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text:
Mithramycin and chromomycin A3 are two structurally related antitumour compounds, which differ in the glycosylation profiles and functional group substitutions of the sugars. Chromomycin contains two acetyl groups, which are incorporated during the biosynthesis by the acetyltransferase CmmA in Streptomyces griseus ssp. griseus. A bioconversion strategy using an engineered S. griseus strain generated seven novel acetylated mithramycins. The newly formed compounds were purified and characterized by MS and NMR. These new compounds differ from their parental compounds in the presence of one, two or three acetyl groups, attached at 3E, 4E and/or 4D positions. All new mithramycin analogues showed antitumour activity at micromolar of lower concentrations. Some of the compounds showed improved activities against glioblastoma or pancreas tumour cells. The CmmA acetyltransferase was located in the cell membrane and was shown to accept several acyl-CoA substrates. All these results highlight the potential of CmmA as a tool to create structural diversity in these antitumour compounds.
Mithramycin 1 (Fig. 1) is an antitumour compound of the aureolic acid family produced by Streptomyces argillaceus (Rohr et al., 1999), which was discovered in 1961 and approved for use as anticancer drug in 1970 (Newman and Cragg, 2007). It has been used clinically for the treatment of several types of cancer, including testicular carcinoma, chronic myeloid leukaemia and acute myeloid leukaemia and also for the control of hypercalcaemia in patients with malignant diseases (Remers, 1979; Skarbek and Speedie, 1981). More recently, renewed interest for 1 is growing because of its potential new applications. It has been found that 1 could be used in combination with bevacizumab, a neutralizing antibody against VEGF (Vascular Endothelial Growth Factor), as a novel antiangiogenic therapy for pancreatic cancer and other cancers (Jia et al., 2007). Moreover, 1 has revealed to be a neuroprotective drug, possessing potential application as neurological therapeutics, and for the alleviation of symptoms underlying β-thalassaemia and sickle cell anaemia due to its activity as inducer of erythrocitic cell differentiation (Chatterjee et al., 2001; Fibach et al., 2003). The antitumour properties of 1 are due to its inhibitory effects on replication and transcription processes during macromolecular biosynthesis by interacting, in the presence of Mg2+, with GC-rich nucleotide sequences located in the minor groove of DNA (Sastry and Patel, 1993; Sastry et al., 1995; Barcelo et al., 2007). As a consequence it blocks the binding of proteins to GC-rich sequences in gene promoters, and inhibits transcription of genes regulated by these factors, such as those regulated by the Sp1 family of transcription factors (Miller et al., 1987; Ray et al., 1989; Blume et al., 1991; Snyder et al., 1991).
Chromomycin A32 (Fig. 1) is an antitumour compound structurally related to 1, but differing in the glycosylation pattern. Mithramycin contains a trisaccharide of d-olivose, d-oliose and d-mycarose, and a disaccharide of d-olivose, while 2 contains a trisaccharide of d-olivose, d-olivose and 4-O-acetyl-l-chromose B, and a disaccharide of 4-O-acetyl-d-oliose and 4-O-methyl-d-oliose attached at positions 2 and 6 of the aglycone respectively. These differences in the stereochemistry and functional group substitutions of the sugars are expressed in the binding properties of these compounds to DNA (Majee et al., 1997; Mir et al., 2003). Thus, the acetoxy groups in d-oliose (sugar A) and l-chromose B (sugar E) in 2 provide additional H-bonds with the 2-amino groups of G-bases and thus adding more specificity to the DNA binding (Silva and Kahne, 1993; Silva et al., 1993; Majee et al., 1997; Chakrabarti et al., 2001). This is also shown in their antitumour activities, where 2 is approximately 10 times more active than 1 against several tumour cell lines (Menéndez et al., 2004a; Pérez et al., 2008). We have cloned and characterized the gene cluster involved in the biosynthesis of 2 (Menéndez et al., 2004a,b; 2006; 2007). It contains the cmmA gene, which encodes the acetyltransferase responsible for transferring both acetyl groups to d-oliose and l-chromose B during 2 biosynthesis (Menéndez et al., 2004b). Inactivation of cmmA leads to the accumulation of 4A,4E-O-dideacetyl-chromomycin A3 (3) (Fig. 1), which differs from the parental compound by lacking the two acetyl groups, and being approximately 100 times less active, which confirms the importance of the acetyl groups for the antitumour activity of 2 (Menéndez et al., 2004b).
In this article we report the use of CmmA to increase the structural diversity of mithramycins and to generate novel mithramycins with antitumour activity. We also show that CmmA is located in the cell membrane and exhibits substrate flexibility using different acyl-CoAs as donor substrates.
Results and discussion
Generation of acetylated analogues of mithramycin by bioconversion
Two sugars (l-chromose B and one d-oliose) in 2 are acetylated at the C4 hydroxyl groups (Fig. 1). These acetyl groups are incorporated into the molecule by the chromomycin acetyltransferase CmmA (Menéndez et al., 2004b). Since these groups greatly increase the antitumour activity of 2, we decided to use CmmA in order to generate acetylated derivatives of 1 with high antitumour activity. To achieve this, we performed a bioconversion experiment by growing the wild-type strain Streptomyces griseus ssp. griseus (chromomycin A3 producer) that was subsequently fed with 1. These cultures were then extracted and analysed by HPLC-MS. In addition to 1, a new peak was detected showing the same absorption spectrum than 1, but with higher retention time and an m/z value in positive mode of 1128 (data not shown). This is in agreement with compound 1 with one acetyl group attached. We also tested if this acetylation event was CmmA-dependent by running a bioconversion experiment using S. griseus C10A as host (cmmA- mutant). No acetylated mithramycin was obtained in these conditions confirming that CmmA is required for this acetylation process (data not shown). This result showed that CmmA was flexible enough to be able to acetylate 1. The bioconversion efficiency using the wild-type strain was rather low (approximately 14%). Most probably this was due to the fact that the acetyltransferase CmmA has more affinity to its genuine substrate and/or was mainly committed to the biosynthesis of 2. With the aim of improving the production of acetylated derivatives of 1, we tested the bioconversion efficiencies as biocatalysts of different mutant strains of S. griseus affected in biosynthesis of 2. These mutants (C60GI, C10GII, G10GIII and G10GIV; Fig. 2) expressed the acetyltransferase CmmA but were blocked in glycosylation steps taking place before the acetylation of sugars, and therefore preventing the synthesis of potential natural substrates for CmmA. When any of these mutants was used in bioconversion experiments with 1 as substrate, higher production of monoacetylated mithramycin 11 was obtained. Since S. griseus C10GIV was more efficient than the other mutant strains in the bioconversion experiments (35% of conversion, Fig. 3A), it was selected as biocatalyst for further experiments.
Our next goal was to generate acetylated derivatives of different analogues of 1, which differ from 1 either in the glycosylation pattern (Fig. 1, compounds 4 to 8) or in the structure of the aglycone side-chain (Fig. 1, compounds 9 and 10). Streptomyces griseus C10GIV was fed with all these compounds independently and the cultures were analysed by HPLC and HPLC-MS (Fig. 3B–H). In all the cases new peaks were detected which corresponded to monoacetylated derivatives, as deduced from the higher retention times and masses of the novel compounds, differing in 42 units in comparison with the respective substrates. These mass changes are consistent with the incorporation of a single acetyl group to the compound. Bioconversions with some compounds led to the formation of several derivatives with the same mass (monoacetylated), but with different retention times (compounds 4, 5 and 6 shown in Fig. 3B, C and D), suggesting that acetylations took place in different positions of the substrates. Moreover, bioconversions with compounds 4, 5, 7 and 9, and also 1 resulted in the generation of compounds with mass differing in 84 units from the corresponding substrates, being consistent with the generation of derivatives with two acetyl groups attached (Fig. 3A, C, E and G). Also, an analogue of compound 5 was obtained with an m/z value in positive mode of 1197 (peak 16 in Fig. 3C), which is consistent with the incorporation of three acetyl groups to compound 5.
Isolation and structure elucidation of selected compounds
Some acetylated compounds were selected for purification and further characterization. Selection was mainly based on the antitumour activity of the parental compounds, but also considering the yields and the possible structural diversity of the acetylated derivatives. In total we purified seven compounds: one derivative from 1 (compound 11, Fig. 3A), five from 5 (compounds 12 to 16, Fig. 3C) and one from 9 (compound 17, Fig. 3G). These compounds were purified by preparative HPLC from cultures of S. griseus C10GIV fed with the corresponding substrates. The acetylated compounds were initially identified by HPLC-MS analyses by comparing the UV absortion spectrum and analysing the mass of the molecular ion. For the new derivative obtained from 1 (compound 11) a molecular formula of C54H78O25 (m/z: 1128) was deduced, indicating the incorporation of one acetyl group into the trisaccharide moiety. Similarly, mass spectra of the five derivatives isolated from 5 revealed three monoacetylated derivatives (compounds 12 to 14) with a molecular formula of C53H76O25 (m/z: 1114), one diacetylated derivative (compound 15) with a molecular formula of C56H80O25 (m/z: 1156) and one triacetylated derivative (compound 16) with a molecular formula of C58H82O26 (m/z: 1198), all of them acetylated in the trisaccharide chain. The compound 17 isolated from 9 showed a molecular formula of C53H76O24 (m/z: 1098), indicating the presence of one acetyl group in the trisaccharide chain again. Final identification of the novel compounds was carried out by 1H mono- and bidimensional NMR experiments (COSY, TOCSY) (see Supporting information). The acetylated positions in the novel analogues were identified by comparing the corresponding spectra with those previously described for compounds 1, 5 and 9 (Wohlert et al., 1999; Remsing et al., 2003; Baig et al., 2008). The chemical structures of the isolated compounds are shown in Fig. 4.
These new compounds differ from the parental compounds in the presence of one, two or three acetyl groups attached at specific positions of different sugars of the saccharide chains. Three different hydroxyl groups in mithramycins were susceptible of being acetylated by CmmA, those located at C3 and C4 of sugar E and the one at position C4 of sugar D. Acetylation at 3E occurred when d-digitoxose was the distal sugar of the trisaccharide chain instead of d-mycarose, as it happens in 5; it is likely that the presence of the C-methyl group at C3 in d-mycarose prevents the access of CmmA to that position. In chromomycins, the 4E hydroxyl group is the most common acetylated position. On the contrary, no chromomycin has been identified yet with an acetyl group at 3E (Menéndez et al., 2004b; 2006). Most probably, the existence of a C-methyl group at C3 in l-chromose B (sugar E) in 2 would prevent the acetylation event. On the other hand, 4D position is susceptible to acetylation in mithramycins but not in chromomycins. This could be explained because sugar D in mithramycins is a d-oliose while in chromomycins is a d-olivose. These two sugars only differ in the configuration of the hydroxyl group at C4, axial in the former and equatorial in the latter, which highlights the importance of the configuration of that hydroxyl group to be acetylated. On the contrary, no compound was produced with acetyl groups in the disaccharide chain of 1 or its derivatives. This is in contrast to what happens in 2 where the 4A position is normally acetylated, and the 3A and 4B positions are occasionally acetylated (Menéndez et al., 2004b). These differences could be also explained by the different sugar configuration of the disaccharide chain in both compounds, consisting of d-olivoses in 1 and in some analogues, and d-olioses in 2. In summary, based on the structures of known chromomycins and acetylated mithramycin compounds reported in this article, it seems that CmmA has preference for hydroxyl groups in axial configuration and not for those in equatorial configurations, unless these are located at the third sugar of the trisaccharide chain; approximately 59% of acetylated mithramycin compounds obtained by bioconversion are acetylated at hydroxyl groups in axial configuration, versus 39% in equatorial configuration.
The generation of different types of acetylated mithramycins indicates that CmmA shows substrate acceptor flexibility, both regarding the position and type of sugar acetylated and also regarding the structure of the side-chain of the aglycone. In this respect, we were able to generate acetylated derivatives of mithramycins independently of the structure and length of their side-chain.
The antitumour activity of the new mithramycin analogues was tested against four tumour cell lines (Table 1). Compilation of the average GI50 (50% of growth inhibition) values showed that all of the compounds were active at micromolar concentrations and most of them were also active at 0.1 µM or lower. In general, all compounds were less active against glioblastoma cancer cells. This was particularly noticeable for compounds 12 and 15. This different behaviour could be ascribed to differences in gene expression patterns that are specific to each cell line (Ertel et al., 2006). Compound 12 showed improved activity against colon cancer cells (about five times higher activity than the parent compound 5). Moreover, compound 14 was particularly active against glioblastoma cancer cells in relation to its parent compound (activity up to five times higher than that of compound 5 from which it derives), and compounds 12, 13 and 16 were better against pancreas tumour cells (about five times more active than the parent compound 5). This could be particularly important since glioblastoma is one of the most common tumours of the central nervous system, and despite many efforts to overcome this aggressive disease the median survival for patients with glioblastoma is only 1 year (Shrieve et al., 1999). On the other hand, the treatment of pancreatic cancer remains elusive; in spite of treatment, < 5% of patients remain alive after 5 years (Chua and Zalcberg, 2008). Therefore, development of new compounds with high activity against pancreatic tumours is currently most demanding Interestingly, most of those mithramycins with improved activities bear one acetyl group (e.g. compounds 12, 13, 14), but there were other compounds with two or three acetyl groups (e.g. compound 16). This indicates that apparently there is no correlation between the number of acetyl groups and the antitumour activity.
In order to in vitro assay CmmA, its coding gene was subcloned in pEM4T under the control of the erythromycin resistance promoter (ermE*p) and expressed in Streptomyces albus. Correct expression of cmmA in this strain (S. albus pEM4TCmmA) was verified by carrying out a bioconversion experiment using as substrate 4A,4E-O-dideacetylchromomycin A33, its natural substrate. This was readily converted into 2, while a S. albus strain containing an empty vector was unable to carry out that bioconversion (data not shown). Next, localization of CmmA in the hyphae was attempted. Mycelia from S. albus pEM4TCmmA and S. albus pEM4T (as control) were fractionated into soluble and membrane fractions and the fractions were analysed by Western blotting using anti-CmmA serum. No positive signals were shown in fractions from S. albus pEM4T (Fig. 5A). However, positive signals were detected in the membrane fractions but not in the soluble fraction of S. albus pEM4TCmmA (Fig. 5A), which is consistent with the location of CmmA in the cell membrane. These fractions were also tested for acetyltransferase activity using an in vitro assay. Again, fractions from the control strain did not shown acetyltransferase activity (data not shown). However, cell-free extracts and membrane but not soluble fractions from S. albus pEM4TCmmA were able to acetylate 3 generating 2, in the presence of acetyl-CoA (Fig. 5B). In this reaction two other compounds were also produced that could correspond to monoacetylated derivatives of 3, since their masses were consistent with the incorporation of a single acetyl group to 3. These results clearly demonstrate that CmmA is located and active in the cell membrane. This location is consistent with the idea that in the biosynthesis pathway of chromomycin A3 acetylation of sugars is one of the latest biosynthesis steps, taking place in close relation to its secretion.
We also tested the possibility of CmmA to use different donor substrates. We screened short straight- and branched-chain acyl-CoAs (C3 and C4). Of all acyl-CoA donors tested only C3 chain acyl-CoAs (isobutyryl- and propionyl-CoA) were utilized by CmmA, whereas longer-chain-length acyl-CoAs (isovaleryl-, isobutyryl-CoA and β-methylcrotonyl-CoA) were not. When isobutyryl-CoA was used, a new peak with a higher retention time and an m/z value in positive mode of 1169 was produced (Fig. 6A), which is consistent with the incorporation of an isobutyryl group to 3. When the donor substrate was propionyl-CoA, two new compounds were produced that showed higher retention times than 3 (Fig. 6B), and with m/z values in positive mode of 1155 and 1211. These masses were consistent with the incorporation of one and two propionyl groups to 3 respectively. Propionyl- and Isobutiryl-CoA were not used with the same efficiency by CmmA, being the former better substrate than the latter. As shown in Fig. 6, the use of propionyl-CoA in the reaction led to the complete acylation of compound 3, with the generation of derivatives with one and two propionyl groups. However, when isobutiryl-CoA was used approximately half of compound 3 remained non-acylated and only one derivative with one isobutiryl group was generated. Isobutiryl-CoA differs from propionyl-CoA by the presence of a methyl group at C2, which makes it bulkier and thus probably interfering in its interaction with the active site of the enzyme. These experiments demonstrate that CmmA has some tolerance towards its donor substrate. CmmA shows similarity to other acyltransferases involved in tailoring steps of antibiotics biosynthesis. Some of these acyltransferases (MdmB, AcyA and Asm19) have also shown considerable substrate tolerance for the acyl donor, but they were quite specific towards the acceptor substrate (Hara and Hutchinson, 1992; Arisawa et al., 1994; Moss et al., 2002). On the contrary, CmmA is also able to acylate different substrates and at different positions, showing broader acceptor substrate flexibility. This highlights the potential of CmmA as a tool to create structural diversity and therefore to generate novel antitumour compounds.
Strains, culture conditions, plasmids and DNA manipulation
Several strains were used as biocatalysts for bioconversion experiments: S. griseus ssp. griseus ATCC 13273, chromomycin A3 producer, and the S. griseus mutant strains C60GI, C10GII, G10GIII and G10GIV, each one affected in a glycosyltransferase gene of the chromomycin biosynthesis gene cluster (Fig. 2) (Menéndez et al., 2006). Streptomyces albus J1074 (ilv-1, sal-2; Kieser et al., 2000) was used as host for expression of cmmA. Several S. griseus and S. argillaceus recombinant strains were used to prepare substrates for bioconversion experiments and enzyme assays (see Fig. 1): S. griseus C10A to produce 3 (Menéndez et al., 2004b); S. argillaceus ATCC12956 for 1; S. argillaceus (pMP3*BII) for 4 and 5; S. argillaceus (pFL845) for 6; S. argillaceus (pMP1*BII) for 7; S. argillaceus M7U1 (pFL845) for 8 (Baig et al., 2008; Pérez et al., 2008); and S. argillaceus M7W1 for 9 and 10 (Remsing et al., 2003; Albertini et al., 2006). For sporulation on solid medium, the organisms were grown at 30°C on plates containing A medium (Fernández et al., 1998), supplemented with 25 µg ml−1 thiostrepton when appropriate. For growth in liquid medium, the organisms were grown either in TSB medium (trypticase soya broth; Oxoid) or in R5A medium (Fernández et al., 1998), supplemented with 5 µg ml−1 thiostrepton when appropriate. pEM4T was used for expressing genes in Streptomyces (Menéndez et al., 2006). Escherichia coli DH10B (Invitrogen) was used as a host for subcloning. Plasmid pQE60CmmA was used as source of cmmA (N. Menéndez, unpublished). DNA manipulations were performed according to standard techniques for E. coli (Sambrook and Russell, 2001) and Streptomyces (Kieser et al., 2000).
PCR amplification and gene expression
For amplification of cmmA, pQE60cmmA was used as DNA template and the following oligoprimers: 5′-GGACTAGTCCTAGGGGACGAAAGAGGCAGGATG-3′ (SpeI and AvrII sites underlined) and 5′-GCTCTAGATCAGTGGTGGTGGTGGTGGTG-3′ (XbaI site underlined). PCR conditions were as follows: 100 ng of template DNA was mixed with 30 pmol of each primer and 1.25 units of Pfx DNA polymerase (Invitrogen) in a total reaction volume of 50 µl containing 2 µM each dNTP, 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.8), 2 mM MgSO4 and 0.1% Triton X-100. The polymerization reaction was performed in a thermocycler (MiniCycler; MJ Research) under the following conditions: an initial denaturation of 2 min at 94°C; 30 cycles of 15 s at 94°C, 30 s at 55°C and 1 min at 68°C; after the 30 cycles, an extra extension step of 5 min at 68°C and another of 15 min at 4°C were added. After PCR amplification, the amplicon containing cmmA was digested with SpeI and XbaI, treated with Klenow polymerase and subcloned into the EcoRI site pre-treated with Klenow polymerase of pEM4T. In the resultant plasmid (pEM4TcmmA), cmmA is under the control of the ermE*p.
Streptomyces griseus wild type or the appropriate mutant strain was cultivated in TSB liquid medium with shaking at 30°C for 24 h. One hundred microlitres of the resultant cultures were used as pre-inoculum to inoculate Erlenmeyer flasks containing 5 ml of R5A liquid medium. After 24 h of incubation, 15 µg per ml of the substrate (final concentration) was added, and further incubation was carried out for 24 h. In the case of mutants S. griseus C10GIII and S. griseus C10GIV, cultures were supplemented with 2 µg ml−12 (final concentration) in order to induce expression of chromomycin biosynthesis genes (Menéndez et al., 2006). After incubation, samples (1 ml) were extracted with 1 volume of ethyl acetate: formic acid (100:1). Organic extracts were dried under vacuum, and residues were eventually dissolved in a small volume of methanol before HPLC analysis.
HPLC analysis and purification of novel compounds
Chromatographic analyses were performed by HPLC-MS as previously described (Olano et al., 2009), except that bidimensional chromatograms were extracted at 280 nm. Acetylated derivatives were obtained in bioconversion experiments in R5A medium supplemented with 15 µg ml−1 of the precursor molecule. Approximately 1.2 l of culture was prepared to obtain 4E-O-acetyl-mithramycin 11, whereas the derivatives of 5 were obtained from 5 l of culture. In both cases, the cultures were centrifuged and the supernatants were filtered and applied to a solid-phase extraction cartridge (Sep-Pak Vac C18, 10 g, Waters). The retained material was eluted with a mixture of methanol and 0.05% trifluoroacetic acid (TFA) in water. A linear gradient from 0 to 100% methanol in 60 min, at 10 ml min−1, was used. Fractions were taken every 5 min and analysed by UPLC. Fractions containing the desired compounds were neutralized with 3 ml of 0.1 M phosphate buffer (pH 7.0) and evaporated in vacuo. The resulting extracts were redissolved in a small volume of a mixture of DMSO and methanol (50:50) and the products were purified by preparative HPLC using a SunFire C18 column (10 × 250 mm, Waters). Compounds were chromatographed with mixtures of acetonitrile or methanol and 0.05% TFA in water, in isocratic conditions optimized for each peak, at 7 ml min−1. During all purification steps, the peaks were collected on 0.1 M phosphate buffer (pH 7.0). The solutions obtained were partially evaporated in rotavapour to reduce the concentration of the organic solvent and then applied to a solid-phase extraction cartridge (Sep-Pak Vac C18, Waters), washed with water to remove salts, eluted with methanol and finally lyophilized.
An alternative method was carried out for the purification of 4D-O-acetyl-mithramycin SK 17. In this case, 2 l of R5A medium for the bioconversion experiment was supplemented with 50 µg ml−1 of the precursor molecule. The culture was centrifuged and the supernatant was filtered and solid-phase extracted (Sep-Pak Vac C18, 10 g, Waters). A linear gradient of methanol and water (0–100% methanol during 60 min at 10 ml min−1) was applied for elution of the retained material. Fractions obtained were analysed by HPLC and those containing the acetylated derivative were pooled and dried in vacuo. The extract was redissolved in 5 ml of methanol and the purification procedure was carried out in a preparative column (XBridgeTM Prep C18, 5 µm, 30 × 150 mm, Waters) with isocratic elution using MeCN and 0.1% TFA in water (45:55), at 20 ml min−1. As TFA was used in the mobile phase of the purification, the peak was collected on 0.1 M potassium phosphate buffer (pH 7.0) and diluted fourfold with water to be desalted and concentrated by solid-phase extraction, being finally lyophilized. The yields for the different compounds were as follows: 3.1 mg of 11; 1.7 mg of 12; 4.6 mg of 13; 1.0 mg of 14; 1.8 mg of 15; 2.5 mg of 16; and 5.2 mg of 17.
Structure elucidation and characterization of new mithramycin derivatives
The structures of the new mithramycin analogues were elucidated by NMR spectroscopy, employing 1D 1H, 13C and homonuclear 2D 1H COSY/TOCSY experiments. All NMR data were recorded in acetone-d6 at 298 K using a Bruker Avance Ultrashield Plus 600 spectrometer (600 MHz for 1H, 150 MHz for 13C experiments). Typical parameters for 2D experiments were: COSY/TOCSY, 256 and 2048 points in F1 and F2, respectively, 48 transients each. NMR experiments were processed using the program Topspin 1.3 (Bruker GmbH, Karlsruhe, Germany).
Determination of antitumour activity
The antitumour activity of the mithramycin derivatives was tested against four tumour cell lines. Quantitative measurement of cell growth and viability was carried out using the MTT tetrazolium assay (Mosmann, 1983). Values correspond to the average of three independent assays.
To determine the location of the CmmA protein, S. albus pEM4TcmmA was grown in TSB medium at 30°C with orbital shaking for 48 h. Cultures were centrifuged and the mycelium was washed twice with 50 mM Tris-HCl (pH 7.5), 1 mM EDTA and 20% glycerol. The mycelium was disrupted by two passes through a French press at a pressure of 10.3 MPa. DNA was broken by ultrasound (six pulses of 15 s each with intermittent cooling on ice water) in a MSE ultrasonic disintegrator at 150 watts and 50 kHz. Unbroken cells and debris were removed by centrifugation at 12 000 g for 30 min. Samples were collected from the resultant supernatant and were named ‘cell-free extracts’. The supernatant was further centrifuged at 69 000 g for 1 h. The resulting supernatant was designated ‘soluble fraction’. The pellet containing the cell membranes was resuspended in a small volume of the above-mentioned buffer and was named ‘membrane fraction’. Protein estimation in the different samples was carried out by measurement of absorbance at 540 nm by a protein-dye binding assay using bovine serum albumin as standard (Bradford, 1976).
Polyacrylamide gel electrophoresis and Western analysis
Protein analysis was carried out by polyacrylamide gel electrophoresis in the presence of SDS (Laemmli, 1970). Western blotting was performed as described (Burnette, 1981), using the developing kit ECL plus Western Blotting Detection System (Amersham Biosciences). A peptide (ARPGDSTRSFYRRC) corresponding to the second predicted cytoplasmic loop of CmmA was synthesized to produce anti-CmmA serum (SigmaGenosys).
In vitro assays of CmmA
Acetylation assays were conducted in a total volume of 50 µl in Tris-HCl 50 mM pH 8, EDTA 1 mM and glycerol 20%, including 100 µM 3, 130 µg of protein from the membrane fraction (or the corresponding cell fraction to be tested) and 500 µM acetyl-CoA. After incubation at 30°C for 2 h, the reaction products were extracted twice with 50 µl of ethyl acetate acidified with 3 µl of 0.4 N HCl. After phase separation, the organic phase was recovered, the solvent evaporated and the residue were suspended in 50 µl of methanol prior analysed by HPLC. Assays were also carried out using other acyl-CoA donors (2 mM final concentration): isovaleryl-CoA, isobutyryl-CoA, β-methylcrotonyl-CoA, propionyl-CoA and succinyl-CoA.
This work was supported by grants (to C.M.) from the Spanish Ministry of Education and Science (BIO2005-04115 and PET2005-0401) and from the Spanish Ministry of Science and Innovation (BIO2008-00269). B.G. was the recipient of a predoctoral fellowship from the Spanish Ministry of Science and Innovation. Instituto Biomar S.A. is acknowledged for helping us with the antitumour assays.

domain: chemistry


text:
Im 29 and suffered a mini stroke at the start of the Month 2/10/17, ive been put on clopidogrel and simvastatin by the hospital and signed off from work by my doctor untill end of the month, ive had a mri, ct scan and awaiting a echocardiogram and 48hr ecg, i feel very tired and seem to only be able to be up and about on my feet for a certain amount of time before my head starts to feel funny sort of fuzzy and makes me feel not normal, i feel down and generally not myself, Is that a normal thing to be feeling after a TIA (i know its not a full blown stroke which im counting my blessings for) I have a follow up appointment at the TIA clinic at the end of November but im wondering if i should see about getting signed off from work untill ive had that appointment, i work in a busy shop where im on my feet all day if i cant even manage to walk around a supermarket for half hour without feeling funny what hope have i got of running a shop.
The good news is that sufferers from TIAs recover much quicker than the full blown strokes. And usually have no permanent damage. A few very bad TIAs seem to take six or even twelve weeks of recovery.
The simple answer is yes, get signed off until the November appointment.
I know that TIAs are small compared with full strokes, but they are still nasty and sometimes serious ailments.
Also, do take the medication and any medical advice seriously. You do not want the TIA to lead to further problems.

domain: medical


Hundreds in Baltimore protest Freddie Gray’s death in police custody
Crowds are gathering in front of a police department calling for those involved to be punished.
— Colin Campbell (@cmcampbell6) April 22, 2015
The Baltimore Police Department identified the six officers involved in Gray’s arrest on Tuesday as Lt. Brian Rice, 41, Officer Caesar Goodson, 45, Sergeant Alicia White, 30, Officer William Porter, 25, Officer Garrett Miller, 26, and Officer Edward Nero, 29.
According to Baltimore PD Captain Eric Kowalczyk, three of the officers initially approached Gray on bicycles, one made eye contact with Gray, while another officer joined in the arrest and one drove the police van.
— Ben Swann (@BenSwann_) April 22, 2015
The officers were suspended with pay, pending the investigation’s outcome. Five of the officers have provided statements to investigators. “They have completely cooperated with the investigation from day one,” Michael Davey, attorney for the officers, said at a news conference.
“What we know is that he was healthy before he came in contact with the police, before he was unlawfully arrested, he was a healthy 25-year-old man,” William Murphy, attorney for the Gray family, told RT. “And after the police unlawfully arrested him, there was a severe injury to his spinal cord and he died from those injuries. Those are facts. That’s what we know.”
— Anya Parampil (@anyaparampil) April 22, 2015
Gray passed away on April 19. On Monday afternoon, city officials admitted the 25-year-old had requested an inhaler while in police custody, but said calls for medical assistance were not made for another 42 minutes. An ambulance was dispatched to the scene to take Gray to a local hospital, where he slipped into a coma and eventually died.
In his police report, Officer Garrett Miller said that Gray was stopped after fleeing “unprovoked upon noticing police presence.” Miller said a knife was found clipped to Gray's pants pocket and he was taken into custody.
“During transport to Western District via wagon transport the defendant suffered a medical emergency and was immediately transported to Shock Trauma via medic,” Miller wrote in his report.
There was another person inside the van, but as he is a “witness in a criminal investigation” the police have refused to disclose his identity. According to Murphy, however, that person had no access to Gray. “Someone severed his spine,” Murphy said. “It wasn’t Mr. Gray. That just leaves the police department.”
“At this point, the family just wants the truth. They want answers,” said Murphy. “They want to know exactly how Mr. Gray was injured, when he was injured, why he was injured, why he was arrested unlawfully.”
— Anonymous (@YourAnonNews) April 22, 2015
Murphy also called on the Baltimore PD to release any 911 calls about the incident, as well as to reveal whether there were any cameras in the police van. Baltimore law enforcement officers do not currently wear body cameras. In November, Mayor Stephanie Rawlings-Blake said she would veto a city council bill that would require police officers in the city to wear body cameras while on duty.
“Were the police talking amongst themselves at the beginning of this incident?” asked Murphy. “Throughout the incident? If they were, we want those radio transmissions.”
He noted that the officers have had enough time to coordinate their version of events, while “we will never hear Freddie Gray’s side of the story.” The Maryland Law Enforcement Officers' Bill of Rights says that a police officer under investigation who may face disciplinary action may not be interrogated until 10 days after the incident in question.
Murphy pointed out that the police lacked probable cause to arrest Gray in the first place. Running away from the police was not a crime, nor was carrying a knife, he said.
“The only reason that he could have possibly been arrested is running while black.”


text:
According to the Bureau of Labor Statistics , there were 270 workplace suicides in 2013, an 8% increase from 2012.
Since suicide can be an issue in the workplace, it is important that employers are prepared to recognize warning signs and know how to handle both the employee and any resulting situations, should they arise.
It is rare for a suicidal employee to actively reach out for help. While the intent to harm oneself may not be clearly stated, there are several warning signs that may be observed by supervisors or coworkers, including:
- Significant loss, including loss of job, social status, finances, or close personal relationships.
- Significant changes in behavior or mood; withdrawing from friends, family, and coworkers.
- Loss of interest in regular pursuits, including job, family, hobbies, and activities.
If warning signs are observed, the QPR Institute recommends that three steps be taken:
- Question: Ask the person if he or she is thinking of committing suicide.
- Persuade: Ask the person to reconsider and seek immediate help.
- Refer: Provide the person with references for experts who are equipped to handle the situation. Do not try to handle the situation yourself. The company’s Employee Assistance Program (EAP) or a hotline such as 1-800-273-TALK are good starting points.
This is a sensitive subject and managers are advised to proceed very cautiously. Occasionally, an employee may be making a joke in poor taste that leads someone to make the wrong conclusion, but there may be times when an employee is in serious trouble and someone at the workplace feels morally obligated to help. It can be difficult to know exactly when help should be offered without intruding on an employee or coworkers privacy.
Suicidal thoughts or attempts also get into areas where assistance or job protection may be mandated under various regulations. One example is job-protected time off for treatment under the Family Medical Leave Act (FMLA). Another example may be reasonable accommodations for ongoing therapy sessions to deal with mental issues that are protected by the Americans with Disabilities Act (ADA).
If any of your employees are showing concerning behavior, please begin by seeking human resource guidance, such as Nextep’s HR team, to discuss the employers ethical and legal obligations, as well as additional resources for the next steps.

domain: psychology


text:
Its all hand made by me. I put the shed up virtually single handed, made the throw out of scraps, made the tented ceiling out of some old curtains, painted everything, did the stencilling and stamping, designed and made the mirror and table out of old bits of wood and painted the designs on them all using a few emulsion match pots. Finished it off with my old belly dancing belts. The whole Moroccan theme cost £72 - inspired by a Moroccan style lamp I bought for £ 14.99
The shed was bought on a sudden whim and put up by me in an afternoon about 4 years ago(with the help of an extra pair of hands for one roof section which I just couldn't manage by myself. 2 weeks ago I decided it needed a makeover.

domain: design


A lawsuit recently filed in a West Virginia federal court could have lasting implications on past auto accidents in New Jersey. The suit alleges that Ford Motor Company essentially put profits ahead of safety in allowing millions of vehicles to be on the road despite having dangerous design defects that left them prone to sudden accelerations.
The plaintiffs in the suit are Ford owners from 14 states. They are seeking class action status. The vehicles identified in the suit include the 2008-2010 Taurus, 2007-2010 Edge, and the 2004-2010 Explorer.
Many people remember that Japanese automaker Toyota recently experienced legal troubles due to similar problems. It recently settled a lawsuit with the survivors of a Utah couple killed in 2010 when a Toyota Camry suddenly accelerated on I-80 and crashed into a wall. Toyota also reached a $1.1 billion dollar settlement with a class of Camry owners who claimed that the scandal caused them to lose considerable value in their vehicles. Toyota continues to negotiate settlements with people who were injured in various accidents across the country.
While no reports have arisen about acceleration problems injuring Ford drivers, the lawsuit could affect liability in New Jersey accidents involving Fords. Essentially, a Ford driver accused of speeding into traffic or causing an accident may have a viable defense if it can be proven that the car was defective. Further, Ford could be liable to New Jersey drivers who were previously injured if it can be proven that Ford allowed defective cars on the road.
Source: WRCBtv.com, Lawsuit claims Ford ‘design defect’ can cause sudden acceleration, March 31, 2013


Five Apollo 11 technologies that NASA still uses 50 years after the first man landed on the moon
The technology and inventions that NASA developed along the way are still at play today. Most of them are outdated, but a few have managed to stand the test of time.
Here’s a quick quick at the Apollo technologies that are still in use 50 years after the first Moon landing:
Space blankets then
A scientist at Goddard Space Flight Center examines the thermal blanket that will protect the Integrated Science Instrument Model’s Electronics Compartment of the new James Webb Space Telescope (Source: NASA)
Space blankets were probably the most popular of all the spin off inventions from the Apollo 11 mission. Space is cold, really cold, so NASA wanted to send some blankets with heavy insulation to keep the astronauts warm.
They built the blankets themselves by layering metalized sheets of lightweight mylar, the most effective material for insulation by weight and size.
Space blankets now
Space blankets, also known as emergency blankets, are made out of an insulation first developed by NASA in 1964 (Source: AFM Inc.)
Back then, the requirement was limited to facing extreme temperatures in space. But, soon, the blanket made its way into emergency kits, camping gear and a key tool for firefighting.
Even some clothes use the material to build insulation.
Digital flight controls now
Cruise control in cars (Source: Wikimedia)
It was only after NASA used it for Apollo 11 that the agency and its partner spent years developing it for the aviation industry. Now, it’s used for cruise control, anti lock brakes and electronic stability control systems.
Food safety then
Looking to ensure the absolute safety of prepackaged foods for spaceflight, NASA partnered with the Pillsbury Company to create a new, systematic approach to quality control (Source: NASA)
When astronauts take food up into space, it has to be free of microbes or else they could get really sick. So, again, NASA enlisted the help of a partner — Pillsbury — to help them come up with a solution.
They soon came to the conclusion that the existing quality control methods just weren’t enough to tackle the problem.
Pillsbury could have gone the way of spot-checking the final products to make sure they could go up in space, but instead, the company decided to revamp their entire manufacturing setup instead.
Pillsbury called it the Hazard Analysis and Critical Control Point (HACCP) system.
Food safety now
Pillsbury Doughboy tours the Range Factory (Source: YouTube/GE Appliances)
Since Pillsburry had already come up with the system for NASA’s Apollo 11 mission, they decided to implement the same process in their own factories as well.
Soon, the government asked other food sectors like poultry, seafood and juice producers to follow the HACCP system as well.
Today, the same principles from HACCP form the new regulations that are coming into place to be applied across the entire food industry.
Rechargeable hearing ads then
High Energy Density Silver-Zinc Cells (Source:NASA, SBIR)
During the course of the Apollo 11 mission, NASA was able to build silver zinc batteries for its command module. They weren’t rechargeable but they were the lightest known battery couple.
So, NASA got to work trying to make the silver zinc batteries rechargeable.
Rechargeable hearing ads now
Bose's SoundSport Free wireless headphones (Source: Bose)
The question wasn’t just about having rechargeable batteries but batteries that were small enough to be plugged into a hearing aid machine.
Till before the Apollo 11 mission, the batteries of hearing aids had always been disposable. The zinc batteries batteries, which are small enough, aren’t rechargeable and lithium-ion batteries, though rechargeable, couldn’t be shrunk down to the required size.
Using the silver zinc technology, hearing aids can hold enough power to last a full day and the batteries are durable enough to be recharged up to 1,000 times before they lose their performance.
The same batteries are what make noise-cancelling wireless earbuds a reality.
An illustration of the fuel- and electricity-carrying umbilicals that extended from the gantry to the Apollo spacecraft. In the 1960s Taylor Devices developed shock absorbers to help safely dislodge the umbilicals from the spacecraft during launches. (Source: NASA Spinoff)
The same shock absorbers and computers that were used during the Apollo era now keep buildings and bridges from falling.
The fluid shock absorbers can be found all over the world, especially in earthquake prone regions, reinforcing the infrastructure.


text:
Occasions by Hakkasan Review
Occasions by Hakkasan Review
We review a delivery box from the Michelin-starred dining hotspot
How I miss restaurants – particularly those treat night ones with all the anticipation and excitement they entail. There’s the perfectly crafted cocktails, wine lists as big as bibles, and a menu so full of incredible choices that you can’t possibly decide. There’s also the atmosphere, gorgeous surrounds, and, of course, someone else to serve, cook and clear up.
Not long to go until they reopen but, in the meantime, a meal delivery box is your next best option, and several of the capital’s top restaurants – Michelin-starred winners no less – have conjured up some incredible feasts so that you can enjoy your favourite dishes at home with minimal effort on your part.
Occasions by Hakkasan is one such offering. A few years back I had a stunning dinner at the original restaurant in Hanway Place – there’s now two in London and several worldwide serving its high-end take on Cantonese classics. Achingly cool, Hakkasan is all low lighting, dark panelling, and deep house music giving it a clubby vibe. The cocktails are fabulous and the food will blow your mind. How on earth can you package that into a home delivery box for two? They’ve certainly given it their best shot. But it doesn’t come cheap at £175. So, what do you get for your money?
Impressively, they’ve nailed the excitement and sense of occasion. A weighty black box arrives, you open the lid and in front of you is a beautifully designed menu. Delve in, and you’ll find chopsticks, incense sticks and a link to the Hakkasan play list. There’s recipe cards and a link to a short video tutorial on how to cook the dishes before you.
We dimmed the lights, crafted some cocktails, lined up the music, lit the incense and set to work. The package includes a reusable bamboo steamer to cook the dim sum course – this is no ordinary dim sum with beautiful multi-coloured creations of langoustine, halibut, king crab, and wild mushroom. There’s some cute jars of dipping sauces to go alongside everything, and caviar to adorn some of the dim sum. Simply steamed, they were all mouthfuls of heaven.
For mains, there is grilled Chilean sea bass in honey and a dish of smoked beef ribs that you pop in the oven. The main course requires a bit of timing but no real skill – you just need to juggle the stir-frying of the noodle dish and baby broccoli separately while the meat and fish cook. And what an incredible feast it was – the fish was huge and moist, the beef unctuous, and we loved the little garnishes, which not only looked pretty but really added to the flavours and textures such as pine nuts and seaweed to top the broccoli.
Dessert is a chocolate soufflé – pop in the oven but again make sure you time it correctly. Ours rose perfectly, and looked Insta-worthy incredible topped with the dried raspberries and gold chocolate disk. Wow – they were so good we wondered if we could get them to deliver more.
It was one of the most fun nights we’ve had in lockdown. And while there’s no waiter and you need to do your own clearing up, there’s also the bonus that you don’t have to order an Uber home. And we were rather glad after such a feast that we could just kick back and chill, listening to the Hakkasan sound track.
While it’s certainly not cheap, for that special date night this really is a brilliant idea, and it would also make a gorgeous gift for an anniversary.
• The Occasions by Hakkasan box costs £175 for 2 people and is available nationwide. We tried the Signature Menu – but you can also customise your order with other dishes. There’s a vegan option, too.
• Orders can be made via https://hakkasan.com/mayfair/occasions-by-hakkasan/, with deliveries available on Thursdays, Fridays, Saturdays and Sundays; orders placed before 12pm can be delivered the next day within London.

domain: food


text:
May 13, - Watch Four hot girls play a game, loser strips and more. online on of each game has to perform a forfeit, ranging from the tame to the sexy.
StipHilo Free demo of a live hilo strip game. Magnetic Billiard Put all balls to billiard pockets with the help of a magnet. Jack Ways-2 Collect tokens to get Black Jack.
Claus on a Demonic Adventure Hot and sexy Mrs. Claus collects diamonds for her wet pussy.
BomBilliard Burst the bomb, to open sexy video on a strippiny table. PokerPool-4 This time billiard table has 52 pockets with cards.
Poker3Some-2 To discard some your card, you must cover some opponents card Billiard does stripping a girl game need cues - just make a splash and balls will move to the pockets. Billiard balls roll down to the center of the dimple.
Just press on the table to move balls. A lot of girls are waiting for you in cells of JowBlob castle. If you gamw to see the strip show - clear the screen from cards.
Magnetic Billiard-2 Push white balls to the billiard pockets with metal ball. Magnetic Billiard Metal ball follows your magnet on a billiard table.
Crossroad X Drive your car to the next street, to see the beautiful girl stripping on a crossroad. Hypno Games follows Ash on an adventure with his new companion, a busty.
Anime Maid Do you want to bang stripping a girl game sexy anime maid?
Then you are in luck, because this game w. This sexy reporter has to get h. Satomi is an adventure video game done in the style of the Japanese hen.
Aim the cannon at the pirate ship, and the sexy pirate girl strippinng. Blackjackets Blackjackets is actually quite a unique and fun little porn game.
As it pans out. Sweet Anais You brought a girl back home from the bar, and got her undressed to her lingerie. Abduction This guy is a dangerous creep.
He is kidnapping sexy girls and fucking them with.
News:Ever wanted to reach into the phone or through your monitor and just strip the tech support that isnt helping you on the other side? Well heres your chance!
Leave a Comment

domain: entertainment


text:
ITZY‘s Yeji isn’t just loved by fans, she’s also adored by professional makeup artists and hairstylists!
Recently, ITZY’s makeup artist, Jo Sun, and hairstylist, Ryu Ji, sat down for an interview with YouTube channel AYO.
Jo Sun revealed that Yeji is the K-Pop idol they love experimenting on the most. Because of her luxurious features, she can pull off anything they try on her.
The subject of experiments = Yeji. She can pull off any concepts, so we get to try something new more often.
— Jo Sun
Being able to pull off a concept is one thing, but actually being willing to try it is another…and Yeji is open to whatever they want! She isn’t picky at all with the hair and makeup they give her.
She likes everything, because anything looks good on her. She likes anything.
— Jo Sun
It’s just as Jo Sun said. Yeji really does look stunning in anything and everything! Whether it’s fierce and vibrant makeup…
…or simple and sweet.
Learn more about K-Pop hair and makeup in the full video below.


text:
News Corp is a network of leading companies in the worlds of diversified media, news, education, and information services.
Virus deaths to stay high in summer & 80% on ventilators will die, NY Gov warns
Trump campaign starts petition to demand left-wing media air daily briefing
Socialist Sanders says Trump ‘minimized virus danger facing US’
‘Real tragedy’ as healthy family of 4 all killed by coronavirus in a week
Florida FINALLY locked down, months after 1st case detected during Super Bowl
Healthy ‘hand sanitizer queen, 42, dies of virus as lungs filled with fluid
Emotional NY Gov Cuomo says ‘I couldn’t protect my own brother’ from virus
Europe's youngest virus victim, 12, ‘sent home by GP who said it was allergy’
Donald Trump claims Iran 'planning sneak attack on US troops' in Iraq
Europe death toll hits 30,000 as France, Germany & Spain have deadliest days
Greece make hand sanitizer using bootleg booze flogged to Brit tourists
Doc gives family end of life plan over fears she'll die from virus before wedding
‘Doomsday’ castle with its own GOLD MINE could be perfect place to self-isolate
Amazon workers plan another walkout & say virus not taken ‘seriously’
Nurse ‘strangled doc girlfriend, 27, to death thinking she gave him virus'
Second man dies from virus after going to famous Miami Beach Winter Party
Sex workers with virus in Tokyo red light district 'could've infected hundreds'
Surgeon General says 30 days won’t be long enough to stop virus in US
Congressman Devin Nunes says it's 'overkill' to close schools to stop virus
K-pop star sparks fury by claiming to have coronavirus as 'April Fool's joke'
Dad led cops on high-speed chase after ‘abducting toddler & shooting 3 relatives'
Tormented' woman killed herself after perv secretly filmed her in shower
Dad 'blacked out on alcohol and can't remember killing baby and girlfriend'
Calls to shut down China's cruel wet markets as they keep selling bats & dogs
Obama blasts Trump for 'denying warnings of pandemic and climate change'
Woman pees in cop car, coughs in officer's face & shouts 'I have coronavirus'
FBI bungled 'ALL secret court filings' & blundered beyond Trump-Russia
Airbnb customer’s leg balloons ‘twice the size’ from horror spider bite in bed

domain: news


text:
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“A very special thank you to my Insanely talented wedding stylist Tanya and her beautiful team who created us the most magical wedding ceremony and reception I could of ever dreamed of! When you burst into tears because you are so overwhelmed by how incredibly beautiful it looked you know you got the right people to do the job! (I can’t wait to see the professional ones of the reception these photos don’t do it justice) Thank you Sugared Style! You turned my dreams into a reality by creating something even more spectacular then I ever could of imagined it to be. Highly highly recommend!!! 10/10″
– Alyssa and Bardon
“Just sending a little note to tell you how thankful I am for everything you did for us on Friday. You literally blew me away! When I saw what you had put together, I just couldn’t believe how perfect it was. It is like you took my dream wedding out of my head and made it happen. You are an absolute super star. How did I get so lucky to have you work your magic for us?!
Every little detail was perfection. The rings above the bridal table were out of this world. As were the quotes, the flowers, the candles, the LOVE sign and the stunning antique dresser. I still can’t get over it. Absolutely, incredibly, beautiful.
You were a dream to work with and nothing was too hard. You are not only my ‘wedding stylist’ but my friend and always will be.
Thank you for making my dreams come true – I just couldn’t have done it without you.”
– Kate Evans
“I’ve been thinking about what to write to you and I honestly don’t know where to begin. You made every single part of our wedding day perfect!
From our first meeting a few months before the wedding I instantly felt at ease and the weight lifted from my shoulder. Although we only hired you as an ‘On the day coordinator’ you went above and beyond without any hesitation!
I really want to thank you for everything you did – but in particular for making our ceremony perfect when behind the scenes was chaos, for driving all over the city picking up our bits and pieces, for your beautiful styling skills and for saving my night when my dress broke mid reception! You knew instantly when something went wrong and was there before I even had a chance to worry! And you did it all with an 8 week old baby!! You are super woman!
Please pass on our huge thank you to your wonderful team and your amazing hubby. Everyone in your team were just a dream to work with.”

domain: finance


text:
Do you want to contribute to top quality medical research?
Do you want to work with systems biology in chronic pain?
We invite applications for a joint post doc position in Prof Camilla
Svensson's team that explores cellular and molecular mechanisms underlaying
chronic pain states. The position will be placed in the Department of
Physiology and Pharmacology at the Karolinska Institutet, which has a
collegial, international and dynamic research environment. The Svensson lab is
part the Center for Molecular Medicine (CMM) located on the premises of the
Karolinska Hospital. We offer an exciting, open, and collaborative research
work environment and project, where you can make a difference.
The Postdoctoral Fellow that join the team will be investigating pain
mechanisms in chronic pain conditions. The project is focused on
autoantibodies and mechanisms by which they induce and sustain pain. The work
will be performed in vitro as well as by analyzing samples from patients and
healthy controls. You will also be working with animal models to investigate
pain mechanisms related to the findings in the pain patient samples. You will
be working closely together preclinical and clinical researchers and
biostatisticians with a mission to increase our ability to study pain from a
You will work in a creative, inspiring and vibrant research environment. You
will be part of a research team that is dedicated to undertaking translational
research with the ultimate goal to uncover cellular and molecular reasons
behind pain chronicity and contribute to the improvement of life quality for
individuals suffering from fibromyalgia.
For more information please visit: https:// ki.se/en/fyfa/molecular-pain-
You are a creative, hardworking and enthusiastic team player. You have a PhD
in immunology, neuroscience, biomedical science, or another relevant field.
You have a strong background neuroscience, immunology or neuroinflammation.
Documented practical experience with immunocytochemistry, primary cell
cultures, libraray preparation for single cell and bulk RNA sequencing and
data analysis or animal handling are considered strong merits.
You can work independently but also have the interpersonal and communication
skills to succeed at working in a multidisciplinary team. You have a deep
sense of responsibility, taking initiatives and have capacity and flexibility
to work with researchers and students with different expertise.
You have a very good written and spoken English and is a good communicator,
both when it comes to presentations and preparation of publications. You have
good recommendations from previous supervisors/teachers.
Scholarships for postdoctoral qualification can be established for foreign
researchers who place their qualifications in Sweden. The purpose of
scholarships for postdoctoral qualification is to promote internationalization
and contribute to research qualification after a doctorate or equivalent.
A scholarship for carrying out postdoctoral research can be granted for a
maximum of two years within a four year period following the receipt of a
doctoral degree or equivalent.
To be eligible for a postdoctoral scholarship, the person must have obtained a
doctorate or a foreign degree deemed to be equivalent to a doctorate.
Applicants who have not completed a doctorate at the end of the application
period may also apply, provided that all requirements for a completed degree
are met before the (intended) start date of the post doctoral education.
The head of the department determines whether their previous training and
scholarly qualifications correspond to a Swedish doctorate or higher.
Type of scholarship
The amount is tax free and it is set for twelve months at a time, paid out on
a six months basis. In exceptional cases, shorter periods may be acceptable.
An application must contain the following documents in English or Swedish:
A complete curriculum vitae, including date of the thesis defence, title
of the thesis, previous academic positions, academic title, current
position, academic distinctions, and committee work
A complete list of publications
A summary of current work (no more than one page)
The application is to be submitted on the Varbi recruitment system.
Want to make a difference? Join us and contribute to better health for
|First day of employment
||According to the agreement
|Last application date
||17.Jan.2022 11:59 PM CET


text:
Inclusive Programming - Graduate Assistant
The Office of Equity and Inclusion — The Office of Equity and Inclusion (OEI) works to meet the University’s commitment to diversity, equity, and inclusion with an integrative model of leadership. In collaboration with other university offices, divisions, strategic plans, and staff, the OEI supports an institutional climate of Ignatian Citizenship by balancing opportunities for individual enrichment and reflection on how one’s social identities influence choice and responsibilities and collaborative work for justice. The Office fosters companions who think of themselves as part of something bigger and promotes equity and inclusion as the goal of an intentionally inclusive society.
Graduate Assistant —Inclusive Programming
The Office of Equity and Inclusion is seeking a graduate student who not only possesses a deep passion for promoting diversity, equity, and inclusion (DEI), but also has a strong commitment to advancing these values across the university. This position will provide the chosen candidate with a unique opportunity to gain valuable professional experience, while also contributing towards creating a more inclusive and equitable environment for all.
|Hours and Wages
This assistantship is for the 2024-2025 academic year, starting in August 2024 and ending in May 2025.
Hours: This role will work 225 hours per semester, approximately 15 hours per week.
|Application and Contact Information
To apply, please submit the following materials:
For questions, please contact Rhona Little, Diversity and Inclusion Specialist, Office of Equity and Inclusion.
email@example.com or 410-617-2082

domain: sociology


text:
Heartworm disease is a prevalent problem for dogs in our area. For this reason, we recommend that owners learn a little bit about the disease, treatment, and prevention.
Heartworms are not always a problem until they reach their adult size when they can be up to twelve inches long. As adults, they prefer to live in the right side of the heart and the lungs. For this reason, most dogs will have both lung disease and heart failure.
When a dog has heartworms, it is necessary to treat him or her. However, the treatment can be dangerous because the worms can cause problems as they die. It is very important to watch the dogs carefully during this process. Some dogs may need a second treatment.
Fortunately, heartworms are preventable. We recommend that you prevent heartworms instead of dealing with the disease and treatment. Here at Springmill Pet Wellness Clinic, we offer a once a month chewable tablet that will prevent heartworms and also help with intestinal parasites, keeping your dog and your family even healthier.
Please contact us to get your dog started with a heartworm test and heartworm prevention today.


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TULSA - A traffic alert for morning motorists -- a rollover accident has closed part of Peoria Avenue in midtown Tulsa.
The northbound lanes have been closed around the 51st Street area since about 5:45 a.m.
Police say a pickup truck traveling southbound on Peoria slammed into an SUV that was leaving QuikTrip.
Two people suffered minor injuries.
Police expected cleanup to take some time.
Copyright 2012 Scripps Media, Inc. All rights reserved. This material may not be published, broadcast, rewritten, or redistributed.
Also in the headlines
The swarm of earthquakes shaking central Oklahoma continued Saturday afternoon as a 4.5 magnitude quake struck near Jones, Okla.
Pictures of the first winter storm of the season, Dec. 5 and 6.
The live musical performance of the Sound of Music starring Carrie Underwood aired on NBC December 5, 2013.
Glow Downtown holiday light tour kicked off the Christmas season with festivities at Guthrie Green, The Deco District and the BOK Center, Dec. 1


- What We Do
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When Shanna Rainey was a mother to two young children, she began using methamphetamine.
The SPLC and its allies have filed a lawsuit challenging the Trump administration policy that has separated and traumatized thousands of migrant families.
Ashley Buford could not hold back the tears when she saw her brother behind a chain-link fence with barbed wire on top.
The following statement is by Southern Poverty Law Center Interim President and CEO Karen Baynes-Dunning following reports that Iranians and Iranian Americans are being detained and questioned at U.S. ports of entry:
Our work in 2019 year was challenging – and highly successful – in many ways.
It was pouring rain the day Willie Parker left William E. Donaldson Correctional Facility after 36 years in prison.


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Discover more from Tread by Lee Hedgepeth
LIVE UPDATES: Alabama set to execute James Barber
The state abandoned its last two attempts at lethal injection after issues accessing veins.
Alabama plans to begin its attempts to execute James Barber at 6 p.m. today.
Tread’s Lee Hedgepeth will witness the execution at Holman Correctional Facility in Atmore, Alabama, should it proceed. Live updates will be posted below, with newer posts closer to the top.
If you are reading this article in your e-mail, you must click the story on Tread’s website for live updates.
1:56 a.m. — James Barber has been executed by the State of Alabama. His time of death was 1:56 a.m., according to a prison official.
1:00 a.m. — Members of the media are headed to Holman.
12:40 a.m. — The nation’s highest court has cleared the way for Barber’s execution, but the state hasn’t yet begun to move press to Holman.
When the state is ready to proceed, members of the media will be moved by van inside the gates of Holman prison, where the state’s death row is located.
12:15 a.m. — The U.S. Supreme Court has denied James Barber's application for a stay of execution. The court's 3 liberal justices dissented, saying the court shouldn't allow Alabama to let James Barber be its "guinea pig."
“The Court should not allow Alabama to test the efficacy of its internal review by using Barber as its ‘guinea pig,’” the justices wrote. “It should grant Barber’s application for a stay of his execution.”
Prison officials haven’t yet begun to move the media by van inside Holman’s gates.
12:00 a.m. — The traditional midnight deadline for Alabama execution warrants has passed, but a recent rule change allows prison officials until 6 a.m. to begin the state’s lethal injection of James Barber.
11:00 p.m. — Alabama was scheduled to execute James Barber five hours ago, but state prison officials are still waiting on a Supreme Court ruling as to whether his lethal injection can move forward.
Unlike in the past, there is no midnight deadline for executions. Instead, prison officials have until 6 a.m. on Friday to begin the lethal injection.
9:30 p.m. — Still no word from the U.S. Supreme Court, who will ultimately decide Barber’s fate today. His execution was tentatively set to begin at 6 a.m., but the state has until 6 a.m. Friday to carry out the lethal injection.
8:00 p.m. — Prison officials are still waiting on a decision from the U.S. Supreme Court before proceeding.
Linked here is the execution protocol that applies to Barber, which was released as part of his lawsuit against the state.
7:22 p.m. — Reporters set to witness the execution of James Barber are still in a holding pattern at a small media center outside Holman Correctional Facility in south Alabama, awaiting a ruling by the U.S. Supreme Court on whether Alabama’s lethal injection of James Barber can proceed. So far, federal courts have rejected Barber’s appeals.
Alabama has until 6 a.m. to execute Barber after a rule change extended the state’s traditional midnight deadline.
Despite Alabama's complaints about time constraints surrounding executions, often blaming it on the condemned, however, they chose to tentatively set James Barber's execution to begin at 6 p.m., 18 hours into the 30-hour execution timeframe set by Gov. Ivey.
6:31 p.m. — A federal district court has ruled that Barber’s attorney must be allowed to bring pen and paper to witness her client’s execution. The judge, though, denied the lawyer’s request to bring a watch into the facility, citing an ADOC policy banning jewelry.
6:15 p.m. — Kelly Betts, public information officer for the Alabama Department of Corrections, provided the following written information related to Barber’s activities over the last day.
The witnesses for the victim requested anonymity. There were four witnesses for the condemned inmate: Glen Barber, brother; Elizabeth Bruenig, friend; John Gallo, attorney, and Mara Rose Easterbrook Klebaner, attorney. Five members of the media were witnesses as well as several state witnesses.
On July 19, 2023, Barber had 10 visitors and six phone calls. On July 20, 2023, he had 22 visitors and 2 phone calls. On July 20, he refused breakfast, ate snacks, drank beverages, and had a final meal of Loaded Hashbrowns, Western Omelet, Spicy Sausage, and White Toast.
6:05 p.m. — An Alabama prison official confirmed to Tread that the state will not yet move forward with James Barber's execution, which was tentatively scheduled for 6 p.m. There is technically no stay in place preventing the lethal injection, but we're awaiting a Supreme Court ruling in the case.
5:05 p.m. — James Barber has just filed his reply to Alabama's brief to the U.S. Supreme Court. He is asking the court to stay his execution, scheduled to begin in less than an hour. His lawyers wrote that Alabama misrepresented the issue in the case.
5:00 p.m. — James Barber's final visitors have just left, a source inside the prison told Tread. Barber is likely being moved back to the death cell now, where he'll wait for the U.S. Supreme Court to decide whether it will allow Alabama to move forward with his execution at 6 p.m.
3:15 p.m. — The State of Alabama has filed its opposition to Barber’s request for the U.S. Supreme Court to stay his execution. Lawyers for the state argue the court shouldn’t take the case at all.
“Petitioner James Edward Barber is entitled to neither a stay of his execution nor a grant of his petition for a writ of certiorari,” Alabama’s brief said.
11:46 a.m. — James Barber has applied for a stay of execution from the U.S. Supreme Court.
“James Edward Barber is scheduled to be executed starting at 6:00 pm CT today by a method that is very likely to cause him needless pain and suffering,” Barber’s lawyers wrote in the filing.
Barber's fate now lies in the hands of the court’s nine justices.
10 a.m. — Will James Barber soon become the state’s “guinea pig?”
Alabama’s previous two attempts to execute its citizens through lethal injection led to the repeated poking and prodding of the condemned men — Alan Miller and Kenny Smith — but did not lead to their deaths. Difficulty establishing vein access by members of the execution team led to the state’s failure to carry out its grim mission, officials would later confirm.
In the wake of those failures, Barber argued that an attempted execution by lethal injection would violate the Constitution. Instead, Barber requested that his death come by nitrogen suffocation, an untested, unregulated method of execution allowed by Alabama law.
A federal court, however, rejected Barber’s argument, writing that changes to the death penalty process — for example, an effective 6-hour extension of the timeframe executioners will have to establish vein access — mean that the man’s situation differs substantially from those of Smith or Miller. The Eleventh Circuit affirmed that view with Wednesday’s opinion, leaving only the U.S. Supreme Court the last place Barber could seek relief.
In her dissent, Pryor wrote that Barber’s appeal would likely succeed if he were allowed to seek evidence from the Alabama Department of Corrections related to its three-month “review” of the execution process that came after the botched executions.
“After a three-month ‘review’ of its procedures— conducted entirely internally, entirely outside the scope of any court’s or the public’s scrutiny, and without saying what went wrong or what it fixed as a result—ADOC swears it is ready to try again, with Mr. Barber as its guinea pig,” Pryor wrote on Wednesday.
Barber was convicted and sentenced to death for the 2001 murder of Dorothy “Dottie” Epps in Harvest, Alabama.
In the decades since, Barber said he’s received something he never expected to find — forgiveness.
“I did not deserve it. I could not earn it. But I could not and will not ever take it for granted,” Barber recently said.
Barring action by the U.S. Supreme Court or Gov. Kay Ivey, Alabama is scheduled to begin its attempts to execute James Barber at 6 p.m.
Tread by Lee Hedgepeth is a reader-supported publication. To receive new posts and support my work, consider becoming a free or paid subscriber or simply making a one-time donation.

domain: politics


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Thursday, May 1, 2008
(Directed by David Hare, UK, 1985)
A young man crashes a dinner party given by a middle-aged schoolteacher for her friends. Being British, they are too polite and all believe he came with or knows someone, hence his presence. The next day, the man shows up again and explains that his attending the previous night's dinner was a hypocritical lie, since he didn't know anyone and they all pretended that they did. He suddenly places a gun in his mouth and commits suicide while the teacher watches in shock.
This is the start of David Hare's film Wetherby, focusing on the aftermath and investigation of the suicide of the young man, John Morgan (Tim McInnerney) and simultaneously an investigation of the life of the schoolteacher, Jean Travers (Vanessa Redgrave).
The main reason to see this film is Vanessa Redgrave's excellent, heartfelt performance as a woman who has, in response to a tragedy earlier in her life, has nullified her feelings to the point where she just simply exists. She proudly declares, at one point in the film that she believes she is alone, not lonely and treasures that fact. The point is that she and the friends around her, the Pilboroughs (played by Dame Judi Dench and Ian Holm) are empty and hurting as well. To get by, there are the dinner parties, the pub drinks that Jean shares with Stanley Pilborough where they play elaborate games of one-upmanship and the endless activities that Marcia, Stanley's wife arranges. Into this arrangement of
her semi-lived life comes Karen, a friend of the man who committed suicide. Karen disturbs Jean with her listless approach to living and disaffection and just hangs on. The investigation becomes complicated with a local constable (Tom Wilkinson) asking too many questions. In flashbacks, Jean reveals that she has had a love affair with a long dead man, who died as a result of a stupid chance of events.
Hare's film owes a lot of its structure to Alain Resnais' Muriel, although not having seen that unavailable film, it is hard to comment on the similarities. Hare's film is quite moving and ambitious, being hard to pin down the central mystery on why a young man kills himself over societal rules, but I almost want to believe that the core of the film is that his act forces the schoolteacher to reexamine her life and somehow move on. As one character says in the film, 'Life is messy'.
This is Redgrave's film, her performance lights it up extremely. With this and Karel Reisz's film of Isadora Duncan, this is her best. See it for her.


- Client Sector
- Catalyst Retainer Filter
As they further established their name as filtration problem-solvers, Croft quickly found they were increasingly called upon to apply their specialised knowledge in manufacturing custom-made filters for a host of industrial sectors.
Word of the company's speedy response to filtration problems - and their ability to develop specialised and bespoke resolutions - quickly spread through the petrochemical, food, chemical, pharmaceutical, marine, construction, transport, automotive and printing industries.
Their entry into the nuclear supply chain as a tier 4 supplier was very much a result of their ability to tailor a product to each client's specific needs. Croft were called upon to supply a filter for a catalyst retainer: prior to their involvement, the existing French-manufactured filter had needed to be lifted out of its container and inverted in order to drain it.
However, Croft re-engineered the filter, and not only solved the drainage problem, but also effectively removed a hands-on function from the process, saving both time and cost.
Neil Burns, a partner in Croft, said: "The nuclear industry needs to engage with small specialists across the supply chain who know what they are talking about. A small redevelopment or refinement by an expert can save thousands, or often hundreds of thousands of pounds, not to mention countless working hours. And the amazing thing is that, like us at Croft, manufacturers don't have to be nuclear specialists in order to seize a nuclear opportunity - the nuclear industry is another 'specialised' area of work for us in the same way that food or automotive industries require specific understanding of the manufacturing challenges they face."
' A small redevelopment or refinement by an expert can save thousands, or often hundreds of thousands of pounds, not to mention countless working hours.'


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【I'd like to direct swaps :The Little Prince👑/Mickey or Minnie Mouse/strawberry🍓/coffee☕/rose🌹/The Little Mole(Kretk)/letter “N”/birthday card🎂/cake/anything about Italy or Italian/vocal music/July/Leo/Husky...for my friend's birthday!🎊you can go to my Facebook profile→photos→albums,there're most of my cards for swap(FB🔍Xiaocan Zheng)if you're interested,please PM(Messenger)】
I graduated from college last year and have been preparing for the postgraduate entrance examination these months. Writing and receiving postcards can make me relax.Therefore, I attach great importance to every postcard.
I like reading, collecting stamps, traveling, practicing calligraphy and listening to music(my favorite singer is Zhou Shen周深. His voice is very unique and various🎶🔍YT)
I enjoy any card, but especially cards like:
-giant panda/red panda
-GF/FOTW/PWE/MOTW/CCUN/HPC/WT/COTW/WF/Icons/LouPaper states/Blue Cat WT/GF Large Letter/WonderPost/WPD Postal Hug
𝕋𝕙𝕖 𝕤𝕖𝕣𝕚𝕖𝕤 𝕔𝕒𝕣𝕕𝕤 𝕀 𝕙𝕒𝕧𝕖↓
-maps(from origin)，also love the fictional maps(e.g Harry Potter, the Hobbit, Genshin)
-SpongeBob -Tom and Jerry
-Fantastic Beasts and Where to Find Them
-local myths and legends
-any lovely/warm/same color system cards
-If you find no appropriate card for me, you could choose to send some stamps, new or used, or cut from the envelope.
I'd be very happy and excited if the stamps are about the above too. I really love collecting stamps🥰
But any card will be most welcome! And envelopes, letters, sealing wax or stickers,etc. are all welcome. Come as you like.
Don't know what to write? You could tell me about yourself, where you live、your hobbies、your daily life/food，your favorite book/movie/game…tell me something about the postcard or stamps you chose(name、history、origins、emblematic quality、math、story…）also，you can write a proverb in your native language and translate it,I'm very interested in it .
I'm looking forward to your postcard.

domain: artistic


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One half of the team behind the Lincoln
Continental 2025 Concept that drew plenty of attention at the Detroit Auto Show has come up with a new styling proposal named simply the Chrysler
Review. Colin Bonathan is a student a Lawrence Technology University and his portfolio of work was recently updated with these computer generated images. Whether these are of the final design or a work-in-progress is unknown.


Razing Ginsberg’s To Build A Non-Beat HotelAugust 13, 2013
Constructed in 1906 and empty since Ginsberg’s Dublin Pub vacated a few years ago, the owners of the one-story building on the northwest corner of Bay and Mason are working on plans to raze the building and build a boutique hotel on the site.
The proposed four-story hotel has been designed with 15 rooms with two roof decks for guests. No parking would be provided on-site. And with a bit of retail space on the ground floor, the new building would rise a total of 40 feet in height.
Having been constructed over 50 years ago, a Historic Resource Evaluation will need to be prepared for the existing building at 400 Bay before it may be razed. And of course, there’s always a chance a neighbor or two might Howl.
Comments from Plugged-In Readers
I’m glad to hear that this site is being developed.
Note to future guests: if weather proves uncooperative to hang out on the roof deck, Secrets bookstore is just a hop, skip and jump away.
Is this outside the THD zone of malevolence? This is exactly the kind of thing they have tried to block in the past.
Anybody got drawings of the new building? It could be a nice addition to the neighborhood if done right.
^ bay street is nowhere near the tl.
great news about the parking, great project, great scoop!
Why would no parking for this hotel be “great news?” Not exactly a location with easy access to out of towners without a car. But the shopping center across the street has a ton of roof parking – not sure if overnight is an option though.
You can’t Beat a good pun like that one at the end. Well played.
1. Who needs parking in this location! I lived around the corner for years. F-line streetcar, buses galore, all within 1-2 blocks. Heart of Fisherman’s Wharf. The nearby Hilton (or Sheraton? Can’t remember they seem so similar) used to rent out parking in its garage because – guess what – so many guests did not arrive by car. We rented an extra space there. It probably still does, but I moved out of the neighborhood in 2007 so can’t say for sure.
2. This would be some freaking historic resource. It’s an old metal industrial building that was converted to a funky bar. I don’t think SF Heritage will have a problem when this goes bye-bye.
@DaneSF: it’s great news because it’s a boutique hotel with few rooms and the building itself doesn’t warrant another cutout on the street for underground access. I’m sure management will have something arranged for parking nearby for those who choose to show up with a car. Many boutique hotels in the city operate like this.
As for transit options, the F-line and 30-Stockton are really all you got, neither of which is particularly fast, or reliable, in the case of the F-line. Cable car for those who want to shell out even more money the SF “experience.” Then again, this hotel is geared to tourists and all three of these options cover the major destinations.
Ginsberg’s vacated “a few years ago”?? Seems more like 15-20. My friends and I used to joke it was the only Jewish-Irish pub in SF.
Glad to see something is going to be done with this site.
Your friends obviously didn’t know about O’Greenburg’s.
Used to hang there in the 70’s. Leonard & Vic the Ginsberg owner/bartenders were the best fun and Sinova’s lunches were great, She also made a terrific fresh salsa. In addition to being our after work drinking hole, it was a terrific place to spend a Sunday afternoon listening to live music.
I think we should keep the building there and convert it into a micro craft brewery and pub / restaurant. Play off of the historic qualities of the building for the theme of the restaurant brewpub. It could be called Mason Bay Brewing Company. To me this is a no-brainer, and would be plenty successful with the current growing national interest in craft brewing, especially in California. This way, if there is any need to do a historical value assessment to find out whether this fancy hotel can be built, this cost can be avoided since the original structure could be retained for the brewpub operation. Anyways, there are plenty of hotels in that location already, and that location is not a great one for a special boutique hotel since the only things it is close to is fisherman’s wharf, Trader Joe’s, and Safeway.
UPDATE: Designs For Boutique Hotel To Replace Ginsberg’s Pub
Anyone know where I can get an old T-Shirt for Ginsburg’s? Arriving on the scene a bit late.
Me too! I had one, but a former girlfriend got chocolate on it, couldn’t get it out, then threw it away–all without telling me. I spent a lot of time looking for it, as I thought I’d lost it. LOL.
Finally something for ‘plugged in’ readers to be surprised by: it’s now a vacant lot (or maybe a vacant hole, with a fence around it…hard to tell). Then again maybe they shouldn’t be surprised: demolition too often being the whole play rather than just the first act; which goes a long way toward explaining why proposals aren’t always greeted with coos of approval…rendering euphoria notwithstanding.
Comments are closed.


This vendor’s goal is to monitor performance, detect outages, and track usage of its large scale, SaaS application. In order to do this, it needed to collect and correlate application logs, web logs, and reference information stored in relational databases. The vendor’s development team lacked the time and expertise to build a custom solution using open source frameworks such as Hadoop. X15 Enterprise was implemented and provided the following benefits:
- Eliminated data silos by collecting all structured and semi-structured data into a single platform
- Enabled instant discovery of application issues
- Provided insight into application performance and usage
- Improved application development and testing
- Accelerated time to value
“We went from raw data to having real-time visibility into our operations in days.”
VP Product Development
This Independent Software Vendor (ISV) offers a broad portfolio of products to thousands of customers, which makes application and infrastructure monitoring extremely critical. It’s development team is responsible for monitoring performance, detecting outages, and tracking usage of the SaaS application. In order to do this, the team needed to collect and correlate application logs, web logs, and reference information stored in relational databases.
Lacked domain expertise in real-time analytics on Hadoop
The development team lacked the time and expertise to build a custom solution using open source frameworks such as Hadoop. While it had significant experience working with structured data, the team lacked domain expertise in correlating unstructured log data. Moreover, monitoring large volumes of streaming log data requires real-time correlation, which is challenging. The team had no prior experience with Hadoop which made building a custom solution a long and risky proposition.
The X15 Solution
X15 Enterprise enabled application monitoring within days
The development team ultimately selected X15 Enterprise. Application logs, web logs, and reference information from relational databases are ingested into the X15 platform as they become available. The team leverages X15 querying and reporting capabilities to build visualizations for monitoring the SaaS application.
Eliminated data silos by collecting all structured and semi-structured data into a single platform
The X15 platform discovers data structure upon ingestion and dynamically adjusts it over time to eliminate the need to specify rigid schemas upfront. This is critical, because the logs from applications and infrastructure can vary greatly. The team was able to collect all log data and reference data in the X15 platform in days.
Enabled instant discovery of application issues
The X15 platform supports continuous querying of data as it becomes available. The development team created queries to continuously search for error conditions and trigger alerts as soon as an incident occurs. This reduced mean-time-to-resolution (MTTR) of issues and improved customer experience.
Provided insight into application performance and usage
With X15, the developers created live dashboards to monitor application performance and usage in production. They can proactively plan infrastructure requirements and generate reports for stakeholders.
Improved application development and testing
The development team also leverages the X15 platform to monitor test environments with new releases of the application. Live dashboards are used to monitor performance and alerts are triggered when errors are detected in the logs.
Accelerated time to value
The X15 platform supports a visual query builder, an intuitive search language, ANSI SQL, and regular expressions to analyze data. The development team was able to leverage its SQL experience to query and report on the data without requiring any training. This dramatically accelerated the time to value of the project.