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Functional blockade of α5β1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells
Luciano Vellón1,
Félix Royo1,
Rune Matthiesen2,
José Torres-Fuenzalida3,
Alicia Lorenti4 and
Luis A Parada1, 5Email author
© Vellón et al; licensee BioMed Central Ltd. 2010
Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration. Here, we investigated the genomic events occurring during this process induced by functional blockade of α5β1 integrin in liver progenitor cells.
Cells treated with a specific antibody against α5β1 integrin exhibited cell spreading and scattering, over-expression of liver stem/progenitor cell markers and activation of the ERK1/2 and p38 MAPKs signaling cascades, in a similar manner to the process triggered by HGF/SF1 stimulation. Gene expression profiling revealed marked transcriptional changes of genes involved in cell adhesion and migration, as well as genes encoding chromatin remodeling factors. These responses were accompanied by conspicuous spatial reorganization of centromeres, while integrin genes conserved their spatial positioning in the interphase nucleus.
Collectively, our results demonstrate that α5β1 integrin functional blockade induces cell migration of hepatic progenitor cells, and that this involves a dramatic remodeling of the nuclear landscape.
Wound Healing Assay
Transwell Assay
Cell Scattering
Hepatic Progenitor Cell
Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration. Metastasis seems to arise from the same genetic program that instructs cells to detach, adhere, and migrate through extracellular matrices, crossing tissue boundaries and escaping death due to an unsuitable tissue context [1]. The Hepatocyte Growth Factor/Scattering Factor 1 (HGF/SF1) is the paradigmatic example of a molecule that induces cell scattering with optimal spatial and chronological coordination. This process takes place through a complex network of signaling pathways triggered by the HGF/SF1 tyrosine kinase receptor, Met, which includes the Grb2-Ras-Mitogen Activated Protein Kinases (MAPK), the PI-3'K, and the Signal Transducer and Activator of Transcription (STAT) cascades [2]. Integrins are thought to be essential for cell migration and penetration of the basement membrane, in addition to playing a major role in cellular adhesion to the extracellular matrix (ECM) and certain cell surface proteins. These adhesion receptors also convey a series of mechanical and biochemical extracellular stimuli in signaling cascades that favor cell migration and proliferation [3, 4]. Interestingly, growth factor and integrin-emanating signals can interact to promote cell migration. For instance, c-Met signaling can be modulated by the α6β4 integrin when co-expressed on the cell surface [5], and HGF/SF1, conversely, can regulate the adhesive status and aggregation rate of αvβ3 integrin in epithelial cells [6].
The genome is highly organized within the cell nucleus [7]. Indeed, chromosomes and genes exhibit cell type specific preferential positioning, and this non-random distribution of genetic elements in the interphase nucleus is related to genome function [8]. Genome organization has been broadly investigated, in particular during cell differentiation and tumorigenesis. For example, the stem cell specific genes Nanog and Oct4 acquire differential positioning in the nucleus as their expression levels change during differentiation of human embryonic stem cells [9]. Additionally, changes in the spatial distribution of cancer related genes have been shown in a cell-model of breast cancer during induced malignant evolution [10]. However, much still remains to be elucidated regarding the genomic events associated with cell migration.
MLP29 cells are murine liver progenitor cells that respond to HGF/SF1 treatment with a well characterized sequence of events that resemble the cell invasion program, including cell scattering, migration, proliferation, and tubular morphogenesis [11]. Considering that α5β1 integrin is one of the main ECM receptors of hepatocytes, and that changes in integrin-mediated contacts between the cell and ECM are necessary for cell migration [12], we speculate that functional blockade of α5β1 integrin may result in cell migration, and that the changes in the cell microenvironment will be sensed and transduced into specific nuclear responses. Here, we investigated the molecular and cellular genomic events associated with cell migration triggered by the functional blockade of α5β1 integrin in liver progenitor cells.
Integrin expression profile and adhesion properties of hepatic progenitor and HCC cells
We first assessed the cell membrane expression of β1 integrins in MLP29 and Hep16 cells by flow cytometry and immunofluorescence analyses. Cytometric analyses showed that this integrin sub-unit is expressed in MLP29 and Hep16 cells. However, flow cytometric detection of β1 levels demonstrated significantly higher levels of β1 integrin-types in MLP29 cells compared to Hep16 cells, used as a control because of their low β1 integrin expression levels (Figure 1A). To determine the adhesive capacity to different components of the ECM, we performed cell adhesion tests to FN, VN, LMN and COL-I using the MTT assay. HCC cells exhibited high adhesive capacity to all these components of the ECM, whereas, MLP29 cells displayed high adhesion to FN and COL-I, and low adhesion to VN and LMN (Figure 1B), thus making the MLP29 cellular model suitable for the study of β1 integrin-mediated events.
Β1-integrin expression and adhesive properties of MLP29 and Hep16 cells. (A) Quantitative analysis by flow cytometry revealed that most (90%) of cells were positive for β1 staining and that the intensity levels (Geo Mean fluorescence) of β1 expression was higher in MLP29 than in Hep16 cells. The experiment was performed three times with similar results. (B) MTT assay was used to assess MLP29 and Hep16 cell adhesion capacity to different components of the extracellular matrix, including FN, VN, LMN and COL I. To prevent nonspecific cell adhesion, plates were blocked with BSA. Cell adhesion to the different substrates was estimated by measuring the optic density at A570 nm. The data presented summarize the mean (±SD) of three independent experiments, each performed in triplicate.
α5β1 integrin functional blockade induces cell scattering and migration in hepatic progenitor cells
Considering that integrin α5β1 is one of the main ECM receptors of hepatocytes, we wondered whether specific disruption of the α5β1-mediated cell-ECM interactions would trigger invasive-like cell growth. In order to substantiate our hypothesis, MLP29 cell cultures were treated with a specific antibody against α5β1 integrin and HGF/SF1 as control, owing to its capacity to induce cell scattering and migration. By microscopic inspection, we observed that untreated MLP29 cells grow in tightly packed patches, mimicking the structure of an epithelial sheet, whereas blocking α5β1 caused the loss of cell-cell junctions and induced cell spreading throughout the culture dish, similar to the cell scattering induced by HGF/SF1. These morphological changes involved reorganization of the cell cytoskeleton, from cortical F-actin structures to parallel bundles disposed along the axis of the cellular prolongations (Figure 2A). Moreover, immunoblot and immunofuorescent detection showed that functional blockade of α5β1 integrin induced down-regulation of E-cadherin, demonstrating that effectively the process involved cell-cell dissociation (Figure 2A).
α 5 β 1 integrin functional block induced spreading and scattering of MLP29 hepatic progenitor cells. (A) Immunofluorescent detection of F-actin in MLP29 cells showed that the cells grow in packed islands, while after the treatment with a specific α5β1-functional blocking antibody the cells undergo spreading and scattering. Higher magnification (252×) of untreated and treated MLP29 cells shows the characteristic actin microfilaments reorientation associated with cytoskeleton re-organization. Immunofluorescent and immunoblot detection of E-cadherin in MLP29 cells following α5β1 functional blockade showed that the expression levels of E-cadherin decreased at the cell-cell junctions in cells treated with the specific functional blocking antibody against α5β1, which was corroborated by immunoblot analysis. (B) Representative images of the phospho-MAPKs arrays. Activation of the different members of the MAPKs family was identified by means of a key provided with the kit. MLP29 cells were treated with the α5β1 functional blocking antibody or HGF/SF1, in the presence or absence of the specific MEK inhibitor U0126. Cells were lysed and total protein (20 μg) was resolved by SDS-PAGE and analyzed by immunoblot for ERK1/ERK2 MAPK and phospho-ERK1/ERK2 MAPKs. Blots were then reprobed with an antibody for β-actin as a control for protein loading. Results are representative of at least three independent experiments.
To specifically test cell migration we performed the wound healing and the transwell assay. In the wound healing assay, the same fields on a confluent cell monolayer were pictured right after scratching (0 hours) and 20 hours later. Computational analysis of the images showed that MLP29 cells treated with the α5β1 function-blocking antibody migrated towards the scratch faster than the control cells (p < 0.005). HGF/SF1 also induced migration, but to a lesser extent (Figure 3). In the transwell assay, MLP29 cells treated with HGF/SF1 or anti-α5β1 integrin antibody were placed on top of filters coated with ECM formulations, but migration through filters was not statistically significant after 72 hours. These results suggest that different mechanisms for cell migration may be used in these two types of assays (see below). We further characterized this process by determining the expression levels of β1 and β3 integrin sub-units, which are known to be associated with the invasive capacity of several epithelial tumor types. Moreover, we assessed the level of EpCAM, AFP and CK19, all biomarkers expressed in the liver stem cell niche during liver regeneration [13]. Immunofluorescence with antibodies against these proteins showed that the expression level is higher after both treatments (Additional file 1). In addition, these changes were associated with an increase in metabolic activity (Additional file 2). Furthermore, flow cytometric analysis of DNA content showed that α5β1 functional blockade did not induce drastic changes in the progression of MLP29 cells through the cell cycle (Additional Files 3 and 4).
α5β1 integrin functional blockade induced migration of MLP29 hepatic progenitors. α5β1 functional blockade induced morphological modifications and triggered cell motility in MLP29 cells, in a manner comparable to HGF/SF1. Cell migration was calculated as the mean of six different measurements along the scratch and is expressed as percentage of the wound width covered by the migrating cells. The data presented summarize the mean (±SD) of three independent experiments.
α5β1 integrin functional blockade of hepatic progenitor cells triggers cell signaling pathways involved in cell motility
We checked the activation status of the different members of the MAPKs family in response to α5β1 functional blockade or stimulation with HGF/SF1. Using a MAPK array kit, we determined that both treatments induced the long-term activation (twenty hours) of p38 MAPK, partly matching the hyperactivation of the ERK1/2 and p38 MAPKs cell transduction cascades of the highly invasive Hep16 HCC cells (Figure 2B). Activation of ERK 1/2 MAPKs was also observed, however, the peak of this kinase phosphorylation occurred two hours after α5β1 integrin functional blockade. These effects were reversed by the MEK pharmacological inhibitor U0126 (Figure 2B).
Gene expression profile of migrating liver progenitor cells
To characterize the global genome transcriptional response to functional blockade of α5β1 integrin, we performed GeneChip array analyses (Affimetrix®, Mouse430A_2) of well annotated genes. The data from three independent experiments were collectively analyzed using the R package "AFFYLMGUI" (http://www.bioconductor.org). Each experiment included RNA samples from MLP29 cells subjected to functional blockade of α5β1 integrin, HGF stimulation, and untreated control cells. A total of 2671 genes changed their expression level during cell spreading induced by α5β1 functional blockade, whereas only 191 genes changed their transcriptional status after HGF/SF1 stimulation, when a p-value <0.05 was considered significant in the statistical comparisons between treated and control cells. Because genes are unequally distributed on chromosomes (gene density), we calculated the ratio between the number of differentially expressed genes per chromosome and the total number of genes mapping to the same chromosome (http://www.ensembl.org/index.html). This analysis showed that de-regulation of genes is evenly distributed throughout the genome. Gene ontology (GO) enrichment analysis demonstrated that during cell migration induced by α5β1 functional blockade, more genes were up-regulated than down-regulated. Most of the up-regulated genes were within the Biological Processes (BP) and Cellular Components (CC) categories (Table 1; Additional File 5). The number of up-regulated genes, found in many categories, can be narrowed down to seven, and up-regulation in the expression of the Transforming Growth Factor-β2 (Tgfb2) and Hypoxia-Induced Factor 1 alpha (Hif1a) genes appear to be involved in many of the observed cellular responses (Table 2 and Additional file 6). Down-regulations occurred more frequently within the Molecular Function (MF) compartment, specifically genes involved in the regulation of chromatin stability, such as Sfpq, Dido1 and Sap18 (Additional file 7).
Most significant up-regulations in MLP29 cells upon α5β1 integrin functional blockade
Biological Processes (BP)
GO description
# array
#DEGs
P(GO)
1,15E-05
Aromatic compound metabolic process
Response to external stimulus
Regulation of biological process
Response to stress
0,000407675
Biosynthetic process
Cellular Components (CC)
Extracellular region
Extracellular space
Most significantly up-regulated genes (p < 0.0005) in MLP29 cells upon α5β1 integrin functional blockade
GO-description
Tgfb2+
Sfpq- Dido1- Ncoa5- Sap18- Traf3- Hif1a+ Maff+ Bnc1+ Tgfb2+ Gpx1+
Regulation of biological processes
Sfpq- Hif1a+ Tgfb2+ Gpx1+
Mgat2- Ncoa5- Nmt1- Hif1a+ Tgfb2+
Ide- Lman2- Vnn3+ Tgfb2+ Slpi+
Next we assessed the transcriptional response of gene sets involved in cell adhesion and migration. The results are presented in Figure 4 as color-encoded plots in which a p-value close to 1 indicates statistically significant higher mRNA levels of all genes included in the set, and a p-value close to 0 indicates significantly lower levels. The functional blockade of α5β1 induced up-regulation of several sets of genes involved in cell adhesion, whereas the response to HGF/SF1 stimulation was less pronounced, similar to untreated cells (Figure 4A). The gene sets involved in cell migration exhibited a pattern of gradual change in expression levels among the three types of samples. As expected, untreated cells exhibited significantly lower expression levels of these genes compared to the other two groups of treated cells, whereas the treatment with HGF/SF1 induced a slight increase, and α5β1 functional blockade resulted in a more pronounced up-regulation of cell-migration genes (Figure 4B), among them β1 and β3 integrins. However, before regarding the results obtained for HGF/SF1 as not significant (p-value 0.05 < p < 0.95), it must be recognized that the permutation analyses were done across all samples, including the α5β1 inhibition which displays much stronger regulation of cell migration genes. Most important, these results clearly demonstrate that α5β1 functional blockade triggers invasive-like cell migration.
Gene expression profiling of MLP29 hepatic progenitor cells after α 5 β 1 integrin blockade and HGF/SF stimulation. (A) (B) Plots showing the changes in the expression of gene sets involved in cell adhesion and migration, respectively. Data from three independent experiments were pooled and subjected to permutation analyses to assess the expression level changes of gene sets obtained from the Molecular Signatures Database. The resulting data are presented as color-encoded plots in which a p-value close to 1 indicates significant up-regulation, and a p-value close to 0 indicates a significant down-regulation.
Distinct expression profile of chromatin-remodeling and transcription factors
A total of 32 genes, belonging to the Smarc (SWI/SNF-related, matrix-associated, actin-dependent regulators of chromatin) family of chromatin remodeling factors were represented in the array and the analysis of their expression data revealed that functional blockade of α5β1 resulted in a larger number of differentially regulated genes than HGF/SF1 stimulation (Figure 5). We also analyzed the expression level of transcription factors, and our results demonstrate drastic changes in the expression level in integrin inhibited cells, whereas stimulation with HGF/SF1 presented a more restricted response (Figure 5). More importantly, the transcriptional responses to these two treatments are clearly distinct, indicating that the genomic effects exerted by the disruption of cell-ECM interactions differ from those induced by soluble regulatory factors (HGF/SF1), possibly because cells also apply traction to their integrin receptors, in addition to the integrin ligation-induced signaling.
Changes in the expression level of genes encoding chromatin remodeling and transcription factors in MLP29 cells during migration. (A) Changes in the expression level of genes encoding chromatin remodeling factors were plotted according to their level of significance as a color-encoded map, in which a p-value close to 1 indicates significant up-regulation, and a p-value close to 0 indicates a significant down-regulation. (B) Changes in the expression level of transcription factors following the treatment with α5β1 integrin blocking antibody or with HGF/SF1 for 24 hours.
Cell scattering is associated with nuclear architecture remodeling
To determine whether cell migration involves spatial genome reorganization, we analyzed the distribution of all centromeres by fluorescent in situ hybridization (FISH) using a pan-centromeric DNA probe. By qualitative inspection of microscopic images, we observed that centromeres were distributed throughout the nucleus, but forming the typical aggregates composed by the centromeres of two or more chromosomes (chromocenters) (Figure 6). For quantitative analysis, we determined the volume occupied by these nuclear domains in control, anti-α5β1 and HGF/SF1-treated MLP29 cells. The mean volume of the chromocenters decreased drastically following α5β1 functional blockade (~6 μm3/chromocenter) or HGF/SF1 stimulation (~4 μm3/chromocenter) in comparison to untreated control cells (~8 μm3/chromocenter), while the average nuclear volume remained unchanged (~250 μm3) (Figures 7A).
Change of the nuclear organization of MLP29 hepatic progenitor cells. Quantitative FISH was performed with a pan-centromeric probe. Computational analysis of the spatial organization of chromocenters showed marked variations in their average volume, whereas the nuclear volume remained unchanged in MLP29 cells following α5β1 blockade or stimulation with HGF/SF1. 3D computational reconstruction of the image stacks and quantitative analysis showed that the average fluorescence intensity of the Me3H3K9 foci remained unaltered. However, the number of foci increased concomitantly with a reduction of their volume after both types of treatments. Immunofluorescent detection of Ac3K9/14 H3, on the other hand, revealed that cell scattering is associated with an increase in the level of acetylation of histone H3 at K9.
Change of the nuclear organization of MLP29 hepatic progenitor cells. (A) Bar graphs summarize the mean (±S.E.) volume of at least 1000 chromocenters analyzed and the average nuclear volume. (B) Bar graphs summarize the number per nuclei and the mean volume (±S.E.) of at least 500 Me3H3K9 foci analyzed. (C) The level of Ac3K9/14H3 expressed as the mean (±S.E.) of the fluorescence intensity of 100 nuclei analyzed for each treatment.
To further explore the nuclear effects of α5β1 block or HGF/SF1 stimulation we analyzed the pattern of histone H3 trimethylated at lysine 9 (Me3K9H3) and acetylated at lysine 9 and 14 (Ac3K9/K14H3) by immunofluorescence analysis. Mouse cells have the majority of Me3K9H3 localized to prominent clusters of pericentromeric heterochromatin [14] and, consistent with previous reports, microscopic inspection of MLP29 cell preparations revealed that Me3K9H3 localized to chromocenters (Figure 6). However, detailed 3D analysis revealed that α5β1 functional blockade increased the average number of foci per nucleus, and this was concomitant with a decrease in their volume (Figure 7B). The changes in centromeres and Me3K9H3 foci spatial organization were not related to alterations in the nuclear volume due to technical artifact (mean nucleus volume ~250 μm3) or variation of the mean fluorescence intensity.
Since acetylated histone H3 exhibited a homogeneous nuclear distribution (Figure 6), rather than a focal pattern, we analyzed the levels of Ac3K9/K14H3 by flow cytometry and western blot, in addition to immunofluorescence, in order to determine the protein expression level and measure the mean intensity of Ac3K9/K14H3 more accurately. Interestingly, α5β1 functional blockade or HGF/SF1 stimulation significantly increased the levels of Ac3K9/K14H3 (Figure 7C; Additional files 8 and 9). Altogether, these observations indicate that alterations of the α5β1-mediated cell-ECM interactions during cell migration influence the overall spatial and functional organization of the nucleus.
Positioning of genes encoding integrins during cell scattering
Taking into account that α5β1 functional blockade and HGF/SF1 stimulation resulted in up-regulation of the genes encoding the integrin β1 and β3 sub-units; we asked whether these genes undergo repositioning. To this end, we analyzed the nuclear radial position of each gene independently by interphase 3D DNA-FISH and the results from 50-70 nuclei per experiment were plotted on cumulative distribution graphs. The preferential radial position of Itgb1 and Itgb3 was between 80% and 100% of the relative radius in control cells. Pair-wise comparisons by Kolmogorov-Smirnov statistical tests, demonstrated that these genes did not change their position upon α5β1 functional blockade or HGF/SF1 stimulation (p > 0, 05) (Figures 8A and 8B). Considering the increase in the expression of β1 and β3 integrins associated with cell scattering, these results suggest that the Itgb1 or Itgb3 radial positions do not depend on gene activity.
Radial position of integrin genes during migration of MLP29 cells. Three-dimensional FISH analysis was performed with BAC clones for the Itgb1 or Itgb3 loci and the position of fluorescent signals was determined using computational programs for image analysis. Left panels, 3D reconstruction of MLP29 cell nuclei subjected to FISH analysis with a probe for the Itgb1 and Itgb3 genes. Right panel, absolute radial position of these genes in control and treated cells. Scale bar = 3 μm.
Invasive cell growth occurs not only during malignancy, but also takes place under physiological conditions during embryonic development and organ formation. In the present work, we demonstrate that functional blocking of α5β1 integrin induces cell dissociation and motility, and that this program involves changes in the structural and functional organization of the genome. Cell scattering induced by functional blockade of α5β1 integrin was statistically demonstrated by the wound healing assay, but the transwell assays did not show invasive capacity. Gerlitz et al. have reported that overexpression of the C-terminal domain of the histone H1E accelerated cell migration in the wound healing assays, but inhibited cell migration as measured by the transwell assay [15]. Likewise, our results for this test are contrasting, and this may be due to the fact that the experimental procedure includes the pre-treatment of the cells with the α5β1 functional blocking antibody, which may inhibit their adhesion to components of the transwell membrane and consequently cells can not invade. Alternatively, different migration mechanisms may be used in these two assays: In the wound healing assay, the cells use mesenchymal-like polarized motility and the transwell assay implies amoeboid-like movements [15]. Cell migration was accompanied by the activation of ERKs 1/2 and p38 MAPKs cell signaling pathways and a drastic increase in metabolic status. ERK 1/2 and p38 MAPKs phosphorylation occurs during migration of different cell types in response to various growth factors such as VEGF, EGF and TGF-β, and to components of the ECM [16]. Functional blockade of α5β1 in MLP29 cells, on the other hand, was able to up-regulate the expression of the hepatic stem/progenitor cell markers EpCAM, AFP and CK19, all shown to be associated with migration of epithelial and embryonic stem cells in vitro [17]. Furthermore, EpCAM participates in intercellular and cell-ECM interactions, and is expressed during liver regeneration [18].
Gene expression profiles documented here strongly support the notion that α5β1 integrin-mediated cell migration was associated with a larger number of differentially regulated genes than the process induced by HGF/SF1. Moreover, the number of up-regulated genes was higher than the number of down-regulated genes. These data fit very well with the results obtained from human fibroblasts which exhibited extensive down-regulation of genes when seeded onto low adhesion surfaces, which reduced cell scattering [19]. Following functional blockade of α5β1 integrin or stimulation with HGF/SF1, the most significantly up-regulated genes fell into the GO categories concerning response to external stimuli, biosynthesis and cell growth, and in particular genes involved in cell adhesion and migration. We also detected drastic changes in the expression of several members of the SWI/SNF family of chromatin remodeling complex. This consist of approximately ten ATP-dependent components and are thought to regulate genome function by altering the structure of chromatin [20].
One key question in cell biology is how cells reorganize their genome in response to mechanical conditioning. We detected that migration of MLP29 cells involved morphological changes, cytoskeleton reorganization, and nuclear architecture remodeling. The FISH analyses for the β1- and β3-integrin subunits revealed that the radial position of these genes within the interphase nucleus did not change. Meaburn and Misteli analyzed the radial distribution of four genes related to cell survival, mobility and migration, namely AKT1, VEGF, ERBB2, and FGFR1 in a cell-model of breast cancer. They found that, except for VEGF, the other loci underwent repositioning during tumorigenic differentiation [10]. However, it has also been shown that positional changes of chromosome territories only occur transiently at the beginning of the G1 phase, and then arrangements are stably maintained from mid G1 to early prophase during cell cycle [21], Our results are in agreement with this, and are supported by the flow cytometric analysis of DNA content which showed that MLP29 cells did not progress through the G1 phase of the cell cycle after α5β1 functional blockade. Furthermore, Harnicarova and collaborators have reported that nuclear arrangement of the c-Myc gene and its transcripts was conserved during enterocitic differentiation of HT-29 cells [22]. In contrast to this conserved spatial positioning of β1- and β3-integrin gene loci, FISH analysis with a pan-centromeric probe revealed that the average number of chromocenters per nucleus increased, while their mean volume decreased following α5β1 functional blockade and HGF/SF1 stimulation, indicating that the spatial distribution of individual centromeres changes in response to the forces generated during cell migration. In close agreement with these results, a study on the position of centromeres of human chromosomes 3, 11 and 16 showed that the distance between centromeres decreases when fibroblasts are seeded onto matrices resembling 3D structures, compared to flat 2D surfaces, which generate different tensile forces and activate distinct signaling cascades [19]. We also found that during MLP29 cell migration, the volume of Me3K9H3 foci decreased concomitantly with an increase in their average number. These findings, although expected because Me3K9H3 localizes preferentially to pericentromeric heterochromatin, corroborate the data obtained by FISH with pan-centromeric probes demonstrating chromocenter disaggregation during cell scattering. It has been shown that progressive chromocenter clustering occurs during cell differentiation and that this is associated with increasing levels of DNA methylation [23]. Our immunostaining assays with antibodies against Me3K9H3 showed that the nuclear fluorescent intensity level was the same after functional blockade of α5β1, suggesting that cell migration may occur without changes in the methylation status of histone H3. Most important, these experiments demonstrated that the architectural changes of chromocenters associated with this program are completely opposite to those leading to terminally differentiated or quiescent cells. Thus, it appears that cell scattering involves significant spatial reorganization of the heterochromatic blocks of the genome associated with substantial changes in the expression level of genes encoding for chromatin remodeling factors.
We provide evidence that the functional blockade of α5β1 integrin in hepatic progenitor cells induces cell scattering and cytoskeleton reorganization in a manner similar to stimulation with HGF/SF1. This process is accompanied by activation of the MAPK pathway and an increase in the expression levels of the early HCC markers, Ep-CAM, AFP and CK19. Gene expression arrays showed a massive change in the expression of chromatin remodeling and transcription factors. These functional changes of hepatic progenitor cells are associated with conspicuous structural reorganization of the cell nucleus.
Cell Culture and treatments
Mouse hepatic progenitor MLP29 and mouse HCC Hep16 cells were maintained in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS) at 37°C in a humidified 5% CO2 atmosphere. For experiments, after cell plating, the old medium was replaced with fresh medium containing low FBS (0.1%) and the cells were maintained under this serum starvation condition for at least 16 hours. Then the cells were treated with 10 μg/ml of a specific function-blocking anti-α5β1 integrin antibody (clone BMB5) purchased from Chemicon-Millipore (Temecula, CA, USA) or 20 ng/ml of HGF/SF1. When appropriate, 20 μM of U0126 (ERK inhibitor) was used for 24 hours. Control experiments were carried out by adding equal amounts of DMSO, ethanol or an IgG control Ab.
Antibodies and reagents
MEK1 and MEK2 specific inhibitor U0126 was purchased from Calbiochem (San Diego, CA), dissolved in DMSO, and stored in the dark as a 10 mM stock solution at -20°C. The components of the ECM, fibronectin (FN) and vitronectin (VN) were purchased from Sigma (St Louis, MO), laminin (LMN) was purchased from Chemicon (Temecula, CA) and collagen type I (COL I) was purchased from BD Biosciences-Europe (Erembodegem, Belgium). hrHGF was purchased from Peprotech (Rocky Hills, NJ). Specific antibodies against β1 and β3 integrin subunits, CK19, Acetyl histone H3 (Lys9/14), and tri-Methyl histone H3 were purchased from Chemicon-Millipore (Temecula, CA), and E-Cadherin antibody was from R&D systems. Phospho p42/p44 MAPKinase antibody (Thr 202/Tyr204), total MAPKinase; phospho PKB/Akt (Ser 473) and total PKB/Akt antibodies were purchased from Cell Signaling Biotechnologies (Beverly, MA, USA). β-actin, Ep-CAM and AFP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Pan-centromeric STAR-FISH™ probe were purchased from Cambio (Cambridge, UK).
Sub-cellular localization of β1 integrins, Ac3K9/k14H3, Me3K9H3 and phospho-ERKs was assessed by immunofluorescence. Briefly, the cells were fixed in 4% paraformaldehyde (w/v) in PBS, rinsed in TBS and then permeabilized in 0.5% TBS-Triton X-100 (T-TBS 0.5%) for 10 minutes at RT. After blocking with 2% BSA in TBS for 10 min at RT, the cells were incubated with the primary antibody for 1 hour at RT. The detection was performed with secondary antibodies conjugated to FITC or Texas Red. To study the actin cytoskeleton and morphological changes, the cells were stained with phalloidin conjugated to Texas-red. After exhaustive washings with cold TBS, the slides were mounted with Vectashield containing DAPI (Vector Laboratories, CA, USA). Images were acquired with an Olympus BX61 epifluorescence microscope or a Leica DMIRE-2 confocal microscope.
Flow cytometric evaluation of RGD-binding integrins
MLP29 hepatic progenitor and Hep16 HCC cells were seeded and allowed to attach for 24 hours. Cells were then harvested by scraping, subsequently counted, and then incubated with antibodies against the α5β1 heterodimer and β1 integrin subunit (3 μg/500 × 103 cells/100 μl) for 1 hour at 4°C. After that, cells were washed twice with cold PBS containing 1% FBS, and incubated with a CY2-conjugated secondary antibody for 30 minutes at 4°C. Cells were then analyzed in a FACScanto II flow cytometer (Becton Dickinson). FACS Diva software (Becton Dickinson) was used for data acquisition and analysis.
Flow cytometric analysis of Ac3K9/K14H3 and DNA content
MLP29 hepatic progenitor cells were treated with the α5β1 blocking antibody or HGF/SF1 for 6 hours, and then the cells were harvested. For Ac3K9/K14H3 detection, cells were fixed in 1% paraformaldehyde for 15 minutes and stored in 70% ethanol at -20°C. After fixation, cells were permeabilized with PBS-0.25% Triton X-100 for 10 minutes, washed with cold PBS-1% FBS and incubated with an anti-Ac3K9/K14H3 antibody for 2 hours at room temperature. Then, the samples were washed twice and incubated with a FITC-conjugated secondary antibody. For DNA staining, the cells were washed again and treated with RNAse A (0.2 mg/ml) in PBS at 37°C for 20 minutes. Propidium Iodide (20 μg/ml) was added to the cell suspension, and incubated 30 minutes at RT protected from light. FACSDiva software (Becton Dickinson) was used for data acquisition and analysis.
Cell adhesion studies
Cell adhesion was assessed by the MTT assay using the CellTiter 96® Non-Radioactive Cell Proliferation assay (Promega Corporation, WI). Briefly, MLP29 and Hep16 cells were seeded at 50 × 103 cells/well in 96-well plates coated with FN (10 μg/ml), VN (1 μg/ml), LMN (10 μg/ml), COL I (0.05 mg/ml) and BSA (0.2 mg/ml) as a control, and allowed to attach for 1 hour. After extensive washings, the medium was aspirated carefully and the dye solution added. After 3-5 hours of incubation, the reaction was stopped with the "Solubilization/Stop" buffer provided with the kit. Following overnight incubation at 37°C, absorbance at 570 nm was determined with a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA, USA).
Cell migration and cell invasion assays
To study cell migration we performed the wound healing assay. MLP29 and Hep16 cells were plated seeded on six wells plastic tissue culture dishes and grown to confluence. Then, the cell monolayer was scratched with a p200 pipette tip, washed twice, and incubated in growth medium with 0.1% FBS containing 10 μg/ml of α5β1 functional-blocking antibody or 40 ng/ml of HGF/SF1 for 20 hours. Microscope images were taken from at least three fields for each experiment. The distance covered by the migrating cells was calculated as the mean of six different measurements along the scratch using the Gimp 2.0 image software.
To assess cell invasion, we used the QCM™ 24 wells Cell Invasion Assay from Chemicon-Millipore (Temecula, CA, USA) according to manufacturer's instructions. Briefly, 500 × 103 MLP29 or Hep16 serum starved cells were loaded into an insert containing an eight μm pore size and polycarbonate membrane coated with ECMatrix™ in the presence of α5β1 functional-blocking antibody or HGF/SF1 at the concentrations stated above and incubated for 48 and 72 hours. Invaded cells at the bottom of the membrane were detached, lysed and detected by the CyQUANT™ GR Dye in a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA, USA).
Immunoblotting analysis of ERK1/ERK2 MAPK, p-ERK1-ERK2 MAPK, AKT, pAKT, integrin sub-units and histone acetylation
Cells were cultured in a serum free medium for 16-18 hours and then stimulated with HGF/SF1 or treated with α5β1 functional-blocking antibodies for up to 24 hours. After treatment, cells were harvested, washed twice in PBS, and lysed with the Cell Lysis Buffer (Cell Signaling Biotechnologies) and 1 mM phenylmethylsulfonylfluoride for 30 minutes on ice. The lysates were then cleared by centrifugation (14000 rpm for 15 minutes at 4°C). Protein concentration was determined using the Pierce protein assay kit (Rockford, IL, USA). For analysis of ERK1/2 MAPKs and Akt, equal amounts of protein (20 μg) were resuspended in 5× Laemli buffer (10 minutes at 100°C), resolved by electrophoresis on a 10% SDS-PAGE, and transferred onto nitrocellulose membranes (Amersham Biosciences). For analysis of histone H3 acetylation, 50 μg of protein was resolved on a 15% SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were then blocked with TBS-T (25 mM Tris-HCl, 150 mM NaCl (pH 7.5), and 0.05% Tween 20) containing 5% (w/v) non-fat dry milk and incubated overnight at 4°C with specific antibodies. After incubation, the membranes were washed in TBS-T, and HRP-conjugated anti-rabbit, anti-mouse or anti-goat antibodies were added for 1 hour as secondary antibodies. Immunodetection was performed using the Western Lighting chemiluminescence reagent from Perkin Elmer (Boston, MA, USA).
Cells were fixed following protocols designed for the preservation of the three dimensional structure of the nuclei. For centromere analysis, the cells were fixed in 4% paraformaldehyde in PBS 0.3× (w/v) for 10 minutes and then permeabilized in PBS/Triton X-100 0.5% for 20 minutes at RT and transferred to 20% Glycerol/PBS. After 1 hour, the cells were further permeabilized by freezing and thawing cycles in liquid nitrogen. The probe was dissolved in 50%formamide/20% dextran-sulfate, denatured and incubated along with the denatured cell-targets in a humid chamber at 37°C overnight. For locus-specific 3D FISH, the fixation of the cells was performed according to the protocol described previously [8], with slight modifications. Briefly, the coverslips were fixed for 10 minutes in 4% paraformaldehyde in PBS (w/v). After permeabilization in 0.5% saponin (w/v) PBS/Triton X-100 0.5% (v/v) for 30 minutes, the slides were washed in PBS for 2 minutes at RT and treated with 0.1 N HCl for 20 minutes. Next, the cells were washed with PBS and 2 × SSC for 2 minutes, treated with RNAse A, transferred to 2 × SSC/50% formamide 0.1% NaAz and stored at 4°C. We used probes prepared with the BAC clones RP23-303A11 y RP23-343N12 specific for the Itgb1 or the Itgb3 genes, respectively. The probes were labeled by nick translation with dUTP conjugated with biotin (Roche) or digoxigenin (Roche). Before hybridization, the probes were pre-denatured at 80°C for 5 minutes, then denatured along with the target DNA at 75°C for 5 minutes and incubated at 37°C for 72 hours in a humified chamber. For the post-hybridization washings, the coverslips were immersed in 50% formamide/2 × SSC, 1 × SSC, and 4 × SSC/tween-20 0.1% (v/v) (4T) each for 5 minutes at 47°C. Following one more wash with 4T at RT, the coverslips were blocked with 3% BSA in 4T (wt/v), washed in 4T at RT for 5 minutes and incubated for 1 h with antibodies against biotin or digoxigenin in blocking solution. Then the coverslips were incubated with the respective secondary antibodies for 1 hour, washed twice with PBS for 5 minutes and mounted with Vectashield containing DAPI (Vector Laboratories, CA, USA).
Image acquisition and analysis
Stacks of images scanning the whole nucleus were acquired with an axial separation of 250 nm using a laser-scanning microscope Leica DM IRE2 (Leica Microsystems Heidelberg GmbH). For quantitative analysis of chromocenters and Me3K9H3 foci, regions of interest (ROI) including each nucleus were directly generated on the z-stack using the Volocity.4.3® software (Improvision, Image, Processing and Vision Company Limited) and then the intensity level, volume and center of mass of all objects contained within each ROI was automatically recorded. Between 500-1000 elements (chromocenters or Me3K9H3 foci) per treatment were analyzed. For statistical analysis, the "proportions test" was used to compare the average number of elements per nucleus for each treatment. For the comparison of mean volumes and mean fluorescence intensity, we applied the "t-student" test. Fluorescence intensity of Ac3K9/K14H3 was directly measured on the Z-stack using the Volocity.4.3® software (Improvision, Image, Processing and Vision Company Limited).
To determine the 3D radial position of any given signal, the shortest distance from the center of mass of the nucleus to the periphery, which included the center of mass of the fluorescent signal, was directly measured on the z-stack using the Volocity.4.3® software. The absolute distances from the nucleus center to the gene were normalized as a fraction of nuclear radius, to account for natural variations in nuclear size that may influence positioning. Cell nuclei were subdivided into five concentric shells each corresponding to 20% of the nuclear radius, and the radial positioning data was binned into these five sub-domains. Graphs were made using Microsoft Excel Software. For quantitative measurements, 50-70 nuclei from multiple experiments were analyzed. Statistical differences (p < 0.05) between the distributions of a gene in different conditions were determined using the 1D Kolmogorov-Smirnov test.
Microarray data acquisition and analysis
RNA was isolated from cell plates subjected to the different treatments using the RNAeasy Mini kit (Qiagen) and treated with RNAase-free DNAase (Qiagen). The cDNA was synthesized from 1 μg of RNA using the Reverse Transcriptase kit (Promega) following the manufacturer's recommendations. Expression profiling was performed using the Affimetrix GeneChip ® technology, following the protocols recommended by the manufacturers. Affymetrix raw files (.cel, Mouse430A_2) obtained from untreated control cells, treated with anti-α5β1 antibodies, and stimulated with HGF/SF1 were collectively analyzed by using the R package "AFFYLMGUI" (http://www.bioconductor.org) [24]. Data were background corrected by the rma method [25]. The p-values obtained by testing for differentially expressed genes were calculated using "AFFYLMGUI" and exported as text. Extraction of GO terms for all genes on the Mouse430A_2 chips was used for functional analysis. Hypergeometric p-values for testing over representation of genes in GO categories were calculated as described in Masseroli et al., and the p-values were corrected for multiple testing by the Holm method [26, 27]. The gene set enrichment analysis was done as described previously [28, 29]. The permutation statistics for the heat-plots were calculated for each gene set. This was done by considering the expression level for all genes of each set defined by Molecular Signatures Database. The p-values for the heat-plots were calculated as ndown/N for down-regulated genes and as 1-nup/N for up-regulated genes, where ndown is the number of times a permutated data set resulted in a down regulation that was stronger or equal to the one observed for the unpermutated data. nup was calculated in a similar way for up-regulated genes. N was the total number of permutation which was set to 10000. To assess if a specific gene set was up- or down-regulated for a specific sample class the expression values from a specific class were compared with the expression values in all other sample classes [30]. The calculation of 1-nup/N for up-regulated gene sets allows co-plotting both up and down-regulated gene sets in the same heat-diagram. Mouse genes were mapped to human genes by using HomologGene available at NCBI. Microarray data is publicly available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE23853, Gene Expression Omnibus (GEO) accession number GSE23853.
We would like to thank Dr. David Gisselsson for his critique of the manuscript. This work was supported by funding from Fondo de Investigación Sanitaria (ISCiii), Grant # 05/1117 to LAP; Department of Industry and Department of Health of the Basque Government. FR is a CIBERHED postdoctoral fellow associated with CIC bioGUNE.
12860_2010_526_MOESM1_ESM.TIFF Additional file 1: MLP29 cells were treated with a specific functional blocking antibody against α 5 β 1 integrin or stimulated with HGF/SF1 for 24 hours. Then immunofluorescent detection of invasiveness and hepatic stem cell (HSC) biomarkers showed that cell migration was associated with an increase in the expression levels of β1 and β3 integrin sub-units. The treatments also increased the expression of the HSC markers Ep-CAM, AFP and CK19, all related to the invasive growth of epithelial cells. (TIFF 2 MB)
12860_2010_526_MOESM2_ESM.TIFF Additional file 2: Following treatment with the α 5 β 1 blocking antibody, viability of the cells was determined using the MTT reduction assay. The effects of exposure of the cells to the α5β1 specific antibody or stimulation with HGF/SF1 were analyzed by generating concentration-effect curves as a plot of the fold-time increase in the fraction of surviving cells versus antibody concentration. The data presented summarize the mean (±S.D.) of three independent experiments each performed in triplicate. (TIFF 88 KB)
12860_2010_526_MOESM3_ESM.TIFF Additional file 3: Flow cytometric analysis of the DNA content of hepatic progenitor cells was performed following treatment with an α 5 β 1 -specific antibody in the presence or absence of the MEK inhibitor U0126. α5β1 inhibition slightly increased (statistically non-significant) the cell sub-population in the S phase of the cell cycle and this is concomitant with a decrease in the percentage of cells in the G0/G1 sub-compartment of the cell cycle. These effects on the S-phase were not reversed upon inhibition of the proliferative MAPK pathway. (TIFF 799 KB)
12860_2010_526_MOESM4_ESM.TIFF Additional file 4: Flow cytometric analysis of the DNA content of hepatic progenitor cells was performed following treatment with an α5β1-specific antibody in the presence or absence of the MEK inhibitor U0126. The percentages of cells in each sub-compartment of the cell cycle are plotted as a function of the different treatments. One experiment, representative of three independent experiments, is shown. (TIFF 103 KB)
12860_2010_526_MOESM5_ESM.TIFF Additional file 5: Frequencies of up- or down-regulated genes in the three main GO categories: Cellular Components (CC), Biological Processes (BP) and Molecular Function (MF). (TIFF 96 KB)
12860_2010_526_MOESM6_ESM.TIFF Additional file 6: Most significantly up-regulated genes within the biological processes and cellular components GO categories in MLP29 cells upon α5β1 integrin functional blockade. (TIFF 506 KB)
12860_2010_526_MOESM7_ESM.TIFF Additional file 7: Most significantly down-regulated genes in MLP29 cells upon α5β1 integrin functional blockade. (TIFF 377 KB)
12860_2010_526_MOESM8_ESM.TIFF Additional file 8: Flow cytometric and immunoblot analyses of Ac3K9/14 H3 in the context of DNA content of MLP29 cells treated with an anti-α5β1 specific antibody. Bivariate cytograms indicate the levels of expression of Ac3K9/14 H3 in each sub-compartment of the cell cycle. Bar graphs show the percentage of FITC-positive cells. The data presented are representative of three independent experiments. For immunoblot analysis, treated MLP29 cells were lysed and total protein (50 μg) was resolved by SDS-PAGE and analyzed by immunoblot for Ac3K9/14 H3. (TIFF 411 KB)
12860_2010_526_MOESM9_ESM.TIFF Additional file 9: Flow cytometric and immunoblot analyses of Ac3K9/14 H3 in the context of DNA content of MLP29 cells treated with HGF/SF1. Bivariate cytograms indicate the levels of expression of Ac3K9/14 H3 in each sub-compartment of the cell cycle. Bar graphs show the percentage of FITC-positive cells. The data presented are representative of three independent experiments. For immunoblot analysis, treated MLP29 cells were lysed and total protein (50 μg) was resolved by SDS-PAGE and analyzed by immunoblot for Ac3K9/14 H3. (TIFF 419 KB)
12860_2010_526_MOESM10_ESM.pdf Authors' original file for figure 1
12860_2010_526_MOESM19_ESM.pdf Authors' original file for figure 10
LV performed the cell experiments, image analysis and drafted the manuscript. FR, JT-F participated in statistical and image analyses. RM performed the microarray-data statistical analysis. AL assisted with cell experiments. LP conceived and coordinated the study, performed the FISH experiments and wrote the manuscript. All authors read and approved the final manuscript.
Cytogenomics, CIC bioGUNE-CIBEREHD, Par. Tec. Bizkaia Ed. 801 A, 48160 Derio, Spain
IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Portugal
Institute for Basic Sciences and Experimental Medicine, Hospital Italiano, Potosi 4240, 1199 Buenos Aires, Argentina
Unidad de Traducción Clínica - Ingeniería de Tejidos, Austral University, Pte Perón 1500, 1635 Pilar, Argentina
Institute of Experimental Pathology, Faculty of Health Sciences, National University of Salta, Bolivia 5010, 4400 Salta, Argentina
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text:
Екте гейтост () або (), «коричневий козячий сир» — норвезький напівтвердий бруност, виготовлений виключно із козячої молочної сироватки. Сир має солодкий, карамельний смак та коричнюватий колір. Сироватку кип'ятять, цукри, що містяться в молоці, карамелізуються і сир отримує характерний смак і колір.

Посилання

 Gjetost  на Koch-Wiki
 TINE Ekte Geitost

Норвезькі сири
Сири з козячого молока
Напівтверді сири

redpajama_set_name: RedPajamaWikipedia


Gewürztraminer from Chile is quite a rare beast, but here is a great example offering all of the aromatics, floral tones and exotic style you would expect from this unique grape variety.
Tasting Note : Perfumed, rose petal scents on the nose. On the palate the wine is rich yet elegant, with hints of lychee, orange blossom, citrus fruits and spice. The texture is rounded yet fresh.
Food Pairing : Thai curries, salads with fruit, creamy prawn cocktail, gingery stir fries.
More info : 'Gewurz' in German literally means 'spice', and this grape variety certainly delivers that. Its unique flavour profile makes it unmistakeable in blind tastings, and suitable for matching with dishes that would not pair well with most wines. Most commonly found in Alsace and Southern Germany, this grape performs surprisingly well in Chile's Loncomilla Valley, where it forms part of the del Pedregal vineyard holding. This wine is unoaked and cool fermented to retain it s aromatic flavours, and its small amount of residual sugar enhances its exotically fruity style.


text:
0122-17 New York Times Crossword Answers 22 Jan 17, Sunday
CROSSWORD SETTER: Dan Schoenholz
THEME: Mishmash
Each of today's themed answers starts with a common phrase. The last word in that phrase is the first half of a "mishmash", a repetitive phrase in which the first half uses the vowel I and the second half uses the vowel A. The second half of the "mishmash" is added to the original phrase, giving us our themed answer:
23A. Witty British judge? : POWDERED WIG WAG (from "powdered wig" & "wigwag")
38A. Three-legged race, e.g.? : JOINED AT THE HIP HOP (from "joined at the hip" & "hip-hop")
55A. Nail? : FINGER TIP TOP (from "finger tip" & "tip-top")
66A. "America"? : "OF THEE I SING" SONG ("Of Thee I Sing" & "singsong")
81A. Grant a girl permission to dis Drake? : LET HER RIP RAP (from "let her rip" & "riprap")
98A. Ability to score at Madison Square Garden, e.g.? : NEW YORK KNICK KNACK (from "New York Knick" & "knickknack")
117A. Diving disaster? : TRIPLE FLIP FLOP (from "triple flip" & "flip-flop")
19. Baseball-like game : ONE-A-CAT
One-o'-cat, or more properly "one old cat", is an abbreviated form of baseball with a home plate and just one base.
22. Et ___ : CETERA
The Latin phrase "et cetera" translates as "and other things". The term is usually abbreviated to "etc."
23. Witty British judge? : POWDERED WIG WAG (from "powdered wig" & "wigwag")
"To wigwag" is to signal with a light or flag using coded movements. The wigwag system was developed by Major Albert J. Myer of the US Army in the 1850s.
25. Conquistador Cortés : HERNAN
Hernán Cortés (also "Hernando Cortez") led the expedition from Spain to Mexico that eventually led to the fall of the Aztec Empire.
30. Pizza, e.g. : PIE
Pizza was invented in Naples where it has a long tradition that goes back to Ancient Rome. During an 1889 visit to Naples, Queen Margherita of Savoy was served a special pizza that was created with toppings designed to mimic the colors of the Italian flag. The ingredients of tomato (red), mozzarella (white) and basil (green) can still be found together on menus today, on a pie usually named Pizza Margherita after the queen. I do love basil on my pizza …
33. Ortiz of "Ugly Betty" : ANA
"Ugly Betty" is a drama-comedy show that originally aired on television from 2006 to 2010. The show is based on a telenovela soap opera from Colombia called "Yo soy Betty, la fea". The title role of Betty Suarez is played by America Ferrera
34. Site of Spaceship Earth : EPCOT
Spaceship Earth is perhaps the structure that comes to mind when we think of Epcot in the Walt Disney World Resort. It is the large, white, 18-story geodesic sphere.
37. Language akin to Thai : LAO
Lao is the official language of Laos. Lao is also spoken in the northeast of Thailand, but there the language is known as Isan.
46. "Mr. Blue Sky" band, for short : ELO
The Electric Light Orchestra (ELO) is a symphonic rock group from the north of England.
47. World's most voluminous river : AMAZON
The Amazon River of South America is the world's largest in terms of volume, and accounts for an amazing one-fifth of the world's total river flow. Perhaps even more amazing is that there are no bridges across the Amazon! There isn't even one, mainly because the river flows through tropical rainforest where there are few roads and cities.
48. Chapter in early 20th-century history: Abbr. : WWI
Prior to the outbreak of World War II, what we now know as World War I was referred to as "the World War" or "the Great War".
49. Property inheritor, legally speaking : ALIENEE
An alienee is one to whom ownership of property is transferred, alienated.
53. Julie of TV's "Big Brother" : CHEN
As well as co-anchoring "The Early Show", Julie Chen has been host of the American version of "Big Brother" since it graced our screens in the year 2000.
The "Big Brother" television franchise started out in 1999 in the Netherlands. The term "Big Brother" comes from George Orwell's novel "Nineteen Eighty-Four".
60. Girl with a ball : DEB
"Deb" is short for "debutante", which translates from French as "female beginner".
61. Sound investments, in more ways than one : CDS
A certificate of deposit (CD) is like a less-flexible and higher-paying savings account. Instead of depositing money into a savings account and earning interest periodically, one can open a CD. With a CD one deposits a minimum amount of money but must leave it there for a specified length of time. In return for committing the funds for a fixed period, one is given a higher interest rate than a savings account and can redeem that interest and the initial deposit when the term has expired. CDs are relatively low-risk investments as they are FDIC insured, just like savings accounts.
The compact disc was developed jointly by Philips and Sony as a medium for storing and playing sound recordings. When the first commercial CD was introduced back in 1982, a CD's storage capacity was far greater than the amount of data that could be stored on the hard drive of personal computers available at that time.
62. ___ Minor : ASIA
Asia Minor is also known as Anatolia. It is the geographic part of Asia that protrudes out into the west, towards Europe, and is roughly equivalent to modern-day Turkey.
66. "America"? : "OF THEE I SING" SONG ("Of Thee I Sing" & "singsong")
"Of Thee I Sing" is a musical by George and Ira Gershwin that premiered on Broadway in 1931. "Of Thee I Sing" is about a politician running for president who falls for the "wrong" woman.
The patriotic song "America" is also known by its first line, "My Country, 'Tis of Thee". The song was written by Samuel Francis Smith in 1831, and was the de facto national anthem of the country until "The Star-Spangled Banner" was declared the official anthem in 1931. The melody of "My Country, 'Tis of Thee" is identical with the British national anthem, "God Save the Queen".
My country, 'tis of thee,
Sweet land of liberty,
Of thee I sing;
Land where my fathers died,
Land of the pilgrims' pride,
From ev'ry mountainside
Let freedom ring!
71. Hindi word for "spice mix" : MASALA
Masala is the Hindi word for "mixture", and describes a mixture of spices. A dish named "masala" uses the spices incorporated into a sauce that includes garlic, ginger, onions and chili paste. Who doesn't love Indian food? Yum …
74. Brief second? : ASST
An assistant (asst.) might be referred to as a "second".
75. ___ generis : SUI
"Sui generis" is a Latin expression meaning "of its own kind". The term can be used in a number of fields, and in philosophy it refers to an idea which cannot be included in a wider concept, and idea of its own kind.
76. Theological inst. : SEM
Originally, a "seminary" was where plants were raised from seeds, as "semen" is the Latin for "seed". The first schools labelled as seminaries were established in the late 1500s. Those first schools were more likely to be academies for young ladies back then, rather than for trainee priests.
79. What Cubs fans get carried away by? : EL TRAIN
The Chicago "L" is the second largest rapid transit system in the US, with the New York City Subway being the largest. The "L" is also the second oldest, again with the New York City Subway system having the honor of being around the longest. Note that the official nickname for the system is the "L" (originally short for "elevated railroad"), although the term "El" is also in common use (especially in crosswords as "ELS"). The L is managed by the Chicago Transit Authority (CTA).
81. Grant a girl permission to dis Drake? : LET HER RIP RAP (from "let her rip" & "riprap")
"Riprap" is rock or rubble that is laid along a shoreline to protect against erosion.
Drake is the stage name of rapper Aubrey Graham from Toronto.
86. Fortify : GIRD
The phrase "gird your loins" dates back to Ancient Rome. The expression describes the action of lifting "one's skirts" and tying them between the legs to allow more freedom of movement before going into battle. Nowadays, "gird your loins" (or sometimes just "gird yourself") is a metaphor for "prepare yourself for the worst".
90. 1990s Indian P.M. : RAO
P. V. Narasimha Rao was Prime Minister of India from 1991 to 1996. Rao is seen by most as the leader who transformed his country's economy into the market-driven engine that it is today.
91. Week, on Martinique : SEMAINE
The island of Martinique in the eastern Caribbean is actually a part of France, and is referred to as an "overseas department". As such, Martinique is part of the European Union and even uses the euro as its currency. The island is fully represented in the French National Assembly and Senate, just like any department within France. It's sort of like the status of Hawaii within the US.
93. Game for the goal-oriented? : SOCCER
Soccer (also known as "association football") is the most popular sport in the world. The term "association football" was introduced in 1863 in England, with the name chosen to distinguish the sport from rugby football. The term "soccer" started to appear about 20 years later in Oxford, as an abbreviation for "association".
98. Ability to score at Madison Square Garden, e.g.? : NEW YORK KNICK KNACK (from "New York Knick" & "knickknack")
Madison Square Garden is an arena in New York City used for a variety of events. In the world of sports it is home to the New York Rangers of the NHL, as well as the New York Knicks of the NBA. "The Garden" is also the third busiest music venue in the world in terms of ticket sales. The current arena is the fourth structure to bear the name, a name taken from the Madison Square location in Manhattan. In turn, the square was named for James Madison, the fourth President of the US.
102. Mouse's resting place : PAD
The first computer mouse was invented at the Stanford Research Institute in 1963, by one Douglas Engelbart. Sadly for him, his patent ran out before mice became standard equipment on computers, so he never made any money from his amazing invention.
104. French ___ : FRY
"French fries" are called "chips" back in the British Isles where I grew up. In France, they're called "pommes frites" (meaning "fried potatoes").
105. Title at Topkapi Palace : AGA
The magnificent Topkapi Palace in Istanbul was a residence for the Ottoman sultans for over 400 years, starting in 1459. During its heydey, the palace complex was home to a staggering 4,000 people. The name "Topkapi" translates as "Cannon Gate".
109. Piano dueler with Donald in 1988's "Who Framed Roger Rabbit" : DAFFY
Daffy Duck first appeared on the screen in "Porky's Duck Hunt" in 1937. In the original cartoon, Daffy was just meant to have a small role, but he was a big hit as he had so much sass. Even back then, Daffy was voiced by the ubiquitous Mel Blanc.
"Who Framed Roger Rabbit" was released in 1988, a clever film featuring cartoon characters that interact directly with human beings. The most memorable cartoon characters have to be the goofy Roger Rabbit, and the vampish Jessica Rabbit. The film is based on a novel written by Gary K. Wolf called "Who Censored Roger Rabbit?" There is a prequel floating around that has never been produced, and it's called "Who Discovered Roger Rabbit".
112. Quiz bowl fodder : TRIVIA
Trivia are things of little consequence. "Trivia" is the plural of the Latin word "trivium" which means "a place where three roads meet". Now that's what I call a trivial fact …
114. Like Serbia and Croatia : BALKAN
The Balkan Peninsula in Southeast Europe is usually referred to as "the Balkans". The region takes its name from the Balkan Mountains located in present-day Bulgaria and Serbia. "Balkan" is Bulgarian for "mountain".
127. Speakers' spots : ROSTRA
A "rostrum" (plural "rostra") is an elevated platform, particularly one for public speaking. The original rostrum was the platform used by public speakers in the Forum of ancient Rome.
2. Only woman to sing lead vocals on a Beatles song : ONO
"The Continuing Story of Bungalow Bill" is a John Lennon song that was recorded by the Beatles. The title character was inspired by a young American who was visiting the same ashram the Beatles were staying at in India while studying with the Maharishi Mahesh Yogi. The American stayed in a bungalow next to the Maharishi, and was dubbed "Bungalow Bill" by Lennon and co. There's a line in the song "Not when he looks so fierce", which is sung by John Lennon's wife Yoko Ono. That line marks the only time that a woman provided a lead vocal for a Beatles song.
4. Sierra ___ : MADRE
"The Treasure of the Sierra Madre" is a 1927 novel by German novelist B. Traven. The book was made into a famous 1948 movie of the same name by John Huston, starring Humphrey Bogart and Walter Huston (director John's father).
6. H.O.V. lane user : CARPOOLER
In some parts of the country one sees high-occupancy vehicle lanes (HOV lanes). Out here in California we refer to them as carpool lanes.
9. Chest pain : ANGINA
"Angina pectoris" is the more complete name for the chest pain that we usually refer to as "angina". The term can be translated into English as "a strangling feeling in the chest".
10. Grist for analysts : RAW DATA
When grain has been separated from its chaff, to prepare it for grinding, it is called "grist". Indeed, the word "grist" is derived from the word "grind". Grist can be ground into a relatively coarse meal, or into a fine flour. The names can be confusing though. For example, the grist from maize when ground to a coarse consistency is called "grits", and when ground to a fine consistency is called "corn meal". There is an idiomatic phrase "grist for one's mill", meaning something used to one's advantage. The grinding mechanism, or the building that holds the mechanism, is known as a "gristmill".
11. Californie, e.g. : ETAT
In French, "Californie" (California) is an "état" (state).
12. Gaming giant : SEGA
Sega is a Japanese videogame company headquartered in Tokyo. Sega actually started out 1940 in the US as Standard Games and was located in Honolulu, Hawaii. The owners moved the operation to Tokyo in 1951 and renamed the company to Service Games. The name "Sega" is a combination of the first two letters of the words "Se-rvice" and "Ga-mes".
13. I, to Izaak : ICH
"Ich" is the German for "I", as in "Ich bin ein Berliner" (I am a Berliner), the famous words of support uttered by President John F. Kennedy in 1963 in a speech in West Berlin. The supposed translation of "Ich bin ein Berliner" as "I am a jelly doughnut" … that's just an urban myth. President Kennedy's use of German was perfectly correct.
14. Word for a name-dropper? : NEE
"Née" is the French word for "born" when referring to a female. The male equivalent is "né".
15. 1960s sitcom set in the 1860s : F TROOP
Relatively few people outside of the US saw the American sitcom "F-Troop", which was made in the sixties. I remember watching the show as a young lad because it was picked up by the Irish national television service. The only other country that showed "F-Troop" was Australia.
24. Lorna of literature : DOONE
The novel "Lorna Doone: A Romance of Exmoor" was written by Richard Doddridge Blackmore. R. D. Blackmore was an English novelist, very celebrated and in demand in his day (the late 1800s). His romantic story "Lorna Doone" was by no means a personal favorite of his, and yet it is the only one of his works still in print.
28. Codger : GEEZER
Geezer, codger and coot are all not-so-nice terms for an old man.
31. Thyroid need : IODINE
The thyroid gland is found in the neck, just below the Adam's apple. The gland produces several thyroid hormones, some of which control the rate at which the body uses energy i.e. the body's rate of metabolism.
36. N.L. Central squad, on scorecards : CIN
The Red Scare (i.e. anti-communist sentiment) following WWII had such an effect on the populace that it even caused the Cincinnati baseball team to change its name from the Reds. The team was called the Cincinnati Redlegs from 1953-1958, as the management was fearful of losing money due to public distrust of any association with "Reds".
38. Half of a swinging couple? : JANE
Jane Porter is the love interest in the "Tarzan" series of novels penned by Edgar Rice Burroughs. Famously, Jane was played by Maureen O'Sullivan on the big screen, opposite Johnny Weissmuller as Tarzan. In the movies, Jane's family name was changed from Porter to Parker. Also, Porter is an American in the books, and Parker is an Englishwoman in the films.
40. Alternative to Cinemax : TMC
The Movie Channel is owned by Showtime, which in turn is subsidiary of CBS. The channel's name is often abbreviated to "TMC", although this is informal usage.
52. One lifting spirits? : TOASTER
The tradition of "toasting" someone probably dates back to the reign of Charles II, when the practice was to drink a glass of wine to the health of a beautiful or favored woman. In those days, spiced toast was added to beverages to add flavor, so the use of the word "toast" was an indicator that the lady's beauty would enhance the wine. Very charming, I must say …
57. Chi follower : PSI
The Greek letter psi is the one that looks a bit like a trident or a pitchfork.
59. Diesel in movies : VIN
Vin Diesel is the stage name of actor Mark Sinclair Vincent. He was born in New York City with his twin brother Paul. The twins never knew their father, and their mother is an astrologer. Vincent was given the nickname of "Diesel" by his friends early in his life, as he was said to have a bottomless supply of energy.
64. Shivering fit : AGUE
An ague is a fever, one usually associated with malaria.
67. Key locale: Abbr. : FLA
A "key" (also "cay") is a low island offshore, as in the Florida Keys. Our term in English comes from the Spanish "cayo" meaning "shoal, reef".
69. Manhattan, e.g.: Abbr. : ISL
The island we know as Manhattan was inhabited by the Lenape Indians when the first Europeans explorers arrived in the area. According to the logbook of one of the officers on explorer Henry Hudson's yacht, the island was called "Manna-hata" in the local language, from which the modern name derives.
70. They're dubbed : SIRS
Kneel, and the Queen might "dub thee a knight" if you're lucky. "Dub" is a specific term derived from Old English that was used to mean "make a knight". As the knight was also given a knightly name at the same time, "dub" has come to mean "give someone a name".
71. Mullally of "Will & Grace" : MEGAN
77. It lies between Cleveland, O., and Buffalo, N.Y. : ERIE, PA
Erie is a city in the very north of Pennsylvania, right on the southern shore of Lake Erie. The city takes its name from the Erie Native American tribe that resided in the area. Erie is nicknamed the Gem City, a reference to the "sparkling" Lake Erie.
80. Made out : NECKED
The term "necking" applies to kissing and caressing. I like what Groucho Marx had to say on the subject: "Whoever named it necking was a poor judge of anatomy."
82. Showy in a cheap way : TACKY
Something "tacky" is "in bad taste". The term derives from the noun "tackey" that was used in the early 1800s to describe a neglected horse.
93. Measly amount : SOU
A sou is an old French coin. We use the term "sou" to mean "an almost worthless amount".
94. Guitar Hero activity : RIFFING
A riff is a short rhythmic phrase in music, especially one improvised on a guitar.
Guitar Hero is an amazingly successful series of video games, first published in 2005. It is the third best selling franchise of video games, after Mario and Madden NFL. Sales have dropped in recent years though, and there are no plans for further releases.
96. Wolf (down) : SNARF
"To snarf down" is to gobble up, to eat voraciously. "Snarf" is a slang term that is probably related to "scarf", which has the same meaning.
99. Mantle, e.g. : YANKEE
Mickey Mantle only played professional baseball for the one team, spending 18 years with the New York Yankees. Mickey Mantle memorabilia is highly prized, especially since he retired from the game in 1969, and even more so since he died in 1995. The only other player memorabilia said to command a higher price is Babe Ruth's. Mantle holds the record for the most career home runs by a switch hitter, as well as the most World Series home runs.
108. Racer Yarborough : CALE
Cale Yarborough is a former NASCAR driver and owner. Yarborough was the first NASCAR driver to appear on the cover of "Sports Illustrated".
110. Fig. on a periodic table : AT WT
The atomic weight (at. wt.) of an element is the mass of one atom of the element, relative to the mass of an atom of carbon (which is assumed to have an atomic weight of 12).
111. Mrs., abroad : FRAU
In Germany, a "Mr." (Herr) is married to a "Mrs." (Frau), and they live together in a house (Haus).
112. Bedouin shelter : TENT
Bedouin tribes are Arab ethnic groups that predominantly live in the Middle East, in desert areas. Bedouin tribes tend to be nomadic, not settling permanently in one location.
113. ___ facto : IPSO
"Ipso facto" is Latin, meaning "by the fact itself". Ipso facto describes something that is a direct consequence of particular act, as opposed to something that is the result of some subsequent event. For example, my father was born in Dublin and was an Irish citizen ipso facto. My son was born in California and is an Irish citizen by virtue of being the son of an Irish citizen ("not" ipso facto).
116. ___ Yost, 2015 World Series-winning manager : NED
Ned Yost is the manager of the Kansas City Royals, and a former Major League Baseball catcher. Yost played baseball at high school in Dublin, California, just a few miles from where I am now right now.
118. Mauna ___ : LOA
Mauna Loa on the "big island" of Hawaii is the largest volcano on the planet (in terms of volume). The name "Mauna Loa" is Hawaiian for "Long Mountain".
119. Poland's main airline : LOT
LOT Polish Airlines is the country's flag carrier. Founded way back in 1929, it is one of the oldest airline in the world that is still operating. "Lot" is a Polish word meaning "flight".
120. Start of the Lord's Prayer : OUR
"Our Father …" ("Pater noster" in Latin) are the opening words of the Lord's Prayer, which is probably the best-known prayer in the Christian tradition.
121. Education support grp. : PTA
Parent-Teacher Association (PTA)
1. Inconceivably vast : COSMIC
7. Hard looks : STARES
13. Stream, as of revenue : INFLOW
21. Flowery : ORNATE
26. Copies, informally : REPROS
27. It shows who's who or what's what : ID TAG
29. Perform a full-body scan? : OGLE
32. Quest of 25-Across : ORO
38. Three-legged race, e.g.? : JOINED AT THE HIP HOP (from "joined at the hip" & "hip-hop")
44. School chum, say : OLD PAL
51. On point : APT
54. "One of the most civilized things in the world," per Hemingway : WINE
55. Nail? : FINGER TIP TOP (from "finger tip" & "tip-top")
58. Consider anew, as a decision : REVISIT
65. A- : NINETY
87. Page (through) : LEAF
92. Alias inits. : AKA
95. Keeps in the loop, in a way : CCS
97. Worn out : DONE IN
103. Take a timeout : PAUSE
106. Egg container : SAC
107. Religious image : ICON
117. Diving disaster? : TRIPLE FLIP FLOP (from "triple flip" & "flip-flop")
122. Maintain : ALLEGE
123. Bawdy : WANTON
124. Gently show the door : EASE OUT
125. Give a new tournament ranking : RE-SEED
126. Pulls on, as heartstrings : TUGS AT
1. Bluecoat : COP
3. Darn things : SEW
5. Drink commonly served with a spoon-straw : ICEE
7. Farm females : SOWS
8. Lateral opening? : TRI-
16. From one side to the other : LENGTHWISE
17. Kind of history : ORAL
18. Ebb : WANE
20. Grammy-winning drummer ___ Lyne Carrington : TERRI
30. Opposite of ruddy : PALLID
33. Embrace : ADOPT
35. Bus. card info : PHONE NO
37. Don't work too hard : LOAF
39. Goes by : ELAPSES
41. "That'll be the day!" : HAH!
42. Take responsibility for something : OWN IT
43. Atheist's lack : PIETY
45. Place to hang tools : PEGBOARD
50. Leave a good impression? : ETCH
54. Jet measure : WINGSPAN
56. Think tank product : IDEA
63. Reeling : IN SHOCK
68. They'll take your measure : TAILORS
72. Hard to tell apart : ALIKE
73. Informal measures of popularity : STRAW POLLS
78. Nut : MANIAC
83. Salmon roe, by another name : RED CAVIAR
84. "Don't worry about me!" : I'M OK!
85. Await resolution : PEND
88. Relative of "Aargh!" : ACK!
89. Wetland : FEN
100. Some vaults : CRYPTS
101. Like cats, typically : AGILE
106. Secure spots : SAFES
107. Certain steel beam : I-BAR
115. Common thing to lie about : AGE
Posted on January 22, 2017 October 4, 2017 Categories Dan Schoenholz6 Comments on 0122-17 New York Times Crossword Answers 22 Jan 17, Sunday

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: Vagrant failed on all commands with a same error "code converter not found" I installed vagrant 1.3.4 on my Windows 7 laptop then tried to type vagrant init from command line.  It failed with below error.  I tried other commands (even vagrant --help) but still receive a same error.  I tried googling for a while to no avail.  Is there anyone who can shed light on why this error happens?  Any input is appreciated.  Thanks
C:/HashiCorp/Vagrant/embedded/lib/ruby/site_ruby/1.9.1/rubygems/specification.rb:268:in `[]': code converter not found (
UTF-16LE to Windows-1258) (Encoding::ConverterNotFoundError)
    from    C:/HashiCorp/Vagrant/embedded/lib/ruby/site_ruby/1.9.1/rubygems/specification.rb:268:in `block in _all'
    from C:/HashiCorp/Vagrant/embedded/lib/ruby/site_ruby/1.9.1/rubygems/specification.rb:267:in `each'
    from C:/HashiCorp/Vagrant/embedded/lib/ruby/site_ruby/1.9.1/rubygems/specification.rb:267:in `_all'
    from C:/HashiCorp/Vagrant/embedded/lib/ruby/site_ruby/1.9.1/rubygems/specification.rb:410:in `each'
    from C:/HashiCorp/Vagrant/embedded/lib/ruby/site_ruby/1.9.1/rubygems/dependency.rb:216:in `find_all'
    from C:/HashiCorp/Vagrant/embedded/lib/ruby/site_ruby/1.9.1/rubygems/dependency.rb:216:in `matching_specs'
    from C:/HashiCorp/Vagrant/embedded/lib/ruby/site_ruby/1.9.1/rubygems/dependency.rb:238:in `to_specs'
    from C:/HashiCorp/Vagrant/embedded/lib/ruby/site_ruby/1.9.1/rubygems/dependency.rb:256:in `to_spec'
    from C:/HashiCorp/Vagrant/embedded/lib/ruby/site_ruby/1.9.1/rubygems.rb:1231:in `gem'
    from C:/HashiCorp/Vagrant/bin//../embedded/../embedded/gems/bin/vagrant:22:in `<main>'

A: After searching for a while, this seems to be an issue with Ruby and I found the answer from this here:
Error installing Rubygems on ruby command prompt in Win7
I did the same, changing my locale from Vietnam to US and issue resolved.

A: You can add chcp 65001 > nul at the top of vagrant.bat after @ECHO OFF

redpajama_set_name: RedPajamaStackExchange


text:
Home Seco/Vacuum moves into larger quarters to accommodate its growing business
Seco/Vacuum moves into larger quarters to accommodate its growing business
With its recent move, Seco/Vacuum's office space increased by 50 percent, and shop floor space by nearly 300 percent. (Courtesy: Seco/Warwick)
Seco/Vacuum, a Seco/Warwick Group company, moved its office and shop to a new space. After their fifth year in the North American market, they decided it was time for a move.
The new location stays at the group's birth city — Meadville, Pennsylvania — but the place is different. At a nearby business park in Meadville, Seco/Vacuum will open a new chapter in the company's history.
The move was necessitated by continuing new business growth, a requirement to accommodate additional engineering and production staff, and the need to enhance manufacturing capacity to meet customers' aftermarket support and service demands.
"Our team needed a new home to manage our expansion efficiently," said Peter Zawistowski, Seco/Vacuum's managing director. Our prior space served us well for the past couple of years, but to accommodate new employees and increasing customer demands, it was time to move on. Our new offices are much more conducive to teamwork, while affording us generous meeting room space for customer collaboration. The new shop area will not only help us grow our manufacturing capabilities, but it will also increase our parts and service support and provide a research and development facility to test new technologies."
"Seco/Vacuum's move to larger administration and manufacturing facilities certainly validates our commitment to the North American region as a primary marketplace for Seco/Warwick's proven technologies. We look forward to continuing customer acceptance and growth in the region," said Sławomir Woźniak, CEO of Seco/Warwick Group.
2022 marks the fifth anniversary of the Seco/Vacuum brand. In the most recent full year of financial reporting ended December 31, 2021, the company wrote orders for 40 percent of Seco/Warwick Group's total international vacuum furnace business. The company's portfolio of customers continues to grow in the first half of 2022 for both new equipment as well as support for its installed base.
Seco/Vacuum offers all-vacuum technologies that are engineered to run clean and cool, operate more efficiently than legacy furnaces, produce cleaner parts, and reduce environmental impact by eliminating toxic emissions.
MORE INFO www.secowarwick.com
Previous articleArmil CFS launches new furnace product
Next articleL&L receives order for two retort box furnaces for CMC use
Optimizing Gas Supply for Industrial Lasers
Going viral: Outside-the-box research into sterilizing N95 masks for re-use
Heat Treating Education
Combustion behavior and mechanism of Ti14 titanium alloy
Sintering experiments under way on International Space Station
Innovation for saving energy and improving output
Principles of pyrometry
Optimizing Temperature Measurement in Heat-Treatment Operations
Improved Materials and Enhanced Fatigue Resistance for Gear Components


text:
Bandsintown, Google's Waze Partner To Get You To The Show On Time
Global concert discovery and marketing platform Bandsintown has partnered with Google's Waze navigation app to help concertgoers find the quickest route to the show.
After fans buy tickets for the concert, they can click a "Plan Trip in Waze" button directly on the event to get directions.
Waze's real-time, community-sourced navigation helps drivers finding shortcuts and avoid unexpected road closures, construction, or anything else that might stop fans from getting to the show on time.
"We want to bring people together via live music," explains Bandsintown Managing Partner Fabrice Sergent. "Bandsintown sends millions of fans to concerts and adding Waze to the Bandsintown experience adds another technology layer to make such experience smoother."
"We always want to make sure drivers reach their destination in the quickest and safest way possible," said Adam Fried, Head of Global Partnerships at Waze. "Partnering with Bandsintown through our Transport SDK will make it easy for concert-goers to arrive at their destination on time – spending more time enjoying their favorite band and less time worrying about issues on the road."


As it's Cape Town Fashion Week I thought you should meet a fabulous South African fashion designer Michelle Ludek. Michelle spent time overseas honing her creativity and skills in Canada and Europe before returning to South Africa to fulfill her dream of creating ready-to-wear garments for women designed by women. Every one of Michelle's creations are a perfect combination of fabric and form and I'm definitely ready to wear them!In fact I think that if Genevieve MCC were a dress Michelle would have probably designed it.


Correcting the body's biochemistry may prevent violence. Metals affect the brain, including too much or too little nutrient trace minerals, such as copper. The studies of William J. Walsh, among others, show the connection between biochemistry and violence, indicating possible treatments.
"Acts of violence in the workplace or schools often are not as random as they appear to outsiders. Parents of violent children have been telling doctors and educators for years that their children were born with unique, disruptive, angry, defiant personalities. William J. Walsh, PhD, senior scientist at Health Research Institute and Pfeifer Treatment Center, and one of the leading U.S. researchers in violent behavior, backs them after 25 years of scientific exploration.
Articles and protocols for dealing with dental materials such as mercury, dental amalgam, and nickel.


text:
One Account
Volunteering for Hackney launches to help grow grassroots groups
Volunteering for Hackney 4
Volunteering for Hackney
Hackney,
Local organisations are being asked to take part in a new Council initiative to help small voluntary groups make a big difference in their communities.
The Volunteering for Hackney initiative was launched at a Volunteer Centre Hackney event at Hackney Museum on Tuesday 20 June by Mayor of Hackney, Philip Glanville, and Cabinet Member for Energy, Sustainability, and Community Services Cllr Jon Burke.
Around 50 people attended from organisations including literacy charity Hackney Pirates, repair and reuse project Hackney Fixers, cooking school Bags of Taste, anti-hate crime trust Stop Hate UK, and sports clubs Hackney Wick FC and Mini Mermaids.
The project was developed after extensive consultation undertaken by the Council in 2015 identified that residents wanted to get to know each other, contribute to their community and take greater responsibility for looking after the environment.
The Council is looking for groups to participate in Volunteering for Hackney, and in return will offer structured, strategic support. This includes everything from setting a Facebook page or a website, to filling in grant applications, learning how to get the best out of volunteers through the right training and setting up as a registered charity.
Hackney Council, Volunteer Centre Hackney and Hackney CVS, who have joined forces to operate the initiative, want to ensure that small community organisations and groups that operate at a very local level, are being fully enabled by the Council and other public services to help them thrive and grow.
I am passionate that community action is central to responding to some of the biggest challenges we as a borough face. When I was elected last September I added greater focus on volunteering in the Cabinet to reflect the challenges community groups are beginning to face. I also pledged to create a new network to support more volunteering and community involvement right across the borough. I was proud to launch this initiative, Volunteering for Hackney, and hope many grassroots organisations and activists can get involved.
Philip Glanville, Mayor of Hackney
The material inequalities that now exist in Hackney present a real challenge to maintaining the social cohesion that makes the borough such a special place to live. We know that volunteering and greater social participation in our community helps to reduce the sense of isolation and resentment that inequality can produce. Volunteering for Hackney will seek to harness the incredible enthusiasm and resourcefulness of our grassroots voluntary organisations to combat these challenges. We want to ensure that small community organisations and groups that operate effectively at a very local level, despite few resources, are able to recruit greater numbers of volunteers and increase the impact of their activities.
Cllr Jon Burke, Cabinet Member for Energy, Sustainability and Community Services
Get involved and find out more by contacting: communitypartnerships@hackney.gov.uk ; 020 8356 4066 or visiting: www.hackney.gov.uk/community-partnerships.
Find out more about volunteering opportunities on the Volunteer Centre Hackney website.
London Fields Lido to expand for first time since 1932
SEND co-design group presents recommendations
Local volunteers sought to help plant edible woodland on 1 February
Project SEARCH interns begin their new placement
New plan to improve mental health support and services in City of London and Hackney
Hackney's ninth School Street launched at William Patten School


text:
if [ -z "$1" ]; then
  echo "Usage: $0 target"
  exit 0
fi

TARGET="$1"

export CFLAGS="-m32 -Os -fno-exceptions -fno-asynchronous-unwind-tables -fno-unwind-tables -ffunction-sections -fdata-sections -Wl,--gc-sections -Wno-error=unused-but-set-variable -Wno-error=format-security"
export MAKEFLAGS='-j 4'
cd ~/src; rm -rf binutils-"$TARGET"-build; mkdir binutils-"$TARGET"-build; cd binutils-"$TARGET"-build/
time { ../binutils-2.22/configure --disable-libquadmath  --disable-libquadmath-support --disable-nls --enable-no_exceptions --enable-no_rtti  --disable-ppl-version-check --disable-cloog-version-check  --disable-foo --without-fp --disable-intl --disable-gprof --disable-ld --target="$TARGET"-linux; make; make; } || exit 1
for f in gas/as-new binutils/objdump binutils/objcopy; do
  strip -s --strip-all --remove-section=.comment --remove-section=.note $f
  upx -qqq --best --ultra-brute $f
  du -hs $f
done

redpajama_set_name: RedPajamaGithub