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Quorum sensing of microalgae associated marine Ponticoccus sp. PD-2 and its algicidal function regulation
Wendan Chi1,
Li Zheng1, 2Email author,
Changfei He1,
Bin Han1,
Minggang Zheng1,
Wei Gao1,
Chengjun Sun1, 2,
Gefei Zhou3 and
Xiangxing Gao4
Quorum sensing (QS) systems play important roles in regulating many physiological functions of microorganisms, such as biofilm formation, bioluminescence, and antibiotic production. One marine algicidal bacterium, Ponticoccus sp. PD-2, was isolated from the microalga Prorocentrum donghaiense, and its N-acyl-homoserine lactone (AHL)-mediated QS system was verified. In this study, we analyzed the AHLs profile of strain PD-2. Two AHLs, 3-oxo-C8-HSL and 3-oxo-C10-HSL, were detected using a biosensor overlay assay and GC–MS methods. Two complete AHL-QS systems (designated zlaI/R and zlbI/R) were identified in the genome of strain PD-2. When expressed in Escherichia coli, both zlaI and zlbI genes could each produce 3-oxo-C8-HSL and 3-oxo-C10-HSL. Algicidal activity was investigated by evaluating the inhibitory rate (IR) of microalgae growth by measuring the fluorescence of viable cells. We found that the metabolites of strain PD-2 had algicidal activity against its host P. donghaiense (IR 84.81%) and two other red tide microalgae, Phaeocystis globosa (IR 78.91%) and Alexandrium tamarense (IR 67.14%). β-cyclodextrin which binds to AHLs and inhibits the QS system reduced the algicidal activity more than 50%. This indicates that inhibiting the QS system may affect the algicidal metabolites production of strain PD-2. Our study indicated that a QS-regulated algicidal system may play a potential role in the process of red tides disintegration. QS might be a potential way to control red tides.
AHLs
Algicidal activity
β-Cyclodextrin
Microalgae-associated bacteria
Quorum sensing
Quorum sensing (QS) is a cell density-dependent system for information transfer among bacteria. It enables the bacterial cells to sense changes in cell density through the concentration of signal molecules called autoinducers (AI) released by bacteria themselves (Guo et al. 2011). When the bacteria grow to a high cell density, the concentration of AI could reach a threshold and bind specifically to a receptor protein. Then, the complex will activate the transcription of target genes and regulate specific functions including biofilm formation, bioluminescence, the secretion of virulence factors, and sporulation (Davies et al. 1998; Greenberg et al. 1979; Nealson et al. 1970). In addition to directly affecting gene regulation, increasing evidences show that cross-kingdom QS signaling between associated bacteria and their hosts also confers other ecological functions indirectly, such as growth-retarding effect of the rotifer (Gambello and Iglewski 1991), the settlement of seaweed zoospores (Joint et al. 2007), and carospore liberation of red macroalgae (Singh et al. 2015). Therefore, QS systems and their regulation functions in bacteria have become hot topics in the fields of marine environment, aquaculture, medicine, and bioengineering.
To date, the most studied intercellular AI molecules are N-acyl-homoserine lactones (AHLs), which can be detected by biosensors, gas chromatography–mass spectrometer (GC–MS), and high-performance liquid chromatography-mass spectrometer (HPLC-MS) (Li et al. 2015). These AHLs are generated by an autoinducer synthase, the product of a luxI type gene, and the receptor is a cognate luxR type protein. Though there is an increasing number of AHL synthetase genes with some conserved regions in the genomic databases,the overall similarity between luxI homologs from different genera is rather low (Fuqua et al. 1996). Therefore, it is necessary to determine the profiles of AHLs produced by different luxI homologs to better understand the QS system.
Red tides are natural phenomena that are caused by the explosive proliferation of microalgae (Guzmán et al. 2016). Most red tides result in serious environmental problems that are harmful or toxic to humans, fish, shellfish, marine mammals, and birds (Hu et al. 2016). The algicidal bacteria that are associated with red tide algae play an important role in controlling their host's biomass. In the initial and developmental stages of red tides, algicidal bacteria show no algicidal activity. In the alage blooming stage, the growth of algicidal bacteria was promoted by the nutrients that are organic materials released by the algae. Subsequently, the bacteria begin to produce algicidal compounds that kill their host cells. It is interesting to find that some algicidal bacteria such as Cytophaga strains J18/M01 (Mirsutani et al. 1992) and A5Y (Imai et al. 1993), Flavobacterium strain 5N-3 (Imai et al. 1992), and Flavobacteriaceae strain S03 (Roth et al. 2007) must reach a cell density of at least 106 cells mL−1 before they show any algicidal activity. Li et al. (2014) found that Shewanella sp. Lzh-2 must reach a bacterial cell density threshold of at least 108 cells mL−1 before any algicidal compound can be detected. There are also reports that some microalgae could inhibit bacterial quorum sensing activity (Castang et al. 2004). Because algicidal activity is density-dependent, we hypothesize that the algicidal activity of the microalgae associated bacteria may be regulated by QS system and the interaction between microalgae and their associated algicidal bacteria could be mutually restrictive. This probably is an important factor for maintaining the dynamic balance of phycosphere.
It has been proven that QS systems in some bacteria can regulate the production of secondary metabolites, such as antibiotics, including algicidal compounds. One marine bacterium, MS-02-063, belonging to the γ-proteobacteria, produces a red, algicidal pigment (Nakashima et al. 2006). It is interesting to note that pigment production was also inhibited by adding β-cyclodextrin (β-CD), which forms a complex with AHLs in media, as a QS-quenching substance (Morohoshi et al. 2013). Although it was suggested that the algicidal pigment production was controlled by AHLs-mediated QS, this study neither confirmed the presence of a QS system at the genetic level nor did it find any AHLs molecules. In a subsequent research, Guo et al. (2016) found a bacterium Aeromonas sp.GLY-2107 which could produce two algicidal compounds against Microcystis aeruginosa. They proved that the production of the two algicidal compounds was controlled by QS system. However, this bacterium was screened from freshwater and was not associated with microalgae M. aeruginosa. To date, there has been no report on the QS regulated algicidal activity investigated through the interaction between microalgae and their associated bacteria.
In this work, The QS systems of Ponticoccus sp. PD-2, which is a marine microalgae-associated bacterium with algicidal activity, were confirmed by detecting AHL molecules and annotating luxI type autoinducer synthase and luxR type receptor genes. We analyzed the AHL synthetase functions by introducing LuxI homologes into Escherichia coli. The algicidal activity of strain PD-2 was evaluated and compared after adding β-CD into the culture medium. The purpose of this study was to understand AHL-based QS systems in Ponticoccus sp. PD-2, and to prove the hypothesis that the microalgae associated bacterium may kill the host through QS system. We expect to find a novel way to control red tides by biological treatment via enhancing QS activities.
Strains and growth conditions
Ponticoccus sp. PD-2 used in this study was obtained from China General Microbiological Culture Collection Center (CGMCC 1.16027). It was isolated from Prorocentrum donghaiense. Strain PD-2 was grown in marine broth 2216E (Xu et al. 2012) at 27 °C with shaking at 150 rpm, unless otherwise indicated. E. coli BL21 (DE3) was used to propagate recombinant plasmids and to overexpress zlaI and zlbI proteins. For E. coli strains, the bacteria were grown in Luria–Bertani (LB) medium (Britstein et al. 2016) at 37 °C, with shaking at 150 rpm. Transformed cells were grown in LB broth supplemented with 100 μg mL−1 ampicillin (Sigma-Aldrich, St. Louis, MO, USA). The microalgae strains included red tide species P. donghaiense CCMM1007, Phaeocystis globosa CCMM5010, and Alexandrium tamarense CCMM1002 (CCMM: Culture Collection of Marine Microalgae). All the indicator microalgae strains were courtesy of the Institute of Oceanography, Chinese Academy of Science and deposited in CMBGCAS. f/2 medium (Guillard and Ryther 1962) was prepared with seawater collected from the Qingdao shore, and it was filtered through a 0.45-μm pore membrane. The medium was autoclaved at 121 °C for 20 min. The algae were cultured in f/2 medium at 25 °C under cool-white fluorescent lamps (100 μmol photons m−2 s−1) with a 12:12 light/dark cycle, with shaking three times per day.
The biosensor Agrobacterium tumefaciens KYC55 (pJZ372) (pJZ384, pJZ410), which was provided by Professor Hill at the Institute of Marine and Environmental Technology, University of Maryland, USA. The reporter strain KYC55 (Karina et al. 2013) was prepared by growing it in 1× ATGN minimal medium (Tempé et al. 1977) with 100 μg mL−1 spectinomycin dihydrochloride pentahydrate, 4.5 μg mL−1 tetracycline, and 100 μg mL−1 gentamicin at 30 °C, with shaking at 180 rpm. A subculture was made in the same medium with an initial 1:500 dilution of the cells. The subculture was grown under the same conditions and harvested when an optical density at 630 nm (OD630) reached 0.35. The cells were harvested by centrifugation at 15,000×g for 10 min. The cell pellet was suspended in sterile 30% glycerol to a calculated OD630 of 12. One-milliliter aliquots of the cells were stored at −80 °C.
AHL standards (C6-HSL, 3-oxo-C6-HSL, C8-HSL, 3-oxo-C8-HSL and 3-oxo-C10-HSL) were purchased from the Cayman Chemical Company (Ann Arbor, Michigan, USA). Chromatographic-grade methanol and 99.9% ethyl acetate were purchased from Sigma-Aldrich (Buchs, Switzerland). The standards were dissolved in methanol at a concentration of 1 mM and stored at −20 °C.
Extraction of metabolites of strain PD-2
Bacterial cell suspensions were centrifuged at 12,000 rpm for 5 min. One hundred and fifty milliliters of the cell-free supernatant solution was adjusted to pH 7 with 6 M HCl solution, preserved overnight at 4 °C, and then extracted according to the procedure described previously (Cataldi et al. 2004, 2007). Briefly, 150 mL of the cell-free supernatant solution was extracted twice with 150 mL of ethyl acetate each. The combined organic phases were evaporated by rotary evaporators at 37 °C. The residue was redissolved in 200 μL of ethyl acetate and stored at 4 °C.
Analysis of the AHL profile
Bioautography analysis
All AHLs produced by strain PD-2 were screened using the A. tumefaciens KYC555-based biosensor system developed by Zhu et al. (2003). The reporter overlay was prepared by cooling 90 mL of 0.6% agar to 40 °C and adding 5 mL of 20× AT salts (15 mM (NH4)2SO4, 0.6 mM MgSO4·7H2O, 0.06 mM CaCl2·2H2O, and 0.0071 mM MnSO4·H2O), 5 mL of 20× AT buffer (79 mM KH2PO4, pH 7.0), 1 mL of 50% (w/w) glucose, 200 μL of 20 mg mL−1 X-gal, and 1 mL of the prepared the KYC55 cells. Extracts were spotted onto C18 reverse-phase thin-layer chromatography (TLC) plates (Mallinckrodt Baker, Philipsburg, NJ, USA) and developed with 60% methanol as the mobile phase. The TLC plates were dried, sterilized with ultraviolet light, and placed in an autoclaved Petri dish. The AHLs reporter overlay was poured on top of the plate. The plate was incubated at 30 °C overnight to allow the developing of blue colonies that indicate the existence of AHLs.
GC–MS analysis
The GC–MS analysis was performed according to Cataldi et al. (2007). The analysis of AHLs in the extracts from bacterial cultures was performed by a GC system that was interfaced to a single-quadrupole mass spectrometer (7980A/5975C, Agilent Technologies, Santa Clara, CA, USA). Samples were injected with the split mode (5:1) into an HP-5 MS capillary column, 30 m × 250 μm internal diameter and 0.25-μm-thick film coated with 5% PhMe siloxane. High-purity helium was used as carrier gas at a flow rate of 1 mL min−1. The GC injector temperature was set at 200 °C. The oven temperature program was optimized to hold at 150 °C for 3 min and then increase by 28 °C min−1 up to 280 °C. The transfer line temperature was adjusted to 280 °C. Mass spectrometry conditions were as follows: the electron ionization source was set to 70 eV; the emission current was 500 μA; the MS quad was set to 150 °C; and the MS source was set to 230 °C. The mass spectrometer was run both in full-scan mode (m/z 15–800) and the chromatography analysis performed in SIM mode (m/z = 143).
Analysis of AHL synthases in strain PD-2
The genome sequence of strain PD-2 was sequenced and analyzed by Majorbio Co., Ltd. (Shanghai, China). The whole genome of strain PD-2 was submitted to GenBank (AWRV02000000) (Zheng et al. 2015). From the annotated sequences of Ponticoccus sp. PD-2, two clusters of luxI and luxR homologues were identified. After analyzing the candidate genes related to AHL production we found two AHL-sythase genes. The first AHL-sythase gene named zlaI is located in contig 600 with a length of 870 bp. The second AHL-synthase gene named zlbI is located in contig 616 with a length of 639 bp. The two AHL-related genes (luxR family transcriptional regulator), zlaR and zlbR, were located in the upstream position of zlaI and zlbI, respectively. Each cluster of the luxI and luxR genes is contiguous. The two QS (luxI/R homologs) genes were named zlaI/R and zlbI/R respectively. Comparative analysis of the zlaI/R and zlbI/R sequences and the closest related sequences were performed in the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov), and homologous proteins were retrieved using the protein Basic Local Alignment Search Tool (BLASTP). Sequences were aligned using DNASTAR.Lasergene.v7.1 software. A phylogenetic tree was constructed with MEGA 7.0 software (Kumar et al. 2016), using the neighbor-joining algorithm with 1000 permutations.
DNA manipulations
The complete sequences of the zlaI and zlbI genes were amplified by polymerase chain reaction (PCR). Primers with the following sequences were used: zlaI-F (5′-CGGGATCCATGAATGGAGTGGTCAAAAT-3′), zlaI-R (5′-CCCTCGAGTCAGGCCCGCTTTGCCTTCA-3′), zlbI-F (5′-CGGGATCCATGATCCGCTACATCTACGG-3′), and zlb-R (5′-CCCTCGAGTCAGGCGGCAACCATCTGCT-3′). The 5′-end primer included a BamHI restriction site, and the 3′-end primer included an XhoI restriction site. PCR was performed by incubation for 2 min at 95 °C, followed by 30 cycles of 30 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C, with a final elongation step of 10 min at 72 °C.
Function determination
The amplified zlaI and zlbI genes were cloned into pGEX-6P-1, and the resulting recombinant plasmids were labeled as pGEX-zlaI and pGEX-zlbI, respectively. These two plasmids were transformed into E. coli BL21 (DE3). The sequence of zlaI and zlbI cloned into pGEX-6P-1 plasmid were verified by PCR with primers (F:5′-GGGCTGGCAAGCCACGTTTGGTG-3′; R:5′-CCGGGAGCTGCATGTGTCAGAGG-3′). Then, the transformants were grown on LB plate containing ampicillin (100 μg mL−1) at 37 °C for 24 h with shaking at 150 rpm. The AHLs production profiles of these two transformants were analyzed as described in the bioautography analysis section.
Effects of β-CD on the growth of strain PD-2
The effects of β-CD on the growth of strain PD-2 were tested before determining the effects of β-CD on autoinducer activities. Strain PD-2 was inoculated into 2216E medium and incubated at 27 °C, with shaking at 150 rpm for 20 h. Then, the bacterial cultures were mixed with various concentrations of β-CD solutions (final concentrations of β-CD were 1, 2, and 5 mg mL−1 respectively) and continued incubation at 27 °C for another 64 h with shanking 150 rpm. Growth curves of the bacteria were plotted according to the periodically measured OD630. β-CD at a 1 mg mL−1 concentration, which had the smallest effects on growth, was selected for the subsequent algae-inhibition experiments to minimize the effects resulting from the addition of β-CD.
Assessment of algicidal activity
The algicidal effect of strain PD-2 was assessed as described by Xu et al. (2012). The target species included P. donghaiense, P. globosa, and A. tamarense. The final concentrations of P. donghaiense, P. globosa, and A. tamarense were adjusted to 1.4 × 105, 1.1 × 106 and 2.3 × 104 cells mL−1 at the exponential phase respectively. Strain PD-2 was grown for 20 and 84 h at 27 °C, with shaking at 150 rpm. Metabolites were extracted as described previously. The extracts of the culture were redissolved in dimethyl sulfoxide to a final concentration of 20 mg mL−1. Subsequently, 5 μL of the extracts was added to a 10 mL glass tube containing 5 mL of the algae cultures and incubated at 25 °C. Meanwhile, as a control, 5 μL of an extract of 22l6E medium was added to 5 mL of the algae cultures after 5 day of cocultivation. The algicidal activities were measured by the fluorescence method under the conditions of EX (excitation spectrum) = 463 nm/EM (emission spectrum) = 640 nm.
The inhibition rate (IR, %) was calculated based on the following equation:
$$\text{IR} = \frac{F_{\text{control}} - F_{\text{treatment}}}{F_{\text{control}}} \times {100}{\%}$$
where Ftreatment and Fcontrol represent the fluorescence values in the treatment and control, respectively.
Investigation of the QS-mediated regulation of the algicidal activity of strain PD-2
To investigate whether QS regulates algicidal activity, strain PD-2 was initially grown for 20 h at 27 °C. Then β-CD was added to the culture and the culture was incubated for 64 h at 27 °C, with shaking at 150 rpm. Then, the metabolites were extracted to analyze changes in AHLs production and algicidal activity. The method of detecting the production of AHLs and algicidal activity based on bioautography analysis and assessment of algicidal activity respectively.
TLC overlay assay with the biosensor KYC55 suggested that strain PD-2 produced two AHLs (Fig. 1). The retardation factor (RF) values of lower spot and upper spot were 0.12 and 0.31 respectively. Five standards (C6, OC6, C8, OC8 and OC10) were shown in Fig. 1. According to the RF values of the AHLs, two AHLs matched standards, which were synthetic N-3-oxo-octanoyl-L-homoserine lactone (3-oxo-C8-HSL) and N-(3-oxodecanoyl)-L-homoserine lactone (3-oxo-C10-HSL). GC–MS experiments were performed subsequently to confirm the AHLs of strain PD-2.
Profiles of AHLs produced by strain PD-2 based on bioautography assay. The biosensor is Agrobacterium tumefaciens KYC55. Lane A mixture of synthetic HSL standards: C6 (C6-HSL); C8 (C8-HSL). Lane B mixture of synthetic 3-oxo-HSL standards: 3-oxo-C6 (3-oxo-C6-HSL); 3-oxo-C8 (3-oxo-C8-HSL). Lane D OC10 (3-oxo-C10-HSL) standard. Lane C and E extracts of strain PD-2.There are two blue spots on the plate. This indicate two AHLs existed in strain PD-2. The upper spot matched 3-oxo-C8-HSL standard and the lower spot matched 3-oxo-C10-HSL standard
GC-MS analysis
A chromatography analysis was performed in SIM mode using an ion with m/z = 143 as a marker fragment. The chromatography of the AHL standards were shown in Fig. 2b and c. Through analyzing the MS information, two peaks in PD-2 extracts showed in Fig. 2a of the retention times (RT) were consistent with the standard of 3-oxo-C8-HSL and 3-oxo-C10-HSL at 7.111 and 8.033 min.
GC-MS chromatogram in SIM mode at m/z143. a Extract of strain PD-2. b 3-oxo-C8-HSL standard, RT = 7.111 min. c 3-oxo-C10-HSL standard, RT = 8.033 min
Comparative genomics and phylogenetic tree
Web-based similarity searches using the GenBank database were used to determine the homologous degrees of zlaI and zlbI protein sequences to those of other AHL synthases. The most similar homolog of zlaI, identified using a BLASTP search, was an autoinducer synthase from Sulfitobacter pseuddonitzssschiae (GenBank accession no. WP 037926640.1) with a 77% identity; the most similar homologs of zlbI were autoinducer synthases from Tropicibacter naphthalenivorans and Ponticoccus sp. SJ5A-1(GenBank accession nos. WP 058247237.1 and WP 058863594.1, respectively), with 81% identity. In fact, the multiple sequence alignments revealed that zlaI and zlbI share low homology, but both shared 10 conserved amino acids with other reported autoinducer proteins of Rhodobacterales bacteria as shown in Additional file 1: Figures S1A, B, respectively. Moreover, the phylogenetic tree constructed based on amino acid alignment (Fig. 3a, b) illustrated that zlaI clustered closely with an autoinducer synthase protein from Salipiger mucosus with a bootstrap value of 88% and zlbI was the least phylogenetically related to autoinducer synthase proteins from T. naphthalenivorans and Sagittula stellate with a bootstrap value of 52%. The Fig. 3c showed that zlaR clustered closely to an luxR family transcriptional regulator protein from Salipiger mucosus. Figure 3d showed zlbR clustered closely to luxR family transcriptional regulator proteins from Pelagibaca abyssi and Thiobacimonas profunda with a bootstrap value of 53%.
Phylogenetic tree showed the phylogenetic position of AHL synthases and transcriptional regulator of strain PD-2. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer from the phylogenetic tree. The horizontal talbar at the bottom represents evolutionary distance as 0.2 change pernucleotide position. The numbers at the nodes indicate the bootstrap values as percentage of 1000 replications. Glucose dehydrogenase enzyme (GI:729809030) from Escherichia coli was chosen as the out group for comparison. a The phylogenetic position of zlaI protein. b The phylogenetic position of zlbI protein. c The phylogenetic position of zlar protein. d The phylogenetic position of zlbr protein
Identification of the zlaI and zlbI genes responsible for AHL production
To identify what kinds of AHLs are produced by the zlaI and zlbI genes, the expression of zlaI and zlbI was examined in an E. coli strain that does not produce AHL. The presence of the 870 bp zlaI and 639 bp zlbI genes in the transformants were confirmed by PCR amplification. In bioautography, the KYC55 biosensor detected AHLs from recombinant E. coli culture extracts. In Fig. 4, AHLs signals are observed as blue spots on the plat. This demonstrates zlaI and zlbI encode AHL synthetases in E. coli BL21. The profile of AHLs produced by pGEX-zlaI E. coli BL21 and pGEX-zlbI E. coli BL21 indicate that the zlaI and zlbI genes produced the same AHLs of 3-oxo-C8-HSL and 3-oxo-C10-HSL (Fig. 4). Although this method cannot determine an absolute quantification, the size of a spot on TLC plate may also reflect the concentrations of AHLs to some degree (Deepa et al. 2012). From the size of each blue spot, the zlbI gene produced obviously greater amounts of 3-oxo-C8-HSL than zlaI, thereby demonstrating that 3-oxo-C8-HSL is the main product of the zlbI gene.
Profiles of AHLs produced by PD-2 and recombinants based on bioautography assay. Lane A mixture of synthetic HSL standards: C6 (C6-HSL); C8 (C8-HSL). Lane B mixture of synthetic 3-oxo-HSL standards: 3-oxo-C6 (3-oxo-C6-HSL); 3-oxo-C8 (3-oxo-C8-HSL). Lane C extracts of strain PD-2. Lane D extracts of pGEX-zlaI E. coli BL21. Lane E extracts of pGEX-zlbI E. coli BL21
The effect of β-CD on the growth of strain PD-2
Growth curves with different concentrations of β-CD are shown in Additional file 1: Figure S2. No significant differences were found in the initial 40 h of growth; all the strains entered the logarithmic phase at 10 h and the plateau phase at 40 h. The OD630 values declined after 40 h of incubation with β-CD. The levels of growth inhibition were proportional to the β-CD concentrations. At 84 h, the OD630 values were 0.67, 0.51, and 0.42 at β-CD concentrations of 1, 2, and 5 mg mL−1, respectively. The curve of the control was much more stable (OD630 of 0.7) from 40 to 84 h. The results suggested that the addition of 1 mg mL−1 β-CD had the lowest impact on growth with a growth of inhibition of only 4%, which we considered appropriate for the following experiments.
After the addition of β-CD, the concentrations of the AHLs were detected by bioautography assay (Fig. 5). The spots of samples A (PD-2 cultured for 20 h at low density) and B (PD-2 cultured for 84 h at high density), which did not contain β-CD, were much larger and were deeper blue than C (A adding β-CD, then cultured for 84 h) which was dim blue. This indicates strain PD-2 at low density just produce a small amount of AHLs. When the bacterium continuously grows to high cell density, the AHLs accumulate to high concentration. But after adding QS-quenching β-CD in the low cell density, the concentration of AHLs do not increase. Because β-CD can form complexes with the AHLs that PD-2 produced at high cell density (Morohoshi et al. 2013).
Profiles of AHLs produced by strain PD-2 under different culture condition. The biosensor is Agrobacterium tumefaciens KYC55. Lane A extracts of strain PD-2 cultured for 20 h. Lane B extracts of strain PD-2 cultured for 84 h. Lane C extracts of strain PD-2 co-cultured with 1 mg mL−1 β-CD at 20 h and continuously cultured for 64 h. After overnight incubation, the AHLs production was visually observed as blue spots. There was a positive correlation between the size of the blue spots and the concentration of AHLs
The algicidal effect of Ponticoccus sp. PD-2 on red tide species
Algicidal activities of strain PD-2 were obtained after 5 days of co-cultivation with target microalgae. The date presented were the mean values of IR from triplicates. (a) The microalgae which was co-cultured with the extracts of strain PD-2 cultured for 84 h. (b) the microalgae which was co-cultured with the extracts of strain PD-2 cultured for 20 h. (c) the microalgae which was co-cultured with extracts of strain PD-2 co-cultured with 1 mg mL−1 β-CD on 20 h and continuously cultured for 64 h.
Strain PD-2 shows algicidal effects on all red tide species after 84 h of culturing (Table 1). From the IR value, PD-2 had the strongest algicidal effect (IR 84.81%) on its host, P. donghaiense. Besides its host microalgae, strain PD-2 also had algicidal effects on P. globosa (IR 79.91%) and a small effect on A. tamarense (IR 67.14%). After culturing for 20 h, PD-2 had very little algicidal effects on P. donghaiense and A. tamarense with IR of 20.27 and 18.38% respectively and no effect on P. globosa. The same results were observed when PD-2 was continuously co-cultured with 1 mg mL−1 β-CD for 64 h. Then, the algicidal activities of PD-2 co-cultured with β-CD decreased to IR of 19.45% against P. donghaiense and 16.09% against A. tamarense. Compared to PD-2 without β-CD added, the algicidal activities decrease by more than 50%. In this research, the addition of β-CD reduced the concentration of the AHLs. Thus, the results suggest that the algicidal activities of strain PD-2 are regulated by a QS system.
Algicidal effect of strain PD-2 against various red tides microalgae species
Inhibition rate (%)
Prorocentrum donghaiense
Phaeocystis globosa
Alexandrium tamarense
84.81 ± 2.8
−3.61 ± 3.1
In this study, a Ponticoccus sp. PD-2 strain was isolated from the harmful algal bloom-associated microalga P. donghaiense. Its metabolites had growth inhibition effects not only on its host (P. donghaiense), but also on two other red tide microalgae, P. globosa and A. tamarense. Ponticoccus was first isolated from the coastal seawaters of Korea and has been identified as a new genus belonging to Rosebacter clade (Hwang and Cho 2008). The Rosebacter clade is one of the major marine groups and has members across diverse marine habitats (Buchan et al. 2005). Bacteria of Rosebacter clade also appear in the symbiotic systems of microalgae and may often regulate the algal community (Kodama et al. 2006; Tan et al. 2015).
To our knowledge, this is the first report demonstrating that Ponticoccus sp. has QS functions. In our previous research, we found strain PD-2 could produce AHLs QS signals. Of the diverse AHLs produced by marine bacteria, the most common types are C4-HSL, C6-HSL, 3-hydroxy-C6-HSL, 3-oxo-C6-HSL, C8-HSL, 3-oxo-C8-HSL, 3-oxo-C10-HSL, 3-oxo-c12-HSL and C14-HSL (Berger et al. 2012; Swift et al. 1997; Tang et al. 2013; Zan et al. 2013). The various signal molecules produced by its synthetases (luxI homolog genes) can be investigated by heterologous expression (Gao et al. 2014; How et al. 2015; Morohoshi et al. 2016). To determine which kinds of AHLs are produced by zlaI and zlbI, we expressed zlaI and zlbI in E. coli. 3-oxo-C8-HSL and 3-oxo-C10-HSL were identified in a PD-2 extract and in E. coli overexpressing zlaI and zlbI, suggesting that both zlaI and zlbI can each produce two kinds of AHLs. However, it is rare that two homologous luxI genes can generate identical signal molecules in one strain. Thus, we speculate that the two kinds of signal molecule are synthesized from the same metabolic pathway. In this study, the multiple sequence alignment and the phylogenetic tree (Additional file 1: Figure S1; Fig. 3a, b) illustrated that although there was only a low degree of homology, both of PD-2 and other Rhodobacterales bacteria had conserved regions among AHL synthases. Thus, the zlaI gene and zlbI gene should be new AHL synthetase genes. All the strains shared the 10 invariant amino acids that are characteristic of luxI homologs (Parsek et al. 1997). This strongly indicates a low rate of random mutation for this autoinducer. It also shows that these proteobacteria share a similar QS mechanism and a similar mechanism for regulating the synthesis of AHLs, although the corresponding AHLs regulate different target genes. Through comparing zlaR and zlbR protein sequences with those of other LuxR family transcriptional regulator on NCBI and constructing phylogenetic tree (Fig. 3c, d), we found that there was also a low degree of homology among transcriptional regulator from other bacteria. Thus, the zlaR gene and zlbR gene might also be new transcriptional regulator genes.
Many studies have shown that marine organism-associated bacteria play very important roles when interacting with their hosts. In recent years, a high percentage of marine organism-associated bacteria have been shown to produce QS signals, including bacteria associated with microalgae, sponges, corals, and fish (Britstein et al. 2016; Wagner-Döbler et al. 2005). Thus, studying the QS-mediated interactions between marine bacterial with their hosts has profound theoretical and practical significance regarding the ecological functions of marine bacteria. In fact, many investigations have proven that QS regulates such interactions. The luminous bacterium Vibrio fischeri is the specific light organ symbiont of Euprymna scolopes (Boettcher and Ruby 1990) E. scolopes is a small sepiolid squid (average adult length, 40 mm) that is indigenous to the Hawaiian archipelago, where it lives in shallow sand flats that are associated with coral reefs (Ruby and McFallngai 1992). The squid buries itself in the sand during the day to escape predators, but it comes out to forage in the water column at night (Summers 1983). Additionally, sponges and their associated microorganisms have developed a variety of chemical defense pathways. The production of chemicals which are regulated via QS systems are employed to defend sponges against potentially pathogenic bacteria and fish predators, deter fouling organisms, and degrade surfactants (Guo et al. 2011; Huang and Li 2006).
Cyclodextrins are a family of compounds comprising sugar molecules that are bound together in a ring. Cyclodextrins are well known cyclic oligosaccharides that form complexes with a wide range of organic compounds. Tsukasa proved that α,β-CD could form a complex with an autoinducer in a bacterial culture medium (Ikeda et al. 2002). He found that the addition of β-CD decreased the abundance of AHLs signal molecules by 75%, without inhibiting bacterial growth. But our results indicate that 1 mg mL−1 β-CD had little influence on the growth of strain PD-2, and β-CD could form a complex with AHLs produced by strain PD-2 in the culture medium (Morohoshi et al. 2013). The algicidal activities of strain PD-2 decreased more than 50% due to its QS system was inhibited, this is the first evidence that the QS systems of microalgae-associated bacteria can regulate algicidal activity.
Existing evidence proves that production of algicidal substances can be regulated by QS (Guo et al. 2016; Nakashima et al. 2006), although there is no direct evidence to suggest that the algicidal activities of microalgae-associated bacteria is regulated by QS systems. Consequently, investigations of the effects of QS on the regulation of algicidal functions are needed to increase our understanding of the relationship between bacteria and algae, and QS inhibitors can be used to study QS regulation.
QS:
AI:
autoinducer
AHL:
N-acyl-homoserine lactone
β-CD:
GC–MS:
gas chromatography–mass spectrometer
HPLC–MS:
high-performance liquid chromatography-mass spectrometer
LB:
Luria-Bertani
TLC:
thin-layer chromatography
IR:
inhibition rate
RF:
retardation factor
3-oxo-C8-HSL:
N-3-oxo-octanoyl-L-homoserine lactone
3-oxo-C10-HSL:
N-(3-oxodecanoyl)-L-homoserine lactone
RT:
retention times
PCR:
CCMM:
culture collection of marine microalgae
WC and LZ designed the whole experiment; CH, BH and MZ helped to carry the experiment; WG, CS and GZ helped to analyze the raw data; WC wrote the article and XG helpd on the paperwork. All authors read and approved the final manuscript.
We thank Professor Hill from The Institute of Marine and Environmental Technology, University of Maryland, USA, for providing the biosensor Agrobacterium tumefaciens KYC55 (pJZ372) (pJZ384, pJZ410). We thank the Institute of Oceanography, Chinese Academy of Science, for providing the indicator microalgae strains.
All datasets on which the conclusions of the manuscript rely are presented in the main paper.
This work was supported by The National Natural Science Foundation of China-Shandong Joint Funded Project (U1406404), the National Natural Science Foundation of China (41076108), Basic Scientific Fund for National public Reserch Institute of China (2015T05), Marine science and technology project of Huangdao district of Qingdao city (2014-4-20), Natural Science Foundation of Shandong Province (ZR2014DL011), and the 2012 Taishan Scholar Award. All of these funds have been used for purchasing reagents and data analysis.
13568_2017_357_MOESM1_ESM.doc Additional file 1. Additional figures.
Key Laboratory for Marine Bioactive Substances and Modern Analytical Technology of the First Institute of Oceanography, State Oceanic Administration, No. 6 Xianxialing Road, Qingdao, 266061, Shandong, People's Republic of China
Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, People's Republic of China
School of Life Science of Yantai University, Yantai, 264000, People's Republic of China
National Deep Sea Center, State Oceanic Administration of China, Qingdao, 266237, People's Republic of China
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text:
Stray Kids Scores First Number 1 On Trending Songs Chart With 'Case 143'
stray kids earn their first No. 1 on Billboardit is trending songs chart (dated October 15), powered by Twitter, as "Case 143" surges from #2 following the release of the band's new EP Maxident October 7.
"Case 143" is the lead single from the EP, which is set to hit the Billboard charts next week, October 22.
More from Billboard
Stray Kids are the fourth group to top the most popular songs this year, joining BLACKPINK, BTS and SB19.
Elsewhere on Hot Trending Songs, TREASURE's "Hello" debuted at No. 4, earning the group their fourth entry and second top-five success, following "Jikjin" (peaking at No. 2 in March). "Hello" is the lead single from the band's EP. The second step: chapter tworeleased on October 4.
Additionally, SEULGI's "28 Reasons" opens at No. 19 on Hot Trending Songs, marking his second solo appearance, following his starring turn on Bambam's "Who Are You" (No. 7, January). The song is the title track from his debut solo EP of the same name, which was released on October 4.
Billboard's Hot Trending Songs charts, powered by Twitter and sponsored by Capital One, track real-time global music-related trends and conversations on Twitter, viewable over the past 24 hours or seven days. A weekly 20-position version of the chart, covering activity from Friday through Thursday of each week, is published alongside Billboard's other weekly charts on Billboard.com each Tuesday. The charts highlight the buzz around new releases, award shows, festival moments, musical nostalgia and more. Hot Trending Songs is unique in that it tracks the songs people are talking about, not necessarily what they're listening to.
Keep visiting Billboard.com for the ever-changing chart of trending songs, and check every Tuesday for the latest weekly chart.
Dave claims Top 2 UK Trending Songs with Verdansk and In The Fire
Fredo claims top 2 trending songs in UK
Top 10 Trending Songs You Should Listen To In 2020
Ariana Grande lands top 2 trending songs in UK

redpajama_set_name: RedPajamaCommonCrawl


text:
From Ancient Egypt to Your Dinner Plate...Garlic: It's Good for You! (press release)
Tuesday, August 15, 2006 by: NewsTarget
Tags: health news, Natural News, nutrition
https://www.naturalnews.com/019982_garlic_cancer_research.html
Garlic, recognized for its healing powers in ancient times, is now being rediscovered by medical scientists, who have new evidence of its efficacy against cancer and heart disease. Dr. Richard Rivlin, of NewYork-Presbyterian Hospital/Weill Cornell Medical Center, is guest co-editor of a special March peer-reviewed supplemental issue to The Journal of Nutrition. The issue comprises 35 articles representing the latest research on garlic -- findings that were first presented at a symposium held last year at Georgetown University.
"Medical texts from China, India, Egypt, Greece and Italy mention medical applications of garlic," says Dr. Rivlin, professor of medicine at Weill Cornell Medical College, attending physician at NewYork-Presbyterian Hospital/Weill Cornell Medical Center, and director of the Nutrition Center at the Strang Cancer Prevention Laboratory. "Cultures that developed independently came to the same general conclusions, namely, that garlic could be administered to provide strength and to increase work capacity. Hippocrates, considered the Father of Medicine, used garlic as an essential component of one of his therapies."
Dr. Rivlin believes that while much promising research has been made pointing to the disease-preventive and therapeutic effects of garlic, at the present time, it should be considered complementary medicine, not alternative therapy. "The rapid pace of advances in garlic research provides increasing evidence that garlic has significant potential as a complement to established therapies."
New research on the health benefits of garlic, as published in The Journal of Nutrition special issue, include the following:
* Selenium -- an element found in garlic -- may be instrumental to garlic's anti-cancer properties. ("Cancer Chemoprevention by Garlic and Garlic-Containing Sulfur and Selenium Compounds," by Drs. K. El-Bayoumy and R. Sinha, Penn. State; Dr. J. Pinto, Cornell-Burke Medical Research Institute; and Dr. R. Rivlin, Weill Cornell Medical College)
*Garlic may slow the progression of coronary artery calcification in patients on statin therapy. ("Aged Garlic Extract Retards Progression of Coronary Artery Disease," by Dr. M. Budoff, Harbor-UCLA Medical Center)
*In addition to its cholesterol-lowering potential, blood-pressure-lowering effects, and antioxidant properties, garlic may help moderate levels of homocysteine, an amino acid that is a marker for coronary artery disease. ("Homocysteine-Lowering Action Is Another Potential Cardiovascular Protective Factor of Aged Garlic Extract," by Drs. Y. Yeh and S. Yeh, Penn. State)
*Garlic may inhibit platelet aggregation -- a major contributing factor to cardiovascular disease -- by suppressing calcium mobilization. ("Aged Garlic Extract May Inhibit Aggregation in Human Platelets by Suppressing Calcium Mobilization," by Drs. G. Allison, G. Lowe, and K. Rahman, Liverpool John Moores University, U.K.)
*Garlic may reduce pre-cancerous gastric lesions. ("Factorial Trial Including Garlic Supplements Assesses Effect in Reducing Precancerous Gastric Lesions," by Dr. M. Gail, National Cancer Institute, and Dr. W. You, Beijing Institute of Cancer Research)
*Garlic may suppress progression of precancerous lesions of the large bowel. ("Aged Garlic Extract Has Potential Suppressive Effect on Colorectal Adenomas in Humans," by Drs. S. Tanaka, Hiroshima University, Japan; K. Haruma, Kawasaki Medical School, Japan; M. Yoshihara, Hiroshima University, Japan; G. Kajiyama, Onomichi General Hospital, Japan; K. Kira, Wakunaga Pharmaceutical, Japan; H. Amagase, Wakunaga of America; and K. Chayama, Hiroshima University, Japan)
Health news at FETCH.news


text:
The Royal British Legion
Henley-on-Thames & Peppard Branch ~ BR0753
Poppy Appeal
Fundraising - Sky Dive
Fundraising - March of Nijmegen
Nijmegen - David's Story
Great Pilgrimage 90
Henley-on-Thames Community Poppy Appeal
Your local Poppy Appeal campaign is honoured to be supporting The Royal British Legion. For more than a century, the Legion has been ceaselessly and effectively working to ensure that the past and present servicemen and servicewomen and their families fare better in the face of adversities.
If you have the time and would like to give something back to the Armed Forces for the unique difficulties they resolve and the sacrifices they make for us, please become a Henley and Peppard Poppy Appeal volunteer. In past years our community has generously helped to raise considerable sums and has made a real difference to the Armed Forces community.
The funds raised by our campaign go towards upholding the welfare of the members of the British Armed Forces, veterans and their families, from their first day of service and continues through their life. The funds also help to support British subjects who have served alongside the British Armed Forces such as members of the Brigade of Gurkhas and their families.
The Royal British Legion is the UK's leading Armed Forces charity that also works with many smaller charities that provide specialised support to service personnel in need.
The Poppy Appeal relies on the exuberant spirit of our volunteers, whether it is just to give a few hours a year to collect donations, to help manage the campaign or help fundraise with special activities.
If you could spare a little time to help raise funds towards helping courageous service personnel, please contact your local Poppy Appeal Organiser, Sara Abey at henley@saraabey.com
Here are a few stories showing how the Legion supports the Armed Forces community:
© 2021 Royal British Legion - Henley-on-Thames & Peppard Branch.


I have plans to make a DIY rack as well for my 1081 chassis (that will come up shortly).
My plan was to do them as Neve did in the 70´s with open top and bottoms.
Its more or less just three sides, some ISEP rails and a frontpanel with controls.
The hard part is to choose what kind of connector to use and how to secure it, since the original Amphenol is obsolete.
But once that is solved and i have the 1081 rack drawn up it would be very easy to adjust measurments for the 1073/84 instead.
Well , I will finish 2 channel prototype in next few weeks.It contain 2 jfet D.I. and stepped attenuation between stages.
I'd be interested in 2x 1084. Looks like a great project.
The face plates will be Neve logo'd, right? And not Bee Bird style...?
Actually I dont want to put any logo on it, so if you want , you can add decal latter.1073 name and logo are AMS Neve trademarks , and as some probably know, in 2017 AMS take legal actions against Heritage Audio just for exploiting 1073 name.
You can put me down for 4 - 1084 cases plus boards.
i should be good for 4 1084 cases and boards.
btw , I am going to CNC house in saturday , so thats gonna be start of manufacturing frames.I will try to add few more details like output transformer holder , holes for 1084 capacitor holders...etc.
Sounds awsome, Sarsarist. Does anyone have a prefered power supply design?
My experience is that N modules sound best with regulators from that era, mostly transistor-diode combo.
Also important difference can make PSU filtering , original 33604 have one large 10-15000 cap and psu is rated like something around 5A.Those big caps can sometimes realy kill or smooth transients , and maybe it sound more "vintage" I personally prefer group of smaller values in parallel. In the end of the day , LM317 will do a job , but considering effort to build those beasts , PSU should not be under radar.
Agreed, It's very true that PS contributes hugely to to the overall sound of a unit, it's very easy to think of the unit somehow being separate from its power supply, maybe because of the advent of wall wart universal PS. But of course the whole thing is one circuit from being to end.
any more info on the 1084/1081 frames? Any assumened time frame when these will be ready for purchase?
I ordered them in saturday, so hope that in a 4 weeks from now , they will come out from cnc workshop .After that nickel plating and they will be ready for shipping.
Any interest in 8 module frame housing?
Also , as a first aid I made prototype of small back edge connector with IO xlrs and 24v48v output pot connections.It works nice.
The module frame is of specific interest to me!
Ah! I like your prototype!


text:
Welcome To The Loskop Leisure Route
Loskop Leisure Route
History of Middelburg
Botshabelo History
Anglo Boer War
Loskop Dam History
Steam Locomotive
Loskop Dam Interesting Facts
The dam wall was originally between 1934 and 1938, and was built entirely by hand.
In 1978/9the dam was drained raised by 9 meters to its current height of 54 meters.
The bas is ₊/₋24 meters thick and length is 506 meters long.
The entire dam wall rests on a solid rock base.
The canal entire engineer "Col. DF Roberts" has his remains cemented into the wall.
The canal, which runs down the left hand side of the river measures 480km.
The canal system irrigates about 1600 hectors of farmland; the lowest point of the is only 107 meters below the dam wall.
If the dam wall gates are reopened the maximum amount of water is 29,000 liters per second.
The dam itself is able to supply framers with water for up to three years without rain.
The catchment area of the Olifants River, which flows into the dam , is some 1225 million hectors including areas such as Hendrina, Dacel, Ermelo and Bronkhorstspruit. Included in this catchment area is the Wilger and Klein Olifants River that also contributes to the dam.
On the 15th of February 1996 at 3am dam registered at 117,63%At this point the water flowing from the Oliftants River was 3.2 million cubic liters per hour.
At 100% the dam olds 348 million cubic litters with the surface area of 2385 hectares.
LOSKOP DAM – Early Local Engineering Ingenuity
( An article in The Water Wheel dated September / October 2008 )
Despite being constructed more than 70 years ago , the Loskop Dam , situated across a gorge on the Olifants River , about 32km south of Groblersdal , in Mpumalanga , remains one of the largest dams in South Africa.
Lani van Vuuren reports
The history of Loskop Dam can be traced back to the 1840`s when the first Voortrekkers settled in the Kruis River valley not far from the present dam site. The first farms in the Olifants River valley , including Lagersdrift and Kalkfontein , were pegged out as long ago as 1886. Back then , the farmers cultivated mainly wheat under dry-land conditions.
Notwithstanding the establishments of these farms , due to malaria and cattle diseases like East Coast fever , initial development was slow until the turn of the nineteenth century. In the early Twentieth century , many farmers trekked with their cattle from the Highveld to the Olifants River valley during winter. Each winter they stayed a little longer until they eventually settled in the area in greater numbers. This was particularly during the years 1917 and 1924.
THE RISE OF SMALL IRRIGATION SCHEMES
It was in 1917 that the first private dam in the area was completed on the farm Rooikraal with government assistance. Thanks to irrigation , the wheat crop on this farm increased from 150 to 8000 bags a year. Around 1925 , other small irrigation schemes were completed involving both weirs and pumping water from the river. This led to the establishment of the Hereford Irrigation Board to serve an area of about 10km downstream of the present Loskop Dam.
The proposed Hereford Scheme included an improvement of the diversion weir at Kameeldoring , and also a 41km-long canal extending as far as the Moses River. After a loan of R 70 000.00 was granted by the Land Bank , work started during 1928 under the supervision of the Department of Irrigation. The various contracts were completed in 1930.
The early success of this scheme gave rise to a petition by farmers for the establishment of the Loskop Irrigation District. The old Transvaal Irrigation Department had undertaken a reconnaissance survey of such an irrigation scheme on the Olifants River between 1905 and 1907. However , back then it was recommended that an irrigation scheme should not be implemented until the valley was more densely populated , and not before there was the prospect of a railway service to the area.
CONSTRUCTION OF THE DAM
Construction of the Loskop Dam on the farms Loskop and Vergelegen started in 1934. As was the case with many other government projects at the time , only white married white men were employed on the Loskop Dam construction site. They were paid five shillings a day and provided with free accommodation , food and medical attention.
The number of labourers employed at the end of March 1935 was 440 men. Throughout 1935 and 1936 the Director of Irrigation noted in his report that there was a shortage of labour , especially concerning the construction of the Loskop Dam. The department then asked the Department of Labour to allow single white men on the site as well.
By the end of 1937 the dam was nearly complete , except for a number of minor tasks on the superstructure. Good rains fell in December 1937 and January 1938. This resulted in the dam being filled and it overflowed in January 1938.
( The estimate cost of the entire project was 1.5 million British Pounds.
In June 1935 , a start was made on the canal system , which was eventually completed in 1948 , after an interruption in the work as a result of the outbreak of World War 2. The total length of the canals is 480km.
The Loskop Irrigation Scheme also resulted in the establishment of a town , Groblersdal , laid out on a farm owned by W.J. Grobler. The town was proclaimed in 1938.
RAISING OF THE DAM WALL
Development in the Witbank-Middelburg area necessitated the raising of the dam wall so that the portion of the assured yield which had in the meantime become affected as a result of the construction of upstream dams such as Rondebosch , Witbank and Doornpoort dams could be restored. By 1971 , when the recommendation to raise the dam wall was made , the Loskop Dam system of canals served about 25 000 ha of farmlands.
Between 1974 and 1980 the dam wall was raised by nine meters to a height of 54m above the lowest foundation level.
The full supply capacity of the dam is 362 million cubic meter.
HOLY BURIAL GROUND
The ashes of Lt Col Robert (He was the resident engineer at the Department of Irrigation`s very first hydraulics laboratory near the dam site ) were originally buried on an island in the dam. As the raising of the wall was to result in the inundation of the island , the ashes were removed and reentered in a niche on the left flank of the dam wall.
Year of completion 1938 ( raised in 1979 )
Purpose Irrigation , domestic, industrial
River Olifants River
Nearest town and province Groblersdal , Limpopo
Type Mass concrete
Net storage capacity 348 million cubic meter
Wall height above lowest foundation 54m
Crest length 506m
Material content of dam wall ( original & raised ) 415 000 cubic meter
Type of spillway Uncontrolled
Capacity of spillway 7 750 cubic meter / second
Surface of dam at full supply level 2 350 ha


Shelby Manor Ltd is an assisted living facility in Shelby, NC. Shelby Manor Ltd offers activities at their location for residents. These activities generally allow residents to maintain healthy lifestyles by encouraging movement and socializing with their peers.


text:
Congressional Support Grows for FDA Regulation of Tobacco: Bill Now Sponsored by 55 Senators, 200 Representatives
Statement of William V. Corr, Executive Director, Campaign for Tobacco-Free Kids
Washington, D.C. — As the U.S. House Subcommittee on Health prepares to hold a hearing Wednesday on legislation to grant the U.S. Food and Drug Administration (FDA) authority over tobacco products, bipartisan support for this legislation continues to grow in Congress. The legislation (H.R. 1108, S. 625) now has the support of 55 sponsors in the Senate and 200 sponsors in the House. This bipartisan support from both political parties and both sides of the Capitol demonstrates that Congress has the votes to pass this legislation this year.
This legislation has the strong support a broad coalition of public health, faith and other organizations, the American people, and leading scientific experts:
This legislation has the support of every major national public health organization and more than 500 public health, faith and other organizations across the country. View list (PDF, 29KB).
According to a recent national poll, 70 percent of voters support Congress passing the legislation and 72 percent believe passage of the legislation would be an important accomplishment for Congress. The poll also shows FDA regulation of tobacco is supported across political lines, geographic regions and even by a majority of smokers (View detailed poll results).
Recent reports by the prestigious Institute of Medicine (IOM), a unit of the National Academy of Sciences, and the President's Cancer Panel endorsed FDA regulation of tobacco as a critical element of a comprehensive national strategy to eliminate tobacco use as a significant health problem in the United States.
By enacting this legislation into law, Congress can seize a truly historic opportunity to protect our children from tobacco addiction and save countless lives. There are few actions Congress can take that would make a bigger difference for our nation's health. Every day another 1,200 Americans die from tobacco use and more than 1,000 children become new regular smokers.
Tobacco use is the nation's leading preventable cause of death, killing more than 400,000 people and costing more than $96 billion in health care bills each year. Despite all the death and disease they cause, tobacco products are virtually unregulated to protect consumers' health. This continuing lack of regulation allows the tobacco companies to market their deadly products to our children, deceive consumers about the harm their products cause and resist even the most minimal steps to make their products less harmful.
Among other things, the legislation would grant the FDA authority to crack down on tobacco marketing and sales to kids; require that tobacco companies disclose the contents of tobacco products and reduce or remove harmful ingredients; stop tobacco companies from misleading the public about the health risks of their products; and require larger, more effective health warnings on tobacco products. We urge both the Senate and the House to quickly enact this legislation into law and to reject all efforts to weaken it.


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El metro és el transport públic més utilitzat de Barcelona, dins del recinte es troben més de 2000 suports publicitaris; cartells fixos, projeccions, opis i publicitat dinàmica.

Són milers de passatgers qui visualitzen els diversos spots diàriament, és per això que TMB ha innovat, creant un nou tipus de publicitat, captant així l'atenció dels usuaris.

Sistema de publicitat dinàmica
L'any 2019 la TMB va introduir un tipus de publicitat dinàmica als túnels del metro amb la qual, mitjançant pantalles led, els passatgers poden veure la reproducció d'imatges dels diferents espots publicitaris.

Els espots se situen fixos dins de l'interior del túnel, disposats de manera que els passatgers els visualitzin des de l'interior del vagó. Aquest sistema consisteix en la reproducció de vídeos sincronitzats de 10 segons de durada.

Per aconseguir l'efecte òptic, se situen 300 barres de leds a una cadència de 30 frames/segon, espaiades entre si per 52 centímetres en un tram de 160m aproximadament, pel qual el metro passa a una velocitat d'uns 54km/h. El sistema de percepció humà interpreta els fotogrames com a una sola imatge, d'aquesta manera, en comparació amb els films, el desplaçament del vagó substitueix al moviment de la pel·lícula. Les pantalles es mantenen connectades a un quadre de comandament situats en una cambra de comunicacions a l'estació d'Espanya.

Actualment (2021), el metro disposa de dos suports publicitaris dinàmics situats entre les parades d'Hospital Clínic-Diagonal i Espanya-Rocafort.

Referències

redpajama_set_name: RedPajamaWikipedia


Zainab Balogun is married! And her wedding was such a surprise that even the internet didn't know about it till the big day. We were all innocently scrolling through our feeds when we saw that the movie star and media darling was having her traditional wedding.
We were all shook but pleasantly so. Today, in the spirit of Owambe Saturdays and just because we love us some Zainab Balogun, we're sharing some reasons why she's our favourite 2018 celebrity bride so far.
The element of surprise to the Zainab Balogun/Dikko Nwachukwu wedding made it extra special. It's very difficult for celebrities to keep their personal lives away from the eyes of the public especially in this super curious social media age. So when we see a celeb who manages to pull off a surprise wedding, we give them kudos.
Zainab Balogun is a showstopper on any good day, but on her wedding day, she looked even more stunning. Her outfits were colourful and radiant and just made her gorgeous skin glow even more than usual. We need to get the numbers of her vendors because this kind of aso ebi goodness must be shared.
What's a great wedding without an amazing couple? Seeing Zainab Balogun and her groom gleaming with love in their eyes was enough to make even the stoniest hearts palpitate, and ours did, you can be sure of that. There's really nothing as sweet as true love and we're happy that our Zainab has found it.
What did you think about Zainab Balogun's wedding? Share your thoughts with us in the . below.
Meanwhile, you can check out all the beautiful moments from Zainab Balogun's wedding.


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New releases for 4 March 2016
26 February 2016 25 February 2016 Mark Willsmusic / new releasesLeave a comment
First up for 4 March is a debut album by a band called Mexrissey, an album of Morrissey covers done with a full mariachi band, and it's brilliant! Nada Surf's first for 4 years captures the indie rockers at the height of their considerable powers. The Cat Empire have delivered a lovely, danceable jazzy album in Rising With the Sun, The Coral's Distance Inbetween comes 6 years since their last original studio album, and it's worth the wait. The Feeling's fifth album is self-titled to reflect the rawness of the recording process, which you can hear clearly, Loretta Lynn's Full Circle is a great country album describing her journey through music up to her current 83 years young. The prolific M. Ward, of both She & Him and Monsters Of Folk, is a singer-songwriter of great consistency and he doesn't disappoint with More Rain. Release of the week is Ray LaMontagne's Ouroboros, which is very different from his previous work but well worth a listen.
Click here for more details and to order your copy.
Loretta Lynn / M. Ward / Mexrissey / Morrissey / Nada Surf / new release / Ray LaMontagne / The Cat Empire / The Coral / The Feeling
A Formal Horse best of Beth Hart Björk Bob Dylan Bruce Springsteen David Bowie Emily Barker events exclusive release Father John Misty Foals in-store Joe Bonamassa Kate Bush Led Zeppelin limited edition live music live performance local artist Michael Kiwanuka Mogwai Neil Young new release Nick Cave Nick Cave & The Bad Seeds pre-release offers pre-release of the week pre-sale offers pre-sale of the week promotion Public Service Broadcasting Queen Record Store Day Ride RSD Ryan Adams Southampton special edition special offer The National The Rolling Stones Ty Segall Van Morrison Wilco
Pre-sale of the week: The Cadillac Three – Country Fuzz, out 7 February 14 January 2020
New releases for 17 January 2020 10 January 2020
Pre-sale of the week: Nathaniel Rateliffe – And It's Still Alright, out 14 February 8 January 2020
Christmas and New Year sale! 23 December 2019
The Hundred Records top twenty of 2019 21 December 2019
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© Hundred Records. Registered in England. Number 9153994. Registered address: 11 Chalice Court, Hedge End, Southampton, Hampshire.


Video door phone system is not only used on high-rise residential buildings and apartment buildings, but also on some multi-storey or single-family villas. The installation of video doorbell also improves the home security. To villa users, should we choose a wired or a wireless video door phone?
Wired video doorphone, wireless video door phone, and wireless audio intercom can be used on villa. Wired models are generally cheaper. However, for aesthetic purpose, the wires need to be hidden in the wall. If the wire is aging, maintenance will be very troublesome. Outdoor unit of wireless video door phone must be plugged into an AC or DC power outlet and installed in a relatively fixed position. Indoor monitor can be movable. Transmission range between two devices can be 500 meters. As for wireless audio intercom, its visitor image is invisible; only audio is transmitted. Its penetration is better and the price is cheaper.
There are two ways to power wireless video door phone. One is to use battery; the other is to connect directly with power supply. If power supply is used instead of battery, no pollution to water and soil exists; and it is environmentally friendly. Also installation is easier without wire connection between outdoor and indoor units.
Wireless video door phone is relatively simple to install, but the penetration is not very strong. Generally, it can only penetrate 2-3 brick walls. If the wall is particularly thick, transmission may not be smooth. Therefore, not everyone can use wireless video doorbell. Wired video doorbells have more smooth transmission because of the wiring, but many villa users are not willing to punch holes on the walls and do wirings. If you want to install a wireless model, you should select according to actual situation and structure of your house.
We hope this article to help you choose a video doorbell!
Copyright © Dongguan Tianluo Electronic Technology Co., Ltd.


In Kos you will find the most extended beaches in the whole Aegean.
One of the most famous organized beaches is this of Tigaki, with warm, shallow waters and thin sand. Another sandy beach of 1000 meters length, is this of Lampi, which is found 4km south and west from the town of Kos. Also, 3,5 km from the town at the south, after the port of Kos, you will find the beach Psalidi and Agios Fokas, with small pebbles and crystal waters.
Keeping the same direction for 5-6 km more, at the end of the road and after descending the hill, you will find yourselves at the beach of Thermi, where you can enjoy famous mineral spas, inside a round artificial lake that is surrounded by rocks right near the sea. The warm spa waters come out from the deep ground and create bubbles to the surface.
It is a natural spa with beneficial properties for every age, where body and spirit will relax in just a few minutes. Having as its landmarks the volcanic black pebble and sand, as well as the surrounding rocks, this beach is ideal for pictures of rare wild beauty.
Leaving the town, after Tigaki, where your base is, you can visit many great beaches. In a distance of 3km at west you will find Marmari, mostly with little waves and with white sand, this beach is ideal for surfers. Keeping the same direction we arrive to the famous village Kardamena. There lies a huge sandy beach. Its left side is organized and offers sea sports, while its right side is more quite and calm, with dunes and natural trees shadow.
The next stop is Paradise Beach, with direction to Kefalos. This beach is a miracle of nature, it is endless and it creates a bay of 6 km length. Its waters are shallow but get deeper as you walk in. You will not find a single rock in its bottom, while if you turn your eyes behind while swimming, you will see hills with thousands of cedar and palm trees.
Moving 7 km at northwestern of Kefalos, we meet a beach with beautiful coves, Agios Theologos, which took its name after the chapel of Agios Ioannis Theologos. Finally, at a 5 km distance northern of Kefalos, inside a little bay, you will find Limnionas, a quiet beach and a hospitable shelter for small touristic and fishing boats in case of bad weather.
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text:
package graphedit.view.scrollers;

import graphedit.view.GraphEditView;

import java.awt.Color;
import java.awt.Graphics;
import java.awt.Graphics2D;
import java.awt.Rectangle;
import java.awt.event.MouseAdapter;
import java.awt.event.MouseEvent;
import java.util.Timer;
import java.util.TimerTask;

import javax.swing.JComponent;
import javax.swing.JScrollBar;
import javax.swing.plaf.basic.BasicScrollBarUI;

public class VerticalScrollBar extends JScrollBar{

	private static final long serialVersionUID = 1L;
	private GraphEditView view;
	private final int UP_SCROLL = 0, DOWN_SCROLL = 1;

	private final int interval = 50;
	private Timer timer;
	private Color gray = new Color(237,240,242);
	public VerticalScrollBar(GraphEditView view) {

		this.view = view;
		setOrientation(VERTICAL);
		setUI(new CustomUI());

	}

	class CustomUI extends BasicScrollBarUI {
		@Override
		protected void installListeners() {
			super.installListeners();
			if (incrButton != null) {
				incrButton.addMouseListener(new MouseAdapter() {

					@Override
					public void mouseClicked(MouseEvent e) {
						view.scrollDown();
					}

					@Override
					public void mousePressed(MouseEvent e) {
						timer = new Timer();
						timer.schedule(new ScrollTimerTask(DOWN_SCROLL), 0,interval);

					}

					@Override
					public void mouseReleased(MouseEvent e) {
						timer.cancel();
					}
				});

			}
			if (decrButton != null) {
				decrButton.addMouseListener(new MouseAdapter() {
					@Override
					public void mouseClicked(MouseEvent e) {
						view.scrollUp();
					}

					@Override
					public void mousePressed(MouseEvent e) {
						timer = new Timer();
						timer.schedule(new ScrollTimerTask(UP_SCROLL), 0,interval);
					}

					@Override
					public void mouseReleased(MouseEvent e) {
						timer.cancel();
					}
				});
			}
		}

		@Override
		public void paintThumb(Graphics g, JComponent c, Rectangle thumbBounds) {

		}
		@Override
		public void paintTrack(Graphics g, JComponent c, Rectangle trackBounds){
			Graphics2D g2 = (Graphics2D)g;
			g2.draw(trackBounds);
			g2.setColor(gray);
			g2.fill(trackBounds);
			g2.setColor(Color.GRAY);
			g2.draw(trackBounds);
		}
	}

	class ScrollTimerTask extends TimerTask {

		int direction;

		public ScrollTimerTask(int direction){
			this.direction = direction;
		}

		public void run() {
			if (direction == UP_SCROLL)
				getView().scrollUp();
			else
				getView().scrollDown();
		}
	}

	public GraphEditView getView() {
		return view;
	}

}

redpajama_set_name: RedPajamaGithub


text:
Home2Site Admin2022-04-03T11:23:24-04:00
Caleb Bates2023-01-20T15:33:12-05:00
By Caleb Bates|2023-01-20T15:33:12-05:00January 20, 2023|
Audrie Lamb2023-01-18T14:14:05-05:00
By Audrie Lamb|2023-01-18T14:14:05-05:00January 13, 2023|
Bowling Green Independent Schools meets refugee, migrant families where they are with Teranga Academy
By Audrie Lamb|2022-12-28T15:13:11-05:00December 21, 2022|
Grants available to accelerate literacy of primary students
By Staff|2023-01-27T16:14:42-05:00January 27, 2023|
The Kentucky Council on Postsecondary Education (CPE) is offering grants of up to $55,000 to support colleges of education in implementing the Kentucky Reading Project for Read to Achieve.
KDE Associate Commissioner Kelly Foster to be presented Bob Harmon Service to Education Award at National ESEA Conference
The National Association of ESEA State Program Administrators (NAESPA) announced Jan. 23 that Kelly Foster, associate commissioner at the Kentucky Department of Education (KDE), has been selected to receive the 2023 Bob Harmon Service to Education award.
KDE's Commissioner's Student Advisory Council presents recommendations to increase school safety
By Joe Ragusa|2023-01-25T09:05:41-05:00January 24, 2023|
The Kentucky Department of Education's Commissioner's Student Advisory Council presented a report on potential actions to increase school safety during its meeting on Jan. 24.
2022 Kentucky Teacher of the Year: Because I applied…
By Willie Carver|2023-01-20T13:42:15-05:00January 20, 2023|
Almost two years ago, I quietly received an email that I was nominated for the 2022 Kentucky Teacher of the Year. I remember thinking that I would never be picked because I didn't feel like I had done anything worth celebrating.
Thanking Kentucky's support staff for their vital role in student success
By Jason Glass|2022-12-16T15:35:33-05:00January 4, 2023|
One of the things I miss most being commissioner of education is the ability to be out in our schools every day. While I love my position and the opportunities it gives me to help shape policies and state priorities, the school building is where the rubber meets the road.
Harlan Middle/High School celebrates Family Friendly Schools award
By Charles Morton|2022-12-16T15:39:32-05:00December 20, 2022|
As a way to strengthen local efforts to connect with families and students, the Harlan Independent School District jumped at the opportunity to participate in the Family Friendly Schools initiative.
Kentucky History Education Conference to bring educators together
2023-01-26T17:06:25-05:00By Caleb Bates|
The Kentucky Historical Society in Frankfort will bring history teachers from across the Commonwealth together for the Kentucky History Education Conference: Turning Points in History.
2023-01-30T13:59:40-05:00By Audrie Lamb|


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Home » articles » Bombardier Jones: Dare To Hope – Review
Bombardier Jones: Dare To Hope – Review
August 31, 2020 by Jamie Parmenter
Blending rock, country and folk, Dare to Hope aims to take the listener on a journey through Americana-tinged stories of hope and despair.
Bombardier Jones, aka Ian Jones, knows how to create a sound and spread it through an entire record. It's not easy to develop a coherent and flowing album, with many artists able to create a killer single, but struggle when it comes to thickening out their ideas over multiple tracks. Jones doesn't have this problem, and with a foray of talented musicians in tune with his ideas, new record Dare to Hope delves into a foray of emotions amongst driving rhythms and settled soundscapes.
Great idea kicks off the record and we're thrown straight into classic rock/blues as guitars flow and melodies are mercilessly pushed forward. With a chorus reminiscent of something The Who would come up with in their heyday, it's a catchy opener that sets out the sound for the record well. Golden Hour follows with a more settled, country-tinged sound that bleeds honesty and has a folky undertone to the emotion-laden lyrics: "why can't the dust let the vision narrow and leave me at peace in a stolen time".
As we get further into the record the band are given more space to breathe and show off a flutter of individuality every now and again. Let the Light Shine feels like a live jam, with everyone seemingly taking their moment in the limelight, harmonica whaling, guitar and trumpets working in unison, and drums holding everything together. This is all wrapped in strong melodies and structure that make for an album highlight. The soulful So Many Tears then follows with all the slowness and prowess that allows the track to breathe and build in a meaningful way, whereas Unrecorded Time ups the ante with a rolling drumbeat, once again harking back to that bluesy, country atmosphere to bring out its charm before settling into an Americana-style lullaby.
One thing that could do with a little improvement is the way the vocals are harnessed throughout. Jones has a good voice that fits the music well, but at times you just feel another layer or two, or a more all-encompassing backing vocal could really thicken out the sound and make it hit just that little bit harder. Touch of Her Hand is an example of this with the chorus feeling a little thin for the overall sound or the warmth of Summer's Come could further be heightened with a stronger duet vibe about it.
These thoughts are, however, put to one side with a great album closer on the album-titled track Dare to Hope. Pulling all that has been learned throughout the entire album into a dexterous and dreamy country ballad, this track shows off a sweet and innocent side to the songwriting that works very well. It all rises up into a strong chorus that sends the listener on their way nicely.
Dare to Hope is, at times, gently caressing and at others ruthless and powerful. Wrapped in a classic rock and country bow, it's an album where you get to know the innermost thoughts and feelings of a songwriter unafraid to let it all hang out. It's a showcase of talent with a few really strong ideas, and with a little more tailoring on the vocal arrangements, it could really round off the rich soundscapes within.
Score: 3.5/5
Filed Under: Review
Sun King Records » "Dare To Hope" Review by Vinyl Chapters says:
[…] Read here. […]


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Cleanup to resume at Leviathan Mine Superfund site
GARDNERVILLE, Nev. (AP) – Water quality experts plan to renew treatment of polluted runoff this week from Leviathan Mine, the Superfund site that for years has belched a toxic stew of materials into a Carson River tributary.
This summer's work, which for the first time will include the participation of the Atlantic Richfield Co., could produce a "real breakthrough" in long-term efforts to clean a poisonous blight in the Sierra, an official from the Environmental Protection Agency said.
"This summer well really start to grapple with the long-term questions," Kevin Mayer, EPA project manager, told the Reno Gazette-Journal.
Declared a Superfund site in May 2000, the abandoned sulfur mine has leaked a toxic mix of acids and dissolved metals into two creeks that drain into the Carson River, rendering parts of them incapable of supporting aquatic life.
The mine's current owner, California's Lahontan Regional Water Quality Control Board, is expected this week to commence its third summer of chemically treating chocolate-colored runoff captured in the mines holding ponds, Mayer said. Once treated, the runoff can safely be released into streams, preventing an overflow of toxins such as occurred in the winter and spring of 1998 and 1999.
Mayer said the ponds can probably be emptied this summer at a treatment cost of about $1 million, making an overflow next winter highly unlikely even in the event of an extraordinarily wet season.
About $1 million will also be spent this summer by Arco, the company that owned the mine before it was acquired by California in the early 1980s, Mayer said. Arco's efforts will focus on capturing runoff seeping into creeks downstream from the holding ponds toxins which otherwise enter the watershed without any treatment whatsoever.
Located in Alpine County, Calif., 25 miles southwest of Gardnerville, Leviathan Mine produced copper sulfate from 1863 until 1872, then was reopened for a six-year period to produce sulfur beginning in 1936. From the 1950s to early 1960s, open pit mining was used to extract sulfur until the mine was finally shut down in 1962.


text:
is_enabled(EXAMPLES)
if (EXAMPLES)
	deploy(chapter01-greeting-example BUNDLES shell shell_tui log_service chapter01-greeting-example-client chapter01-greeting-example)
	deploy(chapter04-correct-lookup BUNDLES shell shell_tui log_service chapter04-correct-lookup)
	deploy(chapter04-correct-listener BUNDLES shell shell_tui log_service chapter04-correct-listener)

	deploy("hello_world" BUNDLES shell shell_tui apache_celix_examples_hello_world hello_world_test log_service log_writer)
	deploy("wb" BUNDLES tracker publisherA publisherB shell shell_tui log_service log_writer)
	deploy("wb_dp" BUNDLES tracker_depman publisherA publisherB shell shell_tui log_service log_writer dm_shell)
	deploy("echo" BUNDLES echo_server echo_client shell shell_tui)
	deploy("producer_consumer" BUNDLES producer consumer database shell shell_tui)
	if (NOT ANDROID)
		deploy("mongoose" BUNDLES shell shell_tui log_service apache_celix_examples_mongoose)
	endif ()

    #deploy("locking_example" BUNDLES benchmark_runner reference_benchmark start_stop_benchmark mutex_benchmark math_provider shell shell_tui log_service log_writer)

endif (EXAMPLES)


Ethos briefing proves a big hit with readers of The Lawyer.
This week's edition of The Lawyer carried our briefing on the importance to the Legal Sector of maintaining the security of data in print format.
First established in 1981 and with a print circulation in excess of 28,000, The Lawyer is a must-read title for the UK's Legal Sector. We were invited by the magazine to contribute a briefing on the vital importance of data security in light of recent announcements by the Information Commissioner's Office. The briefing was carried in this week's print edition and was also made available online to The Lawyer's 250,000 monthly visitors.
The response has been tremendous, underlining how legal practices of all shapes and sizes are seeking expert guidance on how to improve the security of the data. We work with over 3,000 companies, many of them in the Legal Sector including Bates Wells Braithewaite, Devonshires and The Bar Council.


text:
News February 05, 2021 at 08:00 am
Weeks before Floyd's death, three of the cops involved similarly roughed up an innocent Black man: video
Francis Akhalbey | Content Manager
Francis Akhalbey February 05, 2021 at 08:00 am
February 05, 2021 at 08:00 am | News
Francis Akhalbey is a Social Media techie, Writer and Content Manager. He loves basketball and dreams to make it in the NBA in his next life. I hope you're reading this LA Lakers! RiP Kobe and Gigi!! 8|24 #MambaMentality
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Derek Chauvin, Thomas Lane and J. Alexander Kueng were filmed roughing up an innocent Black man three weeks before their involvement in George Floyd's death -- Screenshots via the Star Tribune
Three of the same Minneapolis officers who were involved in the fatal George Floyd encounter were filmed roughing up an innocent Black man just three weeks before Derek Chauvin knelt on the 46-year-old's neck till he died.
According to the Star Tribune, Chauvin, together with officers Thomas Lane, J. Alexander Kueng and Luis Realivasquez, were responding to a May 3 call from a woman who claimed she was being held hostage by armed men at an apartment in South Side. Kueng and Lane had just recently joined the force and were on field training.
When the officers arrived at the scene, they met Adrian Drakeford – a Black man who also lived in the apartment building – as he was leaving the premises. Without any provocation whatsoever, the officers reportedly tackled him to the ground and handcuffed him, saying they thought he had a knife. It later turned out Drakeford did not have any knife in his possession. It was rather a knife sharpener and the officers later admitted that. Drakeford was also not one of the hostage-takers.
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In the video of the incident obtained by the news outlet, officer Chauvin can be seen observing Kueng and Lane handcuffing Drakeford – who is on the ground – while bystanders tell them to take it easy on him and also tell them he did nothing wrong. In response, Realivasquez tells the crowd to "back up" and pulls out a can of a chemical irritant. Drakeford's girlfriend and brother also witnessed the incident and tried telling the officers they got the wrong person.
"He didn't do nothing!" his girlfriend said.
Drakeford was later released without any charges filed against him. His brother was arrested and slapped with a misdemeanor charge of obstructing the legal process. That charge was, however, later dropped by the Minneapolis City Attorney "in the interest of justice" following a review. Officers could not also locate the woman who made the call and were unable to establish if she was still being held by the supposed armed men.
"It's not the style of policing you want to see any law enforcement practice," Andrew Gordon, deputy director for community legal services at Legal Rights Center, a Minneapolis-based nonprfit, told the Star Tribune. "Their interest is not necessarily about investigating a crime. … Their interest is to put these kids in their place." Gordon also represented Drakeford's brother.
In an interview, Drakeford's brother said one of the neighbors who was at the scene and was among the people calling out the officers, actually lived at the address the officers were called to. They, however, did nothing.
"They took me to jail and they didn't even investigate the guy they were there for," he said.
On May 25 (twelve days after the incident), Chauvin, Lane and Kueng responded to the 911 report of Floyd attempting to make a purchase at a grocery store with a counterfeit bill. Realivasquez was not involved in the Floyd encounter. Unlike Drakeford, Floyd died in the hands of the three officers.
"The timeline here is troubling," Gordon said. "The same officers are involved in the murder of George Floyd, using some of the same techniques and the need and desire to control people."
Drakeford's incident drew similarities to Floyd's death. Just like Drakeford, Floyd was aggressively tackled to the ground by Chauvin, Lane and Kueng. While Chauvin had his knee on Floyd's neck for almost 9 minutes, he ignored pleas from bystanders despite Floyd also telling him that he could not breathe.
Floyd's death triggered unprecedented nationwide protests against police brutality on Blacks and other minorities as well was racial discrimination in the United States. The protests ultimately went global.
Chauvin, Lane and Kueng are facing criminal charges in connection to Floyd's death. Chauvin faces a second-degree unintentional murder and a second-degree manslaughter charge while Kueng, Lane and another officer involved in the incident – Tou Thao – are charged with aiding and abetting second-degree murder and aiding and abetting second-degree manslaughter.
Prior to the Floyd and Drakeford incident, quite a number of misconduct complaints were reportedly brought against Chauvin. Gordon told the Star Tribune he was surprised Drakeford's wrongful arrest and the officers' failure to get to the bottom of the 911 call, coupled with Chauvin's misconduct complaint history, did not prompt any of their superiors to look into how these officers handle such encounters.


It´s Art Forum time in Berlin until October 3 and i am doing my best to cram as many exhibitions as possible in just 2 days. Yesterday i managed to visit only two of them, starting with Preview Berlin, "The Emerging Art Fair" with an aim to present art, still in its early stage, on the international art scene.
I spent what looks like an insane amount of time admiring Jonathan Delachaux´s eerie paintings at Haas & Fischer gallery. For more than ten years the painter has been working with three fictitious characters; he gave them names, a history (they used to be austistic but are now cured, thank you), took them on his trips around the world and made them accompany other artists. Delachaux forced them to be musicians and as their producer he recorded several CDs.
The gallery had also a selection of Chitra Ganesh´quirky works. At first sight they look like comics staring characters from the Hindu mythology but you read the dialogues you realize that they are meant to puzzle and comment on gender and social issues.
The Galerie Maisterravalbuena had a spectacular painting by Huanchaco, a very pop portraits that glues references to the cultural influences of the US, Europe and China on a very Mexican-looking painting.
Plenty of art pieces to like at the V1 Gallery. Especially Troels Carlsen´s primates. Carlsen examines the grey area between animal kingdom and realm of man, by portraying monkeys as senatorial candidates a century ago.
In other situations the concrete physiological borders are blurred. Wellknown anatomics are revised in a way that doesn't quite correspond with knowledge aquired in primary school's biology-classes. Troels Carlsen manipulates with genes resulting in a fusion between chimpansees and gibbons on one side and the human body on the other. This genetic hybrid makes it difficult to distinguish man from animal and animal from man.
Kunstagenten Contemporary Gallery had a remarkable installation to show off Thorsten Brinkmann´s self-portraits.
At the ArtMbassy booth, i loved Stefano Giuriati and Aldo Giannotti's photographic take on the Carabinieri – Stazione Mobile – the result of a performance made in Munich. Wearing Italian policemen' uniform, they just tried to blend into very German settings in a way that makes us question the concepts of our own homeland and also the concepts of the national borderline inside the European Union (via).
More images from Preview Berlin.
What is the name of the artist with the chicken installation (poules pictures) ?
Any better quality pictures somewhere on the net ?


2019 Ameri-Lite 188rb detailed specs.
Anyone know where I can find detailed specs for the 2019 ameri-lite 188rb, for example I know i want to replace the tires on this trailer (picking up sat.) , but, want to know whats on it before then. I've looked everywhere.
Thanks, but that doesn't say what size tires are on the trailers.
Suggestion; There are a few videos on youtube where salesmen are selling their campers. You could get a look and even ask them what's on it. Ask a salesman. They will talk your ear off.
Chineese no doubt. Surprised mine have gone over 10K w/o a blowout but seems a 13" D reated tire is a bit of a unicorn locally.
Nearly all trailer tires are made in China. Drive within their weight and speed ratings and you'll likely be fine. Keep in mind that MOST are rated for a max speed of 65. Judging by the folk passing me on the highway, that's one that many people exceed, and likely the cause of many blowouts.