text:
Shaping mechanisms of metal specificity in a family of metazoan metallothioneins: evolutionary differentiation of mollusc metallothioneins
The degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu+ or Cd2+. They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (
HpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd2+, Zn2+ or Cu2+ and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd6-HpCdMT and Cu12-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions.
The Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.
Metallothioneins (MTs) constitute a superfamily of genetically polymorphic cysteine (Cys)-rich polypeptides that bind, with high affinity, closed-shell metal ions such as Zn2+, Cd2+, Cu+ and others
Understanding how MTs and other metalloproteins achieved metal specificity through evolution is a key question in the study of their structure/function relationship
So far, all pulmonate MT isoforms have been obtained as native metal-MT complexes purified from snail tissues, where they are primarily involved in the metabolism of the metal ion species bound to the respective isoform
Analysis of recombinant HpCdMT and HpCuMT metal complexes reveals sequence-determined specialization of metal binding
Recombinant expression of
Binding of Zn2+ and Cd2+ by HpCdMT
Mass spectrometric analysis documents that recombinant synthesis of HpCdMT in
Figure 1
Deconvoluted electrospray ionization time-of-flight mass spectography spectra of the different metal-metallothionein (MT) complexes recombinantly synthesized
Deconvoluted electrospray ionization time-of-flight mass spectography spectra of the different metal-metallothionein (MT) complexes recombinantly synthesized: (a) HpCdMT obtained in Cd-enriched medium; (b) HpCdMT produced in Zn-enriched medium; and (c) HpCuMT synthesized in Cu-enriched medium under low aeration conditions. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis of these preparations indicated a respective mean content of 6.2 Cd/MT in (a) and 5.8 Zn/MT in (b) for HpCdMT and of 12.2 Cu/MT for HpCuMT in (c).
Figure 2
Ultra violet-
Ultra violet-
Binding of Cu+ by HpCuMT
The recombinant expression of
Complexes of HpCdMT and HpCuMT with non-cognate metal partners
In contrast to the single, well-defined MT species resulting from the recombinant expression of the
Figure 3
Electrospray ionization (ESI) time-of-flight mass spectroscopy (MS) spectra of the different metal-metallothionein (MT) complexes obtained when recombinantly synthesizing the MT peptides with their 'non-cognate' metal ion
Electrospray ionization (ESI) time-of-flight mass spectroscopy (MS) spectra of the different metal-metallothionein (MT) complexes obtained when recombinantly synthesizing the MT peptides with their 'non-cognate' metal ion. (a) HpCdMT produced in Cu-enriched medium and; HpCuMT in (b) Cd- and (c) Zn-enriched media, where M stands for Zn+Cu, due to the difficulty of ESI-MS to discriminate between these two metal ions. For the Zn and Cd-enriched preparations, the MS spectra show the +4 charge state (intensity
The recombinant synthesis of HpCuMT in Cd2+-enriched cultures led to mixtures of a number of Cd-HpCuMT species with a Cd content ranging from six to more than nine equivalents and also including a sulphide-containing Cd9S-HpCuMT complex (Figure
Therefore, these two MTs behave in accordance with their high metal specificity when recombinantly synthesized by bacteria grown in cultures enriched with the non-cognate metal. The HpCdMT isoform thus rendered several mixed Zn, Cu-MT complexes of different stoichiometries when biosynthesized in a Cu-rich medium. Following an equivalent behaviour, the HpCuMT isoform produced a mixture of species of different stoichiometry when synthesized by bacteria grown on Zn- or Cd-supplemented media. The classification of MTs according to the metal-binding behaviour shown when recombinantly synthesized in cultures enriched with different metals and the correspondence of this classification with other 'metal-specificity' criteria, have been fully reviewed
Stability of the Cd6-HpCdMT and Cu12-HpCuMT complexes is documented by their metal exchange inertness
In order to study the lability/inertness of the recombinantly synthesized Cd6-HpCdMT and Cu12-HpCuMT species and their propensity to exchange their preferentially bound metal ions, an equimolar mixture of these two complexes was allowed to stand for 20 h at 25°C (Figure
Figure 4
Electrospray ionization (ESI) time-of-flight mass spectroscopy (MS) spectra of an equimolar mixture of recombinant Cd6-HpCdMT and Cu12-HpCuMT, recorded at (a)
Electrospray ionization (ESI) time-of-flight mass spectroscopy (MS) spectra of an equimolar mixture of recombinant Cd6-HpCdMT and Cu12-HpCuMT, recorded at (a)
Transformation of HpCdMT and HpCuMT in yeast MT-knockout cells confirms metal-specific roles
In order to advance from metal-specific folding to metal-specific function, the particular performance of the two snail MT isoforms was compared by complementation studies in another heterologous system. Hence, yeast cells deficient in their endogenous MTs (yeast
Figure 5
(a) Copper and (b) cadmium tolerance evaluated by phenotype rescue on DTY4 [metallothionein (MT) deficient] yeast cells
(a) Copper and (b) cadmium tolerance evaluated by phenotype rescue on DTY4 [metallothionein (MT) deficient] yeast cells. Metal tolerance of each DTY4 strain transformed with different MTs, as indicated in the side boxes, is shown as a percentage of the growth rate exhibited in a non-metal-supplemented medium. Cup1 and Crs5 are the two yeast MTs, and MT1 stands for the mouse MT1 isoform.
In marked contrast, when the cells were grown in media with increasing concentrations of Cd2+, tolerance was greatest in the strain transformed with the endogenous yeast
These results show that the two snail MT isoforms also assume metal-specific roles in a heterologous eukaryotic environment (yeast), in accordance with their metal-specific binding preferences revealed by their synthesis in recombinant prokaryotic systems. Significantly, the total equivalence between the features of the metal-MT complexes synthesized in these two hosts (bacteria and yeast) has recently been demonstrated for both cadmium and copper, using the Cup1 MT as a model system
In pulmonate snails, CdMT and CuMT isoforms are products of cell-specific expression
In the midgut gland of the snail
Figure 6
Cell-specific visualizuation of HpCdMT and and HpCuMT mRNA, and isolation of both native expressed isoforms from snail midgut gland tissue
Cell-specific visualizuation of HpCdMT and and HpCuMT mRNA, and isolation of both native expressed isoforms from snail midgut gland tissue.
Pulmonate MT isoform genes display metal-specific transcription patterns
The pattern of metal-specific transcriptional induction of MT isogenes was examined in two pulmonate species: in
Figure 7
Real-time detection polymerase chain reaction (copy number/10 ng total RNA) of messenger RNA (mRNA) of
Real-time detection polymerase chain reaction (copy number/10 ng total RNA) of messenger RNA (mRNA) of
HpCdMT and HpCuMT as prototypes of isoform families which have evolved metal specificity in pulmonate snails by modulation of non-cysteine amino acid positions
HpCdMT and HpCuMT can be considered as prototypes of a series of orthologous genes also present, except from
Figure 8
Amino acid sequence alignments of terrestrial and freshwater pulmonate snail metallothioneins [MTs; CdMT, CuMT and Cd/CuMT]
Amino acid sequence alignments of terrestrial and freshwater pulmonate snail metallothioneins [MTs; CdMT, CuMT and Cd/CuMT]. Protein or nucleotide sequences were obtained from GenBank and are specified in the legend of Figure
The three isoform types share strictly conserved Cys positions in their primary structure, confirming the fundamental importance of the sulphur atoms provided by these residues for metal complex and metal thiolate cluster formation, irrespective of the metal species involved. Besides Cys, a few other amino acid positions, either on the N-terminal tail of the peptides or in the direct neighbourhood of Cys residues, show conserved positions through the members of the three isoform subfamilies (Figure
Table 1
Comparative protein Blast analysis of pulmonate metallothionein (MT) isoform families*
CdMT versus CuMT
CdMT versus Cd/CuMT
CuMT versus Cd/CuMT
*Protein BLAST calculation (blastp algorithm, NCBI tools) for testing similarities between the three pulmonate MT isoform subfamilies, exemplified by CdMT, CuMT and Cd/CuMT from
A nucleotide-based neighbour-joining tree shows that pulmonate MT isoform subfamilies are assembled in three distinct branches and are thus clearly distinguishable from all other mollusc forms represented by the group of Bivalvia (Figure
Figure 9
Nucleotide-based neighbour-joining tree of mollusc metallothioneins (MTs) comprising mussels and gastropods, with
Nucleotide-based neighbour-joining tree of mollusc metallothioneins (MTs) comprising mussels and gastropods, with
Figure 10
Amino acid sequence-based neighbour-joining tree of pulmonate metallothionein (MT) subfamilies represented in clusters of Cd/CuMTs, CuMTs and CdMTs, with
Amino acid sequence-based neighbour-joining tree of pulmonate metallothionein (MT) subfamilies represented in clusters of Cd/CuMTs, CuMTs and CdMTs, with
In MTs, metal binding and metal exchange reactions are mainly governed by the coordination chemistry of thiolate bonding with closed-shell metal ions such as Zn2+, Cd2+, Cu+
The structural features of the resulting MT complexes with different metal ion species arise from the equilibrium between kinetic and thermodynamic requirements
The complete sequential identity of Cys residues and the high degree of conserved positions for other amino acids shared among the three isoform families (Figure
Cell-specific expression may also contribute to enhancing distinct metal-related functionality
After exposure of Roman snails to Cd2+, virtually all of this metal in the digestive tissues was bound to HpCdMT (20) in a similar manner as for CdMT isoforms of other snail species
Overall, the present study, together with the extensive body of evidence provided by our previous work, suggests that the pair of the metal-specific
Animals and metal exposure
Roman snails (
Metal analyses
Metal-enriched salad samples were oven-dried at 60°C for several days. Dried samples (snail tissues: 50-100 mg dry weight; salad samples: 100 - 500 mg dry weight) were wet-digested in screw-capped polypropylene tubes (Greiner, Kremsmünster, Austria) with a mixture of HNO3 (suprapure; Merck, Darmstadt, Germany) and distilled water (1:1) by heating at 70°C for several days. At the end of digestion, a few drops of H2O2 were added to the heated samples. The remaining solutions were diluted to a known volume with distilled water and analysed for metal concentrations (Cd, Zn, Cu) either by flame (model 2380 instrument, Perkin Elmer, Massachusetts, USA) or graphite furnace atomic absorption spectrophotometry (Hitachi Z-8200) with polarized Zeeman background correction (Hitachi, Tokyo, Japan).
Real-time detection PCR
RNA sampling for real-time detection PCR was done in control snails and animals exposed to metals over a 5 day period (see above). This time range was chosen because, in pulmonate snails, maximal induction of the
Cell- and tissue-specific expression of both HpMT isoforms was demonstrated by
Construction of the recombinant expression vectors for wild-type Roman snail MT isoforms
Recombinant synthesis and purification of the metal-HpMT complexes
All HpMT metal complexes analysed in this work were biosynthesized in 2-L Erlenmeyer cultures of the corresponding transformed
For protein purification, cells were re-suspended in ice-cold PBS (1.4 M NaCl, 27 mM KCl, 101 mM Na2HPO4, 18 mM KH2PO4)-0.5% v/v β-mercaptoethanol, disrupted by sonication and centrifuged at 12,000
Analysis of recombinantly expressed and
The recombinantly expressed metal-MT complexes were analysed for element composition (S, Zn, Cd and Cu) by inductively coupled plasma atomic emission spectroscopy (ICP-AES) on a Polyscan 61E spectrometer (Thermo Jarrell Ash Corporation, MA, USA) at appropriate wavelengths (S, 182.040 nm; Zn, 213.856 nm; Cd, 228.802 nm; Cu, 324.803 nm), either under 'conventional' (dilution with 2% HNO3 (v/v)) or under 'acidic' (incubation in 1 M HCl at 65°C for 5 min) conditions
CD spectroscopy was performed using a model J-715 spectropolarimeter (JASCO, Gross-Umstadt, Germany) equipped with a computer (J-700 software, JASCO). Measurements were carried out at a constant temperature of 25°C maintained by a Peltier PTC-351 S apparatus (TE Technology Inc, MI, USA). Electronic absorption was measured on an HP-8453 diode-array ultra violet (UV)-vis spectrophotometer (GMI Inc, MN, USA), using 1-cm capped quartz cuvettes, and correcting for the dilution effects by means of the GRAMS 32 software (Thermo Fisher Scientific Inc, MA, USA).
Molecular mass determination was performed by electrospray ionization mass spectrometry equipped with a time-of-flight analyser (ESI-TOF MS) using a Micro Tof-Q Instrument (Brucker Daltonics GmbH, Bremen, Germany) calibrated with NaI (200 ppm NaI in a 1:1 H2O: isopropanol mixture), interfaced with a Series 1100 HPLC pump (Agilent Technologies, CA, USA) equipped with an autosampler, both controlled by the Compass Software. The experimental conditions for analysing proteins with divalent metals (Zn, Cd) were: 20 μL of the sample were injected through a PEEK long tube (1.5 m × 0.18 mm i.d.) at 40 μL/min under the following conditions: capillary-counterelectrode voltage, 5.0 kV; desolvation temperature, 90-110°C; dry gas 6 L/min. Spectra were collected throughout an m/z range from 800 to 2000. The proteins that contain copper were analysed injecting 20 μL of the sample at 30 μL/min; capillary-counterelectrode voltage, 4.0 kV; desolvation temperature, 80°C; m/z range from 800 to 2000. The liquid carrier was a 90:10 mixture of 15 mM ammonium acetate and acetonitrile, pH 7.0. For the analysis at acidic pH the conditions used were the same as those used in the analysis of the case for divalent metals, except in the composition of the carrier liquid which, in this case, was a 95:5 mixture of formic acid and acetonitrile at pH 2.4. All samples were injected at least in duplicate to ensure reproducibility. In all cases, molecular masses were calculated according to the reported method
Metal replacement titrations were performed by adding the corresponding metal ions (Cd2+ or Cu+) at equivalent molar ratios to the recombinant Zn-HpMT complexes. Titrations were carried out following previously described procedures
Metal tolerance complementation assays in transformed yeast MT-knockout cells
The cDNAs coding for the different MTs assayed - the two snail MT isoforms (snail HpCdMT and snail HpCuMT), the two yeast MTs (Cup1 and Crs5) and the mouse MT1 - were ligated into the
For metal tolerance tests, transformed yeast cells were initially grown in selective SC-Trp-Ura medium at 30°C and 220 rpm until saturation. These cells were then diluted to OD600 0.01 and used to re-inoculate tubes with 3 mL of fresh medium supplemented with CuSO4 added at 15, 30, 45, 60, 75, 90 and 105 μM final concentrations or CdCl2 at 1, 2, 4, 6 and 8 μM final concentrations. These cultures were allowed to grow for 18 h and the final OD600 was recorded and plotted as a percentage of the OD600 reached by the culture grown without metal supplement. For each concentration, and each kind of transformation, two replicas were run.
MT sequence alignment and phylogenetic analyses
MT amino acid sequences used for pulmonate MT alignments were derived mostly from amino acid sequence files or translated cDNA open reading frame sequences published in GenBank (
ATG: anti-thymocyte globulin; CD: circular dichroism; cDNA: complimentary DNA; ESI: electrospray ionization; FPLC: fast protein liquid chromatography; GPD: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione-S-transferase; Hp:
Authors' contributions
ÒP, AP, and SPR carried out the recombinant expression studies, participating in the construction of the expression vectors, the synthesis and characterization of the corresponding metal complexes, and also performed the yeast complementation experiments. ME and MH performed quantitative Real-Time PCR experiments and in situ hybridization, and carried out most of the sequencing work of MT cDNAs from different pulmonate snail species. AB calculated and established the phylogenetic trees. MC and SA designed the study together with RD, supervised recombinant DNA and recombinant protein experiments, yeast complementation studies, as well as analytical, spectrometric and spectrophotometric work, and contributed to the drafting of the manuscript. RD supervised and participated in molecular sequencing and Real-Time PCR as well as in situ hybridization, and performed chromatography and Reversed-Phase HPLC. He was responsible for the protein alignments and assisted in calculation of phylogenetic trees. He designed the study together with MC and SA, and drafted the manuscript. All authors read and approved the final manuscript.
This work was supported by the Spanish Ministerio de Ciencia y Tecnología grants BIO2009-12513-C02-01 to SA, BIO2009-12513-C02-02 to MC and project No. P19782-B02 from the Austrian Science Foundation to RD. Collaboration between the Spanish and Austrian research groups was financed by the 'Acciones Integradas' grants HU2006-0027 (Spain) and ES 02/2007 (Austria). SPR received a pre-doctoral fellowship from the Department de Química, Universitat Autònoma de Barcelona. Authors from the University of Innsbruck are members of the 'Centre of Molecular Biosciences Innsbruck'. The authors from the Universitat Autònoma de Barcelona (UAB) and Universitat de Barcelona (UB) are members of a 'Grups de Recerca de la Generalitat de Catalunya' (2009SGR-1457). We thank the Serveis Cientificotècnics of UB (GC-FPD, ICP-AES, DNA sequencing) and the Servei d'Anàlisi Química of UAB (CD, UV-vis, ESI-MS) for allocating instrument time to us.
Metallothioneins: unparalleled diversity in structures and functions for metal ion homeostasis and more
Metallothionein: An intracellular protein to protect against cadmium toxicity
A family knockout of all four
The elusive function of metallothioneins
Metallothionein expression in animals: a physiological perspective on function
Stoichiometry and cluster specificity of copper binding to metallothionein: homogeneous metal clusters
The four members of the
Domain specificity of Cd2+ and Zn2+ binding to rabbit liver metallothionein2
111Cd NMR studies of the domain specificity of Ag+ and Cu+ binding to metallothionein
Functional differentiation in the mammalian metallothionein gene family. Metal binding features of Mouse MT4 and comparison with its paralog MT1
Chemistry and biochemistry of metallothionein
Standard isolation procedure for metallothionein
Canine hepatic lysosomal copper protein: identification as metallothionein
Occurrence of cadmium in crabs (
How do bacterial cells ensure that metalloproteins get the correct metal?
Purification and primary structure of snail metallothionein. Similarity of the N-terminal sequence with histones H4 and H2A
Primary structures of a copper-binding metallothionein from mantle tissue of the terrestrial gastropod
Metallothionein in snail Cd and Cu metabolism
Metal distribution and metallothionein induction after cadmium exposure in the terrestrial snail
Spectroscopic characterization of metallothionein from the terrestrial snail,
Electrospray ionization mass spectrometry of Zn, Cd and Cu metallothioneins: evidence for metal-binding cooperativity
Localization and quantification of Cd- and Cu-specific metallothionein isoform mRNA in cells and organs of the terrestrial gastropod
Mechanisms of cadmium toxicity in terrestrial pulmonates: programmed cell death and metallothionein overload
Acute toxicity of cadmium and copper in hepatopancreas cells from the Roman snail (
Copper in
Zinc(II), cadmium(II) and mercury(II) thiolate transitions in metallothionein
Cadmium-thiolate clusters in metallothionein: spectrometric and spectropolarimetric features
Optical properties of metallothioneins. Origin of the optical properties of mammalian metallothioneins
Copper binding to rabbit liver metallothionein: formation of a continuum of Copper(I)-thiolate stoichiometric species
Zn- and Cd-metallothionein recombinant species from the most diverse phyla may contain sulfide (S2-) ligands
Independent metal-binding features of recombinant metallothioneins convergently draw a step gradation between Zn- and Cu-thioneins
Evidence of native metal-(S2-)-MT complexes confirmed by the analysis of Cup1 divalent metal ion binding properties
Structural and bioinformatic analysis of the Roman snail Cd-Metallothionein gene uncovers molecular adaptation towards plasticity in coping with multifarious environmental stress
Distinc Metal-binding Configurations in Metallothioneins
Circular dichroism, emission, and exafs studies of Ag(I), Cd(II), Cu(I), and Hg(II) binding to metallothioneins and modelling the metal binding site
Interprotein metal exchange reactions of metallothionein
Metallothionein gene duplications and metal tolerance in natural populations of
Cloning and characterization of a gene coding for a novel metallothionein in the Pacific Oyster
Role of selection in fixation of gene duplications
The molluscan rhogocyte (pore-cell, Blasenzelle, cellule nucale), and its significance for ideas on nephridial evolution
Rhogocytes (pore cells) as the site of hemocyanin biosynthesis in the marine gastropod
Copper-Metallothioneins in the American Lobster,
Three-dimensional solution structure of
Metal regulation and molting in the blue crab,
Short-term metallothionein and copper changes in blue crabs at ecdysis
Copper Metallothionein of yeast, structure of the gene, and regulation of expression
Copper Metallothionein from the fungus
Primary structure and spectroscopic studies of
Copper metallothionein, a copper-binding protein from
Purification of cadmium-binding proteins from related species of terrestrial helicidae (Gastropoda, Mollusca): a comparative study
Metal accumulation in
Metal ion interactions with
The binding of Cd(II) to the hemocyanin of the Mediterranean crab
Subchronic effects of environment-like cadmium levels on the bivalve
Spectroscopic properties of metallothionein
Signalling events for metallothionein induction
Isoform-specific quantification of metallothionein in the terrestrial gastropod
Binding of excess cadmium(II) to Cd7-metallothionein from recombinant mouse Zn7-metallothionein 1. UV-VIS absorption and circular dichroism studies and theoretical location approach by surface accessibility analysis
Micromolar protein concentrations and metalloprotein stoichiometries obtained by inductively coupled plasma. Atomic emission spectrometric determination of sulphur
Retention of thiol protons in two classes of protein zinc ion coordination centers
Recombinant synthesis of mouse Zn3-α and Zn4-α metallothionein 1 domains and characterization of their cadmium(II) binding capacity
A new insight into the Ag+ and Cu+ binding sites in the metallothionein β domain
Superoxide dismutase activity is essential for stationary phase survival in
Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds
Manipulating yeast genome using plasmid vectors
Clustal W and Clustal X version 2.0
The neighbour-joining method: a new method for reconstructing phylogenetic trees
Confidence limits on phylogenies: an approach using the bootstrap


text:
Cave and his beardy Bad Seeds/Grinderman sidekick Warren Ellis have become prolific soundtrack composers of late, as shown by 2009's White Lunar, a retrospective collection that has, to be frank, probably been heard more often than the accompanying films were seen.
The Road, a John Hillcoat adaptation of a Cormac McCarthy novel, is a tale with a post-apocalyptic backdrop, and its themes of "absence and loss" audibly informed the writing of the score: a lesson in atmospherics, even if you never watch the film.
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- Film Music
- Nick Cave
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text:
Citation Edit
Sony Music Entertainment v. Does 1-40, 326 F.Supp.2d 556 (S.D.N.Y. 2004) (full-text).
Factual Background Edit
Using a publicly available database and information gleaned from the P2P service "Fast Track," the record companies plaintiffs were able to determine that Cablevision was the ISP for a number of Internet users believed to be using "Fast Track" to upload and download copyrighted recordings. But in order to determine the names and addresses of "Fast Track" users, the record companies had to sue them as "Does" and then serve a subpoena on Cablevision seeking that information.
Trial Court Proceedings Edit
One of those sued as "Jane Doe" made a motion to quash the subpoena, arguing that it violates her First Amendment right to engage in anonymous speech. Judge Chin acknowledged that the First Amendment does protect anonymous speech. And he agreed with "Jane Doe" that uploading and downloading copyrighted recordings "qualifies as speech entitled to First Amendment protection." Significantly, however, Judge Chin added, "That protection . . . is limited, and is subject to other considerations."
To evaluate the record companies' subpoena to Cablevision, the judge said that the First Amendment interests of "Jane Doe" must be balanced against the record companies' "need for disclosure." Judge Chin concluded that the record companies' needs were weightier.
The judge identified five record company interests that lead to his conclusion: (1) the record companies had made a "concrete showing" of copyright infringement; (2) the subpoena was specific; (3) there were no other ways for the record companies to determine the identities of Cablevision's subscribers; (4) the subpoenaed information was central to the record companies' case, because without it, they could not serve the complaints they had filed; and (5) Cablevision subscribers "have little expectation of privacy in downloading and distributing copyrighted songs without permission," because Cablevision's own subscriber agreement informs subscribers that they may not transmit copyrighted material without authorization, and that Cablevision has the right to disclose subpoenaed information.
As a result, Judge Chin ordered Cablevision to respond to a subpoena served on it, as the Internet service provider for 40 suspected P2P users who have been sued for copyright infringement by the 17 record companies.


text:
PANDANUS - Beach living
House, New Brighton
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If your mind and body crave relaxation, time and space to meditate then Pandanus right on the beach is the place for you. Absorb the peace and solitude of this beautiful holiday home, which has been designed to blend with the surroundings. Interiors have been especially designed and decorated to reflect the contemporary style while maintaining its seaside aesthetics. Only a step from the deck and you can feel the sand between your toes. A few steps more and you are transported to a beach of solitude and sweeping expanse with no sounds other than the surf, the birds, the wind and Mother Nature. If fishing is a passion then don't forget your surf rods. The beach has deep gutters just in front of Pandanus. Pippies and beach worms are plentiful towards low tide or you can buy your bait locally. This is a very special place and we look forward to welcoming you as our guest.
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Lutheran Social Services of the Southwest (LSS-SW) announced that the Rev. Robert E. Duea will serve as interim president and CEO.
Duea will work from the LSS-SW administrative offices at 10201 S. 51st St., Suite 180 in Phoenix. For 27 years, he led Lutheran Family Services of Oregon and LSS of Wisconsin and Upper Michigan. Prior to working with Lutheran agencies, he served as pastor of a congregation in Bethesda, Md. During his LSS-SW tenure, he will focus on defining leadership needs and strengthening LSS-SW's faith-based partnerships.
LSS-SW, a pan-Lutheran organization, was started in 1970 to care for the most vulnerable by showing kindness, doing justice and serving those in need. LSS-SW partners with individuals, congregations and agencies in 10 Arizona counties to offer services and resources that inspire hope and create a stronger, healthier community. To learn more about LSS-SW ministries, call (480) 396-3795 or visit lss-sw.org.


text:
Tuesday, 25th July 2017
Jain temple, Oshwal Centre - Potters Bar, Hertfordshire
At Diverse Ethics, we have developed a unique approach to building bridges and training leaders about the beauty and multi-dimensional potential of diversity - we organise guided cultural experiences, which combine art, music, festive stories and rituals, food and dialogue. Our guests come away with a truly memorable experience, and a deeper understanding of the complexities and opportunities of living and working in multicultural Britain.
You are a unique leader in the UK, and we would like to invite you to such an experience, and meet other like minded people from different organisations. Some of the results of our previous tours have been written up in the form of articles and videos which you can find linked here.
The venue is headquarters of one of the largest Jain communities in UK, the Oshwal Association. You will experience a five hundred year old Ayambil festival, which lasts nine days, and involves prayer and fasting for purification of the soul. The food and recipes are developed in such a way as to give energy and vitality for this quest. The diversity of ingredients and recipes show how deep such a culture is ingrained into everyday life of compassion, service and respect for all living beings. The event is run and managed by volunteers from the community.
Date: Friday, 14th October 2016
Venue: Oshwal Centre, Coopers Lane Road, Northaw, Hertfordshire, EN6 4DG
Time: 11 am to 3pm
Outline - Welcome and guided tour, Festive lunch, Meeting with community, Q & A and dialogue
Host: Oshwal Association of UK
Directions: By car, it is just two miles off Exit 24 of the M25 for Potters Bar. There is plenty of parking. By rail, it is a few miles from Cockfosters Tube station on Piccadily Line, by taxi.
We very much hope you can make it. Kindly RSVP by 30th September 2016.
Dr. Atul K. Shah, Tel 07804294903
Director, Diverse Ethics, A Social Enterprise
Article added on 16th September 2016 at 3:13pm
Tweets by @atulkshah


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This is our second time staying here. Last year we thought it was great so we booked again for couple of nights this year during spring break. Hotel was a little more expensive this time, but it was their high season.
This year our room seemed a little smaller. Bed was fine. Bathroom was nice with a walk in shower and nice amenities. This time we had a chance to visit the pool and hot tub. Pool was very clean. Hot tub was nice as well. Breakfast is included and is much better than your typical continental breakfast. They have on duty staff to prepare omelets, etc as requested. In addition to the normal items, also had fresh fruit and oatmeal. In the evening there is a manager's reception where you can get drinks, sodas, etc in the breakfast area.
View is nice. Hotel is located in an upscale area...not too many options for dining unless you go back into town. Front desk suggested a couple of places for us and they were fine. Upscale shopping plaza located across the street.
If you are going to the airport, it is about a 45 minute drive, but traffic was not bad mid morning.
- Official Description (provided by the hotel):
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Breath-Taking Kodak Ektachrome Infrared EIR Photographs by Aldo Altamirano2013-10-04 16 Share Tweet
We know you're all convinced that Kodak's infrared film is one rare gem in the analogue-loving world, and we're even more sure that the infrared snaps you'll find after the jump will simply blow you away!
Among the most coveted emulsions in the film photography world, infrared films like Kodak's Aerochrome and Ektachrome Infrared EIR are what lomographers' dreams are made of: richly saturated photos dripping in funky colors. The foliage turns red, orange, pink, and purple, depending on the filter used. The result? Surreal landscapes that would surely delight the master surrealist Salvador Dali himself.
The beautiful photos below, taken by Aldo Altamirano with a Nikon F6 (AF Nikkor 50mm f/1.4D with B+W #099 IR filter) and a 35mm Kodak Ektachrome Infrared EIR makes him one of the luckiest photographers ever. Reading his Flickr photo and description of this film, it's certain that he's aware of this as well:
"This is an almost impossible-to-find film, if you are lucky, you may be able to find one roll frozen on eBay for about $60 (US dollars as per 2012). There are many reasons for that; it has been discontinued years ago, it was made in small batches, it only last about a month out of the fridge and it was an expensive film already in its time, about 5 times more than a regular roll."
As for his thoughts on shooting with film, he says:
"To me, film just looks better. The way I see the world is the way it is captured on film— the subtle tones (or the vivid ones), the grain, the contrasts, the imperfections, the atmosphere and mood. There are many reasons why I love film but it's one of those things you have to try to learn to love it and it can't be described with words.
"There is no 'delete' button, no LCD screen to look at, no instant gratification, film makes you slow down in this crazy world where everything needs to be fast.
Like Vincent Laforet said 'As the world continues to move faster and faster, as processors, resolutions, dynamic ranges, and terabytes keep increasing at an exponential rate, sometimes I think we could all do with a roll of 36 exposures.'"
Follow Aldo Altamirano and see more of his Kodak Ektachrome Infrared EIR photos on Flickr!
written by plasticpopsicle on 2013-10-04 #lifestyle #eir #lomography #analogue-lifestyle #infrared-photography #kodak-ektachrome-infrared-eir #kodak-infrared-eir #aldo-altamirano


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COLUMBIA — Two people died and several others were injured in a Caldwell Parish crash Saturday.
Troopers said a car was traveling east on La. 4 east of Columbia when the driver lost control of the vehicle in a slight curve and slid across the center lines into the path of a westbound car.
A 22-year-old passenger in the first car, Jalisa Harrison, of New Iberia, was pronounced dead at the scene. A 7-month-old infant was taken to a hospital in Columbia where he died a short time later.
The driver of the other car, 19-year-old Amber Benoi, of Jeanerette, was taken to a West Monroe hospital with serious injuries.
Benoit was issued a citation for driving left of center.


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Shahrukh Khan responded very well when he was ranked at 3rd postion after followed by Aamir Khan & Salman Khan.
Here is the question:
.@iamsrk how does it feel to be in the 3rd spot behind Aamir-Salman? #AskSRK ?
— दंगल:Christmas 2016 (@Ak5hayP) May 17, 2015
Here is what Shahrukh Khan replied:
@Ak5hayP I am actually in the last spot. The backbencher and that's why i have most fun….
— Shah Rukh Khan (@iamsrk) May 17, 2015


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Before I add my client to access their Campaign Monitor account, I just want to be sure that they cannot see how much I have marked up the prices. Once I provide them access, can they see this information?
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iIDEAS (IDEAS for the Internet) is an integrated suite of Web-enabled applications that implement a new vision of financial management. These applications enable organizations to significantly improve productivity by leveraging the global networking capabilities of the World Wide Web.
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If Love Were Oil, I'd Be About a Quart Low
A veteran of the Divorce Wars speaks out ... on growing up (Happiness is a Red-Headed Cheerleader), first love (She Wore Spaghetti, But I Loved Her Anyway), marriage (Panty Hose on the Show Rod and Raisins in the Rice), and divorce (Loneliness and No Clean Underwear). Grizzard even offers advice to the young (Never Kiss a Lady When Her Mouth is Full of Snuff) and how his mother gave him fresh "hand-squoze" orange juice and spoiled him for the three women he would someday marry -- and divorce. With the down-home humor that has made him one of America's most popular newspaper columnists, and poignant insights into his own life and loves in these modern times, here is Lewis Grizzard at his bittersweet best.
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- This article is a stub. You can help the Grimorum by expanding it.
The young Gargoyles' attempts to help Elisa interfere with a gang war is complicated with the males fighting over the right to court Angela.
- Angela is revealed to have fifteen Rookery sisters on Avalon, which interests the trio.
- Brooklyn asks Goliath when he and the others get their Avalon World Tour. This is the source of the name for the arc.
- Brooklyn would later go on his own version of the tour, in the TimeDancer storyline.
- Lexington pursues Angela just as much as his rookery brothers. This could be considered odd, because according to Greg Weisman, Lexington is gay.
- Angela would eventually choose to mate with Broadway.
- This episode takes place on the same night as Vendettas.
Memorable Quotes
"And one more thing; DON'T CALL ME ANGIE!" Angela complaining to the others about her nickname.
"Don't back off; slow down," Angela telling the trio to, well, slow down.
Episode Guide


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I'm a composer, instrument builder, and field recordist. I write and perform experimental and electronic music, build sound environments, and develop software for music representation and performance.
Recent creative interests have included critical explorations of instruments and found objects, live animated interactive scores, and feedback cycles between performers and stochastic processes or acoustic automata.
My music has been performed in North America, Europe, and Asia. Past collaborators include Nouvel Ensemble Moderne, ensemble mise-en, the NOW Ensemble, Ensemble MotoContrario, and folk duo Undlin & Wolfe. I'm currently a masters candidate in the Digital Musics program of Dartmouth College. I will be pursuing a Fulbright research fellowship in Mysore, India in 2017-18.
(download cv | short bio | long bio)


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Cam Newton's Post-Game Hat Choice Becomes Instant Meme
Sports| | By Mauricio Castillo
unique style. Coming off a 46-27 throttling of the San Francisco 49ers Sunday afternoon, the Panthers star got to the podium of his post-game press conference dressed sharply in blazer and bowtie, but it was his choice of headwear that has the internet ablaze.It is no secret that across all sports the biggest stars are held under the biggest microscope. Everything they do, everything they eat, everything they say is always being analyzed, criticized, and (mis)interpreted. This is also true for their fashion choices. Quarterback Cam Newton of the Carolina Panthers has always had a
Channeling what can only be described as 1920s vintage style, Newton's hat has instantly turned him into a meme as well as its own Twitter handle. Here are just a few examples of how much fun Tweeters are having with this:
Who wore it better?RT for Me.
FAV for Cam Newton. pic.twitter.com/xo1VrWL8k6 — Cam Newton Hat (@CamNewtonsHat) September 18, 2016
I like to be on Cam's head, or at the local state fair handing out prizes for knocking down the glass bottle pyramid. #CarolinaPanthers— Cam Newton Hat (@CamNewtonsHat) September 18, 2016
When you got a football game at 1 but the 1947 Republican National Convention at 5 pic.twitter.com/DnYS0ZRWMO— NFL Memes (@NFL_Memes) September 18, 2016
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"Were you sent here by the devil?" pic.twitter.com/pYOQTdYnYa— Barstool Sports (@barstoolsports) September 18, 2016
September 18, 2016
I thought someone photoshopped that hat on Cam Newton.— Kara Clark (@karaclark) September 19, 2016
It doesn't matter that Newton threw for over 300 yards with three touchdowns in Sunday's win. The important thing is that the internet is always here to make fun of everything.
Remember when Cam Newton wore that hat? Pepperidge Farm remembers. pic.twitter.com/7rcqWtZHJk— NFL Memes (@NFL_Memes) September 18, 2016


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Aaron Paul Gossip
Aaron Paul gossip, latest news, photos, and video.
Jesse Pinkman in the front row
Gareth Cattermole/Getty
This is for Maria who's mad at me because I've yet to email her back about Aaron Paul at TIFF which is not even half as interesting (and mostly hilarious) as these photos of Aaron Paul front row at Burberry (I'll post the full collection to LifeStyle Full Story
Season 5 Episode 8: A dead man's thoughtfulness
Breaking Bad Season 5 Episode 8 recap I had a long intro that I worked on over the week: it was about Mike's loyalty to his crew and Walt's constant demand for gratification and accolades. But once I watched the episode I scrapped it because Breaking Bad moves quickly; yes it is layered, nuanced and complicated, but it also tells a linear story that evolves and grows even if, as viewers, we aren't emotionally done with a character yet. Full Story
Season 5 Episode 3: Cooking Under the Big Top
Frederick M. Brown/Getty
Breaking Bad Season 5 Episode 3 recap A suit walks into a jailhouse to see his client, with Mike posing as his paralegal (that's a pretty brilliant ruse). The client is Dennis, one of 11 from Lydia's list. Dennis swears he won't flip, but there are 9 people left (Chow and his killer were crossed off last week) and if he doesn't someone else will. Full Story
Breaking Bad Season 5 Episode 2: Madrigal
I'd like to rename this episode Prophylactic Measures in honour of Mike. He has a certain eloquence to him, no? Scene opens in a lab, where a Christopher Walken-esque old dude is trying out an array of sauces, while a team in white lab coats anxiously await his opinion. The head scientist is going through the flavours (the entire conversation is in German): versions of honey mustard, French dressing/Ranch fusion (Franch!) and smoky mesquite BBQ are presented. Full Story
Live Free or Die
Kevin Winter/Getty
Breaking Bad Season 5 Episode 1 recap Walt has hair! On his head! He looks so different, dressed in casual, slightly dirty clothes, unkempt beard, no wedding ring and black-framed glasses. He shows the chatty diner waitress a fake ID to get a free , greasy birthday breakfast (and to shut her up). Full Story
We're Running Out of Time Together
I've come to a very sad realization that I can never be with Aaron Paul in real life. It's not because I'm married (on the Freebie 5 physical limits do not exist), but because he loves concerts. I hate concerts. Could we overcome this obstacle? I just don't know. Promotion for the season 5 premiere of Breaking Bad this Sunday on AMC (set your DVR, yo!) is in high gear. Full Story
Breaking Bad Season 5 preview: Season 4 review
"I am the one who knocks." -WW Poison figures prominently this season. Poison is how these people choose to kill because it's underhanded, precise and can be done with a smile. Poison has to be administered by someone close, someone who appears to be an ally. But it's not just literal poison; Walt is a poison, too. Full Story
Aaron Paul is not Jesse Pinkman
(Lainey: Ready for the final season of Breaking Bad? The recaps are coming. But first, every week leading up to the premiere, Maria's posting previews. Then we'll be recapping the new episodes.) Aaron Paul is not Jesse Pinkman. But in my mind he is. Because I want him to be. Aaron Paul doesn't do a ton of press; I don't know if that's by design or by demand, but this two-page New York Magazine piece Full Story
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Category Archives: Running
Thank you Iron Girl!
Disclosure: I received a comp'd entrance fee to run the Iron Girl 1/2 marathon in exchange for this post. For the past four months I've been training to run the Iron Girl 1/2 marathon in Clearwater. I've been thinking about … Continue reading
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February Training Recap and Xtrain Cathe DVD review
Another month done in preparation for Athleta Iron Girl's half marathon in April. February started with big plans of multiple running days and completion of the Nike+ training xbox video game. As the month went on, I kept on my … Continue reading
Posted in Running Tagged Cathe Xtrain DVD review, half marathon training, IronGirl, product review, running 2 Comments
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On January 17, 2013, I passively set a goal to run the Iron Girl half marathon in Clearwater this April. I say "passively" because December through April is our busy season at the market. We work 6 days a week … Continue reading
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text:
Alexandre Gama is a Brazilian publicist and entrepreneur who has broken down barriers and achieved success in a wide array of fields. He worked his way up in the field of advertising, but has accomplishments in a variety of genres including the music industry, fine arts, and luxury sports car manufacturing. He is known for helping turn companies into overnight success stories, and for the many awards his projects have won over the years.
Gama's flagship business is Neogama. It is one of Brazil's 20 largest advertising agencies and experienced massive growth in its first few years of existence. The company has been awarded several Golden Lions over the years by the Cannes Film Festival. It developed into an international enterprise when it merged with British BBH in 2002, but ended that partnership in 2012 when it shifted its focus back to the Brazilian market.
Not only has Alexandre Gama been successful in his many business ventures, he has repeatedly been the first Latin American and Brazilian to do so. He has managed to expand the boundaries of the field of advertising and gain accolades for his country.
Source: https://pt.wikipedia.org/wiki/Alexandre_Gama_(publicit%C3%A1rio)


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Enjoy Your Rotis With Zero Oil Masoor Dal
Enjoy A Protein Rich Whey Soup For A Stronger Lifestyle
Is There Any Natural Way To Heal My Cracked Heels?
What Are the Easy Pathways To Healthy Skin?
Slow Down The Ageing Process With Anti-Ageing Breakfast P...
Enjoy Your Favorite TV Shows With Healthy Roasted Garlic ...
Make Your Bathroom A Virtual Garden
How To Stop Mosquito From Biting You?
What Are The Ways To Comfort A Crying Baby?
Are Baby Creams And Lotions Adequate For Baby Skin?
How To Keep Off The Lizard Through Natural Remedies?
Home remedies for mosquito bites
Organic Ways to Remove Marks...
Turmeric Benefits: The Gold Ingredient...
Roll on essential oils for...
How to Reduce Stretch Marks...
Till now, you have been experimenting with several beauty creams to make your skin and hair look beautiful. You must have succeeded with some products while others may have caused some side effects. Have you ever thought of trying natural essential oils? Yes, you heard it right? Natural oils are an answer to all skin and hair related problems, be it acne, ageing or dry hair. So, add these herbal oils in your daily beauty regime and flaunt your skin and hair wherever you go. And, to top it all, they do not have any side effects as well.
You are now all grown up leaving puberty way behind but the acne problem still persists. You have tried all sorts of creams and treatments to get rid of pimples but they appear again and again. Well, worry not! Just try tea tree oil. It has anti-fungal properties which keep bacteria at bay. Besides, thyme and calendula oil too help in treating acne owing to the anti-bacterial agents present in them. Learn more about the properties of tea tree oil here.
Every time you comb your hair they stretch and cause pain. Also, there is no shine in them. Instead of opting for over-the-counter conditioners, try out argan oil, which is a great moisturizing essential oil. The vitamins and fatty acids present in it nourish hair follicles, thus making your hair soft and shiny. You can try other oils like avocado and castor. Also, coconut oil and hot olive oil deeply nourish the scalp, making your hair soft and smooth.
Coconut oil is a great cooking oil as well. To know about its benefits, click here.
If you have been yearning for long and lustrous hair, then try out ylang-ylang oil. It not only prevents your hair from damage due to salty water but also keeps them strong. Jamaican black castor oil is another great option for good hair growth.
When essential oils are mixed in body scrubs, they not only provide nourishment to the exfoliated skin but also ensure regeneration of new tissues. To make a scrub at home, mix almond oil and sea salt together. Gently exfoliate your dead skin with it. Make sure that salt pieces are not big enough otherwise they may hurt you. This scrub works well for dry and chapped lips as well.
Almond oil has several other beauty benefits. To see how, click here.
If you wish to do away with your face's natural oil, just dab some essential oil on your face. All you need is either grape seed or jojoba oil. Take a dollop of it and massage on your face. Later, wipe your face with warm wet towel. You will definitely feel refreshed after that.
Jojoba oil has several other benefits. To know more, click here.
To get a glowing and marks-free skin, all you need to do in incorporate rose hip and jasmine oils in your beauty regime. Since rose hip oil is a rich source of lycopene and vitamin C, it helps in skin rejuvenation. While jasmine oil works on stretch marks and fine lines and maintains proper elasticity.
Ageing is something which each one of us dreads. We cannot stop the skin from getting wrinkles and fines lines but can delay them for sure. Essential oils like geranium, frankincense and patchouli can really help in fighting ageing signs. Since geranium oil boasts of astringent properties, it therefore tightens the skin. The other two oils too have the same anti-aging effects. However, they also keep natural skin oil under control and can treat eczema and dermatitis.
You need not buy any toners and cleansers from the market to remove makeup. All you need is castor, olive and sunflower oils. Just mix equal proportions of all three oils. Use a cotton ball to apply this mixture on your face. Gently wipe off all the makeup. Rinse your face with lukewarm water.
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Product Spotlight
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Phillip Morris Wins Appeal
Court ruled the company not in violation of unfair competition laws.
RICHMOND, Va. -- A federal appeals court in Richmond, Va., ruled Philip Morris USA does not violate unfair competition laws by exchanging discounts on Marlboros for prime display space in retailers' stores.
This followed the lawsuit by R.J. Reynolds Tobacco Co. against Phillip Morris in April 1999, claiming the company was trying to shut out competition. Lorillard Tobacco Co. and Brown & Williamson later joined the lawsuit against Phillip Morris.
U.S. District Judge Frank W. Bullock Jr. in Durham, N.C., originally ordered Phillip Morris to stop, but in May 2002 dismissed the lawsuit, writing the program, "has induced rivals to compete more vigorously."
R.J. Reynolds appealed and July 2002, and yesterday said it is considering a further appeal, according to reports.


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If you were given a chance to choose the last meal that you will have in your life,what would it be?
Wow! ... Steak and potatoes maybe some sauerkraut
Steak, shrimp, baked potato with sour cream, caesar salad, a cold beer, and for dessert i would want red velvet cake and ice cream. oh and a loaf of french bread with my meal.
Pasta with garlic butter sauce and Parmesan cheese sprinkled on top. Cesare salad with extra croutons. A fruity alcoholic beverage in a big, fancy cup. And for dessert strawberry cheesecake.
About Jessie
Birth: September 15
On Moms.com since: Apr 11, 2014
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Got New Carpet? Keep it Looking That Way
Vacuuming. That's right. Just one little verb will add years to the life of your carpet. There are a few other things you need to do, too, but this is one thing you need to do regularly from the time your carpet is installed until its last breath. Now read on to find out the other ways to stretch your hard-earned dollars.
Preventive Maintenance Sounds as pleasant as pulling a tooth, huh? All it really means is using doormats and taking off your shoes. These two super simple measures cut down on the amount of soil brought into your house and onto your carpet. Doormats trap dirt that you bring in on your shoes, and that's less dirt to soil your carpet and damage the carpet fibers.
Vacuum Regularly It's soil in carpet that breaks down the carpet fibers. Carpet grabs dirt, where it gets trapped, drops down to the pile, and then grinds down the carpet fibers, kind of like fingernails on a chalkboard. The key is to remove that dirt before it causes damage.
Spill, Spot and Stain Removal An untreated spill becomes a spot, and a spot becomes a stain, and each one is increasingly harder to get out of your beautiful floor coverings. We totally get that you can't watch that new puppy or messy toddler or sloppy teen all the time, but do your best to mop up that spilled soda or your dog's liquid gold immediately. If you don't, the dyes could penetrate the carpet and magically reappear for years to come! Use plain paper towels or white absorbent cloths, and always blot – don't scrub! Use spot and stain cleaners, and follow the manufacturer's directions. If the spill was legendary and seeped into the carpet backing, you might have problems with the stain reappearing, but persistence is the name of the game. With pet accidents, be sure to use stain and odor removers that keep Fido from re-soiling the same area. (Unfortunately we don't know of a product to keep your toddlers and teens from making lots of messes.)
Regular Cleaning Most carpet manufacturers recommend cleaning your carpet every 12-18 months in addition to regular vacuuming and spill/spot removal. You're in luck! Simplicity makes a carpet extractor with detergent and pre-treatment, and we make a dry carpet cleaner with pre-treatment, in an original formula and a formula just for pet owners. Simplicity's Healthy Home "Take Your Carpet to the Cleaners" dry carpet cleaner and "Carpets Dirty? I'm Your Solution" extractor detergent will not damage your carpet or erode its stain-resistant qualities.
The Carpet and Rug Institute keeps a list of vacuums that have the CRI Seal of Approval and approved cleaning products and carpet extractors that are good for your carpet – and the environment. Simplicity products have gone through extensive testing to meeting this organization's criteria so that we can feel good about our products, and you can feel good about how well you're maintaining your carpet.
So get moving, and take care of that carpet! You'll have the best looking carpet on the block and the satisfaction of knowing you've stretched that carpet investment wisely.


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Gamblers online
Online gambling is a type of gambling that is done online with the help of internet. The year 1996 witnessed the establishment Gaming Commission of Kahnawake, that helped in regulating gaming activity online from the Territory of Kahnawake (Mohawk) and also helps in issuing gaming licence to various online casinos and poker rooms present all over the world. In the year 1994, Antigua as well as Barbuda together passed Free Trade as well as Processing Act that allow or grants licences to organisations which apply to open casinos online.


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The global pipeline of new GM crops: introduction to the database
- Authors: Alexander J. Stein, Emilio Rodríguez-Cerezo
- EUR Number: 23810 EN (Techical Note)
- Publication date: 5/2009
This database is the outcome of a workshop on the "Global commercial pipeline of new GM crops" that was organised by the European Commission's Joint Research Centre, Institute for Prospective Technological Studies (JRC-IPTS), on 12-13 November 2008 with international experts and stakeholders involved in the development, regulation and commercialisation of GM crops worldwide. The main objective of the workshop and subsequent desk research by the JRC-IPTS was to compile a global pipeline of new GM crops that are currently in the regulatory pipelines of various countries (soybeans, maize, rapeseed, cotton, potatoes, rice and "other" crops) and that are to be commercialised in the short to medium term (until 2015) by private and public technology providers. The database lists the status of individual GM "events". So-called "stacked" GM crops are not listed; the regulatory approach to stacks differs across the world and their situation is discussed in detail in the forthcoming workshop report.
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Powder Mountain's 18 gauge panels are heavier than most utility panels yet are competitively priced. The panels are ideal for horse corrals, round pens, training circles and backyard roping arenas.
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E. crus-galli is an annual grass, culms 30-200 cm, spreading, decumbent or stiffly erect; nodes usually glabrous or the lower nodes puberulent. Sheaths glabrous; ligules absent, ligule region sometimes pubescent; blades to 65 cm long, 5-30 mm wide, usually glabrous, occasionally sparsely hirsute. Panicles 5-25 cm, with few-many papillose-based hairs at or below the nodes of the primary axes, hairs sometimes longer than the spikelets; primary branches 1.5-10 cm, erect to spreading, longer branches with short, inconspicuous secondary branches, axes scabrous, sometimes also sparsely hispid, hairs to 5 mm, papillose-based. Spikelets 2.5-4 mm long, 1.1-2.3 mm wide, disarticulating at maturity. Upper glumes about as long as the spikelets; lower florets sterile; lower lemmas unawned to awned, sometimes varying within a branch, awns to 50 mm; lower paleas subequal to the lemmas; upper lemmas broadly ovate to elliptical, coriaceous portion rounded distally, passing abruptly into an early-withering, acuminate, membranous tip that is further demarcated from the coriaceous portion by a line of minute hairs (use 25× magnification); anthers 0.5-1 mm. Caryopses 1.3-2.2 mm long, 1-1.8 mm wide, ovoid or oblong, brownish (Michael, 2003).
The youngest leaf is rolled, the plant more or less slender with stems up to 90 cm (35.43 inch) tall.stems Erect or decumbent, caespitose.Leaves long, linear, acuminate. Ligule is an oblong hyaline membrane, about 2-3(-5) mm (0.078 - 0.118 - 0.197 inch) long, often truncate and/or fringed.
No auricles, sheath smooth.Panicle more or less protruding or entirely protruding from the uppermost swollen leaf sheath, ovate to oblong, 5 - 8 cm (1.97 - 3.15 inch) long, green.
Spikelets green, broadly lanceolate on short pedicels, shining, 4 -6 mm (0.16 - 0.24 inch) long, strongly laterally compressed.
Glumes 4 - 6 mm ( (0.16 - 0.24 inch) long, fertile lemma lanceolate about 3 mm (0.118 inch) long, more or less lustrous; sterile lemma solitary, about (0.2-)1(-1.8) mm (0.008 - 0.039 - 0.07 inch) long. Glumes subequal, with a broad erose-dentate or entire wing near the tip. Hermaphrodite florets.
Plants in loose tufts, up to 1 m tall, in some especially large types up to 1.5 m. Culms usually erect, sometimes somewhat geniculate at the base. Leaves flat, scabrid or glabrous; auricles long, prominent, linear-falcate, surrounding the culm. Spikes 50-300 mm long, the colour varying from green to purplish or blackish, 10-30 nodes, each bearing three 1-flowered spikelets of which 1, 2 or all 3 are fertile, i.e. two-, four- and six-rowed; the awns normally long and straight, but mutations are found, e.g. 'hooded' and triple-awned forms; rachis brittle or tough. Central spikelet sessile, glumes flattened, usually 10-30 mm long; lemma glabrous or scabrid, mostly near the apex, usually tightly embedding the kernel (though not in naked forms), awn of lemma 30-180 mm long, usually scabrid; anthers 6-10 mm long, yellowish; rachilla short or up to half the length of the palea, with long or short hairs. Lateral spikelets usually sessile, when fertile sessile to pedicellate (3 mm long), when sterile, obtuse to acute or with an awn, almost equally as well-developed as the central one when fertile; glumes 10-20 mm long, more or less flattened.
Perennials. Culms to 3 m high, erect, rhizomatous, nodes waxy. Leaves 45-110 x 0.6-1.2 cm, linear-lanceolate, base rounded, margins serrulate, apex accuminate, involute, glaucous; sheaths to 40 cm long, silky villous at the collar; ligules 4-5 mm long, ovate, membranous. Panicles 20-32 cm long, densely silky white. Spikelets paired, one sessile and the other pedicelled, similar, 3-4 mm long, lanceolate, reddish-brown; callus densely long villous. Lower glume 3-4 x 1 mm, ovate-lanceolate, subcoriaceous, margins hyaline, ciliate. Upper glume 3-4 x 1 mm, ovate-lanceolate, dorsally keeled, margins ciliate. Lower floret empty. Upper floret bisexual. First lemma c. 2 x 1 mm, ovate-acute, hyaline. Second lemma 2-2.5 mm long, linear-lanceolate, hyaline. Stamens 3; anthers yellow or reddish. Ovary oblong; stigma white. Pedicelled spikelets similar to sessile ones.
Job's Tears is a coarse annual herb. The stem is erect, branched, rather coarse, and stout, and 1-2 m high. The leaves are 10-40 cm long, 2.5-4 cm wide, with the base broad and cordate. The spikes are 6-10 cm long, erect and peduncled. The male spikelets are about 8 mm long. The capsules (fruits), enclosing the female flowers and the grains, are hard, bony, white or nearly black, shining, ovoid, about 8 mm long. The chief value of Job's Tears lies in the edibility of the fruit. The berries are also strung as beads, used as rosaries, made into curtains, trays, bags etc.
A monoecious plant. Male flowers in terminal racemes; spikelets, two-flowered glumes nearly equal, herbaceous, terminating in two sharp points; females, axillary in the sheaths of the leaves. The spikes or ears proceed from the stalls at various distances from the ground, and are closely enveloped in several thin leaves, forming a sheath called the husk; the ears consist of a cylindrical substance, a pith called the cob; on this the seeds are ranged in eight rows, each row having thirty or more seeds. From the eyes or germs of the seeds proceed individual filaments of a silky appearance and bright green colour; these hang from the point of the husk and are called 'the silk.' The use of these filaments or stigmata is to receive the farina which drops from the flowers, and without which the flowers would produce no seed. As soon as this has been effected, the tops and 'the silk' dry up. The maize grains are of varying colour - usually yellow, but often ranging to black.


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True Crime with Aphrodite Jones
Road to Nowhere
Season 6 Episode 2
A long haul trucker disappears just before New Years. Thirteen years later a tax fuel auditor goes missing. The investigation into one case expands into another, and police uncover one of the most bizarre and grisly crime scenes they've ever seen.
- 43 min
- Premiered 05/09/2016


text:
Gonzalo Madrigal
He likes to play soccer and listen to music.
He also Likes to play Xbox and eat tamales.
He was born in Riverside and his parents are from Mexico.
His Favorite holiday is Christmas
I met this guy at summer bridge named Gonzalo
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See the Program of Studies for a complete Special Services curriculum. The following are some of the programs that the District offers:
Bartley Academy
The Bartley Academy is a self-contained alternative education program for high school students that co–exists within the traditional high school program. It is located in West Morris Central High School. WM students are provided transportation.
Home Instruction
Home instruction is provided to eligible students.
Behavior Support Program at WMC
Life Skills Program at WMM
Structured Learning Experience at WMC and WMM
Special Services >


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Holiday suggestions please(6 Posts)
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By Mark Kurtzman.
In the diagram below, the game appears drawn. In almost all cases, when serious tournament players compete, they agree to a draw when there are no winning chances. In this game, however, it was the final round of an important championship tournament. And there was a lot of prize money at stake…$7,600 to the winner! Everything seems equal, except White had 1 minute left on the clock in sudden death and black had almost 2 minutes. Black played Rg2+ in this position. White replied with Rc2 hoping for a rook trade, but black played his rook back to g7. The position continued with similar rook moves being played by both sides leading nowhere. White, realizing he would lose the game on time if this would continue, started saying "draw, it's a draw." Black said nothing and did not acknowledge his opponent. The Chief Tournament Director then appeared to observe the final moves of the game. They continued to play and when white realized he had only seconds left, he started to scream, "Draw, you can't win this position. It's a draw!" The opponent continued to play and the White player turned to the tournament director expecting him to intervene, but he did not. White's flag fell and he lost the game on time and the $7,600 prize as well. Is this fair? You make the call…
27th National Chess Congress Philadelphia, PA December 20, 1996


text:
Blue touch paper was a type of paper fuse for explosives. The second and third incarnations of the Doctor used small amounts of explosive wrapped in blue touch paper to destroy locks. (TV: The War Games, Spearhead from Space)
The Fourth Doctor once used a firecracker and blue touch paper to unclog the gas pocket powering the Sacred Flame. He later gave another piece to the Sisterhood of Karn in case it should happen again. When they asked him what the strange symbols meant, he told them, "Light the blue touch paper and stand well back". (TV: The Brain of Morbius)
The Fifth Doctor had Tulung use blue touch paper to set fire to Shaun Brett's monument, killing the Permians trapped inside. (AUDIO: The Land of the Dead)


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|Message view
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|"Gav...." <brightoncomput...@brightontown.com.au>
|PDF output and Dispatcher
|Sun, 09 Mar 2008 02:17:34 GMT
Hi All, This was going to be an email about how PDF was not working with Dispatcher, more accurately that it did not work using Pelt Theme or any theme that does not over-ride FO from /common/ . Usually, when I copy over Pelt Theme for over-riding to my project, I also copy over /common/ just in case I need to change any of that too. This was my downfall here, because changes made to /common/fo recently did not get updated in my local project, so broke PDF output. I then spent 1/2 hr fault-finding and correcting errors in layout-master-set.ft only to find that this had already been done in whiteboard. So, a doh! moment for me, and a heads-up to anyone else that has local copies of themes/common/* , remember to update them, better still, try not to change anything in common, copy it to /pelt/ and change it there instead. Gav...
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Margaret Green Animal Rescue
Business / Club -
0.12 miles away
Attraction -
0.77 miles away
Blue Pool
Attraction -
1.01 miles away
The Blue Poole and Tea House
Business / Club -
1.04 miles away
Corfe Castle
Attraction -
1.21 miles away
Business / Club -
1.27 miles away
The Greyhound Inn
Business / Club -
1.29 miles away
Dorset Cottage Holidays
Accommodation -
1.31 miles away
Dorset Cottage Holidays
Business / Club -
1.31 miles away
Area Information
The village of Church Knowle can be found approximately 2 miles west of Corfe Castle. Build using mainly Purbeck stone, this small village has a 13th century church and manor house as well as an animal sanctuary.
The name Church Knowle comes from the knoll or round burial mound that the village church is built up on.
Getting There
Church Knowle is reached via the A351 from Wareham, turn right just before Corfe Castle
The nearest train stations are Wareham, Poole and Bournemouth
Wildlife and Walks
The area around Church Knowle is ideal for walking and cycling with many miles of footpaths and bridleways.


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ExteriorCurrent Page
For many caravans, their low starting price quickly becomes apparent. Not for this one. KNAUS SPORT has everything that has been making its bigger brother so successful for so many years. This does not only include the high‐quality exterior design, but also a wide range of equipment that makes the SPORT so comfortable and safe.
It does not really matter how big your SPORT is, thanks to the high‐quality chassis and tried‐and‐tested stabilisers, you can easily navigate even the most difficult turns.
The cat's eye back lights in the unmistakeable KNAUS optic.
No annoying insect buzzing noise thanks to the fly screen door.
A gas bottle locker without the obtrusive loading sill. Brilliant.
Cornering safely: The AKS stabiliser keeps you on track.
The standard GRP roof is particularly hail‐proof.
The hot‐dip galvanised chassis is ready for many years' holidays.


text:
Contact Us 1 year ago
Dentures Lab Technician - Advara Dental and Dentures harrisburg, pennsylvania
Job Description:Advara Dental and Dentures is looking for an experienced Lab Tech for our Harrisburg PA dental office. Must have Denture Lab experience. Great pay : Up to 35 per hour depending on experience. Health Insurance and Dental Insurance Paid Time Off Great work environment. Company Description:Advara Dental and Dentures is a full service retail dental office located in Harrisburg, PA.Sour
1 year ago
Harrisburg, PA
Contact Us


text:
General News
SF Again says "No" to Sequoia(Page 1 of 1 pages)
(more...) (less...) , Add to My Group(s)
|Alan Dechert is the CEO of Open Voting Consortium.
Go To Commenting
|/* The Petition Site */
|The views expressed in this article are the sole responsibility of the author and do not necessarily reflect those of this website or its editors.
Most Popular Articles by this Author: (View All Most Popular Articles by this Author)
Blow Struck Against Machine with Secret Voting Software and Invisible Ballots
SF Again says "No" to Sequoia
San Francisco or New York -- which will be first with open source voting system?
Video on Electronic Voting
AB 852 - New OVC-Sponsored CA Assembly Bill


text: There's been so much in the news lately about bad plastics -- including right here in this column -- that I thought I should write about a potentially good thing happening with them.Bioplastics, plant-based biodegradable plastics, are gradually replacing plastics made from petroleum. They comprise only a small portion of the plastics, but as soaring prices and politics make petroleum an expensive and controversial raw material, bioplastics are an increasingly attractive alternative. The Guardian reported recently that the sector is growing by 20% to 30% a year. Major players include NatureWorks, owned by Cargill and Japanese company Teijin Ltd.; Mirel, a joint venture between Archer Daniels Midland ( ADM) and biotech firm Metabolix ( MBLX); Cereplast; Italian firm Novamont and Australia's Plantic Technologies, which has a marketing agreement in the U.S. with Dupont ( DD). The Biodegradable Products Institute has a list of others. The plastic fibers these companies manufacture are indistinguishable from conventional plastics. Chances are good that you've already bought products containing or packaged in bioplastic -- without even knowing it. Toyota ( TM) has used it to make components for the Prius and Raum. Novamont's bioplastic is a component in Goodyear ( GT) Tires. Sony ( SNE) puts bioplastic in its Walkman. Target ( TGT) uses it for its gift cards. The brand that seems to have the most reach so far, Natureworks, has seen its plastic used to pack fresh veggies, salad greens and cut fruit sold at Wal-Mart ( WMT) and Kroger ( KR), in packaging for Green Mountain Coffee Roaster ( GMCR) coffees, and in sheets sold at Target and J.C. Penney ( JCP).


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Disney Insider-Trading Probe: Criminals Do Not Take Note
The Disney insider-trading case is in its infancy, but at this early stage it appears fair to call the alleged scheme ham-handed.
To recap, prosecutors and the SEC claim that a Disney secretary and her boyfriend conspired to trade inside information about the media giant in advance of earnings reports.
Yonni Sebbag, 29, whose girlfriend worked at Disney, allegedly sent letters to 33 investment firms stating he had access to Disney earnings reports before their release and that he was willing to share his information for a fee, according to a pair of complaints filed yesterday. (Here's a copy of the criminal complaint and click here for the SEC complaint.)
Typically, it takes months of meticulous sleuthing to piece together insider-trading plots, WSJ reports in this article on the Disney case.
But in the Disney matter, "the alleged peddlers simply put their offer in writing and sent it out so widely that word of the scheme was bound to reach authorities," according to WSJ's Ethan Smith and John Emshwiller, who offer details about the FBI's sting operation.


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Bella's dress in Twilight for prom? It's not known, but in one of the earlier drafts of the ending of Twilight, Stephenie Meyer wrote that instead of them going to the airport straightaway to meet Edward, they went shopping.
But, since Alice is a vampire and has superspeed, she can probably run or drive and get a nice dress from a big city in half a day.


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Welcome to Naughty Office, where we explore all of your work-related fantasies! Inside, you'll see secretaries getting fucked by their bosses, interns sucking cock, and a whole lot more!
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text:
It Happened on the Night Shift (2 of 5) by Rick White
This content is part of a series.It Happened on the Night Shift (2 of 5)
Series: Hope For The Holidays
Dr. Rick White
Luke 2:8-20
Christmas Shopping Illustration: There are several men in the locker room of a private club after exercising. Suddenly a cell phone on one of the benches rings. A man picks it up and the following conversation ensues:
"Honey, It's me."
"Are you at the club?"
"Great! I'm at the mall doing some Christmas shopping and I saw a beautiful mink coat. It is absolutely gorgeous!! Can I buy it?"
"What's the price?"
"Only $1,500."
"Well, okay, go ahead and get it, if you like it that much."
"Ahhh, and I also stopped by the Mercedes dealership and saw the 2001 models. I saw one I really liked. I spoke with the salesman and he gave me a really good price. . .and since we need to exchange the BMW that we bought last year. . ."
"What price did he quote you?"
"Only $60,000!"
"Okay, but for that price I want it with all the options."
"Great! Before we hang up, something else. . ."
"It might seem like a lot, but I was reconciling your bank account and. . .well, I stopped by to see the real estate agent this morning and I saw the house we had looked at last year. It's on sale!! Remember? The one with a pool, English garden, acre of park area, beachfront property. . ."
"How much are they asking?"
"Only $450,000. . .a magnificent price, and I see that we have that much in the bank to cover. . ."
"Well, then go ahead and buy it, but just bid $420,000. Okay?"
"Okay, sweetie. Thanks! I'll see you later!! I love you!!!"
"Bye. I do too."
The man hangs up, closes the phone's flap and raises his hand while holding the phone and asks to all those present, "Does anyone know to whom this phone belongs?"
Mikey's Funnies, Dec 5, 2000
Introduction: Today in our series Hope For The Holidays we are going to look at the familiar passage from Luke 2, so take your Bibles or look in your notes at we read this passage. (Read Luke 2 ...
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