sports. The focus of these interventions targets a large proportion of the population, but also is less likely to include younger children and the elderly. As the public and medical community become more aware of the potential dangers of head traumas, regardless of severity, expanding injury prevention efforts to include these populations will become increasingly important, and may involve provider awareness concerning head trauma in child abuse, safe play for younger children, and product and home safety.


To further clarify mechanisms underlying the genotoxicity, a comprehensive DNA adduct (DNA adductome) analysis was conducted using DNA samples derived from the lungs of mice exposed to MGT. In total, 30 and 42 types of DNA adducts were detected in the vehicle control and MGT-treated groups, respectively. Principal component analysis (PCA) against a subset of DNA adducts was applied and several adducts, which are deduced to be formed by inflammation or oxidative stress, as the case of etheno-deoxycytidine (εdC), revealed higher contributions to MGT exposure. By quantitative-LC-MS/MS analysis, εdC levels were significantly higher in MGT-treated mice than those of the vehicle control. Taken together with our previous data, it is suggested that inflammatory responses might be involved in the genotoxicity induced by MGT in the lungs of mice.Nanosized-magnetite (MGT) is widely utilized in medicinal and industrial fields; however, its toxicological properties are not well documented. In our previous report, MGT showed genotoxicity in both  Recently, we have reported genotoxic effects of MGTs using in vitro and in vivo assay systems, and clearly demonstrated that MGTs induce genotoxicity in both cultured mammalian cells and mice lungs instilled intratracheally . Using this method, accurate mass values of precursor ions can be obtained, and this is an advantage for identification of chemical structures of DNA adducts. To identify the chemical structures of DNA adducts screened by adductome analysis, we have already made a list of DNA adducts, including m/z [M + H] values of precursor and daughter ions corresponding to more than 250 literature-based DNA adducts  of mice lungs exposed to MGT.Recently, Kanary et al. ,25, estaet al. . Using tucts see . Moreovegpt delta transgenic mice after intratracheal instillation  at tR 20.1 min) had the highest contribution to MGT exposure based on its PCA significance. This was followed by DNA adducts named A4 (m/z 363.17 [M + H] at tR 25.9 min), A10 (m/z 355.23 [M + H] at tR 31.0 min), A14 (m/z 652.37 [M + H] at tR 21.4 min) and A9 (m/z 243.12 [M + H] at tR 31.0 min) revealed higher contribution to MGT exposure (vs. MGT) and are shown in the importance plot  against a subset of DNA adducts observed in these data set was further applied and is shown in the 2D PCA scores plot A and assexposure B. On thence plot . Severalnce plot . A hieram/z [M + H] were compared with known DNA adducts listed in m/z values [M + H] for inflammation-related adducts, ammonium added butano-etheno-deoxyadenosine , etheno-deoxycytidine  and 3-methyldeoxycytidine , respectively. In contrast to this, we could not find identical m/z values [M + H] for the remaining 4 contributors in the DNA adduct list. In order to clarify the formation mechanism of the remaining 4 contributors, A2, A10, A14 and A37, we prepared various in vitro model reactions, including oxidative stress and inflammation, and compared their m/z values [M + H] and tR with each other. As a result, A10 (m/z 355.23 [M + H] at tR 31.5 min) and A37 (m/z 356.24 [M + H] at tR 31.4 min) were seen to correspond to one of the DNA adducts observed in the reaction mixture with oxidized-arachidonic acid (m/z 580.79 [M + H] at tR 25.9 min (A2) and m/z 652.37 [M + H] at tR 31.0 min (A14) could be seen in any of the in vitro model reactions. From these observations, it is suggested that inflammatory responses might exist in the mechanisms behind the increase in mutations by MGT treatment.To identify the chemical structure of DNA adducts detected as the “major contributors” to MGT status, we used the list of DNA adducts constructed by ourselves . Firstlynic acid . No adduIn order to confirm the chemical structure of DNA adducts diagnosed as highly contributing to MGT status, we synthesized authentic 15N-εdC and analyzed it by quantitative LC-MS/MS apparatus ). A peak with a 252.1 ≥ 136.1 transition corresponding to εdC, eluted at the same position as authentic 15N-εdC (255.1 ≥ 139.1), was observed in the lungs of both vehicle and MGT-treated mice . Levels in vitro assay [et al. [et al. [in vivo genotoxicity. It has been reported that MWCNTs damaged mitochondria to increase ROS production and cause toxicity against lung alveolar epithelial cells, A549 [εdC is produced from 4-hydroperoxy-2-nonenal via lipid peroxidation, and is known to be an inflammation-related adduct . Since iro assay , it is lro assay . In addiro assay . Support [et al. , have be [et al. ,31,32,33 [et al. , demonstls, A549 . Similarls, A549 ,15. Takegpt delta mouse study of Totsuka et al., 2014 [2/g . Detailed information, such as particle appearance, dispersed diameter and zeta potential of MGT can be found in the previous report [MGT was purchased from Toda Industrial Co., Ltd. , and this material was identical to those used in the l., 2014 . The decs report .E. coli) were purchased from Sigma Co. . All other chemicals used were of analytical grade and purchased from Wako.NucleaseP1 and HPLC grade methanol were purchased from Wako . Phosphodiesterase I was purchased from Worthington. Bovine spleen phosphodiesterase II, DNase I, Type I agarose, low melting point agarose, and Triton X-100 and bacterial alkaline phosphatase Type III  were obtained from Japan SLC . Animals were provided with food  and tap water gpt mutation frequency was significantly increased in mice lungs treated with multiple doses of 0.2 mg, but not in the 0.05 mg treatment group. In the DNA adduct formation analysis, even though singly treated with 0.2 mg of MGT, the levels of oxidative stress related DNA adducts were significantly increased. Therefore, we thought that a single dose of 0.2 mg MGT/animal was sufficient to analyze comprehensive DNA adduct analysis. Control samples were obtained from the lungs of mice given the vehicle. Mouse lung DNA was extracted and purified using a Gentra® Puregene™ tissue kit . The protocol was performed according to the manufacturer’s instructions except that desferroxamine  was added to all solutions to avoid the formation of oxidative adducts during the purification step. The extracted DNA was stored at −80 °C until analysis for DNA adductome analysis.For DNA adduct analyses, each group of 4 to 5 male ICR mice was intratracheally instilled with MGT at a single dose of 0.2 mg per animal, and sacrificed 24 h after MGT administration. Our previous study  demonstrn = 4) and MGT treated (n = 4) mice were enzymatically digested according to the method of Goodenough et al. [Penicillium citrinum), 10 mM sodium acetate , and ZnCl2  were added, and incubated for a further 3 h at 37 °C. Alkaline phosphatase (from E. coli), phosphodiesterase I (20 U/mL in water) and Tris base  were added last, for an additional 16–18 h at 37 °C. The sample was purified using Vivacon500® , then, the reaction mixture was centrifuged  using Ultrafree®  and the filtrate was used for DNA adductome analysis.Mouse lung DNA extracted from vehicle  equipped with a Xevo QTOF mass spectrometer , instrumented with an electrospray ionization source (ESI) and controlled by MassLynx 4.1 software. Sample injection volumes of 4 μL each were separated on a ACQUITY UPLC BEH130 C18 column  at a flow rate of 25 µL/min. The column temperature was set to 40 °C. Mobile phase A and B were water and methanol, respectively. Chromatographic separation was performed by a gradient elution as follows: 0–5 min, 1% B; 5–10 min, linear gradient to 10% B; 10–35 min, linear gradient to 80% B; 35–45 min, 80% B. MS parameters were set as follows: mass range scanned from 50 to 1000 with a scan duration of 0.5 s , capillary 3.7 kV, sampling cone 40 V, extraction cone 4 V, source temperature 125 °C, desolvation temperature 250 °C. Nitrogen gas was also used as the desolvation gas (flow 800 L/h) and cone gas (30 L/h). All data were collected in positive ion mode. MSm/z. DNA obtained from normal human fetal fibroblast cell line, TIG-3, with internal standard was used as a reference [Relative peak intensity of each potential DNA adduct was calculated as previously described . The releference .m/z), sample name, and the normalized peak intensity was analyzed by S-plot analysis using SIMCA-P+ 11.5 . The method parameters were as follows: Mass tolerance = 0.05 Da, Apex Track Parameters: Peak width at 5% height (seconds) = 15/Peak-to-peak baseline noise = 50, Apply smoothing = Yes, Collection Parameters: Intensity threshold (counts) = 100/Mass window = 0.05/Retention time window = 0.10, Noise elimination level = 6, Deisotope data = Yes.The raw data files obtained from LC/MS runs were analyzed using MassLynx v4.1 and MarkerLynx 4.1 software (Waters). The application detects, integrates, and normalizes the intensities of the peaks to the sum of peaks within the sample. The resulting multivariate dataset consisting of the peak number (based on the retention time and 4 (Wako) and 1.5 mM ascorbic acid (Wako). The DNA modification related to oxidative stress was formed from ctDNA and 10 mM hydrogen peroxide (Wako) in the presence of 1 mM CuSO4 and 1 mM ascorbic acid in 1 mL of 500 mM sodium phosphate buffer, pH 7.4 for 24 h, in atmospheric oxygen at 37 °C. The reaction was terminated by the addition of 1 mM butylated hydroxytoluene (Wako) and 100 µM diethylenetriaminepentaacetic acid (Wako).The DNA modification derived from oxidation of unsaturated fatty acids was performed by incubating calf thymus DNA  with 20 mM unsaturated fatty acids including arachidonic acid (Sigma) and linoleic acid (Sigma) in the presence of 75 µM CuSOn = 5) and MGT treated (n = 5) mice was enzymatically digested, and εdC was analyzed and quantified by the Waters 2795 LC system  interfaced with a Quattro Ultima triple stage quadrupole MS (Waters) using the same procedure previously described [Mouse lung DNA (40 μg) extracted from vehicle . All the calculations were performed using statistical package R. In RF, we confirmed that the out-of-bag misclassification rate was saturated at 0% with 100,000 generated trees with two variables. The data were statistically compared with the corresponding solvent control using the Student’s We have demonstrated that MGTs induce inflammation-related DNA adduct formation in mouse lungs by using comprehensive analysis of DNA adducts, DNA adductome analysis. In PCA analysis, εdC was detected as the “major contributor” to MGT status. Due to the inducible base, the exchange pattern of εdC has been reported to be a C to T transition , being the predominant mutational pattern detected in mouse lungs exposed to MGT . Therefo


It is well-documented that both chemokine (C-C motif) ligand 19 (CCL19) and 21 (CCL21) mediate cell migration and angiogenesis in many diseases. However, these ligands’ precise pathological role in ankylosing spondylitis (AS) has not been elucidated. The objective of this study was to examine the expression of CCL19 and CCL21 (CCL19/CCL21) in AS hip ligament tissue (LT) and determine their pathological functions.The expression levels of CCL19, CCL21 and their receptor CCR7 in AS (n = 31) and osteoarthritis  LT were analyzed via real-time polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). The expression of CCL19, CCL21 and CCR7 in AS ligament fibroblasts was also detected. The proliferation of ligament fibroblasts was measured via a cell counting kit-8 (CCK8) assay after exogenous CCL19/CCL21 treatment. Additionally, the role of CCL19/CCL21 in osteogenesis was evaluated via RT-PCR and enzyme-linked immunosorbent assay (ELISA) in individual AS fibroblast cultures. Furthermore, the expression of the bone markers alkaline phosphatase (ALP), osteocalcin (OCN), collagenase I (COL1), integrin-binding sialoprotein (IBSP) and the key regulators runt-related transcription factor-2 (Runx-2) and osterix were investigated. Moreover, the CCL19/CCL21 levels in serum and LT were measured via ELISA.The mRNA levels of CCL19/CCL21 in AS hip LT were significantly higher than that in OA LT, and IHC analysis revealed a similar result. Exogenous CCL19/CCL21 treatment did not affect the proliferation of ligament fibroblasts but significantly up-regulated the expression of bone markers, including ALP and OCN, and the key regulators Runx-2 and osterix. In addition, the serum levels of CCL19/CCL21 were apparently elevated in AS patients compared to healthy controls (HC), and the expression of the two chemokines correlated significantly in AS patients.CCL19 and CCL21, two chemokines displaying significantly associated expression in serum, indicating a synergistic effect on AS pathogenesis, may function as promoters of ligament ossification in AS patients.The online version of this article (doi:10.1186/1471-2474-15-316) contains supplementary material, which is available to authorized users. Ankylosing spondylitis (AS) is the representative rheumatic disease of seronegative spondyloarthropathy and is characterized primarily by recurrent inflammatory back pain and bilateral sacroiliitis . Hip ankCCL19, CCL21 and their corresponding receptor CCR7 are described to be crucially involved in the adaptive immune system, their primary role being the migration of monocytes/macrophages, mature dendritic cells (DCs) and naive T cells to lymph nodes –9. StudiThe effects of these two chemokines on proliferation, adhesin and/or integrin avidity, invasiveness, endocytosis, differentiation and survival have been comprehensively summarized in a prospective review .Numerous studies have revealed their respective roles in immune diseases, such as rheumatoid arthritis (RA), eosinophilic pneumonia, breast cancer and acquired immune deficiency syndrome (AIDS) –18. A reIn this study, we preliminarily focus on AS patients experiencing severely limited hip activity . First, we detected higher expression of CCL19, CCL21 in LT and ligament fibroblasts. No significant effect on LT fibroblast proliferation was detected upon exposure to these two chemokines. Next, we examined the roles of CCL19/CCL21 in the osteogenic potential of AS ligament fibroblasts. The expression of the bone markers ALP, OCN, Runx-2 and Osterix were up-regulated by CCL19/21 simulation. However, IBSP and COL1 expression was not seen up-regulated. Serum CCL19/CCL21 levels are closely associated, and CCL19 expression appeared to correlate to the Visual Analogue Scale (VAS) Pain Score and disease duration, whereas CCL21 expression did not display this correlation. Our result reveals a novel role of CCL19/CCL21 in ligament ossification and may provide useful evidence for further investigation in AS patients.Patients meeting the modified New York criteria  for AS aThe LT was fully shredded using RNase-free scissors and transferred to a 1.5 ml RNase-free EP tube. A total of 1 ml of TRIzol (Invitrogen) was used for no more than 100 mg of LT. TRIzol was used according to the manufacturer’s instructions. Total RNA was reverse-transcribed to cDNA using a ReverTra Ace qPCR RT Kit . The transcripts were stored at −80°C until further use. For RT-PCR, SYBR Green Real Time PCR Master Mix  was used in a total volume of 20 μL. The primer sequences are listed in Table 2O2 for 5 minutes, followed by blocking with goat serum for 30 minutes at 37°C. The primary antibody rabbit anti-human CCL19 , rabbit anti-CCL21 , rabbit anti-CCR7 , or an isotype-matched irrelevant antibody (R&D Systems) was individually added to the sections at the proper concentration. Following three washes with PBS, the slides were incubated for 30 min in the secondary antibody  at 37°C. The slides were washed 3 times with PBS and then stained with DAB for 5 minutes and hematoxylin for 45 s, followed by rinsing with tap water for 5 minutes. Finally, the slides were processed via alcohol gradient dehydration, xylene immersion and resin blocking. Three fields from each slide were randomly selected, and tissue staining was analyzed using Image-Pro Plus 6.0 software. The scored data were pooled, and the mean density value ± standard deviation (SD) was calculated for each group.The LT samples were immediately fixed using 4% formaldehyde and embedded in paraffin. The samples were deparaffinized in xylene for 20 minutes at room temperature, followed by rehydration using an alcohol gradient. Antigens were unmasked by first incubating the slides in 0.01 mol/L boiling citrate buffer at pH 6.0 for 15 minutes and then cooling at room temperature. Endogenous peroxidase activity was blocked via incubation in 3% H3 tissue blocks. The tissue blocks were centrifuged twice in 15 ml centrifuge tubes in 10 ml of PBS at 1000 r/min. Next, the tissue blocks were digested in 5 ml of PBS and 500 μl of collagenase I  for 5 h at 37°C. Then, 5 ml of 0.25% trypsin was added, and the samples were mixed and incubated at 37°C for 2 minutes. The samples were filtered using 200 mesh filters, and the filtrate was centrifuged at 1000r/m for 5 minutes. The supernatant was discarded. Then, 5 ml of DMEM/high glucose culture medium supplemented with 100 U/ml penicillin , 100 μ g/ml streptomycin  and 12% FBS was added to the centrifuge tube. The samples were mixed by pipetting and transferred to culture flasks in a humidified 5% CO2 incubator at 37°C The passages from 3 to 8 were used. For osteogenic differentiation, the fibroblasts were cultured to confluence and then incubated in DMEM/high glucose medium containing 100 nM dexamethasone, 50 μg/ml ascorbic acid, 10 mM of β-glycerophosphate [The previously described method for primary cell isolation was mentioned performed  with somhosphate , 24 and 6 cells were stained at room temperature via incubation in an anti-CCR7 pAb  or an irrelevant IgG2a isotype control at a concentration of 0.025/ml for 90 min. After two washing and centrifugation steps, the cells were incubated in a PE-conjugated goat-anti-rabbit IgG pab  for 40 min. Then, the cells were immediately analyzed via flow cytometry, and the data were calculated using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences).The expression of CD90 (Biolegend) was identified as a molecular marker of fibroblasts based on FACS analysis as previously described , 26. The3 cells were seeded in a volume of 100 μl DMEM on each well of a 96-well plate. A range of concentrations of CCL19/CCL21  was added to the medium, and the cells were cultured for 48 h. Mesangial (M) cells, which display a proliferative response upon exposure to CCL19 [The proliferation of fibroblasts was examined via a cell counting kit-8 (CCK-8 kit) assay according to the manufacturer’s instructions. Briefly, approximately 2 × 10to CCL19 , were ki6 fibroblasts from one AS LT were seeded on 6-well plates. Five groups were assigned: osteogenic differentiation medium as a positive control, CCL19 . For statistical comparisons of two groups of samples, the Mann–Whitney U-test was used. Alternatively, comparisons of continuous variables were performed using Student’s two-tailed t-test using SPSS 17.0 software; p < 0.05 was considered to be significant. The relative gene expression levels were determined according to the 2We first investigated the expression of CCL19/CCL21 in AS and OA hip LT via real-time PCR and IHC. RT-PCR analysis revealed significantly increased transcriptional expression levels of CCL19/CCL21 , function (BASFI), disease activity (BASDAI) and global assessment  in AS patients. The serum CCL19/CCL21 levels were significantly higher in AS patients than in OA patients and healthy controls (HC) and were higher in OA patients than in HC Figure  in AS paIn this study, we detected increased expression of CCL19/CCL21 in AS hip LT. IHC staining of CCL19/CCL21, as well as their distribution, indicate that vascular endothelial cells and fibroblasts may represent the primary source of CCL19/CCL21. However, in vitro cultured AS ligament fibroblasts secreted only a small amount of CCL19 and no CCL21. This indicates that other cells, such as vascular endothelial cells, are likely the source of CCL19/CCL21. Moreover, inflammatory macrophages in enthesis and inflammatory factors, such as TNF-α, LPS, which regulate the expression of CCL19/CCL21 in synovial fibroblasts , may alsBecause fibroblasts are the principal cell type in LT and are closely associated with ligament ossification in AS patients , we examSyndesmophytes are evident in AS and, ligament fibroblasts are involved in their formation . Many inIn this study, we detected higher levels of serum CCL19/CCL21 in both AS and OA patients compared to HC. CCL19/CCL21 were reported to predict the inflammatory state in some diseases , but we There are some limitations to our study. First, pathological studies demonstrated that multiple cell types, including fibroblasts, periosteal cells, endothelial cells and stem cells, are present in LT, so other cells that express CCR7 should also be considered. Second, all of the patients in our study were admitted for joint replacement surgery and exhibited severely restricted hip activity and a poor BASFI score , and these patients do not represent the entire AS population. Third, there is a significant difference in the average age between the AS and OA groups, which may influence the results. Fourth, the precise concentrations of CCL19/CCL21 in AS LT (n = 10) and peripheral blood are similar to those of RA synovium and peripheral blood , but ossIn summary, we observed higher expression of CCL19/CCL21 in AS LT. The increased CCL19/CCL21 displayed pro-ossification potential but did not affect the proliferation of AS ligament fibroblasts. Moreover, the serum CCL19 levels significantly correlated to the serum CCL21 levels, the VAS score and the disease duration, which reveals its important role in global disease assessment of AS patients.CCL19/CCL21, two chemokine that were higher expressed in AS LT and showed a pro-ossification potential, were also elevated and closed associated in serum samples of AS patients.Common communication: Miao Min Yong.


Ictidomys tridecemlineatus) using confocal and nonconfocal split-detection adaptive optics scanning ophthalmoscopy using 790 nm light. Photoreceptor density, spacing, and Voronoi analysis are consistent with that of the human cone mosaic. The high imaging success rate and consistent image quality in this study reinforce the ground squirrel as a practical model to aid drug discovery and testing through longitudinal imaging on the cellular scale.Ground squirrels are an increasingly important model for studying visual processing, retinal circuitry, and cone photoreceptor function. Here, we demonstrate that the photoreceptor mosaic can be longitudinally imaged noninvasively in the 13-lined ground squirrel ( During induction of anesthesia, the pupils were dilated with one drop each of 2.5% phenylephrine and 1% tropicamide. An ocular speculum was used to keep the eyelids open and saline drops were applied as needed to maintain corneal hydration. Respiratory rate and heart rate were continuously monitored during anesthesia, and a warming pad was used to maintain body temperature during anesthesia and recovery.µW at 790 nm and 48 µW at 850 nm. Measurement of the eye's wavefront aberrations was performed using a custom Shack–Hartmann wavefront sensor  and S-cone opsin  overnight at 4°C. Subsequently, primary antibodies were rinsed 3 × 15 min and 1 × 1 h in cold PBTA. Corresponding secondary antibodies donkey antirabbit 488 and donkey antigoat 647 fluorochromes  were added for a final overnight incubation at 4°C. Samples were then rinsed in 4°C PBTA 3 × 15 min and 1 × 1 h prior to being mounted in 5% n-propyl gallate in glycerol on glass slides, covered with a coverslip , and sealed with nail polish.Squirrel 140301 was euthanized with an overdose of pentobarbital sodium (120 mg/kg) in August, 2015, and the eyes were prepared for retinal whole-mounts using a previously described protocol . ROIs of 80 × 80 µm were cropped from 9 areas of known distance inferior from the ONH. Photoreceptor density, photoreceptor spacing, and Voronoi analysis were calculated as indicated above.Specimens were viewed and images collected using an Olympus FluoView 1000 laser scanning confocal microscope  equipped with an UPlanFLN 40× oil immersion lens, numerical aperture 1.30 as well as a motorized stage . Optical sections were collected at 1-Simultaneous capture of confocal and split-detection AOSLO images revealed the photoreceptor outer segments as bright spots and the corresponding inner segments as mound-like structures, respectively . The int2 (n = 4). The lowest cell density was found adjacent to the ONH, ranging from 40,157 to 53,118 cells/mm2 (n = 4). Density values at other locations agreed with the previously described photoreceptor topography as illustrated in ex vivo whole-mount studies (Long & Fisher, Imaging at several superior–inferior eccentricities relative to the ONH revealed peak photoreceptor density in the visual streak ranging from 84,025 to 93,668 cells/mmµm (Average intercell spacing ranged from 3.61 to 5.34 µm . Photoreµm  and 5C.2, with the lowest cell density of 33,394 cells/mm2, adjacent to the ONH (µm (ex vivo retinal whole-mount ROIs from three locations, and their corresponding Voronoi cells.Retinal whole-mount staining in one 13LGS revealed values consistent with those obtained with AOSLO imaging. Photoreceptor density in the visual streak ranging from 84,340 to 99,638 cells/mm the ONH . Average ONH (µm . The pac ONH (µm . Fig. 6 Ground squirrels, including the species studied here, are increasingly being used for studies of visual anatomy and retinal circuitry because they are diurnal rodents, relying overwhelmingly on cone-mediated vision as a primary sense for survival. Here we apply noninvasive AOSLO to this cone-dominant mammal, and demonstrate the practicality of the 13LGS in studying living cone photoreceptors.ex vivo whole-mounted California ground squirrel retina (Long & Fisher, in vivo data (in vivo and ex vivo at the same retinal location, and taking individual animal eye axial length into consideration when scaling the AOSLO images is needed to validate in vivo photoreceptor mosaic metrics. Photoreceptor type could potentially be determined by size differences, given that S-cones are slightly larger than M-cones, while rods are slightly smaller (Ahnelt, The photoreceptor mosaic metrics in this study are consistent with previous topographical reports in the California ground squirrel (Long & Fisher, ivo data , althoug Ahnelt, , but to  Ahnelt, . The coa Ahnelt, ]. Geomet Ahnelt, , showing Ahnelt,  and 6C, Ground squirrels have near emmetropic optics (Gur & Sivak, The 13LGS photoreceptors seen in this study show many similarities to human parafoveal photoreceptors , includiRecently, intravitreal administration of adeno-associated virus encoding green fluorescent protein was used to characterize bipolar cell anatomy in 13LGS retina (Light et al.,


Brucella abortus strain and two Brucella suis strains isolated from bovine in Zimbabwe. These strains were selected based on their origin and data obtained when using multiplex PCR assays, then sequenced using next-generation sequencing technologies.This is a report of whole-genome sequences of a  Brucella species are small Gram-negative coccobacilli that cause the zoonotic disease brucellosis. The disease has a negative impact on global economy and public health as it causes substantial losses of livestock and affects the livelihood of communities relying on their livestock for survival. Currently eight terrestrial species, Brucella abortus, B. melitensis, B. suis, B. canis, B. ovis, B. neotomae, B. inopinata, and B. microti, as well as two marine strains, B. ceti and B. pinnipedialis, are recognized  paired-end sequencing technology. A Nextera DNA sample preparation kit (Illumina) was used for the sequencing library preparation and DNA fragments in the 500- to 1,000-bp range were selected. The quality of the data was checked with FastQC. After trimming and merging the overlapping sequence reads, de novo sequence assembly was performed using Abyss-pe version 1.3.6 , ZW046 (batch number 46 at CVL), and ZW053 (batch number 53 at CVL), that were isolated from bovine in Zimbabwe. Isolates were selected on the basis of the data obtained with multiplex PCR assays (Bruce-ladder , and AMOon 1.3.6  with k-mon 1.3.6 . The ordon 1.3.6 . Totals in 9-941  and all CP009094; CP009095; CP009096; CP009097; CP009098; CP009099.Genome sequences of chromosome 1 and 2 for ZW043, ZW046, and ZW053 are available in GenBank under the following respective accession no.:


Here we describe a dynamic programming approach for model-free sequence comparison that incorporates high-throughput chemical probing data. Based on SHAPE probing data alone, ribosomal RNAs (rRNAs) from three diverse organisms – the eubacteria E. coli and C. difficile and the archeon H. volcanii – could be aligned with accuracies comparable to alignments based on actual sequence identity. When both base sequence identity and chemical probing reactivities were considered together, accuracies improved further. Derived sequence alignments and chemical probing data from protein-free RNAs were then used as pseudo-free energy constraints to model consensus secondary structures for the 16S and 23S rRNAs. There are critical differences between these experimentally-informed models and currently accepted models, including in the functionally important neck and decoding regions of the 16S rRNA. We infer that the 16S rRNA has evolved to undergo large-scale changes in base pairing as part of ribosome function. As high-quality RNA probing data become widely available, structurally-informed sequence alignment will become broadly useful for de novo motif and function discovery.Discovery and characterization of functional RNA structures remains challenging due to deficiencies in  Despite the clear functional importance of structure in RNA molecules, it remains very difficult to correctly identify and annotate similar RNA structures because their sequences are often poorly conserved even for RNAs that form very similar higher-order structures. A solution is to use a metric that identifies structural motifs but this, too, is difficult because RNA structure modeling based on sequence alone is generally not very accurate. In this work, we use SHAPE chemical probing to obtain model-free information about RNA structure and then exploit this information to align RNAs by sequence. We show for ribosomal RNAs that sequence alignments based on SHAPE experimental information alone are as accurate as those that actually use sequence information. In addition, we identify regions in the 16S ribosomal RNA that form conserved secondary structures that are different from currently accepted models. These differences, rather than being errors, reveal sites of conformational flexibility that may underlie mechanistic functions in ribosome assembly and translation regulation. We anticipate that structure-informed sequence alignment and structure modeling will become broadly useful tools in RNA function analysis. RNA is a central participant in gene expression and regulation . Howeverab initio or concurrent structure prediction. RNA chemical probing is structurally robust and is not limited by the current, relatively poor, understanding of RNA structure. The SHAPE structure probing approach . Th. Th14]. E. coli to C. difficile and to H. volcanii, respectively; whereas, conventional sequence identity-based alignments using the Needle algorithm had sensitivities of 84% and 72%, respectively. For 23S rRNA, SHAPE-based alignments performed well but not quite at the level of sequence-based alignment  and SIVcpz strain MB897  aligned with a sensitivity of 97% relative to extensively manually curated and hand-annotated alignments (see following companion article in this issue).Pairwise SHAPE-based alignments were also used to generate multiple sequence alignments . AlignmeBoth sequence comparisons and SHAPE data have been successfully used to direct RNA secondary structure modeling . Given tThe SHAPE-defined sequence alignments were incorporated as arguments for secondary structure modeling using the RNAalifold algorithm in the Vienna RNA package –22. RNAaE. coli 16S, 70%; C. difficile 16S, 64%; H. volcanii 16S, 39%; E. coli 23S, 46%). This suggests that these nucleotides are weakly base paired or form alternate structures under the experimental conditions used in this work.Structure models were generated for 16S and 23S ribosomal RNAs  Figs. ThE. coli and C. difficile 16S rRNA predictions improved  undergo a conformational change during conversion of the 30S subunit from an inactive to active conformation  and are  and h44 inset). Given that these alternate structures occur in and near the codon-anticodon decoding site, we propose that these structures form a switch involving low energy base-pairing rearrangements important for regulation of translation.Strikingly, all regions that structure-directed alignment suggests form alternate structures in the 16S rRNA are located close to each other in three-dimensional space, in the neck of the intact small ribosomal subunit , inset. The strategies developed here allow for highly accurate, structure-informed alignments of large  and likeE. coli RNA (DH5α strain) was prepared as described . The supernatant was discarded, and the pellet was allowed to air-dry for 5 minutes. To lyse the cells, 1 mL TRIsure (Bioline) was added to the pellet. The resultant mixture was incubated at room temperature for 5 minutes. The mixture was then transferred to a vial containing 250 μL 0.1-mm glass beads. Cells were lysed using a bead beater over two 90 second pulses, with cells held on ice between pulses. The resultant mixture was extracted with 200 μL chloroform, and the aqueous layer was extracted three times with phenol (pH 8.0):chloroform:isoamyl alcohol (25:24:1), followed by three extractions with chloroform. The RNA-containing solution was exchanged for folding buffer (50 mM Hepes (pH 8.0), 200 mM potassium acetate (pH 8.0), and 5 mM MgCl2) using a pre-equilibrated gel filtration column .S medium  at 37°C d 5% H2)  to an ODH. volcanii cells (strain DS70) were grown to an OD600 of 0.8 and collected by centrifugation . Cells were lysed by incubation in low salt solution . Following lysis, this solution was extracted three times with phenol (pH 8.0):chloroform:isoamyl alcohol (25:24:1), followed by three extractions with chloroform. The RNA-containing solution was exchanged for folding buffer  using a pre-equilibrated gel filtration column .Growth medium was prepared by bringing 600 ml 30% salt solution , 5 g bacteriological peptone , and 1 g yeast extract  to 1 L with deionized water. 2] was added to a concentrated RNA solution  to a final volume of 90 μL. The RNA solution was incubated at 37°C for 30 minutes. Following incubation, 10 μL DMSO (no-modification control) or 10 μL 100 mM 1M7 in DMSO (native 1M7-modified sample) was added to the RNA solution. The RNA solution was then incubated at 37°C for 3 minutes. For the denatured control, 25 μL 4× denaturing control buffer  and 50 μL deionized formamide were added to a concentrated RNA solution , and deionized water was added to a final volume of 90 μL. This solution was held at 95°C for 1 minute, and then 10 μL 100 mM 1M7 in DMSO was added; the combined solution was incubated at 95°C for 1 minute.Determination of SHAPE reactivity by SHAPE-MaP employs three experiment conditions: chemical modification of native RNA, chemical modification of denatured RNA, and a no-modification control. All chemical modifications were performed using 1-methyl-7-nitroisatoic anhydride (1M7) . Chemica2], incubated at 94°C for 4 minutes, and then transferred immediately to ice. Fragmented RNA was purified using a G-25 column (GE) with elution into 1× TE . Following fragmentation, reverse transcription was performed using a 20-μL aliquot of fragmented RNA and 2 μL random DNA nonamers (200 ng/μL). The solution was incubated at 65°C for 5 minutes and then placed on ice. To this solution, 7 μL reaction buffer , 4 μL 60 mM MnCl2, and 5 μL water were added. The solution was pre-incubated at 25°C for 2 minutes prior to adding 2 μL Superscript II (Invitrogen). The reaction was incubated at 25°C for 10 minutes, 42°C for 180 minutes, and 70°C for 15 minutes. Following reverse transcription, the RNA was purified using a G-25 column (GE) with elution into 1× TE.After modification, all three samples were purified by affinity chromatography  with elution into 22 μL buffer. To prepare sequencing libraries, the purified RNA samples were first fragmented; 20 μL of RNA solution was combined with 30 μL fragmentation buffer [250 mM Tris (pH 8.3), 375 mM KCl, 15 mM MgClThe cDNA was converted to a double-stranded DNA library with Illumina platform-specific sequence tags. First, 40 μL of the purified reverse transcription product was used in an 80-μL second-strand synthesis reaction . The product of the second-strand synthesis reaction was purified  and eluted into 12 μL elution buffer. A 10-μL aliquot of the purified DNA solution was then used in a 50-μL end repair reaction . Following end repair, the DNA was purified  and eluted into a final volume of 30 μL 1× TE.To incorporate Illumina platform-specific sequence tags, a dA-tailing reaction was used to incorporate a single-nucleotide overhang at the 3′ ends of the double-stranded DNA. A 15-μL aliquot of purified DNA from the end repair step was used in a 20-μL dA-tailing reaction . Illumina sequences were incorporated using a ligation step with Illumina iAdapters (prepared in house). Immediately following completion of the dA-tailing reaction, 7.5 μL of 5× reaction buffer , 2.5 μL 125 nM DNA adapter, 3.75 μL Quick T4 DNA Ligase (New England Biolabs), and 3.75 μL water were added to the dA-tailing reaction mix. The ligation reaction was then incubated at 20°C for 15 minutes. The ligation reaction was purified twice  with final elution into 20 μL 10 mM Tris (pH 8.0).Illumina libraries were prepared using emulsion PCR , 34. TheThe concentrations of sequencing samples were determined by Qubit High Sensitivity DNA fluorescence assays (Life Technologies) and High Sensitivity DNA Bioanalyzer assays (Agilent). Each sample was diluted to 2 nM and pooled. The pooled library was sequenced using an Illumina MiSeq (300 cycles—PE kit). Sequences were aligned and mutation events counted using the SHAPE-MaP analysis pipeline . SHAPE rx and y began with declaration of matrices D, P, and Q. Each matrix had dimensions m by n, where m and n were the lengths of sequences x and y plus 1, respectively. Considering alignment of (x0…xi) and (y0…yj), Di,j corresponds to the score associated with alignment, Pi,j corresponds to the score associated with alignment that ends with a gap in x, and Qi,j corresponds to the score of alignment that ends with a gap in y. To initialize each matrix, D0,0 was set to 0, Di,0 was set to GOP + i × GEP, and D0,j was set to GOP + j × GEP, where GOP and GEP are the gap opening penalty and gap extension penalty, respectively; P0,j and Qi,0 were set to arbitrarily large negative numbers. Every other cell in the matrix was populated by the following recursion, where s describes a pair-wise comparison score, and xi and yj are the SHAPE values of each sequence at i-th and j-th positions:m and b:b in the recursion, where x'i and y'j were the base identities at positions i and j in sequences x and y, respectively:b is described by the following equation with parameters MATCH and MISMATCH.SHAPE-based alignment was based on the Gotoh algorithm with affine gap penalties . SHAPE-btion see  is descri,j, representing the 3′-most position of the alignment. The next position in the alignment was found using the following comparison: if Di,j was equal to sum of Di-1,j-1 and the score of the pairwise comparison between xi-1 and yj-1, the next position was aligned nucleotides xi-1 and yi-1; if Di,j was equal to Pi,j, the next position was a gap in sequence y; if Di,j was equal to Qi,j, the next position was a gap in sequence x. The trace-back operation was finished when a position was encountered where i = 0 or j = 0.Following population of the matrices by recursion, a trace-back operation was used to find the optimal alignment. The trace-back operation began at position m and b, base-identity scoring parameters MATCH and MISMATCH, and gap penalty parameters GOP and GEP were optimized by exhaustive search over the 16S and 23S rRNA aligned sequence pairs. The best parameter set was then selected based on the average sensitivity across all alignments. In experiments considering only SHAPE values, m = -2, b = 2, GOP = -5, and GEP = -0.25. Scoring function parameters were preserved across SHAPE-only and combined SHAPE and base identity alignments, but GOP and GEP parameters were reoptimized for alignments considering both SHAPE reactivity and base identity. When both SHAPE and base identity were considered, m = -2, b = 2, GOP = -6, GEP = -1, MATCH = 2, and MISMATCH = -2.The SHAPE scoring parameters E. coli and C. difficile and E. coli and H. volcanii were obtained for both 16S and 23S rRNAs. RNA sequence alignments generated in this work were then evaluated by comparison to these alignments. Sensitivities were calculated as the percentage of matched nucleotides in the CRW alignments found in a given alignment.From multiple sequence alignments on the CRW, pairwise alignments between Multiple sequence alignments were generated using T-Coffee . First, To establish a base-line for comparison, sequence-only secondary structure predictions were performed with RNAfold from the Vienna RNA package with a maximum base paring distance of 600 nucleotides . Secondahttp://www.tbi.univie.ac.at/RNA.Multiple sequence alignments were used as input for RNAalifold from the Vienna RNA package . SHAPE dx and y and any base pair in the predicted model at positions xʹ and yʹ were required to meet the following criterion:Secondary structure models were evaluated by calculating sensitivity (sens) as the percentage of base pairs from the CRW covariation model found in predicted structures and by calculating positive predictive values (ppv) as the percentage of predicted pairs found in the covariation model. It should be emphasized that these reference structures are themselves experimental models, and base pairs in these models may show slight local rearrangements in terms of base-pairing partners , 37. To S1 FigE. coli nucleotides are colored by their SHAPE reactivities .This model was constrained by 23S rRNA consensus base pairs based on a SHAPE-based sequence alignment. Predicted pairs that exactly match the accepted covariation model  are show(TIF)Click here for additional data file.S2 FigFull legend is given in (TIF)Click here for additional data file.S1 Dataset(ZIP)Click here for additional data file.S1 Software(ZIP)Click here for additional data file.