text:
Development of an enzyme linked immunosorbent assay for detection of cyathane diterpenoids
Tian Shen1,
Lena M Hof1,
Heike Hausmann2,
Marc Stadler3 &
Holger Zorn1
So-called cyathane type diterpenoids are produced as secondary metabolites by basidiomycetes. Based on their antibacterial, fungicidal, and cytotoxic properties, cyathane type terpenoids represent interesting target compounds in fungal biotechnology.
An indirect competitive enzyme linked immunosorbent assay has been developed for detection of cyathane type diterpenoids. Rabbit polyclonal antibodies were raised against a mixture of striatal A and B conjugated to bovine serum albumin. The conditions for direct attachment of the hapten striatal B to a solid phase by passive adsorption were optimized. The cross reactivities of the striatals A, C and D, of the striatins A and B, and of the erinacines C and P to striatal B were determined. The validation study showed that the ELISA was precise and sensitive. The average IC50 of striatal B was 36.0 ng mL-1 with an inter-assay coefficient of variation (CV) of 13.2% (n = 5). Recoveries from striatal B spiked samples in the assay were in the range of 97.3 – 125.9%. A good correlation between the striatal B concentration measured by the ELISA and by HPLC-DAD (y = 1.1122× – 0.1585, R2 = 0.9942) was obtained from linear regression analysis. The suitability of the ELISA for detection of cyathane type diterpenoids in submerged cultures and fruiting bodies of H. erinaceus was studied. It showed cross reactivity with supernatants from submerged cultures and extracts thereof, but did not show cross reactivity with extracts from fruiting bodies.
The developed method is appropriate for qualitative and quantitative detection of cyathane diterpenoids in complex mixtures. Due to its high sensitivity and specificity, it represents an ideal screening method for discovering new cyathane diterpenoids and new potential producers of them.
Cyathine and allocyathine were the first reported cyathane diterpenoids discovered by Allbutt and Ayer from a static liquid culture of the fungus Cyathus helenae (H. J. Brodie) in the early 1970s [1],[2]. They were proven to be active against actinomycetes, Gram-positive and Gram-negative bacteria, and some fungi, including dermatophytes. Afterwards, various structurally related compounds, so-called cyathane type diterpenoids, were isolated from different basidiomycetous cultures, e.g. striatals and striatins from Cyathus spp., sarcodonins from Sarcodon spp., and erinacines from Hericium spp. (Figure 1) [3]-[7]. All of these compounds share a cyathane skeleton consisting of five-, six-, and seven membered rings and possess antibacterial, fungicidal, and cytotoxic properties. Fungal extracts prepared from C. striatus showed significant inhibitory effects on the NF-κB activation pathway and might be applied for cancer therapeutics [8]. Erinacines promote nerve growth factor (NGF) synthesis, which suggests the application of H. erinaceus or its secondary metabolites for the treatment and prevention of dementia and further neurodegenerative diseases [7],[9]-[12].
Structures of striatals, striatins and erinacines.
Because of their interesting biological activities and their high potential in medicinal and pharmaceutical applications, cyathane diterpenoids have attracted increasing interest in recent years. At present, the most common method for analysis of cyathane diterpenoids is high performance liquid chromatography (HPLC) coupled to diode array and mass spectrometric detection [13].
As a rapid, sensitive and cost effective method, an ELISA specific for cyathane type secondary fungal metabolites may serve as a complementary method, especially in screenings for new producer strains. In the present study, an indirect competitive ELISA for the structure specific detection of cyathane type diterpenoids using polyclonal antibodies was developed, and its potential for the analysis of biological samples was proven.
Optimum coating conditions
Striatal B was chosen as coating hapten in ELISA, because it is more polar than striatal A. In order to increase its solubility in aqueous solution, an equal volume of DMSO was added to the buffers. The optimum coating conditions (best signal-to-noise ratio and concentration dependence) were obtained by coating the plate in PBS/DMSO (1/1, v/v) buffer. More intense signals were obtained by coating at 24°C or 37°C, compared to coating at 4°C. Striatal B was adsorbed more efficiently by coating overnight than for 2 h. Based on these results, the optimum coating conditions for striatal B are summarized as follows: PBS/DMSO (1/1, v/v) buffer, and coating overnight at 24°C. The optimum concentration for coating of the hapten and the dilution of polyclonal antibodies (pAbs) were determined by checkerboard titration to be 5 μg mL-1 and 1:200, respectively, which were used in the following indirect competitive ELISA experiments.
Tolerance against organic solvents
Due to the poor solubility of cyathane diterpenoids in aqueous solutions, the tolerance against organic solvents used to dissolve these compounds was tested for assay optimization. An addition of 5% DMSO, acetonitrile or methanol, respectively, to the pre-incubation solution was found acceptable. Methanol could be used up to 25% without a notable negative effect. High percentages of DMSO up to 30% and of acetonitrile up to 20% resulted in considerable negative effects on the assay. An addition of DMSO >30% or acetonitrile >20% showed a sudden increase of signals. Since methanol reacts with striatals [4], an addition of 5% DMSO or acetonitrile was selected for pre-incubation in the following indirect competitive ELISA.
Figure 2 illustrates a typical standard calibration curve of striatal B. The specificity of the pAbs against striatal B was evaluated by determination of the cross reactivities of several structurally related compounds, such as striatals A, C, and D, striatins A and B, and erinacines C and P. The IC50 values are given in Table 1. All of the tested cyathane diterpenoids showed cross reactivities against striatal B. No color reaction was detected with non-coated plates as blanks.
Calibration graphs for the quantification of striatal B. (a) Example of sigmoidal calibration curve for quantification of striatal B by indirect competitive ELISA. (b) Example of linearity of striatal B calibration curve. The linear working range was found to be 0.001 - 2.5 μg mL-1, y = −0.722 ln(x) – 2.3774, R2 = 0.9904.
Table 1 Cross reactivities of structurally related compounds to striatal B
Assay precision
Intra-assay and inter-assay precisions were determined for several concentrations of striatal B (Table 2). The intra-assay precision was calculated from the extinction difference at 450 nm and 630 nm within one microtiter plate. Less than 10% CV were obtained in every experiment, indicating an acceptable precision. The inter-assay precision was obtained from %B/B0-values of 8 plates on different days. Variations of less than 20% were observed. The average IC50 of striatal B standard was 36.0 ng mL-1 with an inter-assay CV of 13.2% (n = 5).
Table 2 Intra- and inter-assay precision
To assess the assay accuracy, striatal B was added to the sample matrix in several concentrations, and the recoveries of the spiked samples were determined. In parallel, HPLC-DAD was used to analyze the spiked samples. The apparent recoveries of striatal B in the sample matrix as determined by indirect competitive ELISA ranged from 97.3% to 125.9%, while those obtained by HPLC-DAD varied from 95.8% - 110.2%. The limit of quantification was 0.02 μg mL-1 for ELISA, and 0.40 μg mL-1 for HPLC-DAD. A good correlation between the striatal B concentrations measured by ELISA and by HPLC-DAD (y = 1.1122× – 0.1585, R2 = 0.9942) was observed (Figure 3, Table 3).
Correlation between striatal B concentrations in spiked samples measured by ELISA (x-axis) and by HPLC-DAD (y-axis).
Table 3 Recovery of spiked striatal B in sample matrix
Analysis of H. erinaceus
The indirect competitive ELISA was applied to samples prepared from submerged cultures as well as from fruiting bodies of H. erinaceus. The total concentrations of cyathane diterpenoids in supernatants and in extracts thereof are displayed in Figure 4. A similar change of %B/B0-values was observed in both cases during the 8 culture days. %B/B0-values in extracts of fruiting bodies from H. erinaceus were near to 100% and were independent from the sample concentration in the assay.
Analysis of supernatants of submerged cultures of H. erinaceus and extracts thereof during 8 culture days. Control: culture medium without H. erinaceus.
In the present study, striatal B was directly attached as a low molecular weight hapten to microtiter plates. In many studies the use of protein conjugated haptens for immobilization was reported [14]-[16], as the ability of proteins to adsorb to the plastic surface of microtiter plates is much higher than that of low molecular weight haptens. However, the coupling ratio of haptens to carrier proteins may vary depending on the coupling reaction and a waste of valuable haptens during conjugate synthesis is often unavoidable. The involvement of carrier proteins results in several problems. Firstly, it may cause unspecific binding to pAbs and thus reduce specific signals. Secondly, due to the large molecular dimension of carrier proteins, a shielding or masking effect may occur that impedes the binding of pAbs to the conjugated haptens [17]. Some researchers reported about pre-treatments of the plate surface, e.g. by UV irradiation or reagents in order to realize a direct binding of small molecules [18]-[20]. In the present study, the target analyte (molecular weight <500 g mol-1) was directly attached to microtiter plates without pre-treatment of the plate surface. Significant differences between the various coating procedures were found during the study of coating conditions. The pH of the coating buffer significantly affected the coating efficiency. The final pH of the coating buffer PBS, potassium acetate and sodium carbonate after mixing with DMSO was 9.7, 5.8 and 11.1, respectively. An influence of the pH on the coating efficiency has been reported in several publications [21]-[23]. In addition, a decomposition of striatal B was detected in potassium acetate buffer in a time course study by HPLC analysis (data not shown). That might be a reason for the poor attachment of striatal B to the plate in this buffer system. For striatal B, coating at pH 9.7 was more efficient than at pH 11.1.
The presence of organic solvents may influence the performance of an ELISA. Methanol caused the least negative effect, and the use of low concentrations of acetonitrile or DMSO was also acceptable. Similar results have been reported e.g. by [17],[23],[24]. The tolerance against organic solvents allows for the analysis of samples from fungal cultures which require extraction with an organic solvent. The sudden increase of the signals in the presence of acetonitrile and DMSO in high concentrations indicated a false positive signal possibly caused by denaturation of the antibodies and thus an unspecific adsorption to the blocking proteins.
All of the examined cyathane diterpenoids showed cross reactivities against striatal B. As expected, striatin B with IC50 = 109.3% showed the highest cross reactivity. Striatin B and striatal B differ only in the aldehyde group at the C7 ring, and this position of striatal B was used for BSA-conjugate synthesis for immunization. Therefore, striatin B and striatal B possess the same antigenic determinants. Erinacine P showed the lowest cross reactivity with IC50 = 21.1%. Based on the cross reactivities and molecular structures it might be suspected that the resulting antiserum displays the highest affinity toward the cyathane ring moiety.
The recovery experiments suggested that the developed ELISA is accurate and significantly more sensitive than the HPLC-DAD method. It could be used for the estimation of the total cyathane diterpenoid concentrations of biological samples. The assay showed interferences with constituents of the culture media of H. erinaceus, as a matrix effect was observed with the control samples (cf. Figure 4, control). Therefore, the use of organic extracts is preferred for the analysis of cyathane type diterpenoids in fungal culture supernatants. The product concentrations could be estimated during the cultivation period. However, the total concentrations represent the sum of all compounds showing cross reactivity against striatal B. They may thus differ from the individual concentrations determined by HPLC.
No significant interferences (%B/B0-values close to 100%) were observed with crude extracts of fruiting bodies of H. erinaceus. Therefore, the secondary metabolites present in the fruiting bodies, e.g. hericenone and erinacerine, did not cross react with the polyclonal antibodies. These results are in good agreement with a review article about secondary metabolites from H. erinaceus[12]. Up to date, erinacines have been mostly reported from submerged cultures of H. erinaceus, and only traces of erinacines have been found in fruiting bodies [25]. In the present study, the trace concentrations of erinacines in the fruiting bodies were apparently below the detection limit of the ELISA.
The CV% values for the inter-assay precision might be caused by many factors, e.g. the position of the wells in the plate (edge effect), the day to day variation in reagent preparation, the temperature of buffers, the variability in washing procedures, pipetting errors and the loss of enzyme activity of the secondary antibodies after prolonged storage [16],[26].
Polyclonal antibodies against striatal A/B were produced, and an indirect competitive ELISA was developed for determination of striatal B and structural related compounds. The method has shown satisfactory results concerning specificity, sensitivity and accuracy. The application to samples from submerged cultures and fruiting bodies of H. erinaceus indicated that the ELISA may be used for the estimation of total cyathane diterpenoid concentrations in complex mixtures. Due to its high sensitivity and specificity, it represents an ideal screening tool for discovering new potential producers of cyathane type diterpenoids. Further optimization will be needed to improve the inter-assay precision.
Chemicals and solutions
All reagents were of analytical grade unless specified otherwise. Ultra-pure water (produced by arium® 611 VF Water System, Sartorius, Göttingen, Germany) was used for all solutions, media, and HPLC eluents.
Acetonitrile (HPLC gradient grade), calcium carbonate, Edamin® K, 30% hydrogen peroxide (H2O2), and d-mannitol were purchased from Sigma Aldrich (Steinheim, Germany).
Salts, Agar-Agar, bovine serum albumin (BSA), EDTA, α-d(+)-glucose monohydrate, soy peptone, sucrose, 3,3',5,5'-tetramethyl benzidine (TMB), Tween® 20, yeast extract, and organic solvents were purchased from Applichem (Darmstadt, Germany) or from Roth (Karlsruhe, Germany).
Malt extract was obtained from Fluka, Neu-Ulm, Germany. Molasses was obtained from Südzucker, Offstein, Germany. Oatmeal was provided from Dr. Oetker, Düsseldorf, Germany.
Standards of erinacines and striatals, originally isolated from the two selected producer strains, were obtained from the library of pure natural products of InterMed Discovery, Dortmund, Germany (IMD, formerly Bayer Healthcare) and their purity and identity was confirmed by 2D-NMR and HR-MS, prior to their use in the experiments. Additionally, striatals A and B (for immunization) and striatals C and D, striatins A and B, and erinacines C and P (for determination of cross reactivities) were extracted and purified from submerged cultures of the basidiomycetes Cyathus striatus and Hericium erinaceus. The structures were confirmed by comparison to the pure reference compounds and by NMR analysis (cf. Additional file 1: Supplementary material).
Solutions for ELISA:
Coating buffers:
10 mM phosphate buffered saline (PBS), pH 7.4 mixed with DMSO, 1/1, v/v.
0.1 M potassium acetate buffer, pH 4.0 mixed with DMSO, 1/1, v/v.
0.1 M sodium carbonate buffer, pH 9.6 mixed with DMSO, 1/1, v/v.
Washing buffer: 10 mM PBS, pH 7.4 with 0.05% Tween® 20 (PBST).
Blocking reagent (1% gelatin) was purchased from Roche Diagnostics (Mannheim, Germany). Secondary antibodies (goat anti-rabbit lgG peroxidase conjugate) were obtained from Merck (Darmstadt, Germany).
Color development: 0.12 mg mL-1 TMB and 0.05% H2O2 in 0.05 M sodium acetate solution, pH 4.5 (TMB solution). This solution was freshly prepared before use.
Stopping reagent: 0.5 M sulfuric acid.
Fungal strains
Cyathus striatus (STMA07048, isolated from basidiospores of a specimen from a trunk of Picea collected in July 1997 in Stelzenberg, Rheinland-Pfalz, Germany) and Hericium erinaceus (FU70034, isolated from basidiocarp tissue) for submerged culture were obtained from IMD. Prior to the experiments, the identity of both strains was confirmed by microscopic studies and by comparison of their ITS nrDNA sequences with reference data in Genbank. The fungi were maintained on a solid medium containing 20 g L-1 malt extract and 15 g L-1 Agar-Agar.They are deposited in the culture collection of IMD Natural Solutions GbR (formerly InterMed Discovery GmbH under liquid nitrogen. A duplicate strain and the corresponding specimen of the C. striatus material is also maintained at the personal herbarium and culture collection of Marc Stadler.
Fruiting bodies of Hericium erinaceus were obtained from a commercial provider www.pilzgarten.de, Helvesiek, Germany.
Soy peptone, yeast malt, and sugar molasses media were prepared according to [25].
HPLC-DAD
The HPLC system was from Merck Hitachi, Darmstadt, Germany, and comprised a pump (L-7100), an auto sampler (L-7200), an interface (D-7000), and a diode array detector (L-7455, 200 – 600 nm). Column: reversed phase, Nucleosil® 100–5 C18, CC 125/3 mm with a guard column Nucleosil® 100–5 C18, CC 8/3 mm (Macherey Nagel, Düren, Germany).
Flow rate: 0.4 mL min-1 for striatal and striatin analysis and 0.6 mL min-1 for erinacine analysis.
Eluent: Acetonitrile (A) and ultra-pure water (B).
Wavelength: 210, 233 and 254 nm.
Gradient for striatal and striatin: 50% A (0 min) – 94% A (14 min) – 94% A (17 min) – 100% A (19 min) – 100% A (25 min) – 50% A (30 min) – 50% A (40 min).
Gradient for erinacine: 30% A (0 min) – 50% A (15 min) – 50% A (16 min) – 100% A (23 min) – 100% A (38 min) – 30% A (43 min) – 30% A (47 min).
Preparative HPLC
A semi-preparative HPLC system (Young Lin, Hongye Anyang, Korea) equipped with quaternary pump (YL9110S) and dual wavelength UV/Vis detector (YL 9120S), combined with a fraction collector (CHF 122SC, Advantec, Osaka, Japan) was used. A column Kromasil 100 C18, 7 μm, 250 – 20 mm (MZ Analysentechnik, Mainz) with a guard column Kromasil 100 C18, 7 μm, 50 – 20 mm (MZ) was used to isolate the target compounds.
Flow rate: 15 mL min-1.
Gradient for striatals and striatins: 50% A (0 min) – 50% A (10 min) – 80% A (55 min) – 80% A (70 min) – 100% A (80 min) – 100% A (95 min). Wavelengths: 210 and 254 nm.
Gradient for erinacines: 25 A% (0 min) – 25% A (10 min) – 48% A (35 min) – 48% A (45 min) – 55% A (52 min) – 55% A (60 min) – 100% A (90 min) – 100% A (110 min). Wavelengths: 210 and 233 nm.
The ELISA was performed in microtiter plates Immuno Plate Maxisorp F96 (Nunc, Denmark). Incubation steps and color development were performed in an incubator INE 500 (Memmert, Schwabach, Germany). Washing was done by an eight-channel pipette Research Pro 50–1200 μL (Eppendorf, Wesseling-Berzdorf, Germany). The extinction was measured by a microtiter plate reader Synergy 2 (BioTek, Bad Friedrichshall, Germany).
Biosynthesis and isolation of cyathane diterpenoids
Submerged cultivation
Pre-cultures of C. striatus were grown submerged in 250 mL Erlenmeyer flasks containing 100 mL soy peptone medium at 24°C and 150 rpm for 7 days. After that, the mycelium was homogenized with an Ultra-Turrax (IKA, Staufen, Germany) at 10,000 rpm for 10 sec. 40 mL homogenized mycelium were inoculated into a new 1,000 mL Erlenmeyer flask containing 400 mL soy peptone medium and incubated at 24°C and 150 rpm for further 12 days.
Pre-cultures of H. erinaceus were grown submerged in 250 mL Erlenmeyer flasks containing 100 mL yeast malt medium at 24°C and 150 rpm for 7 days. Afterwards, 40 mL homogenized mycelium were inoculated into 400 mL sugar molasses medium in 1,000 mL Erlenmeyer flasks and incubated at 24°C and 150 rpm for further 4 days to obtain erinacine P or 12 days to obtain erinacine C.
Extraction and isolation
After submerged cultivation, the mycelia from 2.2 L culture media were separated from the supernatants by centrifugation at 2,880 g and 4°C for 10 min (Allegra® X-15R, BECKMAN COULTER™, Krefeld, Germany), and extracted twice with 400 mL ethyl acetate. The combined organic extracts were dried over sodium sulfate, and afterwards evaporated to dryness. A brown residue was obtained after evaporation, which was dissolved in acetonitrile and subjected to preparative HPLC. The extracts of Cyathus striatus contained the striatals A, B, C and D. The striatins A and B were prepared by conversion of the striatals A and B by stirring in methanol at room temperature overnight, respectively. The erinacines P and C were isolated from the extracts of Hericium erinaceus.
Production of polyclonal antibodies
The aldehyde function of the striatals and NH2 functions of BSA were used for synthesis of striatal A/B (1/1, w/w)-BSA conjugates. The ratio of striatals to BSA was determined by MALDI-TOF-MS. About 17 striatal molecules were coupled to one BSA molecule (Eurogentec S.A, Seraing, Belgien). The production of rabbit pAbs against striatal A/B was performed by Eurogentec S.A. Two rabbits with ID SA6928 and SA6929 were immunized four times with 200 μg of the striatal A/B-BSA conjugate on day 0, 14, 28 and 56. An additional immunization was done on day 91with 400 μg striatal A/B-BSA conjugate. The final antisera were collected on day 115 and stored at −20°C without further purification until use. The pre-bleed sera of these two rabbits on day 0 before immunization were used as negative control in ELISA. The final antiserum from rabbit SA6928 was used in the following assays because of its higher specificity against striatal B compared to SA6929 (data not shown).
Development of an indirect competitive ELISA
Optimization of coating conditions
Striatal B was directly attached to the plate in several different concentrations. The optimal conditions (buffer, temperature and time) for direct coating of striatal B were studied by an indirect non-competitive ELISA. The incubation temperatures were 4°C, 24°C and 37°C, and the coating time was 2 h or overnight.
Checkerboard titration
The optimal dilution of pAbs and the optimal coating amount of striatal B were determined simultaneously by checkerboard titration (a two-dimensional titration method) by indirect non-competitive ELISA. Striatal B with different concentrations (0.04 to 20 μg mL-1, two fold dilution) was coated to the microtiter plate and then bound to pAbs with serial dilutions (1:50 to 1:6,400, stepwise in two fold dilution).
The tolerance against organic solvents (DMSO, acetonitrile, methanol) used to dissolve the cyathane diterpenoids was examined by indirect competitive ELISA. Different proportions of organic solvent (5 - 50%, v/v, 0% as blank) were added to PBST with a final dilution of pAbs 1:200 and pre-incubated without competing molecules at 24°C for 1 h. The effects of organic solvents on the ELISA system were evaluated by comparing the measured extinctions to those of the blanks.
100 μL of 5 μg mL-1 striatal B in coating buffer 1 were pipetted in wells of a microtiter plate. 100 μL of 1 μg mL-1 BSA diluted in PBS were pipetted in two wells as positive control. The incubation was performed overnight at 24°C.
200 μL of 1% gelatin were added to the wells and incubated at 24°C for 3 h.
Serial dilutions of analyte were prepared in a concentration range of 0.0003 - 20 μg mL-1 in PBST containing 5% acetonitrile or DMSO and a final pAbs dilution of 1:200. The incubation was performed for 1 h at 24°C in 2 mL Eppendorf® cups.
Incubation with pAbs
100 μL pre-incubated analyte were added to each well (in triplicate) and incubated for 1 h at 24°C. For the indirect non-competitive ELISA, 100 μL pAbs with a 1:100 dilution in PBST were used without competitors instead of the pre-incubated analyte.
Incubation with secondary antibodies
100 μL secondary antibodies with a 1:5,000 dilution in PBST were added to each well and incubated for 1 h at 24°C.
Color development
100 μL of the TMB solution were added to each well and incubated for 15 min at 24°C. Without washing step, 100 μL of 0.5 M sulfuric acid were added to each well to stop the color development.
The extinctions were measured immediately by a microtiter plate reader at 450 nm and 630 nm.
Unless otherwise specified, the wells were washed with 300 μL washing buffer PBST three times after each incubation step. The indirect non-competitive ELISA involved all of the steps of the indirect competitive ELISA, except for the pre-incubation of pAbs with competitors.
Striatal B standards and samples were analyzed in triplicates. The differences between extinctions at 450 nm and 630 nm were used for calculation and plotting the sigmoidal curves. %B/B0-values for calibration curves were calculated as follows:
% B B 0 = Δ E − Δ E excess Δ E 0 − Δ E excess ×100
ΔE: the extinction differences of samples at 450 nm and 630 nm.
ΔE 0 : the extinction differences of the upper asymptote of the sigmoidal curve between 450 nm and 630 nm.
ΔE excess : the extinction differences of the lower asymptote of the sigmoidal curve between 450 nm and 630 nm.
Cross reactivity (IC50)
The assay specificity was evaluated by obtaining sigmoidal curves for several structurally related compounds (striatals A, C and D, striatins A and B, and erinacines C and P) as competitors against the striatal B standard. The calibration curve of striatal B standard was measured individually for each plate. The IC50 were estimated by using a linearization of the calibration curves with a logit-transformation of %B/B0-values:
logit % B / B 0 =ln % B / B 0 100 − % B / B 0
Cross reactivity (IC50) = A/B × 100%.
A: concentration of striatal B standard at logit (%B/B0) =0.
B: concentration of competitors at logit (%B/B0) =0.
The linear regression of logit (%B/B0) was used for determination of concentrations in unknown samples.
Acetonitrile was spiked with striatal B at several concentrations (0.001 - 10 μg mL-1) and analyzed by indirect competitive ELISA as well as by HPLC-DAD. The concentrations of striatal B of spiked samples were calculated from linear regression of logit (%B/B0) in ELISA and linear calibration for striatal B in HPLC-DAD. The recoveries of striatal B were obtained by:
recovery % = detected concentration spiked concentration ×100%
Analysis of H. erinaceussamples
The supernatants and extracts of supernatants from submerged cultures, as well as extracts of fruiting bodies of H. erinaceus were analyzed by indirect competitive ELISA:
Sample preparation for pre-incubation of supernatants in ELISA
50 μL of supernatants from submerged cultures (culture day 1 to day 8) were added to 950 μL PBST with a final DMSO proportion of 5% and a final pAbs dilution of 1:200. Culture medium without H. erinaceus was used as negative control.
Sample preparation for pre-incubation of extracts of supernatants
20 mL of supernatants from submerged cultures (culture day 1 to 8) were extracted once with 20 mL ethyl acetate. 15 mL of the ethyl acetate phase was evaporated to dryness, and the residues were redissolved in 1 mL acetonitrile for ELISA and HPLC-DAD analysis. 5 μL of the extracts were added to 995 μL PBST with a final acetonitrile proportion of 5% and a final pAbs dilution of 1:200. Extracts of culture media without H. erinaceus were used as negative controls.
Sample preparation for pre-incubation of extracts of fruiting bodies
32.6 g fruiting bodies of H. erinaceus were homogenized and then extracted with 200 mL ethyl acetate under stirring overnight. The ethyl acetate phase was dried over sodium sulfate and evaporated to dryness after filtration. 1 mg crude extract was dissolved in 1 mL acetonitrile. Several concentration levels (50.0, 10.0, 5.0, 2.5, 1.0 and 0.5 μg mL-1) of the extracts were used in the pre-incubation solution. Acetonitrile was used as blank.
The data sets supporting the results of this article are included within the article and its additional file.
Allbutt AD, Ayer WA, Brodie HJ, Johri BN, Taube H: Cyathin, a new antibiotic complex produced byCyathus helenae. Can J Microbiol. 1971, 17: 1401-1407. 10.1139/m71-223.
Ayer WA, Taube H: Metabolites ofCyathus helenae. cyathin A3and allocyathin B3, members of a new group of diterpenoids.Tetrahedron Lett 1972, 13:1917-1920.,
Anke T, Oberwinkler F: The striatins–new antibiotics from the basidiomycete Cyathus striatus (Huds. ex Pers.) Willd. J Antibiot. 1977, 30: 221-225. 10.7164/antibiotics.30.221.
Anke T, Rabe U, Schu P, Eizenhöfer T, Schrage M, Steglich W: Studies on the biosynthesis of striatal-type diterpenoids and the biological activity of herical. Z Naturforsch C. 2002, 57: 263-271.
Shibata H, Tokunaga T, Karasawa D, Hirota A, Nakayama M, Nozaki H, Tada T: Isolation and characterization of new bitter diterpenoids from the fungusSarcodon scabrosus. Agric Biol Chem. 1989, 53: 3373-3375. 10.1271/bbb1961.53.3373.
Shibata H, Irie A, Morita Y: New antibacterial diterpenoids from theSarcodon scabrosusfungus. Biosci Biotechnol Biochem. 1998, 62: 2450-2452. 10.1271/bbb.62.2450.
Kawagishi H, Zhuang C: Compounds for dementia fromHericium erinaceum. Drugs Future. 2008, 33: 149-155. 10.1358/dof.2008.033.02.1173290.
Petrova RD, Mahajna J, Reznick AZ, Wasser SP, Denchev CM, Nevo E: Fungal substances as modulators of NF-κB activation pathway. Mol Biol Rep. 2007, 34: 145-154. 10.1007/s11033-006-9027-5.
Kawagishi H, Shimada A, Hosokawa S, Mori H, Sakamoto H, Ishiguro Y, Sakemi S, Bordner J, Kojima N, Furukawa S: Erinacines E, F, and G, stimulators of nerve growth factor (NGF)-synthesis, from the mycelia ofHericium erinaceum. Tetrahedron Lett. 1996, 37: 7399-7402. 10.1016/0040-4039(96)01687-5.
Kawagishi H, Shimada A, Shirai R, Okamoto K, Ojima F, Sakamoto H, Ishiguro Y, Furukawa S: Erinacines A, B and C, strong stimulators of nerve growth factor (NGF)-synthesis, from the mycelia ofHericium erinaceum. Tetrahedron Lett. 1994, 35: 1569-1572. 10.1016/S0040-4039(00)76760-8.
Kawagishi H, Simada A, Shizuki K, Ojima F, Mori H, Okamoto K, Sakamoto H, Furukawa S: Erinacine D, a stimulator of NGF-synthesis, from the mycelia ofHericium erinaceum. Heterocycllic Commun. 1996, 2: 51-54.
Ma B, Shen J, Yu H, Ruan Y, Wu T, Zhao X: Hericenones and erinacines: stimulators of nerve growth factor (NGF) biosynthesis inHericium erinaceus. Mycology. 2010, 1: 92-98. 10.1080/21501201003735556.
Krzyczkowski W, Malinowska E, Herold F: Erinacine A biosynthesis in submerged cultivation ofHericium erinaceum: quantification and improved cultivation. Eng Life Sci. 2010, 10: 446-457. 10.1002/elsc.201000084.
Jahn C, Zorn H, Petersen A, Schwack W: Structure-specific detection of plant cuticle bound residues of chlorothalonil by ELISA. Pestic Sci. 1999, 55: 1167-1176. 10.1002/(SICI)1096-9063(199912)55:12<1167::AID-PS77>3.0.CO;2-H.
Xu DM, Yu XY, Liu YQ, Feng JT, Pan LG, Liu XJ, He J, Zhang X: Development of an enzyme-linked immunosorbent assay for podophyllotoxin. Int Immunopharmacol. 2005, 5: 1583-1592. 10.1016/j.intimp.2005.04.006.
Tothiam C, Phrompittayarat W, Putalun W, Tanaka H, Sakamoto S, Khan IA, Ingkaninan K: An enzyme-linked immunosorbant assay using monoclonal antibody against Bacoside A3for determination of jujubogenin glycosides inBacopa monnieri(L.) Wettst.Phytochem Anal 2011, 22:385-391.,
Sathe M, Merwyn S, Ghorpade R, Agarwal GS, Rao MK, Rai GP, Kaushik MP: Design and synthesis of immunoconjugates and development of competition inhibition enzyme-linked immunosorbent assay (CIEIA) for the detection of O-isopropyl methylphosphonofluoridate (sarin): an organophosphorous toxicant. J Hazard Mater. 2011, 192: 1720-1728. 10.1016/j.jhazmat.2011.07.011.
Boudet F, Theze J, Zouali M: UV-treated polystyrene microtitre plates for use in an ELISA to measure antibodies against synthetic peptides. J Immunol Methods. 1991, 142: 73-82. 10.1016/0022-1759(91)90294-P.
Zouali M, Stollar BD: A rapid ELISA for measurement of antibodies to nucleic acid antigens using UV-treated polystyrene microplates. J Immunol Methods. 1986, 90: 105-110. 10.1016/0022-1759(86)90390-X.
Lagace J, Arsenault S, Cohen EA: Alcian blue-treated polystyrene microtitre plates for use in an ELISA to measure antibodies against synthetic peptides. J Immunol Methods. 1994, 175: 131-135. 10.1016/0022-1759(94)90339-5.
Geerligs HJ, Weijer WJ, Bloemhoff W, Welling GW, Welling-Wester S: The influence of pH and ionic strength on the coating of peptides of herpes simplex virus type 1 in an enzyme-linked immunosorbent assay. J Immunol Methods. 1988, 106: 239-244. 10.1016/0022-1759(88)90203-7.
Güll I, Wirth M, Gabor F: Development of a sensitive and reliable ELISA for quantification of wheat germ agglutinin. J Immunol Methods. 2007, 318: 20-29. 10.1016/j.jim.2006.07.025.
Kim YJ, Cho YA, Lee H-S, Lee YT: Investigation of the effect of hapten heterology on immunoassay sensitivity and development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Anal Chim Acta. 2003, 494: 29-40. 10.1016/j.aca.2003.07.003.
Brun EM, Garcés-García M, Puchades R, Maquieira A: Enzyme-linked immunosorbent assay for the organophosphorus insecticide fenthion. Influence of hapten structure. J Immunol Methods. 2004, 295: 21-35. 10.1016/j.jim.2004.08.014.
Bhandari DR, Shen T, Römpp A, Zorn H, Spengler B: Analysis of cyathane-type diterpenoids fromCyathus striatusandHericium erinaceusby high-resolution MALDI MS imaging. Anal Bioanal Chem. 2014, 406: 695-704. 10.1007/s00216-013-7496-7.
Whittington RJ: Evaluation of a simple method for improving the precision of an ELISA detecting antibody in serum. J Immunol Methods. 1992, 148: 57-64. 10.1016/0022-1759(92)90158-P.
The authors thank the BMBF and CLIB 2021 for financial support of the project (FKZ 0315404 A). Part of the work was supported by the excellence initiative of the Hessian Ministry of Science and Art which encompasses a generous grant for the LOEWE research focus `Integrative Fungal Research'.
Justus Liebig University Giessen, Institute of Food Chemistry and Food Biotechnology, Heinrich-Buff-Ring 58, Giessen, 35392, Germany
Tian Shen, Lena M Hof & Holger Zorn
Justus Liebig University Giessen, Institute of Organic Chemistry, Heinrich-Buff-Ring 58, Giessen, 35392, Germany
Heike Hausmann
Department Microbial Drugs, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, Braunschweig, 38124, Germany
Marc Stadler
Tian Shen
Lena M Hof
Holger Zorn
Correspondence to Holger Zorn.
TS purified the cyathane type diterpenoids, carried out the ELISA and drafted the manuscript. LH was involved in the studies on the cross reactivities, and HH was responsible for NMR analyses and data interpretation. MS provided the fungal strains and contributed to the discussion. HZ participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.
Additional file 1: Supplementary material.(DOCX 25 KB)
Shen, T., Hof, L.M., Hausmann, H. et al. Development of an enzyme linked immunosorbent assay for detection of cyathane diterpenoids. BMC Biotechnol 14, 98 (2014). https://doi.org/10.1186/s12896-014-0098-4
Cyathane diterpenoids
Hericium erinaceus
Striatal
Erinacine


Valencia, on the eastern coast of Spain, was a wonderful city for a weekend away from Oxford. The third largest town in Spain, after Madrid and Barcelona, Valencia offers stunning architecture, guaranteed sunshine, fascinating markets and great nightlife. Our students discovered that the traditional Spanish dish, paella, originated in Valencia – definitely one of the highlights of the trip.


Everyone — from secular atheist to Orthodox Jew — can appreciate the wonder and awe of the natural world around them. This deep reverence for nature is deeply rooted in Judaism and forms the basis for two important issues in Jewish ethics: bal tashkhit and tza'ar ba'alei khayim.
Jews have known for centuries that survival depends upon a healthy environment where the food required can be produced, yet the planet suffers destruction at the hands of humankind. The ancient code of bal tashkhit, literally "do not destroy," prohibits wasteful or senseless destruction of resources and the environment.
Instead, Jewish tradition encourages caring for the land, and, by extension, for the entire world. The Torah states that even farmland should observe a 12-month "Shabbat" of rest every seven years. Today, Jewish groups, basing their actions on religious principles, are among those most active in working for a healthier environment.
Bal tashkhit tells Jews to behave responsibly: Drive energy efficient cars when possible, turn off the lights when leaving a room, and avoid squandering food, money, or useable things.
Although Judaism places the role of humans above that of animals, the traditional principle of tza'ar ba'alei khayim, avoiding "the suffering of any living creatures," insists that Jews must respect and honor animals in their care. For instance, the Talmud states, "A person is prohibited to eat until he first feeds his animals" (Berachot 40a), and Exodus 20:10 notes that on Shabbat the animals should rest alongside their humans.
This underlying law has a number of consequences, including the condemnation of hunting and animal fighting for sport, and the rule that animals must be slaughtered as quickly and painlessly as possible. Many Jews today expand this law to avoid any product that came from ill-treated animals.
Jewish tradition affirms the essential wholeness and holiness of the natural world. A Hasidic teaching enjoins Jews to remove their shoes, just as Moses was asked to take off his shoes when approaching the Burning Bush, because the ground he was standing on is holy. All people are always standing on holy ground.


Type the number of units of each size/price catagory into the box. Go to the bottom of each page and click on the "Add items to cart" button.
10% discount from the pound price on orders of 5 lbs or more of an item.


Before a leaf-bud has burst, its whole life acts; in the full-blown flower there is no more; in the leafless root there is no less. Its nature is satisfied and it satisfies nature in all moments alike.
for a workshop on Victorian Flower Painting!
Gone are the days when paintings and sculptures had been the only impressive forms of art available.
The interesting fragrance of roses adds life to the tragic environment. Roses are intended to summon love and strength.


So far I have completed the description stage document, which outlines the current developments in steganography, and the problems and processes that this project will need to tackle.
Over the past week and a bit I have been locating papars that discuss relevant areas of steganography. I have detailed these papars below. Over the next few days I intend to provide summaries of these papers on my blog.
Hopper, N. J., Langford, J. & Ahn, L von. (2002). Provably Secure Steganography.
Zhao, H., Wang, H. & Khan, M. K. (2011). Steganalysis for palette-based images using generalized difference image and color correlogram.
Cao,Y., Zhao, X. & Feng, D. (2012). Video Steganalysis Exploiting Motion Vector Reversion-Based Features.


Whether you're searching for your new favourite pair of jeans, your perfect little black dress or the mother-of-the-bride gown to top all gowns, Butterfly Boutique Millet has something in store for you. From size 2 through 3X, our focus is to make women of all sizes feel beautiful and comfortable in their own skin with stunning fashion and a terrific shopping experience.
We pride ourselves on providing the best service possible, so whether you have something specific in mind or have no clue what you're looking for we are happy to help!


text:
Darko Nestorović (, born 4 April 1965) is a Bosnian professional football manager and former player.

Playing career
Born in Kakanj, SR Bosnia and Herzegovina, Nestorović played for Čelik Zenica, Rad and Rijeka in the former SFR Yugoslavia.
After Rijeka and the breakup of Yugoslavia, he played abroad for Panserraikos in the Greek First Division, FC Ham-Kam(Hamar) in the first league of Norway , FC Golden Hong-Kong in the first league of China, FC Umea in the first league of Sweden. In the first league of Serbia and Montenegro, he played for FC Spartak (Subotica) and FC Mogren (Budva). In his playing career, he was a representative of all national teams of Bosnia and Herzegovina and a youth representative of Yugoslavia in the generation of Darko Pančev, Dragan Piksi Stojković, Mitar Mrkela, Dika Stojanović, and with that generation he qualified for the European Championship. He also remained as FC Celik's all-time top scorer. He played in the UEFA Cup with FC Rad (Belgrade) and was the scorer in a 2-1 victory over FC Olympiakos (Greece) in Belgrade. Abroad, he was a member of the Hong Kong national team.

Managerial career
After ending his playing career, Nestorović stayed in football as an professional manager, starting off at Radnik Bijeljina. After Radnika, he managed Drina Zvornik then Rudar Prijedor.
With Rudar Prijedor, Nestorović achieved the greatest success in the history of the club. In three seasons, he introduced a team from the second league to the first league, and the following year to the premier league of Bosnia and Herzegovina. With the same players, he was in the middle of the Premier League table. He was twice chosen as the best coach of the year in the selection of Sporski Žurnal, as well as by his fellow coaches. Then he managed Sloboda Mrkonjić Grad, Radnik Bijeljina, who he promoted to the Bosnian Premier League by winning the 2011–12 First League of RS.
When he lead FR Radnik Bijeljina, Darko was coach to the Bosnia and Herzegovina U23 Olympic national team, Sutjeska Foča and Rudar, before becoming head coach of the Bosnia and Herzegovina U21 national team on 27 December 2014. Nestorović stayed as head coach until 10 mart 2017.

He then became manager of Kuwait Premier League club Al-Arabi on 13 August 2017, working alongside Kuwaiti manager Mohammed Ebrahim Hajeyah. On 5 September 2018, he became the new manager of newly promoted Bosnian Premier League club Zvijzeda 09.
Nestorović took over the team that was last in the standings with only three points and that everyone had already seen in the second league. In a very short period, he stabilized the team and with five consecutive victories in the championship led the team to the sixth place in the table and saved relegation.Even after making decent results with the club, Nestorović decided to leave Zvijezda on 11 March 2019. On 6 September 2019, he returned to Al-Arabi. Nestorović took over Al Arabi, who were also last in the Kuwait First League table and were in a very difficult situation. He eventually managed to lead the team to third place and also played with Al Arabi in the final of the Prince Cup and was first in the League Cup. Al Arabi played the most beautiful football in Kuwait during that period.

Nestorović once again became manager of Radnik Bijeljina on 24 November 2020. On 16 April 2021, he decided to resign as manager of Radnik for private reasons. On 14 June 2021, Darko became the coach of Kazma, another club in the Kuwait Premier League.
With Kazma, Nestorović was the autumn champion of the Kuwait First League and played in the semi-finals of the Emir Cup. After twenty years, Kazma was at the top of Kuwaiti football.During that period of his stay in Kazma, Nestorović was a candidate for the selector of the National Team of Kuwait.

Honours

Manager
Rudar Prijedor
 Second League of RS: 2007-08

Rudar Prijedor
 First League of RS: 2008–09

Radnik Bijeljina
 First League of RS: 2011–12

References

External links

1965 births
Living people
People from Kakanj
Association football forwards
Yugoslav footballers
Bosnia and Herzegovina footballers
NK Čelik Zenica players
FK Rad players
HNK Rijeka players
Panserraikos F.C. players
Hamarkameratene players
Yugoslav First League players
Super League Greece players
Eliteserien players
Yugoslav expatriate footballers
Expatriate footballers in Greece
Yugoslav expatriate sportspeople in Greece
Bosnia and Herzegovina expatriate footballers
Expatriate footballers in Norway
Bosnia and Herzegovina expatriate sportspeople in Norway
Bosnia and Herzegovina football managers
FK Drina Zvornik managers
FK Rudar Prijedor managers
FK Sloboda Mrkonjić Grad managers
FK Radnik Bijeljina managers
Bosnia and Herzegovina national under-21 football team managers
Al-Arabi SC (Kuwait) managers
Kazma SC managers
FK Zvijezda 09 managers
Premier League of Bosnia and Herzegovina managers
Kuwait Premier League managers
Bosnia and Herzegovina expatriate football managers
Expatriate football managers in Kuwait
Bosnia and Herzegovina expatriate sportspeople in Kuwait

redpajama_set_name: RedPajamaWikipedia


text:
ITELEPORT.NET
portal About technology news
The terrible secret of Betelgeuse, the giant star has swallowed its neighbor
Giant star with an incredibly beautiful name Betelgeuse, which translates from Arabic as the house…
SpaceX blew up his rocket during testing
In 2019 founded by Elon Musk SpaceX company has done a great job. At least…
NASA have discovered an unusual cluster of dark matter
Dark matter — the mysterious substance, haunting scientists for decades. Using the space telescope NASA…
Scientists have discovered thousands of "foreign" stars in our galaxy
Our milky Way is going how to eat several more smaller galaxies. Rather, it already…
What happens if you collide two neutron stars?
Neutron stars — the superdense remnants of stars arising from the explosion of a supernova….
Discovered why Venus has active volcanoes so important to science?
Truly hellish climate of the second farthest planet from the Sun in the Solar system,…
Why the moon has no magnetic field?
The magnetic field of the planet is a unique substance with the help of special…
the Andromeda Galaxy was the same size as the milky Way
All this time, astronomers believed that our nearest Andromeda galaxy about three times more massive…
While we celebrated the New Year in the Solar system broke new comet
When August 30, 2019 the astronomer of the Crimean astrophysical Observatory Gennady Borisov noticed an…
What will the company SpaceX in 2020?
American entrepreneur Elon Musk started the company SpaceX back in 2002 with the purpose of…
Next to Earth found a new earth-like planet
Planet Earth and the Solar system are in a fairly densely populated area of our…
Scientists first recorded the magnetic explosion on the Sun
Astronomers recorded the most powerful explosion on the surface of the Sun, not similar to…
The space debris problem can be solved in the near future
Environmental pollution is one of the most acute problems of our time. We invariably leave…
Found 19 galaxies without dark matter
Recently, scientists found 19 galaxies less the milky Way, in which the number of dark…
Mysterious radio signal has puzzled astronomers. Perhaps a new type of Solar system
For about two months, scientists were observing a part of the sky near the constellation…
A neutron star is that I was looking for more than 30 years may have been detected
The most dense objects in the Universe — neutron stars — quanta collapse into the…
The star collapses into a black hole right in front of the Hubble lens
Distinguished astronomer Carl Sagan believed that through us , the universe knows itself, because we…
NASA has detected two meteors building blocks of life
Despite the fact that today we know a lot about the development of living organisms…
Created the most detailed simulation of the Universe
One of the most difficult to resolve riddles of modern cosmology is the formation of…
Physicists have found proof of the existence of another force of nature
Everything in our Universe operates in the interaction between the four fundamental forces of nature:…
Compiled a complete map of Titan's surface
Titan is the largest moon of Saturn and the only after Earth celestial body, where…
3D printer AI Apple Artificial Intelligence Astronaut black hole Brain cancer Car CERN CES Chang'e-4 China climate Collider DNA Earth Elon Musk Future Genesis global warming GPS Hubble ISS Mars Medecine Microsoft Moon NASA NVIDIA Oculus Osiris-Rex Pluto Robots Roscosmos Rover Soyuz Space SpaceIL SpaceX Tesla Tesla Model 3 Voyager Voyager-2 Windows
Zika virus may help fight brain cancer
Created "live" concrete made from bacteria, the ability to recover
Archaeologists have solved the mystery of one of the most mysterious statues in the world
The Czechs claim to have invented a revolutionary way of charging an electric vehicle
In electric cars, well almost all. What is not very well corrected. And some manufacturers even offer unusual solutions, about which we also wrote. Here...
The company Tesla presented an electric pickup Cybertruck and it is available to order
Tesla is mainly known as a manufacturer of electric car and truck Semi Truck. However, in 2017, Elon Musk spoke about the development of corporate...
The astronauts will help Lamborghini to create a new material for cars
The Italian company Lamborghini does not spend time on the production of cheap cars and focuses on the development of expensive sports models. Accordingly, in...
Flying cars could become a reality in the near future
Probably, many of us flying cars are associated with some sci-Fi movie. The latest research in the field of automotive industry show that flying cars...
How to create a self-driving car after hacking the iPhone
We all know a little bit quirky guys, we feel in a good way saying that "they are not of this world." These guys do...
Cosmonauts return from the ISS and wireless high-speed tunnel Elon musk
Every Monday in the new issue of "News of high technologies" we summarize the results of the last week, talking about some of the most...
Elon Musk spoke about the history of The tunnel Boring and its security
December 18, Elon Musk announced the opening of the first underground tunnel The Boring Company. The test route near the town of Horton, electric allows...
Artificial intelligence has learned to recognize emotions. What it may lead?
What is consciousness and how it appeared?
Artificial intelligence detects breast cancer better than a professional doctor
What kind of column with a voice assistant to buy?
What creates AI? Certainly not for the common good.
Science is inseparable from politics: why the peaceful protests effectively enforced?
Bacteria can come together and create patterns in the form of flowers

redpajama_set_name: RedPajamaCommonCrawl


Learn MoreGet my FREE health resources here!
Great health, great energy, great body.
It is time to prioritize you for a change. (I know you are taking good care of others!) When was the last time you talked with someone about your health and received the personal attention you deserve?
Let me teach you how to discover great health in your body again! Achieve a sustainable weight through breaking habits of a lifetime, nutritional coaching and finding exercise that you love. So that you can be ready, physically, mentally and emotionally – to become the best version of you ever and reach all those as-yet unfilled dreams and desires!
You are unique. And I am also unique in my approach to teaching you how to achieve and maintain better health, energy and confidence in your own skin.
I create a supportive environment that will enable you to achieve all of your health goals, not by dieting, but by regaining trust in your body.
Your lifestyle changes will all be designed around you and for YOU, no one else. You are not the same as your neighbour, best friend or even your sister, so won't be treated as such.
I will not be giving you a full meal plan that you stick to to a tee, lose the weight and then bounce back to where you were before, having learnt nothing. Instead, you will be taught gradually how to eat the right way, whilst still enjoying the foods you love and in a way that works with your lifestyle.
There is more to achieving great health than just eating the right foods and exercising enough. So be prepared to go on a journey with me to places in yourself and of yourself, that you may not realise are even within you – yet.
You will be held accountable by me and supported by me throughout our time together and will have full access to my brains when you need them. During our time together you will learn that there is no wagon, so you can't fall off, there is just life!
Most approaches to nutrition dwell on calories, carbs, fats, proteins. Instead of creating lists of restrictions and good and bad foods, I coach my clients to create a happy, healthy life – in a way that is flexible, fun and free of denial and discipline.
The ultimate goal – coaching you to be the BEST version of yourself in ALL areas of your life. And it all starts with your health.
Get in touch – and lets get started!
Advanced Clinical Weight Loss Practitioner.
Bachelor of Physical Education, majoring in Exercise Prescription, Otago University.
Mother to 3 children, 8 and under.
I will help you discover what your body is really telling you when it comes to the food that you eat, through changing habits, one at a time and get you back into your ideal body.
I hold a Bachelor of Physical Education from Otago University. I will tailor your personalised fitness program to your body, and your lifestyle.
I will work with you one on one and alongside Teagan Fea (RTT Specialist) we tailor the most amazing Coaching Program to your body, mind and lifestyle.
I practice what I preach and will ONLY recommend food, exercise, techniques and products that are an everyday part of my own life. I will never suggest that you do, eat or use anything I haven't tried myself. If it's good enough for you, then it's good enough for me!
Start changing your body, health and life TODAY!


text:
Sun | Sports
— Jan 2nd, 2004
Outback Bowl at Tampa, Fla.
Iowa 37, Florida 17
Gator Bowl at Jacksonville, Fla.
Maryland 41, West Virginia 7
Capital One Bowl at Orlando, Fla.
Georgia 34, Purdue 27, OT
Rose Bowl at Pasadena, Calif.
Southern Cal 28, Michigan 14
Orange Bowl at Miami
Miami 16, Florida State 14
Cotton Bowl at Dallas
Mississippi (9-3) vs. Oklahoma State (9-3), 11 a.m. (FOX)
Peach Bowl at Atlanta
Clemson (8-4) vs. Tennessee (10-2), 1:30 p.m. (ESPN)
Fiesta Bowl at Tempe, Ariz.
Kansas State (11-3) vs. Ohio State (10-2), 5 p.m. (ABC)
Humanitarian Bowl at Boise, Idaho
Tulsa (8-4) vs. Georgia Tech (6-6), 9 a.m. (ESPN)
Sugar Bowl at New Orleans
Oklahoma (12-1) vs. LSU (12-1), 5 p.m. (ABC)
Football 2005-06 Bowl Glance Tuesday New Orleans Bowl at Lafayette, La. Arkansas State (6-5) vs. Southern Miss (6-5), 5 p.m. (ESPN) Wednesday GMAC Bowl at Mobile, Ala. UTEP (8-3) vs. Toledo (8-3), 5 p.m. (ESPN) Thursday Las Vegas Bowl BYU (6-5) vs. California (7... [Read More...]
College Hawaii Bowl Saturday Nevada 49, Central Florida 48 (OT) Nevada 7 21 0 14 7 — 49 Central Florida 17 3 12 10 6 — 48 First Quarter UCF—Marshall 51 pass from Moffett (Prater kick), 13:20. UCF—Ke.Smith 78 run (Prater kick), 9:24. Nev—R.Hubbard 4 run (Jaekle kick), 8:33. UCF—FG Prater 47, 4:08. Second Quarter Nev—Mitchell 1 run (Jaekle kick), 11:32. Nev—... [Read More...]
2005-06 Bowl Glance Ne...
2005-06 Bowl Glance New Orleans Bowl Tuesday, Dec. 20 at Lafayette, La. Arkansas State (6-5) vs. Southern Miss (6-5), 5 p.m. (ESPN) GMAC Bowl Wednesday, Dec. 21 at Mobile, Ala. UTEP (8-3) vs. Toledo (8-3), 5 p.m. (ESPN) Las Vegas Bowl Thursday, Dec. 22 at Las ... [Read More...]
The NFL Career Touchdown Leaders The career touchdown leaders in the National Football League (x-active): 1. Jerry Rice 10 197 1 208 2. Emmitt Smith 164 11 0 175 3. Marcus Allen 123 21 1 145 4. x-Marshall Faulk 100 36 0 136 5. Cris Carter 0 130 1 131 6. Jim Brown 106 20 0 126 7. Walter Payton 110 15 0 125 8. John Riggins 104 12 0 116 9. Lenny Moore 63 48 2 113 10. x-Marvin Harrison 0 110 0 110 11. Barry Sanders 99 10 0 109 12. Don Hutson 3 99 3 105 12. Tim Brown 1 100 4 105 14. x-Terrell Owens 2 101 0 103 15. ... [Read More...]
College BOWL GLANCE Tuesday, Dec. 21 New Orleans Bowl Southern Mississippi 31, North Texas 10 Champs Sports Bowl Georgia Tech 51, Syracuse 14 Wednesday, Dec. 22 GMAC Bowl Bowling Green 52, Memphis 35 Thursday, Dec. 23 Fort Worth (Texas) Bowl Cincinnati 32, Marshall 14 Las Vegas Bowl Wyoming 24, UCLA 21 Friday, Dec. 24 Hawaii Bowl Hawaii 59, ... [Read More...]


iPod video downloads are now hot favorites among iPod users. Everyday, an incredible number of video downloads are utilized in PCs and synced with iPods. Video iPod or iPod video may be the name folks share with the new generation iPod which is designed with video viewing capabilities. It is this technology; in conjunction with the vast accessibility to iPod video downloads that pushed up the popularity of iPod videos as probably the most downloaded form of media.
There are lots of online music and video stores to download videos for the iPod video downloader. The most famous is definitely iTunes. You're able to download movies, TV shows and music at a typical charge per download. When you yourself have missed a bout of your favorite TV show on national television, just go there and download that specific episode at $1.99. It's that convenient because the iPod video downloads are available ab muscles next day it's screened. In addition you do not have to worry about having to buy the entire season.
If you are looking for free iPod video downloads which are legal, you can try some of the following sites. They are mainly number of entertaining videos uploaded by people as if you and me or aspiring film producers and public domain videos. Sometimes you will undoubtedly be amused by the kind of iPod video downloads that exist on this planet.
Watch Free Movies Online: If you're a supporter of classic movies, you would love the free video downloads.
Singingfish: This can be a video search engine that archives many movies, movie clips and videos. Read the hot favorites section.
SearchforVideo: Another video internet search engine where you will be able to find a lot of iPod video downloads. You will even find a range of videos under different categories like news, movies, music, celebrities, health, TV channels, etc.
The Open Video Project: This is site with a massive number of digital video for sharing.
Vmix: One interesting and wacky site where you will discover a great deal of entertaining and funny videos submitted by amateurs and professionals alike.
No matter where you download, there's something you will need to know. Most video files are not exactly iPod video files but rather AVI, RM, DivX, WMV, ASF and VOB video files. In these instances you would need to convert them into MPEG 4 or H.264 video format to ensure that your iPod to play them. There are many converters on the market, some paid while others free. Key in "free iPod video converters" in the search engines and you ought to be able to discover a few good free video to iPod converters.
Learn more about iPod video downloads and which are typically the most popular sites video lovers are utilizing to download good quality video for his or her iPod at prices even school-going teens can afford.
This content of this short article is provided for the objective of education and illustration only and is certainly not connected with Apple, iTune, or any company or subsidiary of Apple. This informative article might be freely reprinted or distributed in its entirety in any ezine, newsletter, blog or website. The author's name, bio and website links must remain intact and be incorporated with every reproduction.
Next Post Are You A Home Based Business Entrepreneur?


text:
Pay Dues Renew Membership
AORP News
If you have questions about your password, please contact Janet Gross at janetgmarie@aol.com
Want to Join? Click on "Join" on the left of the Blue Ribbon above.
DONATE TO THE AORP LEGAL FUND
DONATE TO THE DOLLARD-SUTER FAMILY FUND
D.C. POLICE MEMORIAL FUND
IRS Letter Ruling with Explanation from our attorneys
Welcome to the Association of Retired Police Officers (DC)
The Association of Retired Police Officer (AORP) represents retired police officers in the Washington, D.C. Metropolitan area. The agencies included are the Metropolitan Police Department (MPD), the U.S. Park Police (USPP), the U.S. Secret Service Uniformed Division (USSS/UD), the U.S. Secret Service (USSS) and the U.S. Capitol Police. The organization was chartered in 1935 and the AORP has a long record of assisting and representing retired police officers and their families.
By joining AORP, you become a member of our police retirement community. Benefits of joining include access to our web site, AORP Newsletter, retirement information and legislation that affects you, links to several web sites that are beneficial to retirees and access to 5,000 plus retired DC Area police officers.
Access to our web site is via a password that only AORP members with a current membership can obtain, so if you qualify consider becoming a member. See the Join page for membership specifics. If you are an AORP member and need a password, go to AORP Web Site Member Access to apply for a password.
Please see the Meetings link for details.
Do you have comments or questions? Write us at
janetgmarie@aol.com
police21stcent@aol.com
In Memoriam (chronological)
Widow's Info
SSA Windfall
© AORP 2000-2018


No blog post today: Rebooting my brain & my schedule!
For a new writer this leads me to wonder if I am ready for a critique group not in terms of what I could get but what I can give.
At one point have you written enough to be a critique partner instead of a beta reader?
You don't have to have "written enough." If you're writing, you can be in a critique group. If you're new to writing/publishing, then you will have something of a learning curve, but that's okay. Because you'll learn as you go.
A beta reader generally is a reader who reads your stuff and gives you some feedback. A critique partner is generally a writer that you swap work with. You critique them; they critique you.
If you're professional-minded about your writing, you're going to give an entirely different perspective on someone's work than a person who's a pleasure reader. Both perspectives are useful.
I have never been able to keep critique partners. I throw my everything into doing them. Maybe I'm too intense.
Maybe. I find the folks who seem to work best together are those who mesh well together, personality wise. For me, I don't have a standing critique partner/group, but I have three or five people I could swap critiques with when it's time. I think finding the perfect group/partner can be hard, just like finding great romantic partners or really great friends. It takes time and some effort.


text:
Justice for Palestine: A Challenge, A Victory, and the Work Ahead
Yesterday New York Governor Cuomo signed an executive order that would punish groups involved in the boycott, divestment, and sanctions (BDS) movement supporting Palestinian rights. He made this shameful, anti-democratic move just before joining the Celebrate Israel parade on New York City's Fifth Avenue. Please join us in denouncing this assault on the First Amendment and our right to boycott.
Sign the petition demanding Governor Cuomo immediately rescind executive order 157
If you are a New York State resident please also call Governor Cuomo's office 1-518-474-8390 and tell him that you want him to rescind executive order 157. Sample script below:
I am calling to ask Governor Cuomo to immediately repeal Executive Order 157. It is unconstitutional to punish those who use boycott and divestment as a means to bring about political change. Boycotts are a constitutionally protected and time-honored nonviolent tactic to achieve social justice.
When we started our boycott campaign against settlement profiteer Ahava Dead Sea Laboratories in 2009, Israeli officials dismissed the efforts of the BDS Movement as inconsequential. Seven years later we can see that our principled work is so effective that the Governor of New York has signed this anti-democratic and unconstitutional Executive Order. He is trying to stifle speech and to punish those using the time-honored non-violent tactic of boycott to hold Israel to account for its violations of human rights and international law. Governor Cuomo may think he can employ this bullying tactic to stop people and groups from using boycott and divestment to end Israel's occupation and its apartheid policies, but he has misread the law and underestimated our commitment to justice and equality.
Sign the petition telling Governor Cuomo to rescind order 157
Our original plan for this message was to celebrate the release of Palestinian feminist parliamentarian Khalida Jarrar after fourteen months in an Israeli prison, and we want to pause to savor this victory. International outcry, including letters from CODEPINK members, about Jarrar's being held in administrative detention for months without charge, resulted in a trial in Israeli military court where Jarrar accepted a plea deal as the means to earliest possible release. (With the hope of actual justice non-existent, the vast majority of Palestinians facing Israeli occupation courts take plea deals.) What were Jarrar's crimes? She admitted to being a member of the Palestinian Front for the Liberation of Palestine (PFLP), a leftist political party that she was elected to represent, and to other offenses such as making a political speech, attending a book fair, and advocating on behalf of Palestinian prisoners. Upon her release last Friday, Jarrar said, "I know that the occupation wants to put all of Palestinian society into prison."
As Israel's occupation enters its fiftieth year, we pledge to redouble our efforts for justice in Palestine. We know how crucial the BDS movement is, and that is why we are pushing to get companies like RE/MAX and Airbnb to stop selling, renting, and advertising homes in illegal settlements in the West Bank and East Jerusalem. Last Friday, CODEPINK and the rest of the Stolen Homes coalition organized actions in 18 cities across the world to pressure Airbnb to stop violating international law by listing homes in the settlements. Read about the actions here.
From our successes with Ahava and SodaStream to our continued work on the RE/MAX and Airbnb campaigns, we know that together we can have an impact. This is why it is so important that we protect our right to engage in boycott as a form or nonviolent action. Sign the petition, and if you are a New York State resident call Governor Cuomo.
Working for freedom, justice, and equality,
Nancy K., Alli, Ariel, and the entire CODEPINK team


text:
Best News Network
NewsAzi - Best News Network
Why index funds are best bets if you want to invest internationally
By Kemal Pasha On Nov 21, 2021
In the prevailing scenario, one can say with confidence that India is one of the fastest growing economies. However, India's GDP represents only 3% of World GDP and roughly the same percentage of global stock market capitalization.
Globally, a lot of innovation is taking place through disruption of various traditional industries. Such innovation and disruption creates investment opportunities across geographies. Such opportunities are very limited in India in the listed space. Hence, to participate meaningfully in these opportunities, investors need to take exposure to several themes or emerging mega-trends outside India.
Reasons for Investing Internationally:
Winners keep changing:
Every country has its own strengths and weaknesses, and reactions to global news flows can be consequently different.
As can be seen in the chart above, no single country/region has been the best performer consecutively for a prolonged time period. In the last 10 years, the US has been the top performer in 6 years but only twice consecutively. International investments reduce single country/region-specific risk at portfolio level.
Correlation benefits:
Correlation shows the strength of relationship between two variables, in this case the stock market performance of one country vis-à-vis the other.
As against the general notion that Indian & US equity markets have high positive correlation, there have been periods of divergent movements, e.g. 2011, 2013, 2015-16, as given in the previous chart. Also, in the last 10 years, the correlation between India & US has been only 0.29.
Hedge against INR depreciation:
International exposure also carries with it exposure to different currencies. Whenever there is any depreciation in INR, the global portfolio could earn higher INR returns, which is depicted in the chart of the US S&P 500 index below.
Ability to participate in global themes:
Though India is one of the fastest growing economies in the world, it still lags behind the advanced economies with respect to innovation, since the major technology centric firms operate globally. E.g. names like Facebook, Amazon, Apple, Netflix, Google, etc. are not listed on Indian bourses.
Popular themes that are emanating across the globe are robotics, artificial intelligence, electric vehicles, industrial automations, e-commerce and cloud computing. Since these themes are expected to shape our future, it makes sense for domestic investors to participate in such global themes.
To summarize, international investing helps in diversifying single country risk, provides correlation benefits, provides currency diversification, and the opportunity to invest in some of the leading global brands & themes.
Modes of International Investing:
International Investing is possible either through domestic Mutual Funds, either through Index funds which mirror global indices, or actively managed funds, or by investing directly in foreign currency through the Liberalized Remittance Scheme (LRS).
Investing via domestic funds is preferable since the money remains INR denominated and is subject to domestic taxation. Funds which invest in international equities are taxed as fixed income mutual funds in India, i.e. subject to LTCG @20% with indexation if held for > 3 years, or subject to STCG @MMR if held for
Benefits of Index Funds in International Investing
For domestic investors, passive funds or Index funds are the best way to invest in international markets.
Index funds aim to replicate the performance of an index. The case for Index funds is pretty much proven in the US, where almost 50% of the assets managed through that mutual fund industry is covered through passively managed funds. One of the key reasons for this is the information efficiency which is prevalent in US & other Developed markets. This is makes it difficult for a global active manager to consistently outperform the benchmark indices e.g. the S&P500, Nasdaq100, MSCI World Index, etc.
Global Index funds are more economical since they do not have the management & operational expenses associated with actively managed funds.
Furthermore, global index funds provide access to global leaders across industries and access to emerging themes or mega-trends.
Amongst the opportunities available for domestic investors:
The Motilal Oswal S&P 500 Index Fund replicates the S&P500 index which includes the top 500 companies in the United States covering all 11 Global Industry Classification Standard (GICS) sectors making it a broad based diversified index. This index includes top 100 non-financial companies and the companies that form the top 10 holdings include Microsoft, Apple, Amazon, Tesla, Alphabet, Facebook, Nvidia & NetFlix.
The Motilal Oswal NASDAQ 100 ETF & FoF offer an opportunity to invest in global technology companies that are part of the NASDAQ 100 Index, which covers the top 100 non-financial companies.
The HDFC Developed World Indexes Fund of Funds tracks the performance of the MSCI World Index which covers 23 developed markets, covers approximately 85% of the free float-adjusted market capitalization in each country, through 1500+ constituents.
(The author, Nitin Shanbhag, is Senior Group Vice President – Investment Products, Motilal Oswal Private Wealth. The views are his own.)
For all the latest Business News Click Here
Denial of responsibility! NewsAzi is an automatic aggregator around the global media. All the content are available free on Internet. We have just arranged it in one platform for educational purpose only. In each content, the hyperlink to the primary source is specified. All trademarks belong to their rightful owners, all materials to their authors. If you are the owner of the content and do not want us to publish your materials on our website, please contact us by email – [email protected]. The content will be deleted within 24 hours.
AziNewsBest NewsBetsBusinessbusiness newsFundsGlobal Index fundsHDFC Developed World Indexes Fund of Funds
Kemal Pasha
Islanders Are Finally Home Sweet Home
Enjin Coin Price Prediction: Where Will the Metaverse Take the ENJ Crypto?
Latest Headline's
Vagina: What's typical, what's not
A Project to Count Climate Crisis Deaths Has Surprising…
Israel Police using NSO's Pegasus to spy on citizens…
Watch: The Great Indian Murder trailer blends mystery,…
Next Generation Sequencing (NGS) Market Registered at CAGR…
myEntropy calculates entropy level of online services file…
Employee Training And Development Categories You Should…
Actor Sanjay Mishra, whose movie Turtle is based on water…
The Secret to Easy Homemade Pizza
© 2022 - News Azi. All Rights Reserved.
Website Design: News Azi
2-Step Verification value
Insert the code if you enabled 2-Step verification for your profile
Send me a code via email
Send me a code via sms
The code has been already sent.


The Bathtub is made to your exterior and interior requirements. The exterior dimensions are important because they are the maximum dimensions of your tub. You should take into account doors opening and closing, nearby cabinets and walk space when you calculate the maximum exterior dimensions. The interior dimensions are the inside of the tub and are customizable to fit two people, a taller person and a soaking angle. The drain location is customizable to fit the area where the faucet fixtures will be placed.


EcoNest identifies homes designed and built respectfully, in appreciation of the harmony and beauty of nature and in a way that uses nature's resources so as to consume less energy, create less waste, nurture our health, and enrich our senses. A bird builds its nest using the materials at hand to create a perfect shelter for its bioregion. It doesn't fly to the next state for twigs nor does it build a home that is bigger than it needs. Instinctively it creates an environment that is nurturing, nontoxic, and free of synthetic chemicals. Like the bird, humans desire shelter that is cozy and nurturing, that satisfies the soul, mind, and body. This is the econest. A true econest also involves a timber frame and walls created with a mix of straw and clay. This unique method of green building avoids the typical problems involved with conventional building, such as industrial waste, excessive chemical usage, and inefficient synthetic insulation. Baker-Laporte and Laporte explain how to avoid the pitfalls of modern homes and how to avoid acute chemical sensitivity, and they focus on how to simply live in harmony with nature. EcoNest profiles ten different homes located around the country, including a floor plan with interior and exterior photos for each. Chapters include Building with Light Straw/Clay, The Elements of Econest Design and Construction, and Eight Common Questions About Econests. Paula Baker-Laporte, A.I.A., graduated from the University of Toronto, School of Architecture, in 1978. She has been active in a wide-ranging residential architectural practice based in Santa Fe, New Mexico, since 1986. Her previous books include Prescriptions for a Healthy House, and she is a contributing author to A People's Ecology: Explorations in Sustainable Living edited by Gregory Cajete, The Healthy House, and The Art of Natural Building. Robert Laporte is the founder of the Econest Building Company and the Natural House Building Centre, and is author of Mooseprints: A Guide to Holistic Building. He served on the original board of directors for the Timber Framers Guild from 1985 to 1987. He is a professional builder specializing in earth, straw, and timber-frame structures. Robert and Paula's econest projects have been featured on HGTV and in several magazines, including Natural Home, Fine Homebuilding, Residential Architect, Organic Style Magazine, Yoga Journal, and The Inspired House.
Download Econest : Creating Sustainable Sanctuaries of Clay, Straw, and Timber (9781586856915).pdf, available at smadeo.com for free.


text:
Корбониколаевка () — село в Новгородковском районе Кировоградской области Украины.

Население по переписи 2001 года составляло 112 человек. Почтовый индекс — 28220. Телефонный код — 5241. Код КОАТУУ — 3523484704.

Местный совет
28220, Кировоградская обл., Новгородковский р-н, с. Петрокорбовка, ул. Молодёжная

Ссылки
 Корбониколаевка на сайте Верховной рады Украины
 Административно-территориальное устройство Кировоградской области

Населённые пункты Новгородковского района


These are Delbards are bare root potted and unlike the bagged roses they have a much longer shelf life. They can be watered unlike bagged stock.
Rankins Roses are Australian agents for the French Delbard roses and we are privileged to be able to offer these flamboyant and revered roses. Their unique collections offer a broad and comprehensive range, with the 'Grand Parfumes', brilliant 'Painters','Bordure' and 'Climbing Collection', 'Roses of Liberty' and the effervescent 'Couture Collection'. They are available in a wide range of colours and perfumes. All will inspire and bring great joy.


Aug 25, 20170183;32;SF Flotation Machine is the representative equipment in the flotation production, mainly used in the flotation of nonferrous metals and rare metals, such as copper, gold, silver, zinc, lead, nickel, etc; and nonmetal materials, such as kaolinite, phosphorus, sulfur, etc.
compound cpopper flotation ll . vertical compound cone crusher christoartfair.nl. mineral processing flotation cellpoud . The mobile type for Alluvial gold mining trommel screen .. ball mill, classifier,ic separator, mixing tank, flotation machine, rotary dryer. Chat Online Henan Yuhong Heavy Machinery Mining Machinery .
cone flotation ll from. CS series hydraulic cone crusher hp series , chrome ore machineriess , crush stone. Manganese ore flotation cells for metal mineral and non metallic mineral plant. . High efficiency lead / zinc iron ore flotation machine china gold mining . china leading manufacturer gold mining mobile.
Cone Crusher Manufacturers For Sale In Italy. mobile stone crusher for sale in italy pneusmpg.be A wide variety of used cone crusher for sale options are available to you, of design, manufacture, sale, and aftersale service in crushing industries. . pe series used jaw cpopper flotation ll plant for sale.
cpopper flotation ll drawing. Lock Cylinder Assembly Machine. 42 products Related Searches for lock cylinder assembly machine floating fish feed . 2 The Seller offer life long maintenance service, only labor cost will be straightening and polishing machines, hydromatic draw looms, copper and.
Fine crushing and screening mobile station. Applications The processing of crushed material, collected gravel, and refined sand, etc.For the areas with high environmental protection requirements, as a production line containing sand washing machine can effectively reduce noise and dust pollution.
371250 products China Bracket, China Bracket Suppliers and Manufacturers Directory ,floating shelf brackets ,metal brackets from China china factory mobile cpopper flotation ll machine in le . 2. More than 30 Machines ensure your hangers delivery time.
cpopper cone flotation ll cone crushing equipment for granite. Sep 16, 2018 cpopper cone flotation ll cone crushing equipment for granite 4 1/4 cone crusher and 5 1/2 cone crusher in crushing plant. copper Copper Ore Processing Plantmining ball mill machines used in glod mines offers .
buy gold mining cone crusher 2242 XINHAI. Ball mill,buy gold mining cone crusher 2242,chromite ore mining and quarry sibanye gold mine contact details Grinding Mill China Gold mining equipment, processing plant,crushing machine large capacity gold mineral crushing Mining Cone Crusher, Wholesale Various High Quality Gold Mining Cone .
ChemIndustrial cone bottom mix tanks liquefy, dissolve, suspend and mix granular materials. Heavy duty, high torque agitators are driven by electric motor/gear drive combinations. Live Chat. mini cpopper mobile flotation ll movable flotation ll p.
When doing a dock flotation calculation, determine your requirements and translate that to the amount of cubic feet of flotation your project will demand. We have provided a quick reference chart that compliments the Dock Flotation Calculation. This chart is an estimation of volumes and the amount of weight the flotation unit should support.
gold machine mobile mini jaw cpopper flotation ll for sale. Rod Mill is a widely used construction equipment, sand gravel, one of the rod mill Sand type is mainly used for feldspar, quartz, minerals and other raw materials, Hammer Plate,Cone Crusher Liner Plate,Blow Bars , PE series,PEX series,PV series,etc.


Right in front of the beating heart of Dutch politics lies a new culinary hotspot, Palmette. Their restaurant is internationally oriented with firm roots in french cuisine. Residing in the old building of the legendary 'Bistro De La Place'. I was responsible to come up with their identity and to design a wallpaper covering the entire restaurant.


Homoeopathy Neals Yard Remedies can be very useful guide, and Homoeopathy Neals Yard Remedies play an important role in your products. The problem is that once you have gotten your nifty new product, the Homoeopathy Neals Yard Remedies gets a brief glance, maybe a once over, but it often tends to get discarded or lost with the original packaging.
2019 Planner Nebula Galaxy Weekly Planner 2019 With Weekly Spreads To-do Lists Inspirational Quotes Funny Holidays And More. Large Pretty NASA Space Galaxy Print Daily Organizer.


As Vice President of Senior and Student Housing, Michael DiGiacomo oversees the management and operations for United Group's student housing portfolio consisting of 1,554 beds and two senior housing properties containing 261 apartments. Mike manages a team of over 25 staff members and also has responsibilities for two local commercial properties.
Prior to his current position, Mike was the Property Manager for Schuyler Commons, part of United Group's senior housing portfolio. Before joining United Group, he was a Property Manager for Gross and Cohen LLC, where he directed operations for off-campus student housing properties in New York and Kentucky.
Mike holds an Associate's degree from Fulton Montgomery Community College and a Bachelor of Science in Liberal Studies from Excelsior College.


Liu Xiaobo, the famous Chinese scholar, dissident and Nobel laureate, is dead. He frequently said China was in need of a moral giant. Little did he realise that for numerous Chinese, he was that very giant himself.


text:
Q: Prove that there are exists two denumerable sets $A and B$ such that ${ab| a \in A, b \in B}$ is enumerable. Prove that there are exists two denumerable sets $A$ and $B$: {$a*b| a \in A, b \in B$}
Set is denumerable if we have an algorithm which can output all elements from this set in finite time.
Set is enumerable if we can't find this algorythm

redpajama_set_name: RedPajamaStackExchange


text:
Burston – wieś w Anglii, w hrabstwie Norfolk, w dystrykcie South Norfolk. Leży 28 km na południe od miasta Norwich i 133 km na północny wschód od Londynu. Miejscowość liczy 538 mieszkańców.

Przypisy

Wsie w hrabstwie Norfolk


Representative Office Formally Opens October 30th.
Wavecom (Nasdaq: WVCM; Euronext Nouveau Marche: 7306), the world's number one provider of integrated wireless modules and a major technology partner for China's fast-growing wireless manufacturing sector, today announced the official opening of its first representative office in China, in Beijing's fast-emerging "Silicon Valley" district of Zhongguancun.
Wavecom has been working with Mainland Chinese customers since 1999. China is the company's largest single market in its largest region. Sales in Asia-Pacific accounted for 87.2 per cent of Wavecom's third quarter 2002 revenues of €171 million.
"Opening a representative office is the latest milestone in our continuing commitment to markets in Asia, and China in particular," said Jean-Charles Andreani, managing director of Wavecom Asia-Pacific Limited.
Wavecom's Beijing Representative Office is located in Zhongguancun, a hub of technology operations to the northwest of the capital that is increasingly regarded as China's "Silicon Valley". The new office gives Wavecom executives closer and speedier access to local customers and China's wireless communications market, particularly in northern and central parts of the country.
The office is headed by Chief Representative Officer Haibo Zhu. "We already have half a dozen local staff and we expect this figure to reach 30 within six months to a year," he said.
Wavecom's WISMOs are found in many of the leading local brand mobile phones sold in China. Prominent handset providers, such as TCL and Soutec, use WISMOs to speed up product development cycles in order to get new, feature-rich products to market more efficiently.
China's mobile market is expected to continue to grow in spite of the global economic downturn. According to the technology consulting company Made for China Insight, handset production is expected to reach 232.8 million a year by 2005, up from 80 million in 2001. By 2005, China could be the source of one third of the world's mobile phones.
Wavecom recently appointed Elcoteq to assemble and test WISMO products at its factory in the Beijing Economic Technological Development Area. The move strengthens Wavecom's manufacturing capabilities in Asia and adds further support to its fast-growing customer base in the region as well as in other global markets. Such a partnership also further demonstrates Wavecom's commitment to the mainland China market.


This is an extraordinary stone. We see here a cross in a field, with to the right a small church that also seems to have a cross on top of it. To the left of the cross in the field, there is a figure standing, surveying the cross, and to his left is a large rock, but what is astounding is the presence of two figures that dominate this landscape. First, in the middle of the stone, seemingly perched on a branch, there is a large bird, and above the bird is what looks like the figure of an angel. I cannot say whether they bode well or ill, but it seems to me that this is a scene of death, of the silence before resurrection, when we are not sure of the whereabouts of the soul, which has left the body, but we trust in this shining symbol, two pieces of wood attached crosswise and sturdily.
Meanwhile, in language, when we become aware of the presence of God, a seminal moment because it changes our perspective, we no longer rely solely on the I, on the self, but we count down from this letter to O, the letter of repentance, the letter of exclamation, of recognition, of the truth finally dawning. O can represent God, it is an eternal figure. This shift in our perspective is revealed in words such as "obey" and "joy". They become exclamations: "O be I!" and "I O I!" (remember that j and y are the semi-consonants that correspond to the vowel i). Whereas before we were in debt (a word closely related to death by a shift in the alphabet, addition of breath), we find that "owe" now spells "O we!" and the debtor's note has become a note of recognition of the other: "I O you!" The debt is lifted by the figure of Christ on the Cross. This is not some abstract religion, this is a person, united with the Father and the Holy Spirit in the Trinity, whom we recognize when we see him: O (an open I).


Get our your tie-die t-shirt and head on down the pond! Jerry Garcie and Grateful dead fans don't miss this one of a kind memorabilia. Will look great next to the lava lamp.
All Celebriducks have weighted bottoms and float up right and come with collectors quality box packaging.


text:
Lyon and Strasbourg fall for Padam Mobility's Demand-Responsive Transport
Padam Mobility News 267 views by Padam Mobility
On November 13th, the cities of Lyon (Sytral) and Strasbourg (CTS) inaugurated with Padam Mobility their Demand-Responsive Transport services. Elected officials and companies already favor the service. The third and the eighth biggest cities in France are thus complementing and modernizing their public transportation networks. With an offer more finely tuned to the demand, tens of thousands of users are preparing to see their mobility improved.
Improving employees' commuting
In Lyon, it is in the Mi-Plaine area, which includes 1375 companies and 20,000 employees among the municipalities of Chassieu, Genas and Saint-Priest, that the Sytral launched TCL on demand. This service is integrated into the existing network, with identical pricing. Padam Mobility is in charge of the Demand-Responsive Transport software (interfaces, management and reservation systems and algorithms) that makes this service effective.
Dynamic Demand-Responsive Transport is a concrete solution that addresses the mobility challenges in large areas. It allows us to refine the mesh of the network and better staple territories to ensure overall consistency. Our top priority is the accessibility of our network, in the broad sense of the word" says Fouziya Bouzerda, president of the Sytral.
Six nine-seater vehicles, powered by natural gas (NGV) from 2020 operated by transit operators Keolis Lyon and Berthelet. They now crisscross the Mi-Plaine area to complement the transportation offer. The companies that are located there are enthusiastic. Getting employees to this extended area was difficult. With the job market being so tense, it's a breath of fresh air.
Opening up suburban and rural territories
In Strasbourg, Flex'hop Z1 covers 13 rural and peri-urban municipalities located in the West and South-West perimeter of the Eurometropolis. Padam Mobility's Dynamic Demand-Responsive Transport service runs daily from 6:00 am to midnight. It is based on a mandatory reservation made from 15 days to 30 minutes before departure. With 80 stops, it offers more than 4000 itinerary possibilities to meet all travel needs.
This new innovative solution will offer a flexible, fast, ecological service tailored to the mobility needs of residents and employees in the less densely populated areas of the agglomeration. It will thus enable periphery-to-periphery connections which are currently difficult with the network of conventional bus lines" details Robert Hermann, President of the Eurometropolis of Strasbourg.
It is a high-quality service, at the same price as a normal ticket. The service works with electric vehicles that are accessible to people with reduced mobility. It is universal both in terms of time schedules and accessibility. The challenge is obviously to be competitive-attractive compared to the individual cars" adds Alain Fontanel, president of the CTS.
On the CTS Facebook page, a user is already enthusiastic:
It's very good for peripheral municipalities. Especially for people with no means of transport! Well done for the initiative!
The eurometropolis has already announced its desire to extend the service, and eventually validate the DRT on all municipalities in the metropolis.
Meet the users of the Demand-Responsive Transport
Tags : 2019
Padam Mobility deploys its Demand-Responsive Transport solutions in Paris region
Demand-Responsive Transport : Why aren't they all equal? The reservation channels
Company values that beat the bullshit detector [A 4-step method]
One step closer to MaaS in Lille
Padam Mobility offers technological solutions to ensure social distancing in transports


Old timers have noticed that the contractor's building on Hanover Avenue transformed into a community center for Overlook Park. But only the oldest, or train buffs, realized that the building was the freight depot and office for the Lehigh & New England Railroad. Lehigh & New England was formed in 1895, primarily as a coal carrier. The line ran from Allentown to Maybrook, New York.
In 1904 it was acquired by the Lehigh Coal and Navigation Company. The line ceased operation in 1961. Among it's infrastructure were impressive bridges across both the Lehigh and Delaware Rivers, both of which were dismantled. Ironic that a remnant of our industrial era is being utilized by the successor of a public housing project.
And why was the traffic light installed there? And why is it still there?
i recall spinosa construction operated out of there for years. the warehouse section behind the shown office portion was demolished and a new addition built for overlook community center. the front has been retained and restored. sorry, don't know about the stoplight.
I really like your site Michael, this is intersting info. I used to follow another local blog site regularly I just can't go there any more. Feel kind of icky when I do. Thanks for being what appears to be a credible source of info.
If I'm thinking of the right place, the stoplight was always there. I believe it remains since there is a new road planned that will eventually connect Union Boulevard to Hanover Avenue at that location. I believe the new road will be called New England Avenue.
Also, wasn't the building you are writing about supposed to become an East Side police station?
Someone inform the blinding genius of the from-out-of-town, resume-building City Bureaucrats who are so much smarter than everyone else that this is the Mid-Atlantic region of the eastern United States.
anon 12:05, i believe there was to be an east side sub station there. not sure about the full station, those plans were under kuhn, whose eyes were bigger than our budget.
anon 12:14, i'm not familiar with the new england ave., but perhaps it was in connection with lehigh & new england railroad.
New england ave... will connect to the new lehigh river bridge? I believe the stoplight was installed due to a pedestrian being struck and killed around that bend, an attempt to slow traffic.
But, City Bureaucrats have (willfully, intentionally?) created their own cloud of controversy and accompanying cynicism.
And that bill has come due with some creditors.
...and wondering where the standard commentary trashing the history of evil, greedy, and, most importantly, rotten and mean-spirited United States that normally greet posts of this nature at this blog are at today?
anon aka 12;14, 1:39 and 1:42. i was somewhat inciting with my last, unnecessary sentence. my original purpose was our railroad history. no less than six railroads serviced our industrial might, which was legendary.
The traffic light remains from the intersection with Dauphin St. It was removed just a few years ago as part of the redevelopment of the area.
New England Avenue will connect with Hanover Ave immediately east of the old station, aligning with Ellsworth St. This new road will run under an existing overpass on Union Blvd and connect with Dauphin St near the location of the American Parkway bridge.
Construction on New England Ave has begun but has stalled as the result of contractor who is alleging soil contamination in an attempt to extort additional money out of the city.
The freight depot was another failed attempt by the City to be real estate developers. They bought the building to make an east side substation, then decided that they could attach the property to their application to the HOPE grant to rebuild the projects and make it more attractive.
The City has a track record of "buying" vacant industrial sites and not doing enough due diligence on the site and getting stuck holding the bag when the work needs to start.
Case in point, the Lehigh riverfront project. The City has, and still is, trying anything they can to acquire and real estate they can down along Front St for the Last 30 years now. They got the old A&B site and were so caught up in the moment they forgot to see where the chemicals from the slaughterhouse were stored (OOPS). Once they started demolition they found numerous tanks underground that had to be removed.
Another case in point on the riverfront is the newly acquired Neuweiler Brewery. The City took this property and is now spending upwards of 35 to 40 MILLION dollars in environmental cleanup (I know because that is the estimate I was given at wholesale, not the special "government only" rate that contractor's have). Then to top it off, they bought the adjacent property to the rear for $800,000 (no that is not a typo). The property has plenty of environmental issues of its own with the former owner's dumping on the site (well burying is more the word). The City would have known more about this, but they were too busy carting the owner of the brewery to jail when he was trying to give them the evidence they needed.
At one time, in my younger "I know better than you" days, Mike and I would argue the City's role in real estate development and you know what, he was right, I was just too young to realize it.
If anyone needs any further proof, just take a look at the various real estate projects the City takes part in. Can't find any? I'll be more than happy to point them out.
Also, in regards to the soil contamination, the City as a public entity must check out any environmental issues and get them squared away before they do anything. I am not 100% sure but I believe that they are legally required to under the law to do so.
Not saying the contractor isn't milking it, I am not here to debate that, I don't know enough about this specific situation.
This is why it is best for the City to not even take title to brownfield sites and just to help put the property owners in touch with buyers. Now of course if it is absolutely necessary, they should just to get something done, if there is a viable, better for the commnity, reuse once complete or if there is an immediate threat to public safety. Usually in the case of the City, neither apply, it is merely the "acquire to control" mentality.


Come and visit our store for other interesting items.
We are the largest independently owned Pawnbroker/ Secondhand Retailer in the South West. We trade, buy, sell and loan, you can also put an item on layby and we're happy to post many items.
Remember to click on See all ads for this seller to see a great range of products we have in store or type Advanced Cash and Gold in the search field.