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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: literature


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❉ ‘Messiah of Evil’ falls somewhere between arthouse and grindhouse, early George Romero and late Mario Bava…
❉ Bowie’s queer sensibility is most closely associated with his music, but how did it manifest in his film career?
❉ George A. Romero, ‘Night of the Living Dead’ creator, dies at 77
❉ A book with a difference for cult film afficionados.
❉ We conclude our look back on Ray Milland’s cult triptych with his second feature for Roger Corman’s AIP studios.
❉ A modern-day witch uses spells and magic to get men to fall in love with her, in a tribute to 1960s Technicolor thrillers.
❉ We review the first two titles in this exciting new series, ‘Martin’ and ‘Theatre of Blood’.

domain: library


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Ellen and Doug go together like two peas in a pod. Just like peas, these two grew up on farms. So it was quite fitting that their reception was held at one. Their wedding was just about as beautiful as their relationship. Each and every thoughtful and carefully handcrafted detail was not lost on me. From the hand painted signs to the seating chart, each detail was awe inspiring. Even the wood used to hold the centerpieces on each table was hand chosen from family farmland before finishing into a fine piece of art.
Water of some kind is needed to grow a plentiful harvest. Ellen and Doug are for sure to grow a bountiful marriage as the rain clouds blessed them on their wedding day (luckily all before the outdoor festivities)! We even momentarily saw a rainbow (not in the images) hovering outside over the town. The rain clouds parted after the indoor ceremony and the most beautiful peaceful sky let in and splashing on the dance floor ensued late into the night.
Trigger Warning – Sweet tooths abort! This wedding reception contained one of the best make your own smores tables I’ve ever been privy to photograph.
And a special thanks to all the vendors
that helped make this day such a joy to photograph!
Dresses – Bridal Elegance
Flower Girl Dresses – Posh Peanut on etsy
Hair – Studio 718
Suits – Mr. Penguin
Rings – Zales
Boots – Cavenders
Flowers – TPM Stems
Reception Decor – DIY
Food – Stone Jug Barbeque
Music – Joe Beck Entertainment
Cake – Kelsey’s Kakes
Tent and Dance Floor – Rental Pros
Transportation – Top Shelf Chauffeur
Ceremony – Trinity Lutheran Church
To see more awesome RJA wedding images check out our wedding gallery.
The latest images hot off of the PLP editing screen.

domain: artistic


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If you're new to Nashville's dining scene, there's no doubt you've had to familiarize yourself with a little restaurant history in order to understand what people are talking about. You've probably scratched your head or smiled and nodded when someone spoke cryptically about "that restaurant that went in after Maude's but before Atlantis" or "the Cajun joint that used to be on Murphy Road and then was out by the DMV before it moved to Charlotte."
Well, now it's your turn to join in the conversation, because a slew of restaurants recently opened in places that, until recently, were known as something else. Now you too can chime in about "the sports bar that used to be a Latin-fusion lounge" or "the brewpub that used to be a nursing home." You can amaze and bemuse your dining companions with teasers such as, "How about that seafood place that used to be a seafood place?"
Here is a first-impression rundown of a few nascent restaurants that witnesses to local dining history can't help but describe in terms of what — or where — they used to be.
When it comes to provenance, here's an unusual one: Cooper Brunk, an alumnus of Mack & Kate's and the bygone and beloved Ombi on Elliston, opened a brewpub in the erstwhile Cornelia House nursing home. The facility was shuttered in 2007 and transformed into a mixed-use development that will soon offer affordable housing for deaf residents. Cooper's anchors the retail component, which houses several boutiques, a bakery and a salon.
Dark and cozy, with comfortable booths and a manly vibe underscored by a deep bench of bottled and draft beers, Cooper's could be described as a fortunate collision of Brown's Diner and Rumours — made for a man but pretty enough for a woman. Or made for beer folks but culinary enough for food people. And with grilled cheeses and root beer floats, it's kid-friendly to boot.
With enough snacks and entrées to satisfy both drinking and dining appetites, Cooper's offers a comfortable balance of bar and restaurant. It's the kind of place where an afternoon of wings, Benton's ham sliders, deviled eggs, pickled okra and dark chocolate stout can morph into a wine-soaked evening of beer-basted game hen with root vegetable pot pie ($16.50) and bacon-wrapped stuffed trout ($17.50).
You'll know you're there when you see the twinkle lights outlining the angular deck that hangs over Eastland Avenue. Located along the burgeoning restaurant corridor that includes Silly Goose, Wild Cow, Eastland Cafe and Rosepepper Cantina, Cooper's gives East Nashville yet another thing to brag about.
Opens 4 p.m. Tuesday through Friday and 10 a.m. Saturday and Sunday.
In terms of sheer wordplay, this newest incarnation of Watermark's coastal-themed sister might be our favorite to talk about. Recently relocated from the Adelicia tower to 12South, Fish & Co. can be described as the seafood place that moved from the former Miro to the former Mirror, which, if you remember, used to be a seafood place. (Laurell's Central Market, which vacated the building in 1999, was a pioneer in the revitalization of the 12South neighborhood.)
In any case, the new Fish & Co. seems to have found a match between concept and location. On a recent Saturday night, servers were learning to navigate the tightly packed tables in the dining room, which has been painted a warm blue-green after its brief recent stint as the mustard-and-brown-bathed Blind Pig. Buzzing with neighborhood traffic, the crowded room had an air of cozy festivity that we never saw in either the Miro District dining room or the Blind Pig concept.
The best points of James Beard-honored Chef Louis Osteen's menu are still available, including bacon-wrapped trout ($24), the crab-cake appetizer ($12), shrimp and grits ($16/$24), oyster BLT with Benton's bacon ($11), and an array of oysters on the half-shell, including Apalachicola, Gulf Coast and Fanny Bay.
Bar opens at 4 p.m. daily.
No moss grows under restaurateur Chris Hyndman. The founder of Virago recently moved the decade-old eatery to the Gulch, where it will anchor his M Street dining complex, alongside Whiskey Kitchen and the long-anticipated Kayne Prime steak house. No sooner had Hyndman launched Virago 2.0 than he shut down Lime, the Latin-fusion showplace in Midtown, and relaunched it in late January as Tavern.
Think Whiskey Kitchen with a dash of sports bar. The new concept twists the layout of Lime, moving the massive central bar to one side and filling the open air space with flatscreen TVs. With more suspended monitors than a small airport terminal, Tavern is a good place to catch a game, because each deep, leather-lined booth effectively has its own tube.
Like its Gulch cousin, Whiskey Kitchen, Tavern boasts a roster of elevated pub food, but not just a reissue of WK's fare. Chef Robbie Wilson maintains separate repertoires at the two joints. Tavern's spin on the bar classics includes fried organic catfish ($14.50), a bucket of mussels with fries ($14.50), fried chicken with cold sesame soba noodles ($14.50), as well as a range of salads and sandwiches.
On our visit, a group at the bar was huddled over an open flame, roasting their own dessert of s'mores. That kind of thing never would have happened at the place that used to be there.
Opens at 11 a.m. daily.
"Business Plan: We're going to have Bud Light binge consumption on *FOUR* levels instead of…
Calling the people responsible for Tootsie's, Rippy's and Honky-Tonk Central ""restauranteurs" is a bad joke…
F*ck this, and f*ck these guys.
For the love of god, please stop with sticking garage doors in every building downtown…
Arrgh, Eastie. You're totally correct. Gin fell out of my text during an edit. My…

domain: food


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Get fitter in your sleep
When you follow a well planned training program over a period of time, your body gets fitter. Most people would acknowledge that. Exercise=fitness. Full stop. Right? Not necessarily.
One of the really important components of fitness, and one that gets overlooked way too often, is recovery. And one of the really important components of recovery is sleep. I’ve written before on how chronic sleep deprivation can inhibit weight loss, and in fact cause us to gain weight, but today I wanted to look at how sleeping helps you to get fitter.
But firstly, we need to look at how training gets you fitter.
A guy named Hans Selye described how the body responds to the stress of exercise, using what he termed the General Adaptation Syndrome (GAS). Basically, the body goes through a set of specific short term responses, and longer term adaptations, after being exposed to a stressor. The body goes through three stages in response to a stressor. Exercise is one such stressor.
Stage 1: Alarm or shock. This is the immediate response to the stress of exercise, and can include feeling flat, sore or stiff and a bit tired. The fitter you are, the greater the stress needed to induce this shock phase. Hence, it’s really important to have a training program that takes into account your individual fitness level.
Stage 2: Adaptation or resistance. The body responds to repeated bouts of training. It thinks, “Crikey, if she’s going to keep doing this to me, I’d better do something about getting stronger”. The body equips itself with the tools to survive further stress of the type you are imposing on it. This can include nervous system adaptations, hormonal changes, and tissue building, just to name a few. You need to have stressed your body sufficiently in stage 1, for adaptations to take place in stage 2.
Stage 3: Exhaustion. This is the stage you never want to reach. It’s simply put, over training. Too much training and not enough recovery. There’s just too much stress for the body to be able to adapt.
The Role of Sleep In Recovery
For anyone at least partially serious about their fitness, harder training is a double edged sword. It can be the key to greater fitness, but can also send your body over the top towards injury and illness. Exercise can cause heaps of stress to joints and ligaments, muscles, the nervous system, and the endocrine (or hormonal) system. And yet, it’s the stimulus your body needs to signal it to get stronger to make itself better able to cope with that stress of exercise.
Recovery takes place anytime you ease off with your workload. You can still actually be exercising whilst you are recovering (most training programs will include a recovery every 3-5 weeks where you ease back on your training-and if yours doesn’t, it should), but it’s when you’re at rest that the greatest training adaptations take place. Sleep is the ultimate rest period in which your body can adapt to all your hard training.
During sleep, hormones such as the rejuvenating Human Growth Homone (HGH) reach their peak. Your immune system gets a recovery boost when you’re asleep, and neurotransmitters in the brain are replenished. There’s an amazing amount of biochemical activity going on inside your body when you are asleep.
Human growth hormone is important for well, yes, growth. It’s also linked with fat loss, muscle tone, immune health and the firmness and elasticity of your skin. Mostly, when HGH receives a natural boost, it’s a good thing, and sleep causes HGH secretion to peak. So, if you’re not getting enough sleep, you’re not getting the maximum levels of HGH your body can produce, and directly adversely effecting your performance.
The other really critical thing sleep promotes is the replenishment of neurotransmitters. Neurotransmitters are chemicals which transmit signals from a neuron to a target cell in the body. They regulate quite a few physical and emotional processes, including mental performance, emotional states, and the pain response. All of these are important for physical fitness. If your neurotransmitters aren’t working effectively, you won’t get much done in any part of your life, let alone your recreational exercise.
With your neurotransmitters under functioning, you’re likely not to be in a great emotional state to train. Day after day you’ll be having “one of those days” where you just don’t feel like you can do much of anything. You wake up just knowing that exercise isn’t going to happen today, unless you have someone drag you kicking and screaming to your workout.
Of course, it’s very easy for me to say get more sleep. If insomnia isn’t an issue, often it’s just a case of not being in bed for long enough that prevents us from getting enough sleep. Turn off the TV, shut down social media channels, and get to bed. And make sure all your electronic devices are on the other side of the door!

domain: health


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Purpose: Along with increasing age and a positive family history subSaharan African ancestry has long been recognized as an important risk factor for prostate cancer. In the United States the incidence of prostate cancer is approximately 60% higher in African-American than in European-American men and the mortality rate from the disease is more than twice as high. The purpose of this review article is to examine specific reports highlighting racial disparity and its possible causes.
Materials and methods: The reports chosen for review of this epidemiology and pathophysiology study were included to demonstrate conditions in which racial differences as well as similarities exist in African-American and European-American men. Reports also include autopsy, biological and clinical studies, and early and late stage prostate cancer.
Results: From the 1970s to the current statistical analysis of the National Cancer Institute Surveillance, Epidemiology, and End Results program African-American men have continued to have a significant higher incidence and mortality rate than European-American men. Autopsy studies show a similar prevalence of early small subclinical prostate cancers but a higher prevalence of high grade prostatic intraepithelial neoplasia. Clinical studies show a similarity in prostate cancer outcome when pathological stage is organ confined but a worse outcome when disease is locally advanced and metastatic in African-American vs European-American men. There is increasing genetic evidence that suggest that prostate cancer in African-American vs European-American men may be more aggressive, especially in young men.
Conclusions: Improving the outcome in African-American men with prostate cancer requires awareness of the epidemiological patterns of the disease and willingness on the part of physicians to implement targeted study initiatives with end points designed to detect the disease early in this population and begin appropriate management. It is proposed that a multi-institutional study should be done to demonstrate the ability to decrease racial outcome disparity by education, aggressive testing and treatment.

domain: medical


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The hardest thing in the world to do is to turn your back on your child, especially when they suffer from bipolar disorder. I have no choice but to walk away from my son and it is ripping out my heart. He attempted suicide Oct 1 and was talking the same stuff at 3 a.m. I have done all I can do and now it’s up to cps.
I will be charged with neglect and the state will put him in a facility or in foster care. For a mother to have do this is so very hard. I have no more choices and it is has come down to be getting a backbone and walking away at this point. I cannot let my child destroy me and himself so I have been given no alternative.
The hospital keeps calling and I refuse to pick up the phone because they want me to take responsibility financially and it’s not happening. CPS knows I have done all I can do for him and it gives me no pleasure to have to be so cold to my child. The problem with being a mother is doing what is best and not feeling guilt.
I feel so bad that I have had to do this to my child but it is for his best no matter how it hurts me. I talked to “C” about the situation and I think I could hear a sigh of relief in his voice. Maybe I am imagining it but I have no doubt that having my son out of the way is what he would like.
When you feel like someone is glad to have your kids out of the way there becomes these undertones of disgust that you feel. I also feel a sense of betrayal from him because I thought he would have common sense to accept my kids but I am starting to think differently.

domain: psychology


Regional Planning Group Seeks Consultant To Perform JLUS For Connecticut Sub Base
By Dan Cohen
Association of Defense Communities
July 29, 2016
The Southeastern Connecticut Council of Governments (SCCOG) is seeking qualifications and proposals from firms to prepare a joint land use study (JLUS) for Naval Submarine Base New London on behalf of its member municipalities — the town of Groton, town of Waterford, town of Ledyard, city of Groton, city of New London and town of Montville. DOD’s Office of Economic Adjustment (OEA) provides grants to state and local governments to conduct a JLUS to support the long-term sustainability and operability of the military installation complex. An award of a grant to the SCCOG to conduct a JLUS is pending approval by OEA.
Interested firms must demonstrate knowledge and experience in community planning, development and land use issues, fiscal impact analysis, economic development, and military installation management and operations.
Responses to this request must be received by SCCOG no later than Friday, Sept. 16, at 4 p.m. Responses received after this deadline will not be considered.


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When you’re searching for a new home or preparing to sell your existing property, it is important that you work with a real estate professional. While you can try to buy or sell a home on your
6 Family-Friendly Activities You Can Do This Fall In The Twin Cities
Dated: September 14 2021
It's that time of year again. Twin Cities families are making plans to celebrate the arrival of the harvest season. Fall is a perfect time for families to enjoy seasonal activities in and beyond the Twin Cities. Here are the top six family-friendly activities we recommend to scratch that Autumn itch.
Frolick in an apple orchard
If part of your annual family tradition involves baking a warm apple pie or whipping up some hearty apple cider, you'll want to take a balmy morning stroll through one of the many beautiful and aromatic apple orchards that dot the Twin Cities.
Minnesota is the home of the Honeycrisp apple and it's one of the best places to pick it, and others, straight from the tree. We recommend local orchards like Afton Apple Orchard in Hastings, Emma Krumbees in Belle Plaine or Apple Jack Orchards in Delano.
Visit a farmers market
Fall is harvest time and the local farmers are excited to showcase an abundance of fresh produce and fragrant flowers. Take a trip to your neighborhood farmers market to support local Twin Cities businesses and turn hearty autumn produce into homey family dinners. Not sure where to start? Here's a list of fall farmers' markets in the Twin Cities that offer a fresh food selection and a fun atmosphere the whole family will love.
Take a scenic drive
Pile your family into the car and take a scenic drive to enjoy the annual changing of the colors. Minnesota is home to some of the most beautiful fall foliage in the country, making a scenic drive a jaw-dropping must this autumn. And the best part is, you don't have to travel too far outside of the Twin Cities to see them. Use this color tracker to plan out your fall leaf-peeping adventure.
One of the most famous fall foliage drives is North Shore Scenic Drive which starts in Duluth and goes up to Grand Portage State Park as it winds along the rugged shores of Lake Superior, running about 154 miles. Make a pit stop at the numerous state parks along the way to enjoy scenic hiking and awe-inspiring waterfalls as you trek through wilderness streams and forested hills.
Another famous scenic drive is the Minnesota River Valley Scenic Byway. This 300-mile scenic route follows the Minnesota River between Browns Valley and Belle Plains and takes you to Ortonville. In addition to even more parks for your hiking and bird watching pleasure, you'll encounter several areas that showcase rich Dakota Indian heritage.
Go for a hike
Enjoy a nice brisk hike on some of the best trails in the state. Whip those hiking boots out and get ready to get them dirty. With about 66 state parks and just as many regional parks, you'll have your pick of the litter when it comes to hiking trails packed with beautiful fall colors and scenic landscapes.
Visit a pumpkin patch
If your kids love carving jack-o-lanterns or helping you out in the kitchen as you tackle the year's first pumpkin pie, take them out to pick the perfect one at a local pumpkin patch. Whether you prefer to carve scary faces into them, bake and eat the seeds, or use them as decorative centerpieces, locally grown pumpkins are a must-have this time of year. You can check out Minnesota Grown's list of 140 pumpkin patches, some of which have carnival rides, corn mazes, petting zoos, and fall festivities to entertain kids of all ages.
Find some Halloween scares and fun
From family-friendly to frightening, Halloween celebrations of all types can be found across Minnesota. Anoka is the self-proclaimed Halloween Capital of the World and celebrates all month long. The Ghosts of Anoka Walking Tour is chock-full of haunted tales about early settlers and pioneers.
For more ghastly fun the whole family can enjoy, don't miss Pumpkin Night in the Park at the Springbrook Nature Center in Fridley. Take part in pumpkin-carving activities, watch fire dancers, play spooky bingo, or hike past more than 1,000 jack-o-lanterns along an enchanted trail.
Or find yourself at Valleyscare in Shakopee, the absolute best for kids or anyone young at heart. Endless streams of activities, all with a different spin on Halloween, include dance shows with famous Halloween figures, Chateau Tours through haunted mazes with a friendly vampire guide, the Sinister Circus with costumed acrobatics, and much more.
What are you waiting for? Head out this weekend with the family and explore all the glory of autumn in Minnesota. Enjoy the fall festivities before the winter chill takes over. Fall is short and sweet, so make the most of it by checking at least a few of these awesome activities off your fall bucket list with the family this season.
Latest Blog Posts
It's that time of year again. Twin Cities families are making plans to celebrate the arrival of the harvest season. Fall is a perfect time for families to enjoy seasonal activities in and beyond the
Buying or selling a home is one of the most significant investments you will make in life. Choosing the right real estate agent to advocate for you in the home buying or selling process is an

domain: travel


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Lovers of first person shooting games will already have a place in their heart for the Android and iPhone to download Deer Hunter for PC free of charge. But now PC owners are also starting to get rather excited because thanks to Android Emulator applications it is now within their grasp as well! Yes, it is true! Deer Hunter for PC has well and truly arrived and we’re here to show you how to get this bad boy up and running in no time at all!
Deer Hunter Game:
This absolutely classic arcade game has become even more popular recently and although playing Deer Hunter for PC is not exactly taxing, the game-play more than makes up for it though! This is ideal for the pick-up-and-play mobile audience and should also transfer nicely to the PC arena. Like a host of other Android games Temple Run 2, Stella for PC and others, this will soon have even more admirers. To enjoy Deer Hunter for PC to the max, you will need to be a great shot because only a head, heart or lungs shot will take that poor deer out of the game. We’ll talk you through how to install the best Android Emulator next and then how to search and Install Deer Hunter on your computer.
How to Download Deer Hunter for PC free | Install Deer Hunter APK:
As we’ve just alluded to, Bluestacks is the way forward for all Android gameplay fans who own PCs, so follow these easy instructions to get your Deer Hunter fix right away:

domain: game


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Asian Flank Steak Recipe
- 1 (1 1/4 pound) flank steak
- 1/4 cup vegetable oil
- 1/4 cup orange juice
- 3 tablespoons soy sauce
- 3 tablespoons honey
- 3 tablespoons rice wine vinegar
- 2 tablespoons minced green onions
- 1 1/2 teaspoons grated fresh ginger
- 1 clove garlic, pressed
- 1/2 teaspoon cornstarch
- 3 tablespoons water
step 1: SCORE steak diagonally across grain at 3/4-inch intervals.
step 2: COMBINE oil and next 7 ingredients in a shallow dish or heavy-duty zip-top plastic bag; add steak. Cover or seal, and chill 8 hours, turning occasionally.
step 3: REMOVE steak from marinade, reserving marinade.
step 4: GRILL steak, covered with grill lid, over high heat (400 to 500 degrees) about 6 minutes on each side or until desired degree of doneness.
step 5: BRING reserved marinade to a boil in a saucepan, and boil at least 1 minute or until reduced by one-third.
step 6: STIR together cornstarch and 3 tablespoons water until smooth. Add to marinade; bring to a boil over medium heat, stirring constantly. Boil, stirring constantly, 1 minute; remove from heat. Spoon over steak.


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Soo Line Trail
One of the most beautiful bike trails around
Grease your chains, secure your helmets and hit what might soon be the longest stretch of paved highway available for free-spirited individuals looking to throw their inhibitions to the wind for an afternoon, a day or even a weekend of bicycling. This is truly one of the most beautiful bike trails around. The Soo Line Trail connects to the Lake Wobegon Trail in Stearns County where you will cross the river on a scenic covered bridge, one of the longest covered bridges in Minnesota! When you go through our town, stop by for some refreshments, lunch or dinner, check out our new community center, or just stop to shoot the breeze with the locals.


County staff presented the final report of a study that will guide amendments to county’s Comprehensive Plan around the Silver Line on Jan. 21.
The Scenario Planning Study is the latest step in a planning process begun in October 2013. Staff studied the tradeoffs and impacts of land uses around the Silver Line. A consultant recommended a “complete community” with the full range of uses and amenities—working, shopping, learning, and playing—close by.
The consultant’s report recommends high-density urban development within walking distance of the planned Metrorail stations, airport-compatible uses to protect Dulles Airport in the long term, embracing data centers in strategic areas, and focusing on a safe, efficient grid network for transportation, among other things. The report is available at on the county government website.


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Would you like to bring your dog to a trusted veterinarian in St. Stephen?
Dogs bring us joy every single day of our lives, so it’s our duty to return the favor by bringing them to a good veterinarian to make sure they get all the care they need. If you’ve been seeking a reliable dog vet near St. Stephen, bring your pup to Rice Veterinary Clinic.
At Rice Veterinary Clinic, all of our experienced veterinary staff are dedicated to providing top-level care to our puppy patients. You can rely on our dog veterinarians near St. Stephen to perform all of these veterinary services:
With loving care from our veterinarians, your dog will be able to enjoy a long and healthy life. Contact Rice Veterinary Clinic today to make a veterinary appointment for your dog in the St. Stephen area!

domain: pet


- Full Description
As computing devices proliferate, demand increases for an understanding of emerging computing paradigms and models based on natural phenomena. Neural networks, evolution-based models, quantum computing, and DNA-based computing and simulations are all a necessary part of modern computing analysis and systems development. Vast literature exists on these new paradigms and their implications for a wide array of applications. This comprehensive handbook, the first of its kind to address the connection between nature-inspired and traditional computational paradigms, is a repository of case studies dealing with different problems in computing and solutions to these problems based on nature-inspired paradigms. The 'Handbook of Nature-Inspired and Innovative Computing: Integrating Classical Models with Emerging Technologies' is an essential compilation of models, methods, and algorithms for researchers, professionals, and advanced-level students working in all areas of computer science, IT, biocomputing, and network engineering.
- Table of Contents
Table of Contents
- Changing Challenges for Collaborative Algorithmics.
- ARM++: A Hybrid Association Rule Mining Algorithm.
- Multiset Rule
- Based Programming Paradigm for Soft
- Computing in Complex Systems.
- Evolutionary Paradigms.
- Artificial Neural Networks.
- Swarm Intelligence.
- Fuzzy Logic.
- Quantum Computing.
- Enabling Technologies.
- Computer Architecture.
- A Glance at VLSI Optical Interconnects: From the Abstract Modelings of the 1980s to Today’s MEMS Implementations.
- Morphware and Configware.
- Evolving Hardware.
- Implementing Neural Models in Silicon.
- Molecular and Nanoscale Computing and Technology.
- Trends in High Performance Computing.
- Cluster Computing: High
- Performance, High
- Availability, and High
- Throughput Processing on a Network of Computers.
- Web Service Computing: Overview and Directions.
- Predicting Grid Resource Performance On
- Application Domains.
- Pervasive Computing: Enabling Technologies and Challenges.
- Information Display.
- Noise in Foreign Exchange Markets.
If you think that you've found an error in this book, please let us know by emailing to email@example.com . You will find any confirmed erratum below, so you can check if your concern has already been addressed. No errata are currently published


Bernie Sanders knows exactly what is at stake in the 2020 presidential race.
“This is not just an election between Donald Trump and Joe Biden,” the Vermont senator explained Thursday. “This is an election between Donald Trump and democracy—and democracy must win.”
That’s not a hyperbolic statement. That’s stark realism, which acknowledges the reality Americans face at a point when President Trump refuses to say whether he will accept the pink slip that polls suggest voters may hand him in November.
Sanders, who was himself an aspirant for this year’s Democratic presidential nomination, is not alone in expressing his concern. Trump dramatically heightened the anxiety level of an already tense election season when he responded Wednesday to a question about the peaceful transition of power by saying, “We’re going to have to see what happens.” Then the president made things worse: “We want to get rid of the ballots, and we’ll have a very peaceful—there won’t be a transfer, frankly. There’ll be a continuation.”
“This statement from the president of the United States should trouble all of us,” warned the American Civil Liberties Union. Senate minority leader Chuck Schumer declared, “President Trump: You are not a dictator, and America will not permit you to be one.” House Speaker Nancy Pelosi urged the president to “just try for a moment to honor your oath of office to the Constitution of the United States.” Even Utah Senator Mitt Romney, the 2012 Republican presidential nominee, chimed in: “Fundamental to democracy is the peaceful transition of power; without that, there is Belarus. Any suggestion that a president might not respect this Constitutional guarantee is both unthinkable and unacceptable.”
But it was actor and activist Mark Ruffalo who spoke for millions of Americans, when he asked, “Okay…so what’s the plan? The man has told us he won’t leave peacefully, he is stacking the court with Justices he knows will rule in his favor, and he is talking about tossing ballots. What is the plan?”
On Thursday, Sanders presented one in a vital speech delivered from George Washington University to people across the country. The senator began by noting that Dan Coats, Trump’s former director of national intelligence, has called for establishing a nonpartisan commission to oversee the election, with an eye toward assuring that it is fairly conducted. In a New York Times op-ed, Coats wrote, “The most urgent task American leaders face is to ensure that the election’s results are accepted as legitimate. Electoral legitimacy is the essential linchpin of our entire political culture. We should see the challenge clearly in advance and take immediate action to respond.”
“I couldn’t agree more. I strongly second Director Coats’s call for this election commission,” said Sanders, who last week joined Schumer in urging Senate majority leader Mitch McConnell to hold hearings to examine election and post-election security issues.
Then, on the chance that McConnell might not do the right thing, Sanders outlined steps that he proposed “we as a people do in the struggle to preserve American democracy”:
“First, it is absolutely imperative that we have, by far, the largest voter turnout in American history and that people vote as early as possible. As someone who is strongly supporting Joe Biden, let’s be clear: A landslide victory for Biden will make it virtually impossible for Trump to deny the results and is our best means for defending democracy.
“Second, with the pandemic and a massive increase in mail-in voting, state legislatures must take immediate action now to allow mail-in votes to be counted before Election Day—as they come in. In fact, 32 states allow for the counting or processing of absentee ballots—verifying signatures for example—before Election Day. All states should do the same. The faster all ballots are counted, the less window there is for chaos and conspiracy theories.
“Third, the news media needs to prepare the American people to understand there is no longer a single election day and that it is very possible that we may not know the results on November 3rd.
“Fourth, social media companies must finally get their act together and stop people from using their tools to spread disinformation and to threaten and harass election officials.
Fifth, in the Congress and in state legislatures hearings must be held as soon as possible to explain to the public how the Election Day process and the days that follow will be handled. As we count every vote, and prevent voter intimidation, everything possible must be done to prevent chaos, disinformation, and even violence.”
That’s not a comprehensive list. The ACLU, the Brennan Center for Justice, Color of Change, and other groups are constantly offering informed counsel on how to conduct free and fair elections, and to count the votes cast in those elections. Hopefully, Biden, vice presidential nominee Senator Kamala Harris, and other top Democrats will embrace and amplify all the best proposals for pushing back against Trump.
But Sanders has begun the pushback by offering a plan that can be acted upon immediately. And as any good planner does, he has made a call to action.
“The American people, no matter what their political persuasion, must make it clear that American democracy will not be destroyed,” said the senator. “Our country from its inception and through the sacrifices of millions has been a model to the world with regard to representative government. In 1863, in the midst of the terrible Civil War, Abraham Lincoln at Gettysburg stated that this government ‘of the people, by the people, for the people shall not perish from the earth.’ That was true then. That is true today. Regardless of what Donald Trump wants, the American people will preserve democracy in our country.”


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Mastering methods is key to learning to cook, argues Hertzmann. Get it right and you’re freed from being a mere recipe-follower. Each of the 50 recipes, based on carrots, cleverly demonstrates a prep or cooking method – julienning, sous-vide, roasting... The principles can be applied to any food. The book (a New Yorker ‘best cookbook’ of 2020) may sound a bit mad but there’s clearly more method than madness here. Brilliantly done.
from the publisher
This is a book for people who open cans and snip frozen bags of produce, and ramp up the microwave rather than cook. It is also for the experienced cook who has never done sous-vide or fermenting. A book for all who dare to tread the boards in the kitchen. Every method presented in this book is approachable for both novice cooks and those with many years experience. He gives prescriptive advice, such as the salt concentration for pickling a carrot should be 3%, but his book is easy to put into action in the kitchen. Some of the methods: Simple slices - Matchsticks, julienne, or bâtonnets. Grating carrots, mandolins, juicing carrots, blending carrots, a new-fangled pickling container, salt fermented carrots, miso-pickled carrots, acid fermentation, Pickling spices, modern, sous-vide cooking - why sous-vide cooking works
Peter Hertzmann believes that learning to cook involves learning about methods, not simply following recipes. His new book 50 Ways to Cook a Carrot, uses the root vegetable to demonstrate a whole battery of kitchen skills, from julienning and braising, to transforming carrots into meringues, foams, and powder. Peter explains the reasons for writing the book, who it’s for – and why carrots…


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Financial, economic as well as political outcomes of the post-summit period will definitely have historic implications for the EU’s integration in the coming seven years, and most probably for several decades to come.
Last month’s summit’s agreements are still on the agendas among the EU-27 leaders regardless the summer vocation. The outcomes will definitely influence the governance and growth structures in the member states; besides, the agreements have opened some new perspectives for a more active and probably a “slightly different” European integration.
Although the final adoption procedures are still ahead by both the EU legislative authorities (i.e. the Parliament and the Council of Ministers) and the member states, the European integration has turned a new page. An analysis of financial perspectives for a new style EU’s political economy is well-worth reading…
“Never before” is a key word that is often used in post-summit’s revelations: first, due to some “technicalities” as the summit was expected to last a couple of days (instead it lasted four days); secondly by the substantial outcomes. The latter is really of a historic dimension: a) the leaders finally agreed on a multi-annual budget (with a new “own resources” structure), which reached the biggest ever in the EU’s history amount – over € 1,2 trillion (which is more than 5 percent of the EU-27 GDP; instead of “regular” about 1 percent); and b) the agreed “rescue package”, the so-called “next generation EU’s” recovery fund totaling € 750 bn to support “the states in need”. The latter fund is being divided into two: €390 bn in grants and €360 bn in loans (though in the summit’s official conclusion it is € 312.5 billion). Thus, in total, the financial injections into a new seven-year stage in the EU’s integration amounts to about gigantic € 2 trillion!
And on top of this, it has to be mentioned that all these -enormous in sizes – financial “packages and funds” have been prepared during just a couple of months!
From politics to economics: towards a “united theory”
First of all, we have to keep in mind the EU’s political guidelines being generally adopted by the EU’s institutions every 5-7 years. Thus, presently, there are 6 Commission’s political priorities adopted for the next seven years at the end of 2019, as they are written in the Commission’s webpage (we leave intentionally the original text intact so that the readers can refer to the priorities of interest): 1. A European Green Deal; 2. A Europe fit for the digital age; 3. An economy that works for people; 4. A stronger Europe in the world; 5. Promoting our European way of life; and 6. A new push for European democracy.
Actually, the seventh priority has been added – pandemic-health care efforts – after a widespread covid-19 effect on all walks of life in the EU member states.
In the political priorities the hierarchy goes from sustainability to digital issues, to economics, to globalization’s effect on EU, to “European values”, and finally – to democracy.
Comparing these political priorities’ reflection with the “financing economy’s” issues in the long-term EU’s budget, MFF 2021-27 (below), one can see how different the political and economic priorities actually are…
The economic-financial “guidelines” in the new MFF (we are aware that economy and finances go closely together) are strikingly different from the political ones: from the “EU single market” (i.e. EU’s internal economic cooperation), innovation and digital, to cohesion and values; to natural resources and environment, to security and defence, to the “neighbourhood and the world”, and finally – to the EU’s administration.
I personally, don’t see much reason for such differences among seemingly “unified” in the EU political, economic and “supporting” financial issues in the EU’s budget priorities; I still think that the all three go hand-in-hand, haven’t they? Isn’t it more feasible to strive for a “unified EU’s political economy” with a sufficient financial support: the European integration is a straight-forward process, which just includes numerous facets of the same doctrine?
Probably, these are rhetoric questions: politicians don’t want to lose fashionable places in the EU’s headquarters, and economists can’t change the old-style governing structures; besides it is often not in their power…
Next generation will pay…
At the end of April 2020, the European Council tasked the Commission to draw a recovery package for the member states; after about two months of work, the “Next Generation EU-plan” package–dubbed NGEU- was presented for approval at the July’s summit.
During four long days and nights – more than 90 hours – the EU-27 leaders agreed on a “generation’s plan” of measures, alongside an important for this plan another focal “pact”, i.e. the MFF-budget; both agreements have signaled to the states and the world that Europe is capable of taking prompt and adequate actions.
The NGEU financing under the “Recovery and Resilience Facility, RRF”, as the main pact’s component, amounts to € 672.5 billion (increased to 750): with € 390 billion for loans (reduced to 350) and € 312.5 billion for grants (later increased to 360). The increased amounts included such additional programs as ReactEU, Horizon Europe, InvestEU, Rural Development, Just Transition Fund and RescEU, which added to the ultimate borrowing about € 78 billion making the whole NGEU € 750 billion (specific figures are seen below). About 70 percent of the grants provided by the RRF shall be committed during the first ten years 2021-22; remaining 30 percent shall be fully committed by the end of 2023.
The EU’s borrowing in the global financial markets will last until 2026 and the repayment shall be scheduled until the end of 2058. But the borrowing shall be repaid (though with a small interest rate) over a longer period of time, approximately between 2028- 2058, hence the name – the “next generation EU”!
Besides, there are several other EU programs that are closely connected to the “generation’s plan” in the so-called “outside the MFF” sources, which include venture capital, international donors and support from the non-EU members, e.g. EEA. The most vital for seven years programs (see below) will totaled to over € 22,3 bn; hence the aggregate “injections” into the EU’s “generational survival”, i.e. MFF + Rescue package + outside MFF programs could come to about two trillions euros!
At the same time, annual GNI-based contributions for some wealthy states (Denmark, Germany, the Netherlands, Austria and Sweden) will be reduced by the following amounts: Denmark by € 377 million, Germany by € 3, 671 million, the Netherlands with € 1,921 million, Austria with € 565 million, and Sweden with € 1,069 million. Is it a “carrot” to silence the “frugal-states”, one wonders? However, these “gross reductions” shall be financed by all other EU member states according to their GNI and consequently, “the generation” …
MFF 2021-27: seven major directions
The multi-annual budget, MFF for 2021-27 is an extremely complicated issue, with several cross-sectoral interactions: thus, in the present analysis we’ve concentrated only on most vital and aggregate MFF’s “headings” visualizing perspective financial allocations for attention to the states’ decision-makers.
These aggregate figures are important to know for all the national governance bodies, as the figures depict the main national growth directions that can be wholly or partially financed from the MFF. Thus, e.g. the “technical support instruments” will improve the EU states’ administrative capacity to design, develop and implement reforms; the EU’s financial support of € 767 million is available for all states.
The MFF for the period 2021-2027 will have the following structure:
1 “Single Market, Innovation and Digital”: about € 133-150 bn (or about € 19 bn/per year); for Horizon Europe program during this period will be about € 76 bn with a special digital program of € 6,7 bn;
Note: final appropriations can be different due to cross-sectoral interconnections among various headings, i.e. could be seen in a “complex table” below.
Research and innovation, R&I: Particular attention will be paid to the coordination of activities funded through Horizon Europe and other EU’s programs, including through cohesion policy’s instruments. In this regard, important synergies will be needed between Horizon Europe (with €76 bn for 7 years) and the EU’s structural funds for the purpose of “sharing excellence”, thereby enhancing regional R&I capacity and the ability of all regions to develop clusters of excellence. Reference to: Summit conclusions, p. 18
2 “Cohesion, Resilience and Values” (which includes a sub-heading for economic, social and territorial cohesion and a sub-heading for resilience and values): € 377, 7 -427 bn – from € 50 to 59 bn yearly, of which 330, 2 bn will be allocated for “Economic, social and territorial cohesion” and 47, 5 bn for “Resilience and Values”;
2.1. E.g. “Connecting Europe facility”, CEF – € 28, 4 bn (21,3 bn for transport, € 5,1 bn for energy and € 1,8 bn for digital issues); The amount of support from the Cohesion Fund to be transferred to the CEF will be € 10 bn; thus the Cohesion Fund allocations of each EU state will be reduced accordingly.
The main objective of Cohesion Policy is to develop and pursue actions leading to the strengthening of economic, social and territorial cohesion by contributing to reducing disparities between the levels of development of the various regions and the backwardness of the least favored regions. Through the European Regional Development Fund (ERDF), the European Social Fund Plus (ESF+) and the Cohesion Fund (CF), it will pursue the following goals: “Investment for jobs and growth” in states and regions, to be supported by all the Funds; and “European territorial cooperation”, to be supported by the ERDF. Cohesion policy will play an increasingly important role in supporting the ongoing economic reform process by the states by strengthening the link to the European Semester. The Commission and the states shall take into account relevant country-specific recommendations during the entire process.
Resources for the “Investment for jobs and growth” will amount to € 322,3 bn and will be divided as follows: a) € 202,3 bn for less developed regions; b) € 47,8 bn for transition regions; c) € 27,2 bn for more developed regions; d) € 42, 5 bn for the states supported by the Cohesion Fund; e) about € 2bn as additional funding for the outermost (art. 349 TFEU); and f) € 500 million for interregional innovations. The amount of resources available for the ESF+ under the “Investment for jobs and growth” will be about € 87 bn; € 175 million of the ESF+ resources for the “Investment for jobs and growth” goal will be allocated for transnational cooperation supporting innovative solutions under direct or indirect management.
Note: Types of regions defined in the following way: a) less developed regions, whose GDP per capita is less than 75% of the average GDP of the EU-27; b) transition regions, whose GDP per capita is between 75% and 100% of the average GDP of the EU-27; c) more developed regions, whose GDP per capita is above 100% of the average GDP of the EU-27.
Resources for the “European territorial cooperation” (Interreg) will amount to about € 8 bn and will be distributed as follows: a) € 5, 7 bn for maritime and land cross-border cooperation; b) € 1, 5 bn for transnational cooperation; c) € 500 million for interregional cooperation; and d) € 271 million for outermost regions’ cooperation.
The ESF+ will provide comprehensive support to youth employment, up- and re-skilling of workers, social inclusion and poverty reduction, including child poverty, by merging existing programmes: the European Social Fund, the Youth Employment Initiative, the Fund for European Aid to the Most Deprived and the Employment and Social Innovation program. Total financial envelope for the ESF+ for the period 2021-27 will be about € 88 bn, of which: € 676 million for the ESF+ under direct and indirect management; € 87, 3 bn for the ESF+ under shared management in the Investment for Jobs and Growth goal. The shared management strand will remain under a sub-heading together with the ERDF and the Cohesion Fund.
In the “resilience” program: there is financial support in the RescEU program of € 1, 1 bn; the EU-wide “health program” shall be established with about € 1,6 bn financing. Besides, the financial envelope for the Creative Europe program will be € 1, 6 bn and the financial envelope for the Justice, Rights and Values program will be € 841 million.
3. “Natural Resources and Environment”: the heading consists of agriculture and maritime policy, as well as environment and climate action with the budget of about € 356, 4 – 401 bn, of which € 258,6 bn will be allocated to market related expenditure and direct payments (about € 35-38 bn per year). Direct payments under regulation 1307/2013 and under the CAP Strategic Plan Regulation will be about € 249 bn. About 40 percent of CAP expenditures shall go to climate actions.
Notes: Direct payment: all EU states will have a “common level” of at least € 200 per hectare in 2022 and all states shall reach at least € 215 per hectare by 2027.
Rural development support: The allocation for EAFRD for the period 2021-27 is about € 78 bn; EU states facing particular structural challenges in their agriculture sector: e.g. in the Baltic Sea region Germany will get € 650 million and Finland € 400 million.
In order to address social and economic consequences of reaching EU climate neutrality goal by 2050, two funds will be created: Just Transition Mechanism and Just Transition Fund. Thus, allocation for the Just Transition Fund for 2021-27 will be € 7, 5 bn. Access to the Just Transition Fund will be limited to 50% of national allocation for the EU states that have not yet committed to implement the objective of achieving a climate-neutral EU by 2050, in line with the objectives of the Paris Agreement, the other 50% being made available upon acceptance of such a commitment.
4. “Migration and Border Management”; the “heading” includes effective control of external borders for ensuring more efficient migration management and a high level of internal security while safeguarding the principle of free movement of persons and goods within the Union. Programs under this heading will help the member states to deliver on a comprehensive approach to migration effectively. Appropriations for this heading will be about € 22, 7 -26 bn, i.e. € 2,3 -3,7 bn per year. Asylum and Migration Fund is established with € 8, 7 bn for 2021-27. The allocation for the Integrated Border Management Fund for the period 2021-2027 is € 5,5 bn, as well as a reinforced European Border and Coast Guard Agency (EBCGA), with a total envelope of € 5 148 million.
5. “Security and Defence”: the level of commitments for this heading will be about € 13,2-15 bn, i.e. € 1,2 – 2,2 bn per year. It will support the Internal Security Fund (with € 1,7 bn), which will contribute to ensuring a high level of security in the Union in particular by preventing and tackling terrorism and radicalization, serious and organised crime and cybercrime as well as by assisting and protecting victims of crime. It will also finance actions dedicated to external migration management in relation to combating illegal migration and trafficking of human beings.
For supporting EU nuclear safety, a specific support will be granted to the decommissioning of the following nuclear power plants: – € 490 million to Ignalina in Lithuania, with an EU contribution rate of 86%; – € 50 million to Bohunice in Slovakia, with a maximum EU contribution rate of 50%; – € 57 million to Kozloduy in Bulgaria with the same maximum EU contribution. In addition, € 448 million for nuclear safety and the decommissioning of the EU’s own installations will be provided.
Major financing from this heading will include a financial contribution of about € 7 bn (about half of the whole commitment in this heading) for the European Defence Fund (EDF) aimed at fostering competitiveness, efficiency and innovation capacity of the European defence technological and industrial base supporting collaborative actions and cross-border cooperation throughout the Union, at each stage of the industrial cycle of defence products and technologies. The programme design will ensure participation of defence industries of all sizes, including SMEs and mid-caps, across the Union, thus strengthening and improving defence supply and value chains.
A financial contribution of € 1, 5 bn will be made to the Connecting Europe Facility to adapt the TEN-T networks to military mobility needs.
6. “Neighbourhood and the World”: allocations in this heading will finance the Union’s external action and assistance for countries preparing for accession to the Union. Stronger coordination between external and internal policies will ensure proper implementation of the 2030 Agenda for Sustainable Development, the Paris Climate Agreement, the EU Global Strategy, the European Consensus on Development, the European Neighbourhood Policy, as well as the external dimension of migration, including the Partnership Framework with third countries on migration.
A modernized external policy will demonstrate EU added value by increasing effectiveness and visibility and making the EU and the states better equipped to pursue their goals and values globally.
Appropriations for this heading will be about € 98, 4 – 110 bn, i.e. about € 13-15 bn yearly; the allocation for the Instrument for Pre-Accession, supporting beneficiaries on their path to fulfilling the accession criteria, will be € 12, 5 bn.
7. “Public Administration”: professional European Public Administration, recruited on the broadest possible geographical basis, plays a crucial role in supporting the Union’s efforts to implement policies and programs in the common European interest with improved effectiveness. Appropriations for this heading, which includes also administrative expenditure of the EU’s institutions and European schools and pensions, will be about € 73-82 bn or about € 10 bn per year. Administrative expenditure of the EU institutions will be about € 7-8 bn per year.
Complex table: total appropriations for 2021-27, in €:
- Single Market, Innovation and Digital: about 150 bn.
- Cohesion, Resilience and Values: about 427; including two sub-headings: Economic, social and territorial cohesion – with 372, 6 bn and Resilience and Values – 54.1 bn.
- Natural Resources and Environment: about 401 bn; including payments for “market related expenditure and direct payments” –291 bn.
- Migration and Border Management: about 26 bn.
- Security and Defence: about 15 bn.
- Neighbourhood and the world: 110 bn.
- European Public Administration: 82 bn.
Total commitment appropriations: 1.210.894
Besides, there are several programs in the so-called “outside the MFF” sources, which include venture capital, international donors and support from the non-EU members, e.g. EEA. Appropriations are envisaged for the following programs in this heading for 2021-27, in mln euros:: = Solidarity and Emergency Aid Reserve: 9.467; = European Globalisation Adjustment Fund (EGF): 1.467; = Brexit Adjustment Reserve: 5.306; and = Flexibility instrument: 6.091.
Total appropriations “outside MFF” – 22.331;
Total: MFF + Outside the MFF programs: 1.232,3 mln.
Tough decisions at tough time…
Remarkable enough, that above mentioned huge financial resources have been agreed on during a dramatic period in the Union’s socio-economic development: GDP for all EU-27 has fallen by about 12 percent already during the second quarter of 2020, which is “by far the sharpest declines observed since 1995”, acknowledged the EU’s statistical agency recently.
“These were by far the sharpest declines observed since time series started in 1995,” Eurostat indicated in a statement, flagging that the April-June period was “still marked by COVID-19 containment measures in most member states.”
Table: The general plan for MFF’s implementation
= May 2020: Commission proposed a revised Multiannual Financial Framework (MFF) 2021-2027, a draft of “Own Resources” decision and some sectoral legislation;
= July 2020: European Council: Political agreement on MFF for 2021-2027 and Own Resources;
= During summer 2020: Consultation with the European Parliament Own Resources;
= Autumn 2020: European Council: final consultations…
= December 2020: Adoption of the revised MFF 2021-2027 with the European Parliament’s consent; adoption of the Own Resources Decision followed by the ratification by all EU states in line with their constitutional requirements;
= January 2021: The start of the MFF 2021-2027 implementation work.
The amounts under the “new generation EU”, NGEU are divided among the following programs: = Recovery and Resilience Facility (RRF) as a NGEU’s main part with € 672.5 billion: of which loans € 360 billion and grants € 312.5 billion.
Plus additional programs: = ReactEU: € 47.5 billion; = Horizon Europe: € 5 billion; = InvestEU: € 5.6 billion; = Rural Development: € 7.5 billion; = Just Transition Fund (JTF): € 10 billion; = RescEU: € 1.9 billion. Total: € 750 billion.
About 70 percent of the grants provided by the RRF shall be committed during 2021-22; remaining 30 percent shall be fully committed by the end of 2023.

domain: finance


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They also occasionally had the musical benefit of one Mark Abraham on
Spasmophone. The Spasmophone is a cobbled together musical invention
with a tuba mouthpiece connected by apparently random plumbing to a giant antique gramophone horn.
There aren't many of these around! Mark was a former member of
Humber Jug, a band which also included well-known
washboard player John Pilgrim, who had featured in several successful skiffle bands.
Humber Jug made 2 EPs, the 2nd of which included Empty Pocket Blues, Stealing, I
Can't Give You Anything But Love, and Mobile Line which is also known as
To avoid the band being "guitar-heavy," Bob took up mandolin and later fiddle, Tony took up the banjo, and Pete jammed a trumpet mouthpiece into an ancient wooden vehicle-inspection lamp handle, thus inventing a sort of wobbly cornet
that the band called a "Lumpit." This line-up developed into the definitive GJB sound, and
they soon started packing North Kent & S.E. London folk clubs and
other venues from about 1969.
In 1972 the four remaining members of the
GJB recorded the album called The Ginger Jug Band. Steve Horne emigrated
to South Africa with his family shortly after, and Bob Jones left to concentrate on his graphic arts career. Mark Abraham rejoined for a while, and John Huttlestone stepped in on guitar and fiddle. Some time later, Geoff
himself left for new pursuits. Tony kept the band going for some years with Charlie Jacobs
and Rick Harper who had returned from college and resurrected his
washboard and jug.
Interest in the GJB started again in 1998 when Geoff Beaumont paid a flying visit to the Broadstairs Folk Festival where he ended up jamming with Tony Petto, Rick Harper and others at a late night session. Tony and Rick were at the festival as part of the Hartley Morris Dance side. Everybody, particularly the original GJB members enjoyed themselves immensely. The following year Geoff was able to stay a little longer and there was a lot more music and even more interest. It was at this time that Kenn Wards started work on the restoration of the old GJB album with the aim of getting it on to an archive CD. The idea of re-forming the band for the following year's festival was mooted. However, it seemed unlikely to take off as the original band members were so widely dispersed and Pete Baston had died some years earlier. However...
Refried GJB is Born
Geoff Beaumont... Fretless Slide Banjo, Jug, and vocals
Add to this line-up the invaluable help of Jayne Petto, Tony's wife, who was there to stop the big drum from sliding out of control and into the crowd by bracing it with her feet!
The line up was later joined by Gideon Payne on harmonica. The Refried Ginger Jug Band played several highly successful pub gigs at the Festival to packed audiences. Interest in the original band was rekindled and stocks of the now available double CD of the original band was sold out within 3 gigs.
Thanks go to Geoff Beaumont, Tony Petto and Suzanne Artus for help with this potted history.
|© 2000 Osiris-AG & GJB|