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In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the 13C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-13C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from 13C-enriched DNA and were designated "anthracene group 1." Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP.
Molecular methods are increasingly being used to explore the microbial diversity of environmental systems without needing to isolate microorganisms from their natural environment, especially because many relevant organisms have proven difficult to isolate from their environmental sources (3, 7, 18). The effectiveness of molecular methods to describe microbial diversity depends on our ability to efficiently extract and purify macromolecules from microbial cells native to an environmental sample (40). Commercially available kits are commonly used to extract nucleic acids from environmental samples by physical and/or chemical lysis of microbial cells, followed by purification of the nucleic acids from cell debris and other organic material. Feinstein et al. (10) recently demonstrated that extracting a soil aliquot only once with a commercial kit can lead to incomplete DNA extraction, thus biasing estimates of the genomic DNA mass yield, the small-subunit ribosomal gene copy number, and the bacterial groups identified; multiple extractions led to broader recovery of organisms in the soil community.
Bioremediation is the primary method of removing polycyclic aromatic hydrocarbons (PAHs) from PAH-contaminated environments (1), but our understanding of the roles of specific organisms within PAH-degrading microbial communities and the metabolic mechanisms responsible for PAH degradation is still developing. Stable-isotope probing (SIP) is one cultivation-independent molecular technique that can link the identity of a microorganism with its metabolic function without isolating that organism from its natural environment (38). DNA-based SIP has been used to identify bacteria capable of degrading aromatic hydrocarbons in PAH-contaminated environments, and in some cases, it has revealed novel bacterial groups (15, 16, 33, 35, 43). Earlier SIP studies of pyrene-degrading bacteria conducted in our lab revealed members of previously uncultivated beta- and gammaproteobacterial groups, neither of which is related to any cultivated genus (16, 35). SIP investigations have also facilitated the isolation of ecologically relevant organisms (15, 19, 36) and have been used to reduce the complexity of community DNA slated for metagenomic analysis (4). To date, SIP of anthracene-degrading bacteria has not been reported.
In many of the DNA-based SIP studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the 13C-labeled (heavy) DNA by density-gradient ultracentrifugation. In the present study, we performed successive DNA extractions on the same aliquot of PAH-contaminated soil either untreated or enriched with uniformly 13C-labeled anthracene to determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene recovery, or the identification of anthracene-degrading bacteria. In addition, we tested the effects of soil loading and multiple extractions on the efficiency of the FastDNA spin kit for soil (MP Biomedicals, Solon, OH).
Soil processing.PAH-contaminated soil was collected from a former manufactured gas plant site in Salisbury, Rowan County, NC. The total PAH concentration was approximately 890 mg/kg, and the anthracene concentration was 32 mg/kg. Large objects were removed by hand. The soil was then sieved through a 10-mm wire screen, blended, and sieved again prior to storage in the dark at 4°C. The processed soil (64% sand, 30% silt, 6% clay, and 15% moisture; pH 7.6) was further prepared by manually removing any remaining small stones and other debris immediately before use in experiments.
Chemicals.Natural abundance isotopomer (unlabeled) anthracene (scintillation grade) was obtained from Eastman Kodak (Rochester, NY). [U-13C]Anthracene was synthesized according to methods to be described elsewhere (Z. Zhang, L. M. Ball, and A. Gold, personal communication). Until the method is published, details of the synthesis can be obtained by contacting M.D.A. at mike_aitken{at}unc.edu. [1,2,3,4,4a,9a-14C]Anthracene (17.3 mCi/mmol) was obtained from Sigma-Aldrich (St. Louis, MO). All other chemicals used were at the highest purity available. All solvents were molecular biology or high-pressure liquid chromatography (HPLC) grade.
Enrichment with anthracene.Soil slurries were prepared in 125-ml flasks containing 1 g of soil (wet weight) and 30 ml of simulated groundwater amended with 0.37 mM NH4NO3 and 0.08 mM K2HPO4. The groundwater was prepared to reproduce the major ion concentrations in the groundwater of Rowan County, NC (0.7 mM CaCl2 · H2O, 0.2 mM MgSO4 · 7H2O, 1.0 mM NaHCO3, 0.06 mM KCl, 1 N H2SO4; pH 7.5) (31) and was filter sterilized through a 0.1-μm-pore-size flowthrough, hollow-fiber membrane water filter (Sawyer Products, Safety Harbor, FL). Four sets of flasks containing the soil slurry were incubated in parallel. For each set, after 2 days of shaking (225 rpm) in the dark at room temperature to reduce the concentrations of native PAHs, the aqueous phase was replaced with fresh nitrogen- and phosphorus-amended groundwater. Duplicate clean flasks were spiked with 625 μg of either unlabeled anthracene (set 1) or [U-13C]anthracene (set 2) (in acetone) to enrich the anthracene-degrading microorganisms. The acetone was allowed to evaporate before the slurry was introduced, and the flasks were returned to the shaker (t = 0). In addition, triplicate flasks containing unlabeled anthracene (set 3) were prepared to monitor the anthracene disappearance by HPLC and to archive community DNA over time. Another set of triplicate flasks containing a mixture of unlabeled and radiolabeled anthracene (20,000 dpm) (set 4) was prepared to monitor anthracene mineralization by liquid scintillation counting of 14CO2 trapped in KOH-soaked filter paper (34). Inhibited controls were prepared by acidifying incubations to pH values of <2 using 200 μl of 85% phosphoric acid.
Monitoring anthracene disappearance.Soil slurry from each triplicate flask in set 3 (1 ml) was mixed with 1 ml of ethyl acetate in each of the triplicate 15-ml conical-bottom glass centrifuge tubes. The tubes were vortexed at maximum speed for 1 min and centrifuged for 5 min at 3,500 rpm. The organic layer of each resulting supernatant was filtered through a 0.45-μm-pore-size nylon filter and stored in a gas chromatography vial at −20°C prior to HPLC analysis. The extracts were diluted with acetonitrile as needed immediately before HPLC analysis. The HPLC system included a Waters 600E system controller (Milford, MA), a Waters 717 Plus autosampler, and a PerkinElmer LS40 fluorescence detector (Beaconsfield, United Kingdom). Analyte standards were prepared from an EPA 610 polynuclear aromatic hydrocarbons mixture stock (Sigma-Aldrich, St. Louis, MO) and used to create a four-point calibration curve for sample quantification. Samples were injected through a 3-μm-particle-size Supelcosil LC-PAH column (Sigma-Aldrich, St. Louis, MO) using a gradient mobile phase of acetonitrile and water and analyzed as previously described (34).
DNA extraction.After centrifugation, DNA was extracted from the soil in each flask from sets 1 and 2 in two 500-mg aliquots of the soil pellet by using the FastDNA spin kit for soil (MP Biomedicals, Solon, OH) according to the accompanying instructions with the following exceptions. Samples were secured horizontally to a bench-top vortexer set at maximum speed for homogenization. After each extraction, fresh sodium phosphate and MT buffers (MP Biomedicals) were added to the Lysing Matrix E tube containing the original soil aliquot, and the extraction procedure was repeated. DNA was eluted from each successive extraction with Tris-EDTA buffer (TE; pH 8.0) into a clean catch tube until seven extractions had been performed. The equivalent extracts of each 500-mg aliquot from a given incubation flask were pooled prior to further analysis; because there were duplicate incubation flasks, there were duplicate series of seven DNA extracts. For the flasks from set 3, the FastDNA spin kit for soil was used to perform a single DNA extraction on a soil pellet resulting from 1 ml of soil slurry containing approximately 33 mg of soil. In a subsequent experiment, DNA was extracted from untreated soil in duplicate aliquots of 33, 100, 250, or 500 mg using the same multiple-extraction procedure described above, except that six successive extractions were performed.
DNA and 16S rRNA gene quantification.The DNA mass yield was quantified with a NanoDrop 3300 fluorospectrometer (NanoDrop Products, Wilmington, DE) using the Quant-iT PicoGreen double-stranded DNA (dsDNA) assay kit (Invitrogen, Eugene, OR). The 16S rRNA gene copy number of a targeted sequence was determined by quantitative PCR (qPCR) using 1 μl of DNA as the template, primers (final concentration, 600 nM) as identified in Table 1, and QuantiTect SYBR Green PCR master mix (Qiagen, Valencia, CA) with the SmartCycler platform (Cepheid, Sunnyvale, CA) in a 25-μl reaction. Primer sets for targeted quantification of several SIP-identified groups were designed and validated as described elsewhere (35), except that sequences were aligned within the myRDP personalized workspace (6). The qPCR temperature program used included 15 min at 95°C, followed by 45 cycles of 15 s at 95°C, 30 s at the annealing temperature (Table 1), and 30 s at 72°C. Data were collected during primer extension, and reaction products were analyzed by melt curve analysis between 65 and 95°C. The r2 value for each qPCR standard curve (cycle threshold versus log gene copy number) was ≥0.995, and the amplification efficiencies of curves from eubacterial and group-specific primer sets were close to 2.0 (Table 1). To compare the abundance of the SIP-identified bacteria in the heavy DNA to their abundance in the light DNA, each group-specific primer set was used to quantify the corresponding sequences in each fraction from one ultracentrifuge tube. Two sets of primers for quantification of sequences for "anthracene group 1" (AG1) were developed. Primer set AG1F/AG1R was used to quantify AG1 sequences in the multiple extraction experiment, while primer set AG1.1F/AG1.1R, which possessed a lower detection limit during creation of standard curves, was used to quantify genes in collected SIP fractions and time course experiment samples.
DNA separation and recovery.DNA extracted from anthracene-enriched samples was mixed with 20 μl of SYBR Safe (Invitrogen, Carlsbad, CA), and the mixture was added to 6-ml polyallomer Ultracrimp tubes (Kendro Laboratory Products, Newtown, CT). SYBR Safe is an alternative to ethidium bromide in CsCl density gradients used in DNA-based SIP assays (25) that simplifies the cleanup of fractions collected from ultracentrifuge tubes because ethidium bromide does not have to be extracted. Inclusion of a fluorescent dye in the CsCl solution allowed us to visualize bands of DNA postseparation, thus locating the approximate positions of fractions containing DNA of interest. The tubes were filled with a cesium chloride solution (ρ = 1.72 g/ml), crimp sealed, and ultracentrifuged (RC70 ultracentrifuge; Sorvall, Newtown, CT) at 175,800 × g and 20°C for 40 h in a TV-1665 vertical rotor (Sorvall). A tube containing 1 μg each of Escherichia coli K-12 DNA from a culture grown in LB broth and a Pseudomonas putida G7 culture grown on uniformly labeled [13C]glucose (Cambridge Isotope Laboratories, Inc.) was included as a control to verify separation of unlabeled DNA from 13C-labeled DNA. DNA bands were visualized with the Safe Imager blue light transilluminator (Invitrogen, Carlsbad, CA) before collection of 24 fractions of 250 μl each from the bottom of each ultracentrifuge tube, as described by Singleton et al. (33). Separation of unlabeled and 13C-labeled DNA achieved in the control tube is illustrated in Fig. S1 in the supplemental material. DNA in each fraction was recovered by ethanol precipitation (30) and resuspended in 100 μl of 0.2-μm-filter-sterilized TE (pH 8.0).
Identification of heavy and light DNA fractions.The DNA concentration and eubacterial 16S rRNA gene copy number in each DNA fraction recovered were quantified, and the eubacterial community profile for each fraction was visualized by denaturing gradient gel electrophoresis (DGGE). PCR for DGGE targeted the V1-V3 hypervariable region of the 16S rRNA gene and was performed with 5Prime Mastermix (Gaithersburg, MD) using 1 μl of DNA as the template and primers 63F-GC and 517R (final concentration, 200 nM each) in a 20-μl reaction as previously described (44). The temperature program was modified such that 10 cycles of touchdown PCR were followed by 15 cycles of conventional PCR. PCR products were loaded onto a 6.5% polyacrylamide gel without denaturant stacked on top of a 6.5% polyacrylamide gel with a urea-formamide denaturing gradient at between 30% and 60% and run for 16 h at 60 V on a DCode system (Bio-Rad Laboratories, Hercules, CA). DGGE gels were poststained with ethidium bromide, and bands were visualized under UV transillumination. Subsequent to identification of sequences recovered in putative heavy DNA from the SIP incubations, selected clonal insert sequences were amplified and analyzed by DGGE to associate the most prominent DGGE bands in the community DNA with specific sequences.
Consecutive fractions in the ultracentrifuge tube with similar DNA concentrations, 16S rRNA gene copy numbers, and community profiles were pooled and identified as the composite heavy or light DNA fraction, depending on the section of the tube from which the fractions were removed. Effort was made not to interrupt a peak when deciding which individual fractions to combine to create composite heavy and light fractions. DNA from the composite heavy fraction of each anthracene-enriched replicate was screened for archaeal rRNA gene sequences using primers 25F (8) and 1492R (22) and for fungal rRNA gene sequences using primers ITS1F (11) and ITS4 (42) before being used as the template to generate a eubacterial 16S rRNA gene clone library.
Clone library preparation and analysis.After identifying the fractions corresponding to heavy DNA in extracts from each [U-13C]anthracene-enriched replicate, PCR was performed with 1 μl of heavy DNA as the template, primers 8F (9) and 1492R (22) (final concentration, 200 nM), and 5Prime Mastermix in a 50-μl reaction. The PCR temperature program used included 10 min at 94°C, followed by 25 cycles of 1 min at 94°C, 1 min at 50°C, and 3 min at 72°C, and ended with one 15-min cycle at 72°C. PCR products were cloned using a TOPO TA cloning kit for sequencing (Invitrogen, Carlsbad, CA), according to the accompanying instructions. Plasmid DNA from a random subset of clones was subjected to restriction analysis prior to sequencing to ensure successful ligation of the insert to the plasmid vector. Inserts were partially sequenced with primer 8F by Functional Biosciences, Inc. (Madison, WI).
Sequences were analyzed by VecScreen (http://www.ncbi.nlm.nih.gov/VecScreen) to remove vector contamination, Bellerophon (13) was used to screen for chimeric sequences, and the RDP Classifier (41) was used to determine the closest cultivated genus to each sequence. Each sequence was compared to those in public sequence databases using BLASTN (2) and RDP release 10.17 (5) to identify closely related sequences. Multiple sequence alignments and guide trees were produced using myRDP (6) and ClustalX (37), respectively, which facilitated the grouping of sequences most similar to one another and to those in GenBank. Rarefaction curves were generated at a 3% sequence distance to test whether clone libraries of adequate size had been generated. The UniFrac significance test (12) was used to determine whether the clone libraries were significantly different from one another. Clones were named to indicate the source soil (Salisbury [SB]) and growth substrate (anthracene [ANT]) and were numbered.
To confirm whether sequences identified in the clone libraries were 13C enriched, we quantified each sequence in fractions spanning the putative heavy and light DNA in selected extracts from the SIP incubations with 13C-labeled anthracene as well as in the extract from the incubation with unlabeled anthracene. To align equivalent fractions from separations of both unlabeled and 13C-labeled anthracene, we also quantified sequences associated with the Acidovorax genus that were identified as degraders of other PAHs in this soil (17) but that were not observed in the clone libraries generated from heavy DNA from the SIP incubations with 13C-labeled anthracene. Thus, Acidovorax genes were expected to be associated with unlabeled DNA fractions, regardless of the incubation.
Anthracene removal and mineralization.Soil slurry was incubated in triplicate with unlabeled anthracene (to follow anthracene removal) or a mixture of unlabeled and radiolabeled anthracene (to follow mineralization) for 20 days. After 3 days, less than 10% of the added anthracene remained in the flasks containing unlabeled anthracene. However, the mineralization experiment continued until the rate appeared to decline at day 20 (Fig. 1), which was then selected as the time to terminate SIP incubations with [U-13C]anthracene.
SIP with anthracene and identification of heavy DNA.After 20 days, seven successive DNA extractions were performed on duplicate aliquots of 500 mg (wet weight) of soil from each of the duplicate flasks containing unlabeled or [U-13C]anthracene. For comparison, six successive extractions were performed on replicate 500-mg aliquots of the untreated original soil sample. Quantifiable amounts of DNA were obtained through the six extractions of the untreated soil (total over 6 extractions = 0.98 ± 0.01 μg/g dry soil), although the number of 16S rRNA genes in the sixth extract was negligible (total over 6 extractions = 2.24 × 108 ± 1.99 × 107 gene copies/g dry soil) (Fig. 2). The pairwise Wilcoxon signed rank test determined that the amount of DNA (P = 0.85) and the number of 16S rRNA genes (P = 0.36) recovered at each extraction step were similar in each of the duplicate extraction series. Both the total DNA (39.0 ± 2.65 μg/g dry soil) and 16S rRNA genes (2.37 × 1011 ± 2.60 × 109 gene copies/g dry soil) were quantifiable through the seventh extraction of anthracene-enriched soil (Fig. 2). The amount of DNA (P = 0.30) and the number of 16S rRNA genes (P = 0.81) recovered at each extraction step were similar in each of the duplicate extraction series.
DGGE analysis of anthracene-enriched samples revealed the same banding patterns for extracts 1 to 4, but additional bands were present in lanes containing amplicons from extracts 5 to 7 (not shown). Based on this analysis, for each incubation flask, the DNA from extracts 2 to 4 (19 μg) was pooled, and the DNA from extracts 5 to 7 (13 μg) was pooled prior to ultracentrifugation. The pooled extracts from each duplicate flask were loaded into separate ultracentrifuge tubes and centrifuged in parallel to duplicate tubes containing DNA from extract 1 (7 μg).
Fractions collected from each of the ultracentrifuge tubes were analyzed by measuring the DNA concentration and eubacterial 16S rRNA gene abundance and by DGGE. In the tubes containing DNA from incubations with unlabeled anthracene, there was a single peak over a range of fractions corresponding to where unlabeled ("light") DNA was observed in the control tube (see Fig. S2A in the supplemental material). No measurable DNA was observed in lower fractions, and 16S rRNA gene abundance was at a background level (2.0 to 2.5 log gene copies) in the range of fractions corresponding to the location of heavy DNA in the control tube. DGGE bands were visible in these lower fractions, but the banding patterns were identical to those of fractions containing unlabeled DNA (see Fig. S3A in the supplemental material). In the tubes containing DNA from incubations with 13C-labeled anthracene, the fractions containing measurable DNA also had quantifiable eubacterial 16S rRNA genes of up to 3 orders of magnitude above the background level (see Fig. S2B to D). Differences in DGGE banding patterns (see Fig. S3B to D) between fractions were used to select the fractions corresponding to heavy DNA in each tube. No archaeal or fungal rRNA genes were detected in any heavy DNA fraction.
Analysis of sequences recovered from 16S rRNA gene clone libraries.A 16S rRNA gene clone library was generated from the heavy DNA recovered from each replicate of extract 1, pooled extracts 2 to 4, and pooled extracts 5 to 7. For each replicate, 32 clones were partially sequenced (192 total), of which 1 containing a vector sequence and 4 containing chimeras were excluded from further analyses. Phylogenetic analysis of the recovered sequences is illustrated in Fig. S4 in the supplemental material, and the major groups corresponding to these sequences are summarized in Table 2. Rarefaction analysis indicated that an adequate number of clones was sequenced (see Fig. S5 in the supplemental material). The pairwise UniFrac significance test (weighted and normalized to account for sequence abundance and branch lengths, respectively) determined that libraries from extract 1 and extracts 5 to 7 were significantly different from one another (P ≤ 0.002); however, all groups identified in extracts 2 to 4 and 5 to 7 were also identified in extract 1. Other library pairs (extract 1 versus extracts 2 to 4 and extracts 2 to 4 versus extracts 5 to 7) were not significantly different from one another (P > 0.1). Sequences clustering with members of the order Sphingomonadales, but that are unrelated to any previously described genus, were abundant in each clone library (14 of 61 for extract 1, 25 of 63 for pooled extracts 2 to 4, and 42 of 63 for pooled extracts 5 to 7) and were designated "anthracene group 1" (AG1). Other well-represented sequences were similar to sequences representing the genera Variovorax (93% similar) and Sphingobium (93%); sequences related to Herminiimonas (99%) and Pigmentiphaga (99%) were found less frequently (Table 2).
Quantification of SIP-identified bacteria.Primers used for quantitative PCR that targeted the 16S rRNA gene were developed to measure the abundances of the major bacterial groups recovered in putative heavy DNA from the SIP incubations with 13C-labeled anthracene (Table 1). Quantitative PCR analyses of unseparated DNA reserved from each of the 13C-labeled anthracene enrichments revealed that there was differential recovery of each SIP-identified group compared to that of the eubacterial community with successive extractions (Fig. 3); the most disparate recovery pattern occurred with AG1 sequences. To test the hypothesis that the extraction patterns of 16S rRNA genes from the different groups are not correlated over the seven extractions, we calculated the correlation between each pair of groups and assessed the significance of the hypothesis that each correlation is 0 using a permutation P value. Specifically, we permuted the data 2,000 times and calculated correlations using the permuted data. The permutation P value is the proportion of permutations in which we observed larger correlations than we observed using the unpermuted data (see Table S1 in the supplemental material). We could not reject the hypothesis of zero correlation for comparisons between AG1 and any other group (P = 0.19 to 0.57), which suggests that the pattern of 16S rRNA gene extraction for AG1 was different from the extraction pattern of each other group. The tests between Sphingobium and Herminiimonas (P = 0.08) and between Variovorax and Herminiimonas (P = 0.10) were marginally significant, suggesting a relatively weak correlation across the seven extracts. Tests between all other pairs were significant (P < 0.02), suggesting similar extraction patterns.
We also quantified growth over time of the SIP-identified bacterial groups during incubations in the presence of unlabeled anthracene. After 3 days of incubation in the presence of unlabeled anthracene, corresponding to when the majority of the added anthracene had been removed, the abundance of the 16S rRNA genes increased by at least an order of magnitude for all of the SIP-identified bacterial groups other than Sphingobium (Fig. 4). We also quantified sequences related to Acidovorax spp. known to be present in the soil but which were presumed not to be associated with anthracene degradation. Acidovorax sequences increased by 4-fold during the 3-day period over which anthracene was consumed, which was significantly lower than the increase in the SIP-identified groups, other than Sphingobium (Fig. 4). The abundances of AG1 and Pigmentiphaga-related sequences did not change significantly after day 3. Variovorax-, Herminiimonas-, and Sphingobium-related sequences continued to increase in abundance between day 3 and day 7, after which there was relatively little change in these sequences.
Each group-specific primer set was used to quantify the corresponding sequences in each DNA fraction recovered from extract 1 and pooled extracts 2 to 4 of the SIP incubation and in each fraction of separated DNA from the incubation with unlabeled anthracene (Fig. 5). Fractions from each of the separations (unlabeled anthracene; 13C-labeled anthracene, extract 1; and 13C-labeled anthracene, extracts 2 to 4) were aligned based on the peak of the Acidovorax sequences in each sample. DNA shifting to lower fractions from incubations with 13C-labeled anthracene compared to those from the unlabeled compound was evidence for incorporation of the label. Compared to the peak fraction in unlabeled DNA, there were shifts to lower fractions (heavier DNA) in the DNA from SIP incubations for AG1 sequences (Fig. 5B), Pigmentiphaga-related sequences (Fig. 5D), and Variovorax-related sequences (Fig. 5F). No such shifts were observed for the Herminiimonas- or Sphingobium-related sequences (Fig. 5C and E, respectively).
Effects of soil loading and multiple extractions on extraction efficiency.Using the FastDNA spin kit for soil, DNA was extracted six successive times from untreated soil in duplicate aliquots of 33, 100, 250, or 500 mg to determine the optimum soil load and number of extractions needed to maximize DNA mass yield and 16S rRNA gene recovery from the same aliquot of PAH-contaminated soil (see Fig. S6 in the supplemental material). With a single extraction, DNA mass yield decreased with an increasing soil load (r2 = −0.94), but soil loads of up to 250 mg did not affect the yield of quantifiable 16S rRNA genes recovered in the first extraction (P = 0.07). Subsequent extractions resulted in further recovery of DNA and 16S rRNA genes, but there was relatively little recovery beyond the second extraction for soil loads of up to 250 mg.
In DNA-based SIP experiments with soil, it is important to recover as much of the 13C-labeled DNA as possible. Feinstein et al. (10) recently demonstrated that multiple extractions of an uncontaminated forest soil led to shifts in the relative abundance of various phyla and in operational taxonomic unit (OTU) composition in pyrosequence libraries between the first and sixth successive DNA extracts. The efficiency of DNA extraction kits used to recover DNA from SIP experiments has not been determined. We performed multiple, successive DNA extractions of 500-mg aliquots of PAH-contaminated soil that was enriched with anthracene to determine whether multiple extractions would affect DNA yield, eubacterial 16S rRNA gene recovery, or the identification of bacteria associated with anthracene degradation by SIP. Compared to a single DNA extraction, multiple DNA extractions maximized DNA yield and the recovery of 16S rRNA genes. Although an initial DGGE profile of each extract suggested that DNA from additional bacteria may have been extracted in later extractions, clone libraries with adequate sequence coverage (see Fig. S5 in the supplemental material) did not support this observation (Table 2). Successive DNA extractions did not result in the identification of additional bacterial groups beyond those identified in the first DNA extraction (Table 2), but there was a significant shift in the relative abundance of the identified genera across successive extracts, specifically from extract 1 to pooled extracts 5 to 7 (P ≤ 0.002). Overall, a group of bacteria within the Sphingomonadales, but not similar to any known genus (designated "anthracene group 1"), and members of the Pigmentiphaga and Variovorax genera were newly associated with anthracene degradation as a result of this study.
Assessment of anthracene-degrading microbial community activity.The activity of the anthracene-degrading microbial community native to the PAH-contaminated soil sample was assessed by measuring parent anthracene removal and radiolabeled anthracene mineralization. Because [1,2,3,4,4a,9a-14C]anthracene is labeled on only one of the two end rings of the symmetrical anthracene molecule, the actual amount of mineralization should be at least twice the accumulation of 14CO2 measured; thus, at least 25% of the anthracene was mineralized over the first 3 days, when the majority of anthracene was removed (Fig. 1). We infer that this would have been the period during which the majority of growth and assimilation of 13C from the parent compound would have occurred during incubations with 13C-labeled anthracene. However, mineralization continued through day 20 (Fig. 1), which might be attributed to the transformation of at least some of the anthracene to one or more extracellular metabolites over the first 3 days, followed by slower mineralization of such metabolites; continuing mineralization between day 3 and day 20 could also be due in part to turnover of previously assimilated carbon. Termination of the SIP experiment at a time point earlier than 20 days would have reduced the chances for dilution of 13C in cellular macromolecules, but allowing enough incubation time for adequate initial labeling of the DNA is also important to consider. In our experience, termination of the SIP incubation once reduced microbial activity is observed (as indicated by a reduced mineralization rate) is a reasonable compromise.
Identification of anthracene-degrading bacteria.Substantial growth occurred over the first 3 days of incubation in the presence of unlabeled anthracene for all of the bacteria identified in putative heavy DNA from the SIP incubations with 13C-labeled anthracene, other than those related to Sphingobium (Fig. 4). Acidovorax organisms known to be well represented in the soil (17), but not found in the clone libraries representing DNA in heavy fractions from the SIP experiment, grew slightly over this period as well; however, the increase in abundance of Acidovorax-related sequences was significantly less than that of all the SIP-identified groups other than Sphingobium. The increases in abundance of sequences related to AG1 and Pigmentiphaga exclusively during the period of anthracene removal in the incubation with unlabeled anthracene (Fig. 4), combined with evidence for 13C enrichment of their DNA in SIP incubations with [13C]anthracene (Fig. 5), suggests that these organisms grew on anthracene itself as opposed to an extracellular metabolite derived from anthracene.
Increases in abundance during the incubation with unlabeled anthracene continued to occur after day 3 for sequences related to Variovorax, Herminiimonas, and Sphingobium (Fig. 4), which may have resulted from growth on any anthracene metabolite(s) that had accumulated over the first 3 days or from slow growth on substrates unrelated to anthracene. Of these three groups, only Variovorax showed any evidence of 13C enrichment of its DNA during the SIP experiment with 13C-labeled anthracene (Fig. 5). Although DNA from Variovorax-related sequences shifted to heavier fractions in SIP incubations, these sequences were spread over a wider range of fractions than that observed for sequences representing AG1 or Pigmentiphaga organisms. Combined with the continued increase in abundance of Variovorax sequences after the period over which anthracene had been removed (Fig. 4), these data suggest that assimilated 13C in Variovorax DNA was diluted with unlabeled carbon over the 20-day incubation period of the SIP experiment.
Unlike Variovorax sequences, neither Herminiimonas- nor Sphingobium-related sequences showed evidence of 13C enrichment of their DNA in the SIP experiment (Fig. 5). The presence of Herminiimonas sequences in the clone libraries representing heavy DNA from the SIP experiment (Table 2) and their significant increase in abundance during the period in which anthracene removal occurred (Fig. 4) are, therefore, puzzling; nevertheless, we cannot conclude that these organisms grew on anthracene. Sequences 99.4% similar to the Herminiimonas-related sequences identified in this study were associated with a phenanthrene-degrading isolate recovered from PAH-contaminated soil from a former coal gasification plant in Iowa City, IA (39), but members of the genus have not been associated with anthracene degradation.
Although Sphingobium-related sequences increased in abundance slightly after 3 days of incubation with unlabeled anthracene (Fig. 4), the greatest increase occurred after the added anthracene had been consumed. This observation, combined with the lack of evidence for assimilation of 13C, suggests that Sphingobium-related bacteria grew on something other than anthracene. The presence of Sphingobium-related sequences in putative heavy DNA from the SIP experiment (Table 2) is likely due to their high initial abundance in the soil as well as their very high abundance at the 20-day time point, corresponding to the termination of the SIP experiment (Fig. 4). Sphingobium spp. have previously been associated with the degradation of PAHs composed of up to 5 rings (14, 21, 29), but only cooxidation of anthracene by a Sphingobium species has been reported (29).
Of the organisms associated with anthracene degradation by SIP, organisms related to AG1 have previously been associated with hydrocarbon-contaminated environments but not specifically with anthracene degradation. Sequences representing AG1 are 98.6% similar to sequences previously recovered from the Rancho La Brea Tar Pits in Los Angeles, CA (20). Variovorax spp. have previously been associated with the degradation of biphenyl (23) and naphthalene (15, 27) but not with anthracene degradation. In the only previous study to associate the genus Pigmentiphaga with PAH contamination, 16S rRNA gene sequences that were >97% identical to those of Pigmentiphaga kullae were recovered from creosote-contaminated soil, but an isolate was not recovered to be tested for specific PAH utilization (21, 27).
Effect of multiple DNA extractions.Similar to the findings of Feinstein et al. (10), multiple DNA extractions did not result in the identification of additional bacterial groups compared to those identified from a single extraction, but there were shifts in relative abundance across the range of extracts. However, a single extraction might have resulted in a much lower representation of sequences belonging to AG1 than that which we observed from the pooled extracts (23% versus 43%, respectively) (Table 2; Fig. 3). The DNA recovered from single extractions of multiple aliquots of soil from the same site is often pooled to reduce the potential effects of extraction bias with such heterogeneous material, but pooling of DNA will also increase the amount of total DNA recovered. Testing of the efficiency of the FastDNA spin kit for soil (see Fig. S6 in the supplemental material) revealed that a single extraction of the maximum 500-mg soil load recommended by the manufacturer was not optimum for the recovery of genomic DNA or 16S rRNA genes from a PAH-contaminated soil. It is likely that at this soil load, natural organic matter and/or contaminants in the soil that were coextracted with DNA resulted in competition for binding sites on the solid-phase sorbent and may also have interfered with PCR of the eluted material (24). We cannot extrapolate our observations to other soils, but we recommend that DNA extraction efficiency as a function of the soil load be determined before extensive experimentation with any given soil and DNA extraction kit.
The benefits of performing multiple DNA extractions on a sample will depend on the downstream applications of the DNA. In an SIP investigation, it is important to maximize DNA recovery, particularly if the heavy DNA in the metagenome is only partially labeled. Partial labeling leads to weaker separation from unlabeled DNA during ultracentrifugation than if the DNA were nearly 100% enriched in 13C. Overall, this study demonstrated that optimizing the recovery of 13C-enriched DNA from an SIP experiment may represent a compromise between the length of the incubations, the number of successive DNA extractions, and the number of soil aliquots for a given soil load.
Funding for this research was provided by the National Institute of Environmental Health Sciences (grant 5 P42ES005948).
We thank Stephen Richardson and Tony Gutierrez of our lab for assistance with HPLC analyses and for troubleshooting DNA separation with SYBR Safe, respectively.
- Received 16 August 2010.
- Accepted 28 February 2011.
- Accepted manuscript posted online 11 March 2011.
↵† Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01942-10.
- Copyright © 2011, American Society for Microbiology


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The Armenian national football squad on Monday will be hosted by Liechtenstein in their sixth—and last—UEFA Nations League group-stage match.
Bookmaker companies consider Armenia the clear favorites in this clash. On the average, they give a bet of 1.33 for Armenia's win; the bet for Liechtenstein winning this game is 11, whereas that for a draw is 5.50.
The Liechtenstein vs. Armenia game is slated for kick off at 11:45pm Armenia time.
In Group 4 of the Nations League Division D, Macedonia top the current table with 12 points, Armenia are in second place with 9 points, Gibraltar are third with 6 points, and Liechtenstein are at the bottom with 3 points.
Solely the winners of each group will advance to the next round.
The copyright for information published on this web site is owned exclusively by Armenian News-NEWS.am information-analytical agency. All information materials published on this website are intended solely for personal use. For full or partial reproduction of any material in other media it is required to acquire written permission from Armenian News-NEWS.am information-analytical agency. Those, who have committed copyright violations, will be prosecuted accordingly.

subset: commoncrawl


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Alex Kurtzman and Roberto Orci, co-writers of director J.J. Abrams' Star Trek reboot, have broken the story for the sequel. The frequent collaborators, who are writing the second Trek screenplay alongside Lost co-creator Damon Lindelof, spoke with Hero Complex and hinted that the next movie could pave the way for another sequel.
Orci said: "I don't know that we've ever thought of it in terms of a trilogy. We thought of the first one as, 'How do we tell how this happened the first time and how do we free it so that it can go on forever without stepping on what came before.' So, if you were thinking of this movie as a second act, yeah, you would think of it as an Empire Strikes Back sort of story, but I'm not sure we're thinking of it as a second act. I can't speak for everybody on that, though."
Kurtzman added that good sequels "find ways to challenge their characters in ways that they couldn't have necessarily been challenged with in the first movie because, as Bob said, the first is always, ultimately, an origin story. So now [with the second] it becomes about this family that's together, so now it becomes about the thing that shakes them up and challenges them."


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From Dwain: The Fahe Loan Committee approved $4.395 million dollars in CLS loans to Fahe Members at their 3/21 meeting. The funds will support Neighborhood Housing Services of Birmingham and Eastern Eight CDC as they build affordable housing and will fund the acquisition and operation of Housing Authority of Mingo County's new residential recovery facility.
APRIL IS FAIR HOUSING MONTH! Check HERE for events & materials. Contact Jackie to share what you are doing to inform and celebrate in your community.
Registration is OPEN! Spring Retreat – April 24-25 ~ Boone Tavern Conference Center, Berea
- Funding Opportunities
- Training Information
- KHC & Partner Updates
- Upcoming Events
- Network Job Postings
Just Transition Fund 2019 Rapid Response Fund – accepting proposals immediately,
continuing on a rolling basis until November
1, 2019. To apply: Contact Mandy
Bridenhagen and send a brief, one-paragraph
summary describing what you seek funding for and why. If invited to submit a proposal, we
will send your organization a link to a short application form.
ARC POWER INITIATIVE – RFP has been released, Application due April 10
HUD – Round 3 YHDP NOFA: Youth Homelessness Demonstration Program is now available. Designed to address systemic responses to youth homelessness and significantly reduce the number of youth experiencing homelessness in up to 25 participating communities (8 of which will be rural). Deadline: Wednesday, May 15, 2019 at 11:59:59 PM EDT
Webinar hosted by the US Interagency Council on Homelessness: Youth Homelessness Demonstration Program: Strategies for Success – review of the YHDP application and strategies for success. Also covered, what to expect if your community is awarded the grant. April 12, 3-4:30 p.m. ET Register
USDA-RD will begin accepting the first round of applications for the Rural e-Connectivity Pilot Program (ReConnect Program) on April 23, 2019, through reconnect.usda.gov.
Deadlines are:
- May 31, 2019, for grants;
- June 21, 2019, for loan/grant combinations; and
- July 12, 2019, for low-interest loans.
Applicants can request technical assistance from USDA by filling out the contact form at https://www.usda.gov/reconnect/contact-us
FHLB Cincinnati: The WELCOME HOME program opened March 1- contact David Brock or Linda Sabino at JCL for details. These funds go fast so please act quickly to reserve a spot for your client!!
Disaster Reconstruction Program (DRP) has been funded ($3.6M) for 2019 on a first-come, first-served basis as long as funds are available, additional information here. Eligible Counties
April 17 – May 14 – NWA Course: Strategic Thinking & Planning View the full course description. Faculty-led online course focuses on why strategic thinking and effective planning are vital to organizational survival and success. Ideal attendees include executive and senior management staff members, board members, and staff members of nonprofit organizations responsible for facilitating a strategic planning process. Virtual Classroom Participant Guide REGISTRATION DEADLINE 4/17
Low Cost/FreeNonprofit Leadership Center Training: Allstate Foundation in cooperation with the Kellogg School at Northwestern University.
IREM LIHTC Compliance Training and HCCP Exam – Thur/Fri, May 9 and 10 – Ohio Capital Corporation for Housing (OCCH) Training Academy will be hosting a two-day training at Lexington-Fayette Urban County Housing Authority, 300 West New Circle Road, Lexington, Kentucky. Review the flier available on OCCH's website. Questions? Lynn Logan at 614-224-8446 / email@example.com.
NeighborWorks America NTI – The last NTI of the year: New Orleans, LA Aug 19-23 (expect registration information late spring)
USDA LINC's Training and Resource Library
Kentucky Housing Corporation (KHC)
Statewide Fair Housing Training: The Lexington Fair Housing Council (LFHC) is offering their annual Statewide Fair Housing Training on Friday, April 12, 2019, from 1-5 p.m. ET, at the Blue Grass Regional Marketplace, 4561 Iron Works Pike, Lexington, Kentucky. Free, Open to the public, limited seating: please register by 5 p.m. ET, on Tuesday, April 9, 2019.
RFP for an Analysis of Impediments and/or Five-Year Consolidated Plan Consultant Due 5 p.m. ET, on Monday, April 22. For more information, please visit KHC's website, under About, Request For.
Weatherization Public Hearing: at KHC, 1231 Louisville Road, Frankfort, Kentucky, on Tuesday, April 16, – 2 p.m. ET, for the U.S. Department of Energy Weatherization Assistance Program state plan. A draft of the state plan is available on the Weatherization Assistance Program page of KHC's website.
Job Opportunity: Paralegal II – Real Estate: Legal Services and Compliance; Salary Range: $37,908 –
Please respond no later than Thursday, April 11. Full details are available on KHC's website, under About, Careers, Job Opportunities.
Homeless & Housing Coalition of KY (HHCK)
We have 38 AmeriCorps slots available for the 2019-2020 program year. NEW THIS YEAR: The 2019-2020 program year will begin on August 1st, 2019. Note: 2018-2019 members will still have until August 31st, 2019 to complete their service hours. Application due May 3
SNAP Proposed Rule Comment Period Extended through April 10 – USDA announced today that the comment period for the proposed time limit rules will be re-opened in response to complaints about technical difficulties with the federal register site, USDA announced that they will re-open the comment period from Monday, April 8 through Wednesday, April 10. The regulations.gov and the federal register sites will accept additional rule comments during this 3 day period.Feeding America and Center for American Progress are also keeping their platforms open for comments through April 10.
USDA: Improvements to Online Tool Single Family Housing (SFH) Direct Eligibility Assessment tool found on the USDA Income and Property Eligibility Site.
Announcement on Recovery Housing in Rural Communities
For program information…
- Farm Labor Direct Loans & Grants
- Housing Preservation & Revitalization Demonstration Loans & Grants
- Housing Preservation Grants
- Multi-Family Housing Direct Loans
- Multi-Family Housing Loan Guarantees
- Multi-Family Housing Rental Assistance
- Rural Housing Site Loans
Consumer Financial Protection Bureau: Notices of proposed rulemaking regarding the Payday, Vehicle Title, and Certain High-Cost Installment Loans Rule (2017 Payday Lending Rule) here. The Delay NPRM proposes to delay the compliance date for the mandatory underwriting provisions available here. You can access the summary and redline here.
Answers to Frequently Asked Questions related to the TRID Rule available here.
Blog: Trends in Home Repair Volunteer Numbers & Recruitment Ideas, a comparison of 20 organizations and tips for increasing volunteer numbers.
Sessions from their Conference: ADVOCACY 101 , Asset-Based Community Development (ABCDs) for Home Repair, Presenter's notes from the session here. Reframing Home Repair Nonprofits for Philanthropy
Foundation for a Healthy Kentucky – access to their data HERE (searchable by location and topic)
April 24-25 – Fahe ED's Spring Retreat – Boone Tavern Conference Center, Berea, KY Register HERE ROOM BLOCK CLOSES THIS FRI. 4/5 – CALL 1-800-366-9358 TO RESERVE!
Sept. 9 -12 – Annual Meeting & Rural Housing Summit ~ Hilton Knoxville
Sept. 12-13 – 2019 Kentucky Afforable Housing Conference (KAHC) – Lexington Convention Center
JOB POSTINGS – Check out a new posting from Frontier: Accounting Clerk; that and more Fahe, Member and Partner job postings HERE
Jobs available: MACED, Rural LISC/RCAC: HERE ReFrame Job Board HERE
Are you interested in Rural Prosperity through the Arts and Creative Sector? Check out this report and resources:A Rural Action Guide for Governors and States
From NRHC: You can find the 116th Congress committee assignments for the House and Senate appropriation subcommittees on: Agriculture and Rural Development; Labor and HHS; and Transportation and HUD HERE
Questions or to express interest in Fahe Strategic Programs? Please email Tyler Johnson
Resources from Previous Webinars/Spring/Annual Meetings can be found HERE
Sign up to receive the Rural LISC Rural e-News each month, click here.
If you have any items you would like to share with the Membership such as job postings, events or news email them to Jackie


text:
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« All Events
On Wednesday August 7th our church will pray over all of our kids, youth, teachers and administrators going back to school for the new year. We will meet at 6:30 PM in the Chapel for this special time of blessing and encouragement. Prior to the service we'll have a fajita bar dinner from Moe's Southwest Grill in the gym from 5 to 6:30 PM for only $5/person. Register HERE or by calling the church office by Friday, Aug 2nd. All are welcome!
710 Mimosa Boulevard • Roswell, GA 30075 • (770) 587-6980 • firstname.lastname@example.com
Weekend Pastor on Call: (770) 344-0319
Service Times: 8:30 AM / 11:00 AM
© 2018 First Baptist Roswell | All Rights Reserved
Powered By Precision Creative


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The new center will enable data sharing and analysis between the organizations, with the goal of creating a synergy between the patient-driven drug-development capabilities of MD Anderson's Therapeutics Discovery division and Boehringer Ingelheim's medicines, according to a press release.
"We could not have chosen a better partner with all its research, translational and clinical expertise in lung and gastrointestinal cancers," said Victoria Zazulina, corporate vice president and global head of oncology, medicine, at Ridgefield-based Boehringer Ingelheim. "Together, we hope to transform the treatment landscape for these diseases by tackling their root causes and drivers, that have so far remained elusive, exploring new and smart ways of killing cancer cells."


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Book with Other Items
Published: 18th November 2016
Number Of Pages: 896
Earn 241 Qantas Points
on this Book
Statistical data analysis is the backbone of sound business decision making, and finding the right tool to analyse a particular business problem is the key. By learning the fundamentals of statistical reasoning and data analysis, you will be on the way to becoming a better manager, analyst or economist.
By providing a framework for solving statistical problems, this seventh Australian and New Zealand edition of Business Statistics teaches skills that you can use throughout your career. The book shows you how to analyse data effectively by focusing on the relationship between the kind of problem you face, the type of data involved and the appropriate statistical technique for solving the problem.
Business Statistics emphasises applications over theory. It illustrates how vital statistical methods and tools are for today's managers and analysts, and how to apply them to business problems using real-world data. Using a proven three-step Identify-Compute-Interpret (ICI) approach to problem solving, the text teaches you how to:
1. IDENTIFY the correct statistical technique by focusing on the problem objective and data type;
2. COMPUTE the statistics doing them by hand and using Excel; and
3. INTERPRET results in the context of the problem.
This unique approach enhances comprehension and practical skills.
The text's vast assortment of data-driven examples, exercises and cases covers the various functional areas of business, demonstrating the statistical applications that marketing managers, financial analysts, accountants, economists and others use. Learning resources such as CourseMate maximise study time to help you achieve the results you want. Completely up-to-date, the seventh edition offers comprehensive coverage, current examples and an increased focus on applications in the real world.
New to this Edition
Audience: Tertiary; University or College
Format: Book with Other Items
Language: English
Number Of Pages: 896
Published: 18th November 2016
Publisher: Cengage Learning Australia
Country of Publication: AU
Dimensions (cm): 27.5 x 21.2 x 2.8
Weight (kg): 1.7
Edition Number: 7
Edition Type: Revised
Earn 241 Qantas Points
on this Book


text:
In one of the most enthralling Round of 16 clashes of the 2018-19 UEFA Champions League, two brilliant sides but with very limited success will take on each other.
For both Tottenham Hotspur and Borussia Dortmund, a win in the first leg is vital considering their tough challenges in the domestic league.
In Germany, Dortmund have a five-point lead over defending champions Bayern Munich, but they are now feeling the heat after bad results in their last three outings.
In England, Spurs are keeping their league title hopes alive but their recent wins have been scrappy. A good performance in the continental competition will boost their confidence. At third in Premier League, with limited resources, they can afford a tough return leg.
Yes, Spurs were knocked out in the same stage of the second-tier Europa League 2015-16 by the Germans but got their revenge last season by winning both the Champions League group stage matches.
All You Need To Know About The Match:
Match: Tottenham Vs Dortmund, Round of 16 first leg, UEFA Champions League
Date: February 14 (Thursday)
Time: 1:30 AM IST; 20:00 PM Local (Wednesday)
Place: Wembley Stadium, London.
TV Guide: Sony Pictures Networks India
Live Streaming: SonyLiv
Likely XIs:
Spurs (3-5-2): Lloris; Alderweireld, Sanchez, Vertonghen; Trippier, Sissoko, Winks, Eriksen, Walker-Peters; Llorente, Son
Dortmund (4-2-3-1): Burki; Hakimi, Diallo, Zagadou, Guerreiro; Delaney, Witsel, Sancho, Philipp, Larsen; Gotze
Key Facts:
This is only the third time Spurs have got this far in the tournament, and have progressed from the Round of 16 just once before.
But, Dortmund are in the Round of 16 for the fifth time, having progressed in three of their four previous appearances.
Prediction: 2-2. Dortmund will have a slight advantage with the hosts missing Harry Kane and Dele Alli, two of their best players. But the presence of free-scoring, creative work-horse in Heung-Min Son will make the encounter an enthralling affair. The same can be said of Raphael Guerreiro, who has scored four Champions League goals for the Germans this season.


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Aqa (a) Biology As2 (Aqa Student Support)
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A revision-style booklet written by senior examiners to match perfectly to particular specifications. Each booklet also contains a sample test and answers to allow students to check their progress. Enzymes Genetic code Recombinant DNA Crop plants Domesticated animals Forensic science
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Ulster County once again has set the example. Now, it is time for others to follow by talking and acting.
The example this time comes in a ban on single-use plastic bags in the entire county, the first one in New York state to pass such a law although a dozen other New York municipalities and counties have passed some form of legislation seeking to reduce their use. Suffolk County allows plastic bags but began imposing a 5-cent surcharge on both paper and plastic bags in January.
The Ulster law takes effect July 15 and applies a 5-cent fee for paper bags. Businesses that violate the law after first getting a written warning are subject to fines of up to $100 for the first offense, $250 for the second and $550 for the third and subsequent violations.
Legislator Tracey Bartels, an independent from Gardiner, was the primary sponsor of the bill, which passed the county Legislature 15-7 last month.
"Every year, New Yorkers use more than 20 billion plastic bags, and just two weeks ago, the U.N. Intergovernmental Panel on Climate Change warned that the world has 12 years to act to prevent catastrophe," Bartels said. "Today, with this bill, Ulster County commits to taking action."
The county executive, Mike Hein, signed the bill into law and said he looks "forward to working together to make this plastic bag ban a reality as we continue to protect our children, our families and our environment for generations to come."
Those who might be tempted to follow the Ulster example will need to do some work to convince legislators who might see this as an unwarranted intrusion into local business practices or who might be sympathetic to the general idea but wary of a ban considering research that shows the carbon footprint, the energy expended and pollution produced, is lower for plastic bags than for paper ones. But plastic bags also have a very visible, very obvious detrimental impact on the environment starting with roadside litter and continuing to the effect they have on landfills.
Any government considering action also should look at an issue that came up in New York City earlier this year — plastic straws. Some chains and stores have voluntarily ended their use after learning about the difficulty of recycling them, their contribution to the growth of landfills filled with things that do not dissolve and even the presence of high levels of plastic fibers in tap water from public supplies.
The importance of the action in Ulster County is not only measured by the immediate impact on the local environment. Just as important is the elevation of the issue in local politics and government and the realization that we do not have to wait for the federal government or other nations to act to have an impact.
Never has the slogan "think globally, act locally" been more important than it is now with an administration in Washington that would prefer that we neither think much at all nor act.
Nearby counties should be putting these ideas on their agendas and inviting informed citizens to join in the discussion.


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Hi all, my question is for the whole community. As an ecologist, former high school teacher and human on planet earth I wonder about the sustainability of the Sous Vide cooking method. Food packaging is already a major burden on our environment, so I ask if you all believe that cooking with the sous vide method is a responsible way to cook.
It's electric, which is great as you can source your power from "green" providers. But the use of disposable plastic bags seems anathema to our current state of affairs on planet earth.
I looked through the forums and see that there is the secondary health concern of chemicals found in plastic bags leaching into the cooked food, and thereby getting ingested by customers.
I also noticed that "silicone, food grade bags" are recommended by the staff at Anova. And I see these available on amazon for seemingly reasonable prices.
I guess my question is, if you use the sous vide method, how many of you use disposable plastic bags vs silicone. And how many of you are concerned this the use of disposables is impactful to our environment?
I use the silicon Stasher brand bags for anything that will fit in them. I'm usually only cooking for two people, so unless I'm doing a roast or cooking something for the freezer they're my go to. The Stasher bags are soft and conform well to the shape of the product in them.
I am currently using ziplock style food safe plastic bags with a one way valve which my manual vacuum pump works with. I have been able to wash them and reuse them so far but once they have become damaged or otherwise worn out and unusable and I have had to discard them I will not be buying any more of those and will be using my electrical vacuum and sealing machine with the two sizes of rolls of plastic that I bought to go with it over 12 months back but have yet to use.
Richard, thank you for raising an important topic and one i try not to think too much about in order to keep my anxiety disorder somewhat under control.
A more significant and broader concern might be the sustainability of what we cook more than the how and the with-what.
Consider how many more people could be fed using sustainable practices instead of consuming the drug-laden Government subsidized GMO-corn fed livestock raised in the contemporary corporate factory-raising method. Who remembers when cattle raising was a solar powered industry using grass?
When i see the images of enormous slabs of meat being cooked and served on Anova's associated Facebook site i often wonder about both health and sustainability issues. Has charing overtaken searing? Has the fattiest meat possible overtaken the formerly more healthy lean meat?
As our planet's population grows we have a substantial amount of rethinking and replanning to do around sustainability.
Comments anyone?
To each his own. I prefer to introduce as little stress as possible to my cooking and eating...worrying about overcooking is about it.
Ahh yes, the beauty of SV cooking. So little to worry about, not even overcooking once you discover your favourite times and temperatures.
SV frees up my time for more significant concerns like the Debt, a declining culture, waste of all kinds, friction in the Middle East, safe drinking water, negative interest rates, and how to keep marauding rabbits out of my garden. Fortunately, all small stuff.
I use a mix depending on what seems most appropriate. I do often use Stasher or SIOChef bags to cook in. But I also sometimes use food saver bags. I use food saver bags when I buy in bulk and portion it out to freeze for storage. I just make the bags a tad longer so if I need to season before cooking I can just cut carefully and then reseal the same foodsaver bag. Best I can tell the reduction in food waste makes the use of the plastics a wash, for me anyway.
Thank you Richard,I really enjoy SV cooking and up until recently utilized vacuum seal bags in all stages of the process. I live in Nova Scotia, a province surrounded by ocean. I see what plastic is doing to our water and it scares the hell out of me. Every time that I finish Sving and put the used bag in our plastic recycling I cringe.I have come up with a process which, although does not solve the plastic waste problem associated with SVing, it helps in a small way.First I season my food item, then freeze it on a pan. Once frozen I then vacuum seal it. This solves two problems. First when vacuuming the frozen item I prevent the juice from being drawn out. Second, because I am placing the item frozen solid in the vacuum bag I am leaving very little food residue in the bag. When it comes time to SV I open the bag take out the still frozen item and place in my recently purchased silicone bag, these are fantastic, and complete the cooking process. The vacuum seal bag is now much easier to clean properly and reuse. I may only get 2 or 3 reuses but every little bit helps.Yes it does take some extra steps and time but I have 3 grandchildren and I really want them to get as much enjoyment out of our oceans as I have. Hopefully this will help in my own small way.
Responsible? Sous vide cooking is possibly the most responsible way to cook. Its very energy efficient, causes no air pollution, and on longer cooks, with sufficiently higher temperatures-does a better job of sanitizing the food through pasteurization. Win-win-win.No power from "green" providers. I'm stuck with the power company which supplies this part of the state.Using food grade plastic bags which can be washed and reused, is more responsible than cooking in aluminum, or non-stick coated pots and pans, which do leach chemicals. It even beats cooking on a flat rock over a campfire, because you don't know what's in the stone. Cooking sous vide means that the temperatures seldom get high enough to cause problems from the plastic bags that contain the food. I use plastic, and wash it for repeated uses, but if the prices of the silicone sous vide bags become more reasonable, I will try them, but I'm happy with the plastic bags until that time.
I agree That plastic is a huge problem that our generation has caused and try to limit the use. Most foods are packaged in plastic. Now thanks to Sous Vide cooked in plastic such a small amountMaybe education for the yuppie set that washing nappies is better than the plastic that will be around after baby has grown. That really annoys meThey cry help the environment. Please.Everything will cause health problems. Be sensible and if we all just did a little to stop the waste and use of toxic stuff.BUT no can't wash nappies the disposables should be banned like single use plastic bags.I love what Sous Vide had brought to my home and have no inhibitions as to a little toxicity for super cooked foods.
When I began sous vide cooking (almost 5 years ago), I purchased a chamber vacuum sealer from Vacmaster (VP215) and some 1000 bags. Maybe 2000. Anyway, this "system" seemed to be the best for my purpose of packaging and sealing sous vide dishes for later use. Most are stored and rotated. The cost of the bags is approximately $30-60 per 1000 depending on the size of the bags. Unfortunately, recycling the plastic is not very practical where I live. Having that many bags available is convenient when you are packaging 30 chicken breasts at a time, 7 large ribeyes, a pile of beef short ribs or 20 pounds of ham. I have been able to help others enjoy SV cooking and helped them package their food as well. I use the chamber vac for sealing other items too, I think recycling is the way to go though. I do believe that reuseable silicon packaging is great for those that cook sous vide occasionally. Up until recently, I made sous vide dishes almost daily so having prepackaged meats or veggies was great (mostly meats). My largest SV meal was three spiral cut hams for a funeral at church. It was perfect. Discussions like this are important. We should continue to make sure our cooking evolves so that it is easier, more efficent, and sustainable, as well as tasting darn good.
I started to experiment with Mason jars using the attachment to vacuum them before putting them in the bath. So far I had pretty good results with moist items, I just add 30 minutes to compensate for the jar warm up time and I let them move around in the bath.
I am just learning Stasher bags but I don't thing they have a product compatible with vacuum sealers. Are they safe at the higher Sous Vide temps (e.gt. 165)While it is aggravatingly time consuming, I re-use various brands of vacuum zip lock bags after soaking them in very hot water and hand washing them. The valves and/or zip lock seals eventually wear out but, depending on brand, I typically get 3-5 uses before having to toss them. Not a perfect solution for the plastic issue but it is a significant reduction.
FYI on bag washing: A while back I made several forms from wire clothing hangers, turned the bags inside out, inserted a form in each bag, and then washed the bags in the dishwasher. WAY easier and certainly more sanitary - except the hangers start rusting inside the bags (which is actually the outside of the bag when being washed). Only a cosmetic issue and does not affect the food (that I know of) but seeing all that rust on the outside as I put food IN the bags started sapping my appetite. Now, if I were more of an entrepreneur I would come up with adjustable non-metallic forms and sell them on the web. I know why Food Saver et al don't want to see or sell something like this it does not seem to be brain surgery for someone more energetic than me :-).
Jack, what are you cooking in Mason Jars?
If it's meat or poultry how effective is the heat transfer to your food?
Poultry and fish, both in a broth/sauce, the heat transfer was effective, but I added 30 minutes or so to make sure that the jar got warm enough on startup.
J. interesting technique. Was the jar completely submerged?
How did you calculate your cooking time?
Just took the regular time and added 30 minutes for the jar warmup. I tried with the 250ml (8oz) jars, the more they are filled the less buoyancy they have. They move around the pot, but at that speed, they won't crack. I'm gonna try with meat soon.
Stasher bags sound interesting, ty for sharing.
J. how long is regular time?
By jar warmup do you mean that you put the filled jars in warm water to start and then started your Anova to get water temperature up to your egg cooking temperature? That would prevent breakage from thermal shock that some of Community experienced.
More from the enviro community, who cannot bear to see people enjoying themselves without pointing out the imagined imminent dangers to mankind. Like those hypocrites currently jet-setting in the Med on a 'global warming' junket where they arrive in private jets and mega-millions yachts to protest about the mis-use of energy by the proletariat. You and I had better cut our energy and quickly, but it's OK for them to use it like gangbusters (Al Gore - when he's not busy inventing the Internet - spends more on heating one of his swimming pools than the average household does on its annual energy bill, for example)
(1) so called 'green' energy is a major swindle. It would not exist without massive subsidies from the rest of us which show up in increased energy bills for Joe Public. Wind and solar are both hopelessly expensive and inefficient compared to clean coal and gas fired turbines which produce 99%+ of the energy today on which modern society thrives. Have you seen the image of an 'environmentally friendly electric car' charging up roadside from a unit powered by - wait for it - a diesel generator? You couldn't make this stuff up.
(2) plastic waste in the oceans is a problem related to inefficient or non-existent waste disposal and recycling facilities in the 3rd world. 95%+ of plastic in the oceans comes from 3rd world countries where people chuck their garbage in rivers - India and China are the main culprits. The solution is not to stop the use of plastic in the first world - where it does enormous good in terms of preserving food for example - but to provide waste disposal systems to the 3rd world. China is embarking on a major clean-up in this regard, which will have hundreds of orders of magnitude greater effect than the virtue-signalling of cutting out straws with your Coke + Big Mac.
(3) the issue of contamination from plastic bags during the sous vide process is possible. The solution is to use food-grade plastic.


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Primary challenge to Amy McGrath's bid to take down McConnell 'might be helpful,' Dem lawmaker says
The knives are out for Amy McGrath.
Rep. John Yarmuth, the only Democrat elected to federal office from Kentucky, hinted Thursday that McGrath, a Marine combat aviator, might not be the best candidate to take down Senate Majority Leader Mitch McConnell next year as a series of unforced errors have already buffeted her campaign in its first two days.
Yarmuth told the Louisville Courier-Journal that McGrath made a "pretty significant mistake" by indicating that she would have voted to confirm Brett Kavanuagh to the Supreme Court on Wednesday. McGrath, who said she opposed Kavanaugh at the time of his contentious confirmation fight, flip-flopped four hours later on social media and said that after "reflection," she would have voted against Kavanaugh.
Yarmuth told the paper that the first days of McGrath's campaign "could have gone smoother," despite raising a whopping $2.5 million in its first 24 hours prior to hitting turbulence. By the end of the third day of her campaign on Thursday, McGrath had tallied over $3.5 million.
"By her own admission she made a pretty significant mistake and corrected it," the lawmaker said. "I hope she prepares a little bit better for the rest of the campaign. I'm sure she will."
"I think a primary might be helpful in this situation."
"I think a primary might be helpful in this situation," Yarmuth added. "Because whoever is going to face off against Mitch McConnell really needs to get their game in shape. Certainly, Amy would be a favorite in any kind of primary, but I think it would help her and if somebody else could beat her they would have demonstrated their strength as well."
Kentucky sports radio host Matt Jones was among the most likely to jump into a primary to challenge McGrath. Speaking Thursday, he said McGrath had experienced key "hiccups," and indicated he was open to running and would decide "fairly soon."
He specifically faulted McGrath for appearing to have "authenticity problems."
"I think she has let the national Democratic Party and the consultants consult her to death. They have literally taken the personality out of her," Jones said at a business summit. "I would say to Amy McGrath, be Amy McGrath. Because at the end of the day if you're Amy McGrath and you lose, then you can look in the mirror and say look I did it. If you're this person they created... you're gonna lose and you're gonna feel bad."
McGrath's campaign launch Tuesday was aided by a breathless NBC News report hours earlier that McConnell's distant ancestors owned slaves -- a revelation blunted by McConnell pointing out that President Barack Obama's ancestors did as well.
In another striking moment, an eager MSNBC anchor also urged McGrath to tell viewers how they could easily donate to her campaign online.
But McGrath, who ran unsuccessfully for the House last year, was quickly criticized for her claim in a televised interview earlier on Tuesday that President Trump's election was similar to the 9/11 terrorist attacks. Then she opened the floodgates in an interview with the Courier-Journal, Kentucky's highest-circulation newspaper.
"You know, I think that with Judge Kavanaugh, yeah, I probably would have voted for him," McGrath said Wednesday.
"I didn't listen to all of the hearings. I don't think there was anything, and I'm not a lawyer or a senator on the Judiciary Committee, so I don't know the criteria," McGrath offered. "But I was very concerned about Judge Kavanaugh, what I felt like were the far-right stances that he had. However, there was nothing in his record that I think would disqualify him in any way. And the fact is when you have the president and the Senate, this is our system and so I don't think there was anything that would have disqualified him in my mind."
Although McGrath called Christine Blasey Ford's accusations of sexual misconduct by Kavanaugh in high school "credible," she reiterated that she did not view them as "disqualifying."
"Well, I mean I think again, I think it's credible but given the amount of time that lapsed in between and from a judicial standpoint, I don't think it would really disqualify him," McGrath said.
Four hours after her remarks were published, McGrath tweeted a mea culpa that immediately drew scorn from both Democrats and Republicans.
"I was asked earlier today about Judge Brett Kavanaugh and I answered based upon his qualifications to be on the Supreme Court. But upon further reflection and further understanding of his record, I would have voted no," McGrath wrote.
She continued: "I know I disappointed many today with my initial answer on how I would have voted on Brett Kavanaugh. I will make mistakes and always own up to them. The priority is defeating Mitch McConnell."
Reaction on social media was unsparing.
"This, my friends, is what we call an unforced error," journalist Yashar Ali observed.
"Take your third position on this later, the night is young," said Jake Wilkins, the communications director for North Dakota Sen. Kevin Cramer.
Read the headline of an article on the left-wing blog Jezebel: "Unfortunately, the Woman Trying to Unseat Mitch McConnell Also Kind of Sucks."
McGrath narrowly lost a House race to an incumbent Republican in Kentucky last year. During that race, McGrath slammed Kavanaugh and suggested she would not support his confirmation -- leading some prominent commentators on social media to charge that McGrath's flip-flop was actually multi-layered.


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** This item is on back-order. The back-order item in any order will be shipped as soon as new shipment arrives. (ETA: 8th – 11th October 2019)
Fruit Pigmented® Natural Vegan Cruelty Free Gluten Free
For All Skin Types | Full Coverage | Matte Finish
Sandstone, a camel nude, semi-matte, opaque & long-wearing lipstick. 100% PURE's innovative formula ensures your lip color always looks vibrant, rich and full, never dry. Formulated with healthy fruit pigments and a nourishing blend of cocoa butter, shea butter and vitamin E to moisturize while providing intensely rich color.


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Elton John may be getting a £5million pay day in his Christmas stocking this year, after being reportedly snapped up to star in the John Lewis advert.
The John Lewis ad has become a new addition to the festive season, and often tells a story about families and friends in the run-up to Christmas day.
This year, claims report that Elton will be both in the ad as well as leading the soundtrack with his classic, Your Song.
It is thought the advert will follow through Elton's life, starting as a young boy with his family and then through jumping through time as he grows up.
At the end of the ad, it'll be Elton as the superstar we now know and love.
An insider told Mail on Sunday: 'It is all very, very charming and the exciting crescendo at the end will be Elton playing his famous piano.'
While this has not been confirmed, a John Lewis spokesman said: 'We are lucky enough to enjoy lots of speculation around our TV ads, much of which is inaccurate, and our one rule is to never comment on any aspect simply because we want to keep the magic.'
The Rocket Man launched a huge 300-date tour in September, and it is reportedly his last ever tour as he begins to wind down his busy schedule.
He was also reportedly offered a spot on the judges' table on The X Factor, but turned it down as he was too busy – leaving the door open for Robbie Williams to take over.
Producers tried to get the Rocket Man to appear, but the singer had to turn it down because he's way too busy.
Furnish told The Sun: 'There was talk about the possibility of a judging role for Elton.
'But he does 100 shows a year and he doesn't have time for X Factor judging roles. We're too busy with Elton's tour, which is starting in September.'
A source told Metro.co.uk: 'Elton has been approached to be a judge in the past – but wasn't this year.
'It must be a case of cross wires although he's always welcome to come on the show because we are big fans and he has performed before.'
If you've got a story, video or pictures get in touch with the Metro.co.uk Entertainment team by emailing us email@example.com, calling 020 3615 2145 or by visiting our Submit Stuff page - we'd love to hear from you.
Got a showbiz story?
If you've got a story, video or pictures get in touch with the Metro.co.uk Entertainment team by emailing us firstname.lastname@example.com, calling 020 3615 2145 or by visiting our Submit Stuff page - we'd love to hear from you.
MORE: Elton John announces 2019 UK tour dates, here's when and where to get tickets
MORE: Elton John celebrates Romeo Beckham's Sweet 16th with rest of the family and we want in


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With the last update, now we can play video html5 via gstreamer. We have new dependencies, gstreamer and gst-plugins-base, if we want full support we need to install gst-plugins-good and gst-libav. We can check the update directly in the commit
Efikamx device
Firefox 38.0.1 ready for CRUX-ARM
After a big research work about this port, finally we have published this new firefox version for ARM. We decided to publish with skia support with a PPC patch. You can check all changes in these commits: Updated to 38.0.1 version Skia activated For any kind of problem, question or suggestion, don't doubt to contact […]
EfikaMX-SmartTop posts
Official device webpage EfikaMX-SmartTop


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. Finding a commercial residence to permit is in no way an easy project. For the start,condo for sale in hoping to meet up with your existing and potential company needs must be deemed carefully with total focus. To get and allow your dream location, you can do this independently or as a result of a realtor to get a cost for the analysis and purchase stages. In amount, this article highlights some of the important techniques found it necessary to have the property searching approach less difficult.
Prior to embarking on the path to getting a industrial residence to permit, it is important to know where your business will suit involving the present time as well as the near future. For that reason, possessing a very clear thought of exactly how much space is necessary and what amenities are needed to fulfill the demands of your organization will all offer an appearance in regards to what kind of house is required. Questions to take into account are how many washrooms can we need? Is it suitable to find within a multiple-storey constructing? And it is the ground work surface suitable for the equipment ahead in place? Understanding and evaluating this sort of variables are essential in terms of developing optimistic perceptions for your clients, commercial residential property for rent employees and suppliers etc. From a area standpoint,houses or condos for rent ease of access will likely feature as another selection purpose.


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1/200 Longueville Rd , Lane Cove NSW 2066
Inspector: IPI Norwest
Phone: 0412-080-857
Here are the details of the Property Inspection that we have completed for this property:
Date Inspection Report Completed: 30/03/2016
Inspection Category: Unit Inspection
Property Address:
1/200 Longueville Rd ,
Lane Cove NSW 2066
Property Type: Unit
Number of Bedrooms: 2
Number of Bathrooms: 1
Parking: 1
Number of Storeys: Multi-storey building
Inspector: IPI Norwest
Phone: 0412-080-857
Request a copy of this Property Inspection Report.
The report is available for resale.
Please click here to request a copy of the Property Inspection Report for this property.
Or complete this quick form.


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Best Pillow for Sleeper
If you have a more compact room, it may cause a little bit of problem because most of those loft pillow kohls adapt a bigger room. Hence, you must do a few hacks in order for your room will probably look a little bit bigger despite the magnitude of this room. To begin with, you are always encouraged to use a mirror into your pillow to ensure it is looks larger. The 2nd solution will be if it is possible, you can make an effort to minimize the number of furniture included in these sets. This way that, apart from accessing bed pillows, it is also possible to adapt the sets to your smaller pillow space.
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A loft pillow kohls could be combined with assorted calming, neutral, and light colors. The white coloring makes it possible for you to have a lot more freedom in picking out the extra furniture away from the place. By way of instance, you may play the coloration of these sheets and pillowcases even though most of them are included at this group. You are able to attempt to receive yourself a brand new one with a suitable coloring to develop a difference and keeping the room to being overly monotone. In this place, normally the big furniture for example storages, pillow, and desk really are whitened. Following that, you may add colors into the little household furniture. You are able to try a bed pillows by adding a daring and bright color such as turquoise and orange in the event that you are looking for an even more modern or pop art theme. The secret is to learn what is the overall design you need to employ from the pillow.
This Loft Pillow Kohls the gallery form Beautyrest Extra Firm Pillow. Hopefully you can find the best inspiration from our gallery here.


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Italy are hoping Daniele De Rossi can show James Bond-like qualities to thwart England captain
Steven Gerrard in Sunday's Euro 2012 quarter-final.
The big-match is as eagerly anticipated in Italy as it is in England, and newspaper Gazzetta dello Sport has come up with an imaginative front page to mark the event.
It features tough-tackling midfielder De Rossi's head superimposed on a Bond-style figure, complete with pistol.
Gerrard is the man in the crosshairs and the tongue-in-cheek instructions make it clear that Gazzetta's reporters believe the Liverpool star is every bit as dangerous as 007's arch-enemy Blofeld.
'Daniele is our agent and for tomorrow evening he has a special mission: OPERATION GERRARD,' reads the headline.
The Azzurri will need De Rossi to be at his best, as they are likely to be without two experienced players for the last-eight clash in Kyiv.
Giorgio Chiellini has been ruled out with a thigh strain, while Thiago Motta may start on the bench because of a hamstring problem.
England boss Roy Hodgson has a fully-fit squad to choose from, after Ashley Young recovered from the shin injury he picked up in the win over Ukraine.
PICTURES: The England team in training


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tag:blogger.com,1999:blog-3011478142547655716.post1287468417450475405..comments2016-04-20T22:06:35.697+01:00Comments on A THIRTY SOMETHING: The Dupe - Loreal v Giorgio ArmaniSarah Phttp://email@example.com:blogger.com,1999:blog-3011478142547655716.post-47190177149856677982016-04-20T13:56:47.782+01:002016-04-20T13:56:47.782+01:00Thanks for this - my Giorgio is almost out, and I ...Thanks for this - my Giorgio is almost out, and I just can't justify the price to myself - it would go on credit. I will try True Match! Fingers crossed! Kristin.https://email@example.com:blogger.com,1999:blog-3011478142547655716.post-32602051803149412102014-04-23T05:45:08.997+01:002014-04-23T05:45:08.997+01:00I love postings like these who compare two or more...I love postings like these who compare two or more products with each other - however, in that cases I'd really love to get some pictures for comparison how the product looked applied on your skin to see for myself. I'm always a bit let down when there's no "proof picture" - although I don't think you're lying of course! It's just, I don't know, it makes it easier to judge for the reader I guess :)
Nonetheless, good post and I'd love to try the True Match foundation now!Sam Murakamihttps://firstname.lastname@example.com:blogger.com,1999:blog-3011478142547655716.post-72866182003697516632014-04-22T22:36:25.437+01:002014-04-22T22:36:25.437+01:00GA Luminous Silk is my ultimate favourite foundati...GA Luminous Silk is my ultimate favourite foundation but I've been using the L'Oreal True Match as well and I completely agree with you, perfect shade, buildable and long lasting, AH-MAZING DUPE! :) xx
hellotashaa.blogspot.co.ukHELLOTASHAhttps://firstname.lastname@example.com


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Ralph Devlin AM QC
Ralph Devlin was admitted to the Bar in 1977 and took Silk in 2005. As a Crown Prosecutor, he appeared in many major Criminal Trials before his appointment to the Fitzgerald Inquiry and Special Prosecutor 1987-1992.
Ralph appeared as lead Counsel for the Medical Board of Queensland in Disciplinary matters, as well as in many Investigative Hearings before the Criminal Justice Commission, in the next decade. He also developed a wide Criminal Defence practice.
In the late 1990's to the current day, Ralph has become the leading Counsel in the prosecution and defence of Environmental Enforcement cases in the Criminal Courts of Queensland-a relatively new Specialty at the Bar.
He was also a senior Surf Lifesaver, serving in a Volunteer capacity as President of Surf Life Saving Queensland (2011-2016) and a member of the Board of Surf Life Saving Australia (2011-2016).
In January 2016, Ralph was awarded 'Member of the Order of Australia' (AM) for 'services to surf life saving and the law.'
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We all need as much luck as we can get this Chinese New year. Fend off the negative vibes with our auspicious colour list this year of the pig. Stick to this colour palette and Chinese New Year luck might just be round the corner!
Blue – Rooster/Monkey/Ox/Dog/Goat
Blue is the perfect cool toned colour for those who are afraid of red this Chinese New Year. For those veering toward a more mild look, try blue for your eyeliner and add that tinge of horoscope magic in your makeup look.
We recommend this lovely beauty by Natasha Denona. The perfect blend of sparkle and vibrance, this eyeliner is sure to be the star of your CNY OOTD. Try it out for some extra glitz this Chinese New Year and watch your luck roll in!
Pink – Rat/Rabbit
Pink is an extremely versatile colour that suits any skin tone. With a variety of shades to choose from, pink adds that tinge of girly or edge to any makeup look.
For a subtle pinkish nude we recommend our girl Kat Von D. Though the formula may be a little drying, it springs to life with a little lip balm applied first on the lip. Pair this with natural eyelashes extensions and a strong cat eye for a balanced and chic look.
Silver – Horse/Tiger/Dragon
Silver highlighter is a great way to bring a slight edge to your Chinese New Year beauty look. Great for fairer skin tones, silver often comes off as mere shine and can add an iridescent quality to your overall look. Our favourite part about adding silver highlight is that it adds a touch of glam without much effort at all!
This miracle product is a hybrid of cream and powder. It adds the perfect touch of sparkle to any cheek and can be applied over foundation. Our Beauty Insider tip is to use this on the inner corners of your eyes. Borrowed from the K-Pop world, this tip helps your eyes pop without any makeup.
Yellow – Snake/Pig
Not for the faint hearted, yellow can be the perfect touch of shine on your lids. We love how it brings out the yellow tones in Asian skin without overpowering the complexion.
This matte masterpiece is the perfect buildable coverage for your eyelids. We love pairing this with a no eyeliner and enough mascara to touch the heavens!


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Log in for pricing
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This 1" wide flat wood rectangle frame has decorative medallions, and is a good choice for smaller sized spaces and mirrors. The Linen White finish on this frame is hand applied giving a clear medium gloss white finish that is perfect for bathrooms, children's rooms or other areas.
Allow 4 Business days before shipping.