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Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The "NiCo" strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.
Over the past 25 years, several techniques and tools have been developed to express and purify recombinant proteins for protein structure-function studies, for the development of new drugs, or simply for the manufacture of enzymes. The most frequently used method for isolating recombinant protein from a cell lysate in a single purification step is immobilized metal ion affinity chromatography (IMAC). In the simplest application of this method, the target protein is tagged with a polyhistidine sequence (typically 6×His), which mediates chelation to immobilized divalent metal ions such as nickel or cobalt. Other studies have demonstrated that peptides with nonconsecutive histidines are also capable of chelation to immobilized divalent metal ions (5) (U.S. patent 7,176,298 [41] and U.S. patent application 2006/0030007 A1).
Escherichia coli is the most commonly used host for high-yield expression of recombinant protein, usually by exploiting the high promoter specificity and transcriptional activity of bacteriophage T7 RNA polymerase. However, several E. coli host proteins also contain nonconsecutive histidine residues exposed to the surface of their ternary structure. In addition, metal binding motifs often mediate binding to nickel- and/or cobalt-containing purification resins. Such host proteins are routinely copurified during IMAC procedures and are therefore referred to as "contaminants." Several metal binding proteins that behave as IMAC contaminants have been identified in recent years. For example, Bolanos-Garcia et al. reviewed this issue in detail by classifying the E. coli metal binding proteins according to their affinity for Ni-nitrilotriacetic acid (NTA) resin by determining the imidazole concentration required for elution (5). Among 17 E. coli IMAC contaminants described, 15 were reported to elute from Ni-NTA at an imidazole concentration of >55 mM, a concentration which is higher than advised for most IMAC column washing procedures. Thus, most of the cited contaminants are eluted only when the imidazole concentration is increased to a level that elutes the histidine-tagged protein of interest. Also, variable amounts of host protein contaminants are detected depending on the expression system used (genetic background of the strain and plasmid) and the culture conditions employed (medium, carbon source, oxygen, temperature, and cell density at the induction time and at the harvest time).
Various techniques for improving the purity of a His-tagged protein of interest have been described in the literature. First, an alternative to imidazole washing and elution is used to elute the protein of interest with an acidic buffer. At pH <6, the histidine side chain in most contexts becomes protonated and loses its affinity for divalent metals. However, the same is true for contaminant proteins enriched in histidine residues. Therefore, low-pH elution may not result in discrimination between elution of contaminants and elution of the protein of interest. Some impurities can also be reduced by adjusting growth conditions (culture conditions, medium composition, and the genetic background of the strain), but this method of addressing the problem is empirical. Secondary chromatographic steps, of course, may be carried out, e.g., size exclusion chromatography (31), protein specific chromatography (Heparin affinity chromatography described by Finzi et al. [10] or immunoaffinity chromatography described by Muller et al. [29]). But these approaches usually require time-consuming optimization procedures that are dependent on the properties of the target protein. Dual affinity tags, which imply the use of a second affinity column, also provide for improved target protein purity. For example polyhistidine has been combined with glutathione S-transferase (GST) or maltose binding protein (MBP) (36, 45). However, important concerns raised by employing double tags are risks of proteolysis or aggregation of the protein after cleavage of the first tag.
Deleting the genes of the most abundant contaminants also may not be a viable solution. For example, SlyD, a peptidyl prolyl cis-trans isomerase, is cited as the most frequent IMAC copurified protein, but E. coli B and C strains lacking the expression of SlyD suffer from a significant growth defect (38, 44). Furthermore, the majority of E. coli contaminants are critical for cell viability, especially under the stressed conditions caused by high-yield protein expression (5). Therefore, E. coli knockout strains for particular contaminants (such as chaperones or other stress response factors) have not been seriously considered as solutions for avoiding contamination of His-tagged target protein. As a new strategy, we engineered BL21(DE3), the most widely used E. coli strain for high-yield expression of recombinant protein, to express the major Ni-NTA binding proteins with an alternative tag. As a result, the tagged contaminants can be rapidly removed either before or after IMAC capture of the target protein.
All strains were routinely grown using Luria-Bertani (LB) liquid broth or agar (27) at 20°C, 30°C, or 37°C. All media were supplemented with appropriate antibiotics as follows: ampicillin at 100 μg/ml and chloramphenicol 4 μg/ml for maintaining the mini F′ plasmid pFOS1-lacIq or 10 μg/ml for maintaining pMAK705 constructs. Tests with BL21(DE3) glmS-CBD were performed on tryptone broth or agar (LT) containing 10 g tryptone, 5 g NaCl, and 15 g agar (22, 42), supplemented with appropriate antibiotics and 200 μg/liter N-acetylglucosamine, when necessary, and incubated at 30°C. The AceE-CBD in vivo activity analysis was carried out using M9 minimal medium agar supplemented with 0.2% Casamino Acids, 1 mM MgSO4, 0.3 mg/ml thiamine hydrochloride, 20 μg/ml tryptophan, 0.2% glucose, and, when indicated, 2 mM potassium acetate. The medium used in fermentations was as follows (per liter): 20 g animal-free soy peptone, 10 g yeast extract, 10 g NaCl, 30 g glycerol, 0.75 g KH2PO4, 0.75 g Na2HPO4, 0.70 g NH4Cl, 0.085 g K2SO4, 0.62 g MgSO4·7H2O, 0.0125 g Fe(III) citrate, 0.015 g MnCl2·7H2O, 0.0013 g Zn(CH3COO)2·2H2O, 0.0025 g H3BO3, 0.0025 g Na2MoO4·2H2O, 0.0025 g CoCl2·6H2O, 0.0015 g CuCl2·2H2O, 0.0014 g Na2EDTA, and 0.05 g PPG Lumulese antifoam.
The fermentation conditions [for the expression of GluRS in BL21(DE3), NiCo21(DE3), and NiCo22(DE3) strains carrying pET21a-gluRS] were as follows. Ten-liter batch fermentations were performed using Bioflo 3000 fermentors from New Brunswick Scientific. The pH was controlled at 7.00 with automatic addition of 28% NH4OH and 46 N H3PO4. The dissolved oxygen was kept above 20% of air saturation using proportional, integral, and differential control (PID) control of agitation (400 rpm to 600 rpm) and mixing pure oxygen with air (the gas flow rate was 0.5 vessel volume per minute [vvm]). The temperature was controlled at 30°C for both growth and induction. Once the culture reached an optical density at 600 nm (OD600) of 8 to 10, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to give a final concentration of 20 μM and the culture was incubated for an additional 3 h.
For shake flask expression of AlaRS in BL21(DE3) slyD-CBD, cells carrying pQE30-alaRS and pFOS1-lacIq (which provides a source of Lac repressor to control expression from the T5-lacO promoter) were grown in LB medium supplemented with 100 μg/ml of ampicillin. After outgrowth at 37°C to an OD600 of ≈0.8, 500 μM IPTG was added and the cultures were incubated for 20 h at 20°C (OD600, ≈3.2 to 3.6; cell pellet, ≈5.5 to 6 g from 1 liter culture).
For shake flask expression of AlaRS in BL21(DE3), NiCo21(DE3), and NiCo22(DE3), cells were grown in 500 ml of LB medium supplemented with 100 μg/ml of ampicillin. After incubation at 37°C to an OD600 of ≈0.8, 200 μM IPTG was added and the cultures were incubated for 4 h at 30°C (final OD600, ≈2.2 to 2.6; cell pellet, ≈2 g).
For shake flask expression of GluRS in BL21(DE3), NiCo21(DE3), and NiCo22(DE3), cells carrying pET21a-gluRS were grown in 500 ml of LB medium supplemented with 100 μg/ml of ampicillin. After growth at 37°C to an OD600 of ≈0.5, 20 μM IPTG was added and the cultures were incubated for 4 h at 30°C (final OD600, ≈2.4 to 2.6). Cells containing pET21a were grown using the same procedure in order to prepare mock lysates for the lysate-mixing experiment (see Fig. 7).
Restriction enzymes and DNA-modifying enzymes were provided by New England BioLabs. Mutagenesis was carried out using a Phusion site-directed mutagenesis kit (New England BioLabs). DNA amplification procedures utilized either Phusion or Taq DNA polymerases.
Bacterial strains and genotypes are listed in Table 1 . Oligonucleotides and plasmids are described in Tables S1 and S2, respectively, in the supplemental material. The pMAK-CBD vector for 3′ end gene tagging was created by inserting the CBD open reading frame (ORF) from vector pTYB1 (New England BioLabs) into the polylinker region of pMAK705 (14). An amino acid linker region which encodes LQASSS(N)10LQS, where the first LQ codons correspond to a PstI restriction site and the last LQS codons contain a SalI restriction site, was inserted. A unique AsiSI restriction site was inserted after the CBD ORF. All allele exchange procedures utilized derivatives of pMAK-CBD containing DNA fragments amplified from the BL21(DE3) chromosome. Genes of interest were cloned into HindIII, SphI, PstI, and/or SacI sites. Downstream DNA (3′ flanking sequence) was cloned into AsiSI, Acc65I, SacI, and/or EagI sites.
To create the strain BL21(DE3) slyD-CBD, pMAKslyD-CBD was transformed into BL21(DE3) and individual clones were grown in LB liquid medium with 4 μg/ml of chloramphenicol (Cm). The allele exchange method of Hamilton et al. (14) was followed to replace the wild-type (wt) slyD gene with the slyD-CBD allele. The strains positive for allele exchange were cured of the pMAK vector carrying the wild-type slyD gene using a coumermycin treatment (8). PCR amplification with primers 4059For and 4060Rev confirmed that the slyD-CBD allele was present at the correct locus within the chromosome of BL21(DE3) slyD-CBD.
To create NiCo21(DE3), the allele exchange procedure was carried out in the same manner as that described for slyD-CBD except (i) can locus analysis was accomplished using primers 4841For and 2187Rev, (ii) analysis of the arnA-arnD locus to confirm the arnA-CBD-arnD allele was accomplished using primers 5032For and 5031Rev, and (iii) analyses to confirm replacement of the wild type glmS gene by glmS6Ala (glms allele with six histidine-to-alanine substitutions) were accomplished using primers glmSdown-Rev, glmS466check-Rev, Hind-glmS-For, and glmS-Sac-Rev.
To create NiCo22(DE3) from NiCo21(DE3), the pMAKaceE-CBD allele exchange construct was utilized. Primers 4845For and 0076Rev were used to PCR amplify the aceE-CBD-aceF locus for sequence characterization.
Mutant glmS constructs were created from pMAKglmS using a Phusion site-directed-mutagenesis kit (New England BioLabs). The glmS2Ala gene has four mutations resulting in alanine codons at positions 62 and 65 (GCTCCTCTGGCT, modified from the wt sequence CATCCTCTGCAT). The pMAKglmS6Ala construct was generated in 3 steps. First, the plasmid pMAKglmS2Ala was amplified with primers His432Ala-Rev and His436Ala-For, followed by ligation to circularize the linear PCR product. The resulting glmS4Ala mutant has the mutated sequence GCTGACATTGTGGC, which results in additional alanine codons at positions 432 and 436. Second, the pMAKglmS4Ala mutant template was amplified with primers His466Ala-Rev and glmS467-For, followed by the ligation of the linear PCR product, to generate the plasmid pMAKglmS(His62-65-432-436-466Ala). Third, the pMAKglmS(His62-65-432-436-466Ala) template was amplified with the primers glmS466-Rev and His467Ala-For, followed by ligation of the linear PCR product, to generate the plasmid pMAKglmS6Ala. The glmS6Ala allele has additional alanine codons at positions 466 and 467 (GCTGCCGCG).
pMS119-pE was generated by PCR amplification of the gene E from bacteriophage x174 genomic DNA (laboratory stock) with the primers Hind-pE-For and pE-Xba-Rev and then cloned into the HindIII and XbaI sites of pMS119 (11).
All the plasmids were confirmed by DNA sequencing using gene-specific primer s1260 (Ptac forward), s1233 (pMAK705 polylinker forward), or s1224 (pMAK705 polylinker reverse).
For the purification of AlaRS from BL21(DE3) slyD-CBD, the cell pellet was resuspended in 30 ml of buffer A1 (50 mM HEPES, pH 7.6, 1 M NH4Cl, 10 mM MgCl2, 7 mM β-mercaptoethanol, 5% glycerol, and 25 mM imidazole) supplemented with 3 ml BugBuster (10×; Novagen) and sonicated. The clarified lysate was loaded onto a 5-ml HisTrap HP column (GE Healthcare Bio-Sciences), equilibrated with 5 column volumes (CVs) of buffer A1. The flowthrough was collected, and the column was washed with 15 CVs of buffer A1. Elution was completed using a gradient from 25 mM to 250 mM imidazole in buffer B1 (50 mM HEPES, pH 7.6, 0.1 M KCl, 10 mM MgCl2, 7 mM β-mercaptoethanol, and 250 mM imidazole). Eleven CVs were collected in 28 tubes of 2 ml each.
For the Ni-NTA affinity test with GlmS, GlmS2His-Ala, and GlmS6His-Ala, cell pellets were resuspended in 6 ml of buffer A2 (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, and 20 mM imidazole) supplemented with 600 μl BugBuster (10×; Novagen) and sonicated. Clarified lysates were loaded onto a 1-ml HisTrap HP column, equilibrated with 5 CVs of buffer A2. After collecting the flowthrough, the column was washed with 15 CVs of buffer A2 and then eluted using a gradient from 20 mM to 400 mM imidazole with buffer B2 (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, and 400 mM imidazole). A pool of eluted fractions was concentrated using a Vivaspin column (Vivascience) and analyzed on SDS-PAGE gel stained with Coomassie blue R-250.
For the purification of AlaRS from BL21(DE3), NiCo21(DE3), and NiCo22(DE3) strains, the cell pellets were resuspended in 20 ml of buffer A2 (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, and 20 mM imidazole) supplemented with 2 ml BugBuster (10×; Novagen) and sonicated. Clarified lysates were loaded onto a 5-ml HisTrap HP column equilibrated with 5 CVs of buffer A2. The flowthrough was collected, and the column was washed with 15 CVs of buffer A2. Elution was completed using a gradient from 20 mM to 400 mM imidazole (10 CVs collected in 25 tubes of 2 ml each) with buffer B2.
For the purification of GluRS from BL21(DE3), NiCo21(DE3), and NiCo22(DE3) strains, a cell pellet of 1 g was resuspended in 10 ml of buffer A3 (50 mM sodium phosphate, pH 8, 300 mM NaCl, and 10 mM imidazole) supplemented with 1 ml BugBuster (10×; Novagen) and sonicated. The clarified lysates were loaded onto a 1-ml Ni-NTA column (Qiagen superflow resin), previously equilibrated with 10 CVs of buffer A3. After batch incubation for 1 h at 4°C with gentle shaking, the flowthrough was collected after a short spin and the resin was washed with 10 CVs of buffer A3. Elution was completed with 5 ml buffer B3 (50 mM sodium phosphate, pH 8, 300 mM NaCl, and 250 mM imidazole).
The elution fractions obtained after Ni2+ affinity chromatography were pooled, when necessary, and incubated with 1, 2, or 5 ml of chitin beads (New England BioLabs), previously equilibrated with 5 CVs of buffer C (50 mM sodium phosphate, pH 7.4, 500 mM NaCl) at room temperature. After gentle shaking for 30 min at 4°C, the target protein was eluted from the chitin bead slurry by gravity flow. The chitin beads were then washed with 5 CVs of buffer C, and a chitin bead sample was removed for analysis of bound contaminating proteins.
Whole cells or purified protein fractions were prepared for SDS-PAGE analysis by mixing 2 parts sample with 1 part 3× sample buffer (New England BioLabs). Samples were analyzed by 4 to 20% SDS-PAGE. Proteins were visualized by Coomassie blue R-250 staining, or proteins were transferred to nitrocellulose membrane (Millipore) for immunoblotting. CBD-tagged proteins were detected by anti-CBD monoclonal antibody (New England BioLabs), and His-tagged proteins were detected by anti-His monoclonal antibody (EMD Biosciences). Horseradish peroxidase (HRP)-linked secondary antibody, and enhanced chemiluminescence reagents were supplied by Cell Signaling Technology.
Protein samples of approximately 1 mg were added to 20 μl of trypsin reaction buffer (50 mM Tris-HCl, 20 mM CaCl2, pH 8) and digested overnight at 37°C with trypsin (New England BioLabs) at a protein-to-protease ratio of 20:1. Online liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) analyses of digested fractions using an Agilent 6330 Ion Trap mass spectrometer with an integrated C18 chromatin immunoprecipitation-nanoelectrospray ionization (C18 ChIP/nano-ESI) interface were performed as described previously (40). Protein separation, digestion, and peptide analysis were repeated in triplicate for each sample. The MS-MS data were analyzed by the Spectrum Mill (Rev A.03.03.084 SR4; Agilent Technologies) search engine using parameters described previously (40), with minor modifications. Data were searched against an E. coli BL21(DE3) database that was supplemented with the three mutant GlmS protein sequences (GlmS2Ala, GlmS4Ala, and GlmS6Ala). The search criteria were set to allow two missed cleavages by a tryptic digest with no other protein modifications. Peptides were validated by using a reverse database search and needed to have a score 2.0 or higher than any reverse score to be valid. Proteins built from these validated peptides scoring 20 or better were considered valid identifications for Spectrum Mill. Proteins identified from one detected peptide using a single spectrum were excluded. The false-positive rate for all Spectrum Mill analyses was less than 1%.
Target protein purity was analyzed using the Caliper LabChip GXII protein assay. Five microliters of protein solution (1:15 lysate mixture after Ni-NTA or chitin columns) was denatured and processed according to the protocol supplied by Caliper LifeSciences. Protein signals between 14 and 200 kDa were analyzed using HT Protein Express Chip version 2 in combination with a Protein Express reagent kit.
In order to confirm the major E. coli metal binding proteins cited in the literature, three different recombinant His-tagged target proteins were overexpressed from a vector in BL21(DE3). An automated AKTA FPLC system was used in combination with a 1-ml HisTrap column to perform a standard fractionation of the cell lysates. Mass spectrometry (MS) analysis of the target protein elution fractions revealed that the following E. coli host proteins also coeluted in significant amounts: DnaK, GlmS, AceE, EF-Tu, ArnA, RnaseE, AtpF, the Rho transcription terminator, CRP, and SlyD (data not shown). Several independent studies, performed using different conditions or different E. coli strains, also reported many of the same contaminants after Ni+2 affinity chromatography (5, 13, 18). The most common E. coli proteins listed in these previous reports were CRP, Fur, ArgE, DnaK, SlyD, GlmS, GlgA, ODO1, ODO2, Can (YadF), ArnA (YfbG), AceE, GroES, and GroEL. Based on these previous studies and our analysis, we chose the following consistent contaminants to tag with the chitin binding domain (CBD) sequence: SlyD, GlmS, Can, ArnA, and AceE. The selection criteria also included the possibility of checking the activity of each tagged protein when expressed from the chromosome.
To create the desired expression strains, we first generated a plasmid encoding each candidate gene fused with the CBD ORF. The respective E. coli gene-CBD constructs were expressed in BL21(DE3) to examine the stability of the respective fusion protein. Anti-CBD immunoblots indicated that each fusion protein was not subject to in vivo proteolysis (data not shown), an outcome that would prevent the removal of the contaminant protein by tag-mediated chromatography. The plasmids encoding CBD fusion proteins were subsequently employed to modify the BL21(DE3) chromosome by homologous recombination at the native E. coli gene locus. The replacement of the native gene with the CBD fusion allele was performed using the allele exchange method described by Hamilton et al. (14). Efficient allele exchange occurs when the exchange vector contains at least 300 bp of homology to both 5′ and 3′ regions flanking the target site on the bacterial host chromosome. Thus, for each allele replacement step, homologous DNA sequences of at least 300 bp were cloned at the 5′ and the 3′ ends of the CBD sequence in the pMAK-CBD vector (see Materials and Methods). E. coli BL21(DE3) derivatives were generated by replacing each candidate allele one by one. The phenotype of each derivative strain was analyzed after each replacement step, and then each strain was tested as an expression host for one or more recombinant His-tagged proteins (E. coli alanyl-tRNA synthetase and/or E. coli glutamyl-tRNA synthetase).
As a proof of principle, we chose first to address the most predominant Ni-NTA contaminant, SlyD. SlyD is a cytoplasmic protein originally identified in E. coli as a host factor required by bacteriophage phiX174 to induce cell lysis. The gene slyD (sensitivity to lysis) was first identified in a genetic selection for E. coli C strains resistant to lysis gene E of phage phiX174 (23). The protein was then isolated and characterized in a further study as a persistent contaminant in immobilized metal affinity chromatography that migrates at the apparent molecular mass of 27 kDa and was also called WHP for wondrous histidine-rich protein (44). With 196 residues and an actual mass of 21 kDa, SlyD is a peptidyl prolyl cis/trans-isomerase (PPIase or rotamase) that provides chaperone activity for proline-limited protein folding (17, 20, 39). The protein is divided into two domains, the N-terminal part (residues 1 to 146) containing the chaperone activity with the peptidyl-prolyl isomerase domain and the C-terminal part of 50 amino acids, particularly rich in histidine and cysteine residues and thus conferring high affinity for divalent cations such as Zn2+and Ni2+ (44). SlyD has been reported as the major contaminant after Ni-NTA purification in several studies (3, 10, 19, 31, 35).
The strain BL21(DE3) slyD-CBD was constructed using the Hamilton allele exchange method (14) and then tested as a host for overexpression of alanyl-tRNA synthetase (AlaRS). AlaRS tagged at its N terminus with 6 histidines was expressed from the pQE30 vector in the control strain BL21(DE3) and in BL21(DE3) slyD-CBD, each carrying the mini-F plasmid pFOS1-lacIq as described in Materials and Methods. The cells were sonicated, and the clarified lysates were loaded onto a 5-ml HisTrap column using an AKTA FPLC system, followed by washing with 25 mM imidazole and elution using a gradient of 25 to 250 mM imidazole. Figure 1 shows Coomassie blue-stained gels with the protein profiles of the elution fractions collected from both strains, in which AlaRS is sequentially eluted. Comparable backgrounds of E. coli proteins are found upon fractionation of both cell lysates, except for one protein that migrates at about 35 kDa in BL21(DE3) slyD-CBD (Fig. 1A and B). This protein was presumed to be SlyD-CBD since wild-type SlyD tends to migrate at 27 kDa and the CBD tag is 7.7 kDa. In fact, a Western blot analysis performed with anti-CBD antibodies on the same BL21(DE3) slyD-CBD fractions confirms the position of SlyD-CBD at about 35 kDa (Fig. 1C). These data indicate that SlyD-CBD is visually detectable as a contaminant of the Ni-NTA fractions and is eluted with 80 to 120 mM imidazole. This result is consistent with the data presented by Bolanos-Garcia et al. (5). Elution fractions 4 to 24 were pooled and then incubated with 2-ml chitin beads for 2 h at 4°C. The resulting flowthrough (FT) fraction and the chitin beads were analyzed by Western blotting, which shows that a significant amount of SlyD-CBD is removed from the protein pool after incubation with chitin beads (Fig. 2). The analysis for in vivo SlyD-CBD activity is presented in "(iv) In vivo activity analysis of each CBD-tagged candidate protein."
GlmS is a 67-kDa l-glutamine:d-fructose-6-phosphate aminotransferase involved in an essential step of bacterial cell wall biosynthesis. The enzyme utilizes d-fructose-6-phosphate and l-glutamine to form d-glucosamine-6-phosphate, which is a precursor for components of the peptidoglycan or lipopolysaccharide. Among 609 residues, GlmS contains 24 histidines, of which 15 are organized in four clusters of at least 3 histidines exposed on the surface, giving the protein a high potential for interacting with Ni2+ and Co2+ cations according to the crystal structure described by Mouilleron et al. (28).
When we replaced the glmS allele with the glmS-CBD allele on the chromosome of BL21(DE3), the resulting BL21(DE3) glmS-CBD strain was nearly nonviable (Fig. 3B). However, complementation of the GlmS-CBD defect was demonstrated using tryptone medium (LT) with addition of 200 μg/ml of glucosamine (GlcN) or N-acetylglucosamine (GlcNAc) (Fig. 3B) (42, 43). GlmS is actually composed of two domains in which the N-terminal domain holds the glutamine amidotransferase activity while the C-terminal domain contains the ketose/aldose isomerase activity. Respective active site residues are located at each extremity of the protein (position 2 for the amidotransferase and position 604 [of 609 residues] for the isomerase) (Fig. 3A). Since our attempt to place the CBD tag at the N-terminal end of GlmS was also unsuccessful, we assumed that the addition of the CBD tag at the N or C terminus of GlmS disturbs the respective active sites located at the termini of the protein. Rather than using a tag to eliminate GlmS from His-tagged protein fractions, we chose to alter GlmS affinity for divalent cations by generating a GlmS mutant in which the most exposed histidines would be replaced by alanines. After examining the three-dimensional (3D) structure of the GlmS dimer, the isomerase active form of GlmS (28), we selected six surface-exposed histidines for mutagenesis (Fig. 3A): histidines 62 and 65 are located in the center of a cluster of 4 exposed histidines, histidines 432 and 436 occupy a central position in another cluster, and finally, histidines 466 and 467 are part of a large cluster of 6 histidines highly surface exposed and generated by the dimer interface (data not shown). The selected histidines (positions 62, 65, 432, 435, 466, and 467) are all poorly conserved residues among GlmS homologs (data not shown).
We first examined the activity of the resulting GlmS6His-Ala protein in a complementation assay using the strain BL21(DE3) glmS-CBD. As shown in Fig. 3B, in the absence of GlcN or GlcNAc, GlmS6His-Ala is able to restore the growth of BL21(DE3) glmS-CBD with the same efficiency as wild-type GlmS. Both proteins, GlmS and GlmS6His-Ala, were expressed from the low-copy-number plasmid pMAK705 (14). Next, to test the affinity of GlmS6His-Ala for Ni-NTA resin, we performed a standard purification on a Ni-NTA column, using BL21(DE3) lysate as a control compared to BL21(DE3) overexpressing GlmS6His-Ala or GlmS from pMAK705. The cell lysates were loaded on a 1-ml HisTrap column using an AKTA FPLC system, followed by standard washing and elution steps (20 to 400 mM imidazole), and the elution fractions were analyzed by Coomassie blue SDS-PAGE and by MS. As an additional control, we included an intermediate mutant protein, GlmS2His-Ala, in which only the histidines at positions 62 and 65 were replaced by alanines. Figure 3C shows that the band migrating at the expected position of GlmS (67 kDa) is detected in the sample overexpressing GlmS (lane "wt"), while the same band is less intense for the GlmS2His-Ala sample (lane labeled "2ala"). Most importantly, overexpression of the GlmS6His-Ala mutant protein does not result in binding and elution of a 67-kDa protein from the Ni-NTA resin [compare lane "6Ala" with lane "−" where cell extract was analyzed from BL21(DE3) carrying an empty plasmid]. Additionally, MS analysis of the Ni-NTA elution fractions supports the SDS-PAGE observations. When analyzing Ni-NTA binding proteins in the control samples, we found 38 times more spectral counts of GlmS when wild-type GlmS is overexpressed from a plasmid than for chromosomal expression in the empty-plasmid control strain. In contrast, overexpression of mutant GlmS6His-Ala from a plasmid results in only a modest increase of GlmS peptide in the imidazole elution fraction (4 spectral counts). These data indicate that the replacement of 6 surface-exposed histidines by alanines significantly decreases the affinity of GlmS for Ni-NTA resin, while the removal of only 2 histidines has a limited effect. The complementation studies show that this mutagenesis does not compromise the ability of GlmS to supply GlcNAc (Fig. 3B).
Replacement of the glmS gene with the glmS6Ala allele was the last step in the construction of the NiCo21(DE3) and NiCo22(DE3) protein expression strains. NiCo21(DE3) additionally contains the slyD, can, and arnA genes tagged with the CBD ORF, while NiCo22(DE3) additionally contains the slyD, can, arnA, and aceE genes tagged with the CBD ORF. The can gene (yadF) encodes a β-class carbonic anhydrase (Can), a zinc metalloenzyme which interconverts carbon dioxide (CO2) and bicarbonate. This protein of 25 kDa exhibits four zinc-binding sites that confer a significant affinity for metal chelating resins. Moreover, Can expression increases in high-density cultivation, during slow growth, or during stress and starvation, in other words, conditions typically encountered during recombinant-protein overexpression (26).
The arnA gene encodes a bifunctional enzyme of 74.3 kDa, a UDP-l-Ara4N formyltransferase/UDP-GlcA C-4″-decarboxylase, involved in the modification of the lipid A required for lipopolysaccharide biosynthesis. Also this modification of lipid A with 4-amino-4-deoxy-l-arabinose confers to Gram-negative bacteria a resistance to the cationic antimicrobial peptides and antibiotics such as polymyxin (6). ArnA (also called YfbG) is found as a recurring contaminant in IMAC, presumably due to the several clusters of surface-exposed histidines detected in the 3D structure of the active hexamer (12).
The aceE gene encodes subunit E1 of the pyruvate dehydrogenase multienzyme complex formed from 12 dimers of subunit E1, 24 subunits of AceF, and 6 LpdA dimers (2). The major role of pyruvate dehydrogenase in the tricarboxylic acid (TCA) cycle is the production of acetyl-coenzyme A (CoA) from pyruvate (37). Although AceE (99.7 kDa) is not essential for viability, its inactivation reportedly leads to disturbance of carbon metabolism (21). The protein displays three magnesium-binding sites that might explain its affinity for metal chelating resins.
For each intermediate strain, we examined the growth rate at diverse temperatures (20°C, 30°C, and 37°C) in order to detect any effect on cell viability. We confirmed the expression and tested the activity of each CBD tagged protein in vivo. In Fig. 4A, the anti-CBD Western blot demonstrates the stable expression of the CBD-tagged proteins in NiCo21(DE3) and NiCo22(DE3). However, we observed that after addition of the aceE-CBD allele, NiCo22(DE3) grew more slowly in liquid culture and on agar plates than BL21(DE3) and NiCo21(DE3) (Fig. 4B). This suggests that the addition of a CBD tag at the C terminus of AceE affects the activity of the individual AceE protein and/or the formation of the pyruvate dehydrogenase complex.
During NiCo strain construction and thereafter, genome stability was verified by PCR and sequence analysis of the loci containing the CBD-tagged genes. When identical DNA sequence tags are present at either 3 or 4 times in a genome, it is certainly possible for recombination to occur, resulting in deletion of intervening sequence to occur. In the case of the NiCo strains, this type of event would result in cell death (and loss of such mutants from the culture) since intervening sequences are extensive and contain essential genes.
To assess the activity of the SlyD-CBD protein, we used an in vivo approach involving the lysis protein E originally expressed from the bacteriophage phiX174 genome. SlyD is required to induce lysis of E. coli C after infection by the phage phiX174 or in other E. coli strains (background B, C, and K-12) when protein E is expressed from a multicopy plasmid (38, 46). Wild-type E. coli is sensitive to lysis in both situations, while a slyD knockout strain is shown to be resistant. The lysis is actually the consequence of the inhibition, by protein E, of MraY, a conserved phosphotransferase involved in the formation of intermediates for the peptidoglycan biosynthesis (4, 25, 47). The lysis phenotype in BL21(DE3) and in the strains carrying slyD-CBD was examined by monitoring OD600 following induction of expression of the protein E cloned on pMS119 plasmid. Figure 5A shows that all the strains display a sensitivity to lysis after induction of the protein E, indicating that SlyD-CBD is active. Lysis depends on lysis protein E, since it does not occur with empty-vector pMS119.
The Can protein is essential for growth under normal atmospheric conditions, even though cells depleted of Can are able to survive when supplied with CO2 or when cynT, a paralog gene of can, is activated upon induction with cyanate or azide (15, 26). The isolated strain BL21(DE3) slyD-CBD can-CBD did not show evidence of a growth defect on plates or under standard liquid culture conditions, suggesting that Can expressed with a C-terminal CBD tag is fully functional (data not shown).
Given that the enzymatic modifications carried out by ArnA confer polymyxin resistance to E. coli, we tested the activity of ArnA-CBD by restreaking the strains on LB medium supplemented with polymyxin B. The assays were performed at a concentration of 2 μg/ml of polymyxin B as described in the literature (7). Figure 5B shows that this is the MIC for NiCo21(DE3) at 30°C, but at 37°C, the strain is fully sensitive to the same concentration. This result suggests that ArnA-CBD is generally less active than wild-type ArnA. Interestingly, NiCo22(DE3), which also expresses ArnA-CBD but contains one more CBD-tagged protein (AceE-CBD), is resistant to polymyxin B at both 30°C and 37°C temperatures. The noticeable phenotype of NiCo22(DE3) is its lower growth rate than those of NiCo21(DE3) and BL21(DE3) (Fig. 4B). We consequently hypothesize that the activity of ArnA-CBD may be compromised and that slow growth allows for this protein to carry out its functions in cell envelope formation.
Depletion of AceE or elimination of its pyruvate dehydrogenase activity results in E. coli dependent on acetate for aerobic growth on glucose medium (21). We therefore performed a plate assay on minimal medium supplemented with glucose (0.2%) with and without potassium acetate (2 mM) to access AceE-CBD activity. As controls, we used two E. coli aceE mutants, the strains CGSC5477 and CGSC4823 (9, 16), which both show a growth defect in the absence of acetate. Despite the fact that NiCo22(DE3) exhibits a lower growth rate than BL21(DE3) and NiCo21(DE3), its growth is not acetate dependent (Fig. 5C). These results suggest that the CBD tag at the C terminus of AceE does not significantly affect AceE activity. If AceE-CBD activity was abolished, NiCo22(DE3) growth could be supported in the absence of acetate by a higher level of expression of the protein PoxB. PoxB is a nonessential pyruvate oxidase whose role of converting pyruvate into acetate and CO2 contributes significantly to growth under aerobic conditions. Abdel-Hamid et al. identified some aceE mutants able to grow without acetate due to an increased activity of endogenous PoxB throughout the growth cycle (with yet a 30% decrease in the growth rate compared to the level for the wild-type strain) (1). Although the nature of the constitutive expression has not been identified, they proposed that the poxB promoter lost its dependence on RpoS, a sigma factor (RpoS, σ38, and σS) required to induce genes in stationary phase (1). To address this uncertainty, we sequenced the poxB promoter of BL21(DE3), NiCo21(DE3), and NiCo22(DE3) and found no mutations in the promoter region of these strains, suggesting that PoxB is still well regulated by RpoS in NiCo22(DE3).
NiCo21(DE3) and NiCo22(DE3) were used to express the E. coli His-tagged alanyl-tRNA synthetase (AlaRS) or the His-tagged glutamyl-tRNA synthetase (GluRS) using two conditions of expression (standard shake flask cultures and batch fermentation). The purification procedure was performed on immobilized nickel using either nickel beads (Superflow; Qiagen) or a HisTrap column managed by an AKTA FPLC system. In both cases, Ni-NTA elution fractions were incubated with chitin beads and the protein profiles of the resulting flowthrough were compared to that of the Ni-NTA elution pool (no chitin incubation). Proteins were characterized by Coomassie blue-stained SDS-PAGE gels by Western blot analysis and by MS.
Glutamyl-tRNA synthetase (GluRS-6His) was expressed in each expression strain using a high-density batch fermentation process carried out at 30°C in rich medium monitored for glucose concentration and oxygen (see Materials and Methods). Figure 6 shows the purification of the GluRS-6His on Ni-NTA resin (Superflow; Qiagen) followed by incubation with chitin beads. The profiles of proteins eluted after Ni-NTA (lanes E) are similar for the three strains (Fig. 6A). After incubation with chitin beads (lanes FT chitin), the collected flowthrough is void of the main contaminants for NiCo21(DE3) and NiCo22(DE3) samples. The Western blot shown in Fig. 6B confirms the removal of the CBD-tagged proteins after treatment on chitin beads (compare lane E and lane FT chitin). Finally, MS analyses of the samples taken after Ni-NTA elution and after chitin flowthrough are consistent with our gel analyses, showing that, first, the CBD-tagged proteins identified in the elution after Ni-NTA are absent in the chitin flowthrough and, second, that GlmS6His-Ala is significantly reduced in the NiCo21(DE3) and NiCo22(DE3) samples compared with GlmS identified in BL21(DE3) (Fig. 6C).
To demonstrate the utility of the NiCo strains for poorly expressed proteins, GluRS was expressed in BL21(DE3), NiCo21(DE3), and NiCo22(DE3) and each cell lysate was then mixed with a "mock" lysate prepared from the same strain grown with an empty expression vector (pET21a). A mixture of 1 ml GluRS lysate to 15 ml empty-vector lysate was chosen so that GluRS would be present at a very low concentration relative to host proteins (see lysate load [L] lanes in Fig. 7). The lysate mixture (1:15) corresponding to all three strains was subjected to standard Ni-NTA chromatography (wash with 8 CVs of 20 mM imidazole and elution with 1 CV of 250 mM imidazole). The Ni-NTA elution samples resulting from NiCo21(DE3) and NiCo22(DE3) were additionally incubated with chitin resin for 30 min. Lane 3 in Fig. 7 shows the protein profile of the Ni-NTA elution sample from the BL21(DE3) lysate mixture, while lanes 6, 7, 10, and 11 show the improvement in purity obtained when NiCo strain elution samples were further processed by chitin incubation. Visual inspection of the SDS-PAGE gel image shows that the NiCo samples (Fig. 7, lanes 7 and 11) have significantly less contaminants than the BL21(DE3) sample. To confirm the improvement in purity, we measured target protein purity using a Caliper LabChip GXII protein assay. Strikingly, the target protein is 90.74% and 87.48% pure after chitin incubation when purified from NiCo21(DE3) and NiCo22(DE3), respectively. When GluRS is expressed in BL21(DE3), the purity is only 56.01% when a standard Ni-NTA purification procedure is followed.
In addition, we performed purification analyses using imidazole elution gradients and found that contaminants are equally problematic compared to single-step elution. His-tagged alanyl-tRNA synthetase (AlaRS) was purified by loading cell lysates on a 5-ml HisTrap column, followed by standard washing with 20 mM imidazole and elution with a 20 to 400 mM imidazole gradient using an AKTA FPLC system. Elution fractions with high concentrations of target protein consistently displayed multiple contaminating proteins. SlyD-CBD and ArnA-CBD were eluted at about 80 to 140 mM imidazole, while AceE-CBD was eluted all along the elution gradient (see Fig. S2 in the supplemental material). Only Can-CBD appears to be washed out before the elution step, as confirmed by Western blotting. Importantly, incubating the target protein pool with chitin beads resulted in the removal of CBD-tagged contaminant proteins (Fig. S2).
NiCo21(DE3) and NiCo22(DE3) were initially designed to remove the most common contaminants found after Ni-NTA purification. In order to determine the efficiency of protein purification on the alternative cobalt resin, we performed a comparison assay between Ni-NTA (Superflow; Qiagen) and cobalt (Talon superflow; Clontech) resins using the standard protocols recommended by the respective manufacturers. The amounts of purified His-tagged protein (glutamyl-tRNA synthetase) were equivalent from both resins, but the cobalt resin often gave elution fractions less contaminated by native E. coli proteins (data not shown). Although SlyD was reported as having a very weak affinity for cobalt affinity resin (24), we found after mass spectrometry analysis that the most abundant contaminant on cobalt resin is still SlyD (data not shown). Among the E. coli proteins copurified with His-tagged GluRS on cobalt, we also identified the elongation factor EF-Tu and the ferric uptake regulator protein (Fur) known to bind Ni-NTA resin.
In this work, we present the characterization of two protein expression strains designed to improve purity of target protein after immobilized metal affinity chromatography (IMAC). BL21(DE3) is the most widely used E. coli strain for protein overexpression and often provides the highest yield of target protein relative to endogenous proteins. For this reason, BL21(DE3) was chosen as the parent for the NiCo strains. NiCo21(DE3) and NiCo22(DE3) were both engineered to express the most common contaminants (SlyD, Can, ArnA, GlmS, and AceE) either with a tag for removal by a rapid chromatography flowthrough step or with mutations to decrease affinity for divalent cations. We have demonstrated that all the CBD-tagged proteins (SlyD, Can, ArnA, and AceE) are efficiently removed from the Ni-NTA elution pool after batch incubation with chitin beads and that the modified GlmS6His-Ala protein has lost its affinity for nickel resin. NiCo21(DE3) and NiCo22(DE3) are thus preferred expression strains for obtaining recombinant His-tagged target proteins with reduced levels of host protein contamination.
We have demonstrated that the addition of a CBD tag at the C-terminal end of SlyD and Can protein does not affect their function in vivo. Importantly, addition of the CBD tag may have favorably altered the affinity of Can for nickel resin since we routinely observed Can-CBD in the 20 mM imidazole flowthrough or wash fractions (see Fig. S2 in the supplemental material), whereas Bolanos-Garcia et al. report that wild-type Can is eluted from Ni-NTA by 55 to 80 mM imidazole (5). In NiCo21(DE3), ArnA-CBD was observed to be partially active at 30°C but apparently inactive at 37°C according to the polymyxin B sensitivity phenotype. However, in the slow-growing strain NiCo22(DE3), the function of ArnA-CBD appears normal at 37°C. One explanation for this inconsistency is that at a higher growth rate, cell envelope formation (and resistance to polymyxin B) demands an ArnA protein with a higher specific activity. Therefore, our results suggest that the CBD tag only compromises and does not abolish the function of ArnA in vivo.
In the final strain NiCo22(DE3), the CBD tag modification of AceE seems to be responsible for the lower growth rate of this strain. However, the lack of acetate dependence for aerobic growth on glucose medium suggests that AceE-CBD is still active. On the other hand, we designed our acetate dependency assay based on data collected from experiments performed with E. coli K-12, since the two available control aceE mutants are K-12 strains (21). K-12 and B strains are known to contain major differences in their respective metabolic pathways, particularly in pyruvate and acetate production (33, 34). For example, in high-density cultures, E. coli K-12 excretes high levels of acetate in the presence of excess glucose concentrations, which affects its growth rate, while E. coli BL21 is much less sensitive to glucose concentration and produces lower levels of acetate (32). It seems that not only are the lower levels of production of pyruvate and acetate in E. coli BL21 the result of more-active carbon metabolism (TCA cycle, glyconeogenesis, glyoxylate shunt, and anaplerotic pathways), but glucose transport may be better controlled in BL21 than in K-12 (30, 33). In conclusion, the acetate assay indicates that AceE-CBD is most likely active.
One advantage of using chitin beads is their compatibility with diverse buffer conditions (HEPES, sodium phosphate, Tris-HCl, 50 mM to 2 M NaCl, 6 M urea, pH 6 to 9, 0.1 to 1% Triton X-100, and high imidazole concentrations) that allow direct loading of the elution fractions after IMAC without performing dialysis. In the experiment using cobalt resin, SlyD-CBD was directly cleared from the elution sample after 30 min batch incubation on chitin beads (data not shown). We routinely use 1-ml chitin beads to remove the CBD-tagged proteins from the Ni-NTA elution pool prepared from 1 g of cell pellet. For large cultures (2 liters or more) or especially when the recombinant His-tagged protein is expressed at a low level, batch incubation in a larger volume of chitin beads may help to efficiently remove all the CBD tagged proteins (1 ml chitin beads for every 0.1 liter of culture is another recommendation). However, increasing the incubation time with the beads (e.g., overnight) did not show any improvement (data not shown).
We show that mutating surface-exposed histidines on GlmS is an effective strategy for eliminating binding to Ni-NTA. However, we have not followed this as a general approach, since we expect that many proteins would not tolerate removal of multiple histidines.
In summary, NiCo21(DE3) is indistinguishable from BL21(DE3) with respect to growth characteristics and the potential for recombinant protein expression. The NiCo strains consistently produced a high yield of recombinant protein in all of our studies, and the most common IMAC contaminants were removed by simply exposing the target protein pool to chitin beads, which are compatible with commonly used IMAC buffers. Therefore, we propose that the NiCo strains are superior alternatives to BL21(DE3) as protein production hosts, and we expect that these strains will perform well under all conditions routinely employed for the propagation of BL21(DE3).
We thank Mern Sibley for introduction of the fhuA2 (T1 phage resistance) allele to BL21(DE3) and for providing the aceE mutant strains, Elisabeth Raleigh and Bill Jack for critical reading of the manuscript, Chris Taron for guidance throughout the project, and Don Comb and New England BioLabs for research support.
†Supplemental material for this article may be found at http://aem.asm.org/.
Published ahead of print on 20 May 2011.
‡The authors have paid a fee to allow immediate free access to this article.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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If you are vegetarian and you want to reduce your overweight, you can get the best weight loss plan for vegetarians. Since it is called as the best, it means that it is safe and healthy for you. There are many ways to get it, whether from the special clinic or you can also get it from Internet. The best weight loss plan for vegetarian is very healthy however it can be very boring. So you need to more creative in making it like developing its variety, moderation as well as balance in the diets you made. In this method you will be free of feeling tired from the exercise you may need to do.
Low Calorie Foods
We all know that vegetarian is a kind of people who do not consume any kind of meat products. Some of vegetarian do not eat all kind of animal flesh and meat but they are eager to eat eggs. They are called as lacto love vegetarians. The other kinds of vegetarian are called as vegan who does not eat any kind of meat including the eggs. The main problem of the vegetarian is that they will get difficulty to find the foods which contain protein. Weight loss plan must emphasize on how people who want to lose their weight still get the foods which contain protein and low of calories. However you do not need to worry since it can be obtained from:
- Seeds
- Nuts
- And soy products
- Green peanuts
- Lentil split peas,
- Soy beans
- Kidney
- And many more
You know Internet can be the great source of anything including the weight loss plan for vegetarians. Just try to visit the website of www.fatlossfactor.com which provides you with many tips of how to reduce your weight. Here you can also find Fat Loss Factor Program Review which can be your direction to choose the best weight loss program whether for common people or for vegetarian. With this program, you will get easier way to lose programs.
The Best Diet for You
Even though your main goal is reducing your weight, when conducting the best weight loss plan for vegetarians, you need to have breakfast during your program. Oatmeal and fruits are the best breakfast for this program. A cup of green tea can help you to reduce your weight too. The best weight loss plan for vegetarian is the best diet you can do.
Please share "Best Weight Loss Plan for Vegetarians: Healthy Diet" if this post beneficial for you. Thanks!

subset: commoncrawl


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Right before the interview he starts teasing me to not mess up!
I mean just look at me
Within minutes of that picture being posted on Eagle's facebook page everyone was commenting that I looked like I was in awe...drooling...starstruck. Yep, sounds about right.
We talked about his Entertainer of the Year win
why he loves coming to Va Beach
(I think YOU brought the heat Luke!)
And he even spilled the beans on where he likes to hang out when he's in town
Here's the full interview
btw did you notice my arm shaking about halfway through the interview from holding up the microphone?!
Ok back to Luke...I just happened to have my cell phone ready at the right moment to capture this
Even my cat can't stop watching
So if you need more Luke...here it is one more time
You're welcome.
P.S. I have the BEST husband...not only does he spend his day off filming my interview, but then he spends his Saturday helping me make GIFS of Luke Bryan.
So as much as I joke about loving Luke Bryan...my Brian is the best :)


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Money Watchers: Dangerous donations
NBC 10 Money Watcher Alison Bologna reports that especially after catastrophic events such as Hurricane Sandy, new schemes were created to entice people to donate money to fake charities.
Describing the con men behind a major charitable fraud scheme former District Attorney Rich White said, "These people were greedy, but on top of being greedy, quite frankly they were stupid too."
The scheme involved 450,000 victims across the U.S. and a whopping $10 million.
The scam involved a pitch like this, "Did you know that police officers have to buy their own bullet proof vests and if they can't afford them they just can't get them?"
That is one of the many false claims solicitors for a bogus charity would make to persuade potential donors to hand over money.
"And convince them that the little they had, the little they had to give, was going to do something good and unfortunately the opposite was true," White said.
And postal inspectors said just pennies on the dollar actually went to any charity, the rest of the money went in the pockets of the telemarketers.
"The owners of the company were living large. They had large homes, recreational vehicles, boats, water skis, and jet skis; things like that," said U.S. Postal Inspector Dean Kowalefski.
The experts offer advice to those who are thinking about donating money to charity.
Do not donate money over the phone, unless you are sure you know the charity; instead, ask them to send you something in the mail.
In this case, experts say, if you ask disreputable telemarketers for something in writing, you're not going to get anything.
"They can't tolerate that practice. For them to be successful they need to get the pressure sales they sell someone right then or they are on to the next one."
And some additional advice from the postal inspectors; never donate cash. If you are writing a check be sure to write it out for the name of the organization and never to an individual.


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We are due to go in March
Whats best to take as
currency-ie Travellers cheques/or use cards as i hear
go touringdont always accept cards for excursions
when you go away for over a month its hard to budget for and would feel uneasy about carrying cash!
Anyone had any experience let us know
Did any one do
EXTRA Excursionswith
GO TOURINGand how where they?? Where they
valuefor money?? or were there better excursions or one could do themselves ?


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Printable Version
Welcome to Pine Bluff Camp Sites - Waterville, Iowa
Located at the edge of Waterville, Iowa on scenic Big Paint Trout Stream! Just blocks away from bar and grill, library, fire department and rescue squad.
Country park with shelter 1 mile East!
Yellow River State Forest just 4 miles East!
12 miles from Lansing, Waukon, or Harpers Ferry!
Amish store 3 miles West!
Pricing: Water, 30 or 50 amp elec. and city sewer.
Campers with Bathrooms Only (or in company of)
April 15 - October 31
$25 + 1.75 tax = $26.75
$150 + 10.50 tax = $160.50
$899.00 + 62.93 tax = $961.93 & $30 elec. deposit
No charge for extra adults, visitors or tents.
Self registration at red roof building.
Every site has:
Wi-Fi, TV, Internet & phone available from
Ace Telephone (888-404-4940)
Water (during season or later)
City Sewer
50/30/20 Electrical (metered for seasonal renters)
Usually Free Firewood at white shed
(limit 5 pieces per night.)
Camp At Pine Bluff Because...
Quieter than most campgrounds (only 10 sites)
High and Dry since purchased in 1987
More economical than most campgrounds
No rock roads to get here!
Contact Information
Pine Bluff Camp Sites
75 E. Main St. (Office: 36 E. Main St.)
Waterville, Iowa 52170
(563) 535-7256
Copyright © 2016 - Pine Bluff Camp Sites | Waterville, Iowa - All rights reserved.
Site designed and maintained by Iroc Web Design Services©.
Your Small Business Web Design Solutions.™


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This may be the most sensitive topic we've ever talked about here on the Fresh Rag Show. Rarely do we talk about the deeper sides of running a creative business. So many artists and makers have gone into those professions because they appreciate the solitude of working with their hands, alone in a studio, satisfied with their solitude while they make stuff.
The more creative people I talk to, the more I realize how many of them would rather be doing something other than being in the spotlight. I see this both on the podcast, and within my own training groups. Of course, not all artists and makers are hard-core introverts, but we all tend to have a bit of it within us, and sometimes it holds us back from reaching our full business potential.
This is the conversation I started with my friend, Kaarin Vembar of Closet Caucus, before I realized we needed to carry the chat over to the podcast.
You are not alone in this battle of personality vs. attention, and your business does not need to suffer, if you know how to manage your own needs and expectations. I won't say we have the answers to the situation that might cause some of you to hold yourself back, but maybe there's some solace in solidarity.
Come join Kaarin and I while we talk through our identity issues.
Relative Links
- Closet Caucus
- Pop Fashion Podcast
- My first chat with Kaarin and Lisa
- Music: Long Time Ago by Street Joy
- Maker of the Week: Alexandra Ackerman


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Illinois Honored Citizens Set of 3 Gold Select
Similar Products Available
Part of the Illinois Commemorative Medallion Program. Three coins are included in this set:
Each reverse featurs the Seal of the State of Illinois.
The coins are minted in .999 pure silver with a proof strike with selective plating in 24K gold. Each comes enclosed in a lucite capsuleand and all three are enclosed in a leatherette presentation box with a certificate of authenticity. A series of coins of distinction.


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2017 Schedule
THE Kumho Tyre Australian V8 Touring Car Series has a fresh five-round calendar for the 2017 series.
The series will again compete at the Virgin Australia Supercars Championship events next year, supporting the V8 Supercars at Phillip Island in April, Winton in May and Queensland Raceway in July with live television coverage on FOX Sports to be included.
The Kumho Series will again make an appearance at Muscle Car Masters in Sydney and will also visit Phillip Island for a second time later in the year, this time with the Shannons Nationals.
The 2017 Kumho Australian V8 Touring Car Series will be contested over five rounds in three states:
|Round One
|Phillip Island, VIC (Supercars)
|April 21-23
|Round Two
|Winton, VIC (Supercars)
|May 19-21
|Round Three
|Queensland Raceway, QLD (Supercars)
|July 28-30
|Round Four
|Phillip Island, VIC (Shannons Nationals)
|September 8-10
|Round Five
|Sydney Motorsport Park, NSW (MCM)
|October 27-29
(Note: Dates include Friday Practice)


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New The LEGO Batman Movie Poster Released for Batman Day
Today is Batman Day and to celebrate the character's 75 anniversary, Warner Bros. has released a new poster for The LEGO Batman Movie. The poster features Will Arnett's Batman springing into action with some simple words of advice, "Always be yourself, unless you can be Batman."
The LEGO Batman Movie is set for release on Feb. 10, 2017 and remember, always be Batman.
LEGO Ninjago Short to be Shown Before Storks
Earlier today, The Hollywood Reporter has released some information regarding next year's The LEGO Ninjago Movie. If you're going to watch the Storks next month, there will be a five-minute animated short shown in the beginning for Ninjago called The Master: A LEGO Ninjago Short. It will feature Jackie Chan as Master Wu who "gets into a conflict with Chicken" voiced by Abbi Jacobson while the short will be narrated by Justin Theroux.
If you remember, the cast of The LEGO Ninjago Movie was announced a few weeks ago with some big name actors/actresses including Chan, Jacobson, and Theroux, among others. Jacobson will also be voicing Nya while Theroux will be voicing Garmadon. The LEGO Ninjago Movie itself will open on September 22, 2017 and it is rumored there will be LEGO Ninjago Movie sets as well as a Collectible Minifigures line to be released in conjunction.
The LEGO Batman Movie 2017 Set Rumors
The rumor mill keeps turning as there is now more in-depth information about The LEGO Batman Movie sets. We already know of two confirmed sets which was revealed during SDCC with The Batmobile (70905) and The Joker's Notorious Lowrider (70906) but it has been reported that there will be about 15 total sets coming out for the film.
Delta Customs over on Instagram has given some more infomation about few more The LEGO Batman Movie sets. First is the new Arkham Asylum which will be at least $100 and around the size of the previous version. The minifigures that are in the set include Batman, Barbara Gordon, Aaron Cash, 2 Arkham Guards, and Harleen Quinzel. In addition, there are also minifigures in jumpsuits which include the Joker, Two-Face, the Riddler, and three more villains.
Next up is a the new movie Batcave. Some details of it include an area where Batman's suits are hanging as seen in the trailer some of which include the Disco suit and the Diving suit. There will also be a walkway which leads to the bat-shaped Bat-Computer which is also seen in the trailer. The Alfred minifigure is reported to be one of the minifigures in the set and is seen trying to escape some Penguin minions. The vehicles of the Batcave include a Batboat and a brick-built duck for Penguin.
In other LEGO Batman Movie set news, there will be a lot of obscure minifigure characters like Tarantula as well as some from the Batman Rogues Gallery such as Zebra Man, Kite Man, and Calendar Man. Also, there will be Mr. Freeze in some sort of exo-suit in a smaller set and it includes Batman and a hostage. Some folks over on Eurobricks has also reported some other sets like a Penguin car and a Catwoman bike set. Finally, The LEGO Batman Movie Collectible Minifigures line is reported to have 20 minifigures instead of the usual 16.
Of course everything reported above, minus the confirmed sets, is purely speculation but Delta Customs has given information before and they turned out to be real in the end so fingers crossed.
Update: Delta Customs has provided more information on The LEGO Batman Movie sets.
There's supposedly a new Batwing that is different from the one seen in the trailer and is shaped as a bat. The cockpit will be the head of the bat and Poison Ivy will be included in the set.
For the Killer Croc set, Killer Croc himself is the size of the Rancor if you have that figure. The set will have various swamp-type builds as well as a red pick-up truck. The minifigures included in the set are Batman, Killer Croc, Zebra Man, and Tarantula.
The LEGO Ninjago Movie Voice Actors Officially Announced
LEGO has officially announced some of the voice actors that will be in next year's The LEGO Ninjago Movie. The list is pretty much the same as the one that came out a few months ago which was revealed from a Warner Bros. catalog. The only difference is the new actors that have been added including Justin Theroux and Olivia Munn as Lloyd's parents. Check out the press release below.
Master Wu – Jackie Chan
Garmadon – Justin Theroux
Lloyd – Dave Franco
Lloyd's mom Koko – Olivia Munn
Cole – Fred Armisen
Nya – Abbi Jacobson
Jay – Kumail Nanjiani
Kai – Michael Peña
Zane – Zach Woods
BURBANK, Calif.–(BUSINESS WIRE)–The main cast has been put together for "The LEGO® Ninjago Movie," a new animated adventure in Warner Bros. Pictures' LEGO franchise. The film will star Dave Franco, Justin Theroux, Fred Armisen, Abbi Jacobson, Olivia Munn, Kumail Nanjiani, Michael Peña, Zach Woods, and the legendary Jackie Chan. "The LEGO Ninjago Movie" is slated to open nationwide on September 22, 2017.
In this big-screen Ninjago adventure, the battle for Ninjago City calls to action young Lloyd, aka the Green Ninja, along with his friends, who are all secret warriors and LEGO Master Builders. Led by kung fu master Wu, as wise-cracking as he is wise, they must defeat evil warlord Garmadon, the Worst Guy Ever, who also happens to be Lloyd's dad. Pitting mech against mech and father against son, the epic showdown will test this fierce but undisciplined team of modern-day ninjas who must learn to check their egos and pull together to unleash their true power.
Jackie Chan ("Kung Fu Panda," "The Karate Kid") stars as Master Wu; Justin Theroux ("Magamind 2," "The Leftovers") is Garmadon; Dave Franco ("Neighbors 2: Sorority Rising") plays Lloyd; and Olivia Munn ("X-Men: Apocalypse") is Lloyd's mom, Koko. Making up the secret ninja crew, Fred Armisen ("The Jim Gaffigan Show," "SNL") voices Cole; Abbi Jacobson ("Neighbors 2: Sorority Rising") plays Nya; Kumail Nanjiani ("Mike and Dave Need Wedding Dates") is Jay; Michael Peña ("Ant-Man," "The Martian") is Kai; and Zach Woods ("Silicon Valley") voices Zane.
Jill Wilfert, an executive producer on the film and Vice President of Licensing and Entertainment for LEGO Group, stated, "We are thrilled with the star-studded voice cast slated for 'The LEGO Ninjago Movie.' Our fans know and love Ninjago characters for their personalities and skills, so it's only fitting that we assemble a diverse and talented cast to bring the world of Ninjago to life on the big screen."
"The LEGO Ninjago Movie" marks the feature film directorial debut of Charlie Bean ("Tron: Uprising"). It is being produced by Dan Lin, Phil Lord, Christopher Miller and Roy Lee, who previously collaborated on the worldwide box-office phenomenon "The LEGO® Movie," along with Maryann Garger ("Flushed Away"). Chris McKay, Seth Grahame-Smith, Jill Wilfert, and Keith Malone are serving as executive producers. The screenplay is by Hilary Winston & Bob Logan & Paul Fisher, story by Kevin Hageman & Dan Hageman and Hilary Winston & Bob Logan & Paul Fisher, based on LEGO Construction Toys.
From Warner Bros. Pictures and Warner Animation Group, in association with LEGO System A/S, a Lin Pictures/Lord Miller/Vertigo Entertainment Production, "The LEGO Ninjago Movie" will be distributed worldwide by Warner Bros. Pictures, a Warner Bros. Entertainment Company.
The LEGO Batman Movie SDCC 2016 Official Trailer Now Available
Earlier today at San Diego Comic Con, Warner Bros. had their panel and showed off all the upcoming movies they have on the slate including The LEGO Batman Movie. The official SDCC 2016 trailer of the movie is now available and it there's all new footage to drool over, especially for all the potential sets that may come out next year.
The trailer also shows Michael Cera as Dick Grayson and him messing around with Batman's other suits like El Murcielago, Glam Bat, and Raggae Man which turns out to be Robin's suit of his choosing minus a couple of pieces here and there. There are rumors that there will be a minifigure series based on the movie so don't be surprised if we see a couple of the suits in the trailer as actual minifigures.
The LEGO Batman Movie will be released in theaters February 2017.
New The LEGO Batman Movie Poster Revealed at SDCC 2016
Warner Bros. has released a new poster for The LEGO Batman Movie in conjunction with the panel that is happening at San Diego Comic Con 2016. The image shows Bruce Wayne taking off his cowl to show off his look, most notably the new hairstyle. There will also be a new trailer very soon so stay tuned for that.
Two The LEGO Batman Movie Official Set Images Revealed
Earlier today, LEGO has published official images of two The LEGO Batman Movie sets which are The Batmobile (70905) and The Joker Notorious Lowrider (70906). We'll see the two sets being displayed at San Diego Comic Con this weekend. Below is a little description of the two sets.
The Batmobile (70905) has 581 pieces and it includes minifigures of Batman, Robin, Man-Bat, and the Kabuki Twins. Batman has a new belt attachment piece while Robin wears some of the new green goggles which we saw before. Man-Bat looks like the same one we've in in a previous set while The Kabuki Twins are brand new. We'll see how the assassins will play part in the movie later on. The Batmobile will retail for $59.99.
The Joker Notorious Lowrider (70906) has 433 pieces and will retail for $49.99. It is the one that we saw an image of yesterday with the character reveal and it includes minifigures of Batgirl, The Joker, and Harley Quinn. All three minifigures have new printing on them and the Lowrider is an interesting looking build as well.


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Linda Walker
Listed by Badger Realty, Llc
Can you believe this price of a Woodland Pines unit with gas heat! Enjoy this 3 level townhouse with master suite upstairs, lots of living space with brick fireplace & large outside deck on main level, three bedrooms and full bath on lower level. Listed below assessed value. Cross country ski trails right on property.
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Skip to comments.Warren Buffett: Criticism is 'ridiculous' (His point is -- tax code treats his secretary unfairly)
Posted on 01/27/2012 11:24:04 AM PST by SeekAndFind
Warren Buffett's secretary, after being pointed out on national television during Tuesday's State of the Union speech, now is facing criticism over her salary and second home.
Debbie Bosanek and her boss both declined Thursday to disclose how much she's paid, saying it's private.
In an interview with The World-Herald, Buffett also said none of the online guesses about Bosanek's salary is right, and the critics are missing his point.
"I'm saying she is being treated unfairly in the tax code, as are tens of millions of others, compared to me," Buffett said. "They shouldn't change the rates on all the other people. They should change mine."
Buffett stuck to his long-held contention that it's unfair for high-income people to pay low tax rates, such as his own 17.4 percent in 2010, less than half Bosanek's 35.8 percent rate. That rate is for everything Bosanek pays to the federal government income taxes as well as payroll taxes for Social Security and Medicare.
Based on that 35.8 percent tax rate, Forbes blogger Paul Roderick Gregory wrote, "She is scarcely the symbol of injustice that Obama wishes her to project. I imagine that there are any number of secretaries who would want her job and her place in the Congress gallery for the President's State of the Union address."
Bosanek, he said, is being portrayed as a "downtrodden woman," but he estimated she makes between $200,000 and $500,000 a year, based on his assumptions about average income tax rates. Other Forbes bloggers disputed the figures, saying Buffett was quoted in the Times of London about five years ago saying he pays his secretary $60,000 a year.
Buffett said Gregory "doesn't have any idea, just zero. If I were to estimate his salary, I'd probably be closer than he is." You can't estimate salaries from tax rates, he said.
Buffett, the chairman and CEO of Omaha-based Berkshire Hathaway Inc., said the issue isn't Bosanek's income, nor is her tax rate unusually high. "They can't attack the facts, so they attack the person. It's ridiculous."
Bosanek said she's not complaining about her salary or the taxes she pays, nor will she apologize for the home she bought last year in Surprise, Ariz., a Phoenix suburb. One blogger announced that she made the purchase "despite a heavy tax burden."
"I just thought it was time to buy a home," she said. "Warren tells me that it will be the best opportunity in my lifetime. Mortgage rates are low and prices have dropped dramatically. Getting a nice home in a great climate for only a $30,000 down payment and a mortgage that has a low interest rate I've been working 37 years and saving for an opportunity like this.
"I share Warren's view about the future of America, and we believe that our country will do just fine. I'm happy to make this investment.
"Hopefully in 10 years, when I turn 65 and Warren turns 92, I will be able to convince him to finally retire so I can retire, after working 47 years, and spend some time where the sun shines in the winter."
Bosanek, while declining to give her salary, said: "I feel like I'm treated fairly at Berkshire."
She said she is neither the lowest-paid nor the highest-paid person among the 21 employees at Berkshire's Omaha headquarters. Their 2010 tax rates ranged from 33 percent to 41 percent, according to Buffett's calculations.
Bosanek said neither she or Buffett ever implied that she was poor or underpaid.
"It's not like I look forward to paying taxes," she said. "But I don't mind paying taxes as long as everybody's treated fairly. . I'm not saying anyone should feel sorry for me or lower my taxes. I never even thought about it until he wrote the article."
Buffett wrote an article last August in the New York Times, calling on Congress to change the tax code so high-income people pay at least as much as middle-income taxpayers and using his secretary as an example.
Obama proposed just such a tax change, calling it the "Buffett rule" and emphasized it in his address on Tuesday by inviting Bosanek to sit in the first lady's box during the speech.
"They needed to pick one person, and I was the lucky person they picked, or unlucky," Bosanek said Thursday.
She said she and her husband, Jerry, looked at houses last June when they took their son, also named Jerry, to visit at the University of Northern Arizona campus in Flagstaff, where he is now a freshman. Her husband drives vans and other vehicles for a company that delivers air freight from Eppley Airfield to destinations in the Midwest. Her father was an Omaha firefighter.
The Bosaneks bought the four-bedroom house in Surprise last summer for $144,000, making a $30,000 down payment and taking out a mortgage on the rest.
In Bellevue, the Bosaneks live in a slightly larger house southeast of South 25th Street and Fairview Road, close to the Tregaron Golf Course. They bought it in 1999, paying $231,274. The tax value is now $217,716.
The Surprise house has a pool, as do many others in the area, and the side yard has an artificial turf putting green with four holes, which was installed before the Bosaneks looked at the house. They spent Thanksgiving weekend there.
"We just had a ball. It was very special for us," Bosanek said, giving their son a break from campus life and staying in their own house and not a hotel.
Bosanek said she is not used to being in the public eye but doesn't mind being used as an example because she agrees with Buffett's views on taxes.
"I'm not whining. I just want to set the record straight," she said. "I'm very lucky. I've got a wonderful job. I work for a wonderful person. I have a wonderful family, and I have a wonderful home in Bellevue and I've got this wonderful new home that, hopefully, I'll be able to pay off someday."
-- The Omaha World-Herald Co. is owned by Berkshire Hathaway.
Instead of raising taxes on millionaires, why not lower the taxes on the rest of us?
—His point is — tax code treats his secretary unfairly—
Message to Warren: Yeah, we get that. We just disagree with you.
So instead of jacking up the rates on those who are prosperous, how about reducing the rates for people like his secretary. What is a fair rate ? A simple flat 10 or 15 % In fact 10% is a very fair number. It is good enough for God, it is more than good enough for the gov't. Flat tax with no deductions except a certain exemption of income before tax is applied. Throw out the whole complex tax code and it would be a simple sheet of paper.
Taxes should be for revenue, not for social engineering.
Hussein and Buffett can criticize and use a lower level employee (whom Buffett could assist financially in any way he likes) as a prop, but when SOMEONE ELSE decides to analyze the fact that she owns two homes and likely makes up to $500,000 a year, well that is (ACCORDING TO HUSSEIN AND BUFFETT APPARENTLY) "ridiculous", eh?
Got hypocrisy?
Hey Warren what would you do if Obozo put a 30% tax on all your income regardless of its source? Would you be making investments that entail risk knowing that 30% of whatever you made in capital gains would be taxed at 30% on top of the 33% federal corporate income tax that comes right off the top of a company's profits? My bet is Buffet would be moving his money offshore in short order.
He isn't opposed to eliminating loop holes.
Recovering_Democrat — you have it exactly right. Add to that the fact that Buffett and his organization owes a billion in back taxes and is fighting it and Buffett becomes the ultimate hypocrite. But then, what else do you expect, he is a democrat
i wonder if their son will get in-state tuition since mom and pop bought their 2nd house there in Arizona?
Blowback is a bitch, ain't it Warren.
My version of the Buffet rule is that all Berkshire Hathaway investors pay the normal 15% capital gains tax, plus a special 65% "secretary tax".
So, his secretary owns TWO homes, and he wants people to see her as being treated unfairly? I own zero homes, but I'm not complaining that the "Rich" do not pay enough in taxes. The argument isn't about whether this group or that pays too much or too little. The argument is that the GOVERNMENT squanders what they take in revenue. The argument is that the GOVERNMENT places too many regulations and restrictions upon people who run small or large businesses, stiffling job creation. The argument is that the GOVERNMENT has become too wieldy, attempting to control ALL aspects of EVERYONES lives. The argument is that GOVERNMENT is the problem.
He's not only a world class rich guy - He's a world class hypocrite.
What he said.
Poor dummy, he puts his secretary in the national spotlight and cries when the light is shined on her
Is that her total tax rate or her marginal tax rate?
If it is her total tax rate and assuming she is married filing jointly with Nebraska income tax and SS+Medicare up to 106,000 and only Medicare above that, I come up with a pay of about $357,000. I ignored deductions and exemptions and any capital gains, even though she should have some. If those were included her income would have to be even higher to get to 35.8% total income and payroll taxes. I also didn't count the employer's half of social security. Either way, she is right about at the bottom edge of the eeeeeeeeeeeeeeevil top 1% of income earners, and I dislike how she is being used as a political bloody shirt comparing the income taxes paid by a 1%-er against a the capital gains of a 0.001%-er.
and then give them less....less services, less Medicare...less SS....
why doesn't Warren pay off his company's $20,000,000,000.00 tax bill, as I recall...
Buffett is such a hypocrite. He is currently fighting the IRS in court over taxes owed and he takes only a 100,000 salary. Precisely to minimize his taxes.
Basically, it's line 60 (tax) + SS+Medicare taxes paid (the full 14.2%) / line 43 (taxable income -- not AGI).
Which is to say all the huffing and puffing boils down to him not paying SS/Medicare on the 60 million in capital gains and dividends.
Well, that and the 14.2% ss/medicare tax is applied before any deductions and credits so an individual with a $100,000 salary, $14,200 in FICA taxes, $85,800 in deductions (that's a lot of kids) and $1,500 in income tax has a 110% tax rate, according to his "math".
Worth noting -- as of 2013, there's a 3.8% medicare tax on investment income in excess of $250,000.
Disclaimer: Opinions posted on Free Republic are those of the individual posters and do not necessarily represent the opinion of Free Republic or its management. All materials posted herein are protected by copyright law and the exemption for fair use of copyrighted works.


text: Friends, Let's honor our fallen fellow Americans by changing our cool HTF logo on every page to one with an American flag (can we make it wave also?) like they typically do during the holidays! Do you agree? It would be a small but meaningful gesture and correspond with national calls for flags to be flown... Lee


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sort by best latest
sometimes therapy doesn't work because the wrong diagnosis was made.
Hi cathylynn99,
I know thats right, as was stated by another hubber, it seems to him we could get enough therapy right here on Hubpages.
Misdiagnosis is surely probable with psychology, the mind being so complex, even if the diagnosis is correct, the treatment can't always be one size fits all.
I pray all who have a need to be helped, will find there helper and the treatment they deserve
My followup to this question is; If we are not truthful, to ourselves and others and we are only willing to let others lead us, but not take an initiative to put words into action, we may be found years later in this same pit we have fallen into.
What motivates me is knowing things will never change if I don't try. My faith in God and his plans to prosper and not harm me also pull me up.


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I Flea Dipped The Dogs Today
Personal Stories, Advice, and Support 2 People
Summer Is RoughIt get's so hot down here in STX and I am the person that always has to bathe the pups each week. I noticed that there was a resistance to the frontline I normally keep the dogs on so I flea dipped the mutts today. I hope that it works. I bought some stuff...
moshimoshi 31-35, F 0 Jul 11, 2010


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Be on the lookout for new beers that come out.\r
There is always somthing new to taste.
Pros: Best beer in Texas
Cons: Get there early. It gets packed very quickly.
Service is horrible, lunch or dinner, doesn't matter. Their servers are inconsiderate and could care less about the customers. I've been there 6 times with different people, it wa...
In ShortThe airy dining room is rustically refined, a mix of whitewash, wood and brick, with burlap grain bags, fans and recessed lighting sharing ceiling space, and beer signs and...
From family friendly trips with kid friendly activities to local plumbers lawyers, spas, and contractors. Judy's Book has millions of listings and reviews for the best and not so great. Whether you're looking for a specific business or just trying to discover great places we make it easy. We're a family oriented site focusing on bringing trusted reviews and recommendations to moms and others. Green and Sustainable places are becoming more relevant to Judy's Book users, have a favorite place? Write a review and tell users why it's Green!


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If you are wanting to serve, we are excited to help you get connected. Once you complete this form, you will receive an invite to join The City. The City is the online tool that all of our serve teams communicate through. From here, one of the serve team leaders will contact you to set up a time to talk and get to know you.
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We as Christians have a tremendous responsibility given to us when the Lord blesses us with a child. By doing so He entrusts us with the responsibility of raising up that child in the way of the Lord and establishing early in his life a foundation built upon Jesus Christ.
It is our desire here at Calvary Chapel Petaluma for the parents of each child being dedicated to the Lord to have a Biblical understanding of what a baby dedication signifies. With this as our goal, we would like you to help us by answering a few brief questions that will be used to assist in preparing you and your child for dedication.
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AOFM students backstage at New York Fashion Week
At AOFM we take our students backstage to work at Mercedes-Benz Fashion Week New York every year. See just one of the many shows our graduates assist on. Don't forget to check out our "Opportunities" section full of work our students have done all over the world.


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Kennewick Man
Kennewick Man
The remains of an ancient human found along a river in Kennewick, Washington, in 1996 set off a heated debate about the ownership and future of the skeleton. Scientists argued that the skeleton, dubbed Kennewick Man, could provide new information about human migration in North America, while Native Americans claimed him as an ancestor and wanted to bury him according to their rites. Forensic anthropological findings and cultural evidence were presented in court procedures over the course of nine years while the fate of the Kennewick Man was debated.
The story of Kennewick Man began in July 1996, when two college students watching hydroplane races found a human skeleton along the Columbia River. The young men turned the remains over to local police, who realized that they were probably very old. The bones were then given to forensic anthropologist James Chatters for evaluation. Chatters reconstructed the skeleton, which was 80–90% complete. He determined that it was from a man who was probably five feet nine or 10 inches and about 40–50 years old when he died. He showed little evidence of arthritis, indicating that he wasn't used to carrying heavy weights and that he might have been a wandering hunter. Dental examinations showed that the skull contained 30 of the 32 teeth and that they were in good shape, indicating that he probably had a diet that included lots of soft foods like meat. He was taller and thinner than most ancient Native Americans and the back of his skull was not flattened from a cradleboard as is commonly observed in skeletons of ancient Native Americans. In addition, the man had a stone spear point lodged in his pelvis and there was evidence of severe trauma to his rib cage that probably limited the use of his arm. Using computerized tomography (CT), Chatters determined that the spear point was serrated and leaf-shaped and typical of the types of spears used between 8500–4500 years ago. He hypothesized that the skeleton was either from a European pioneer who had been attacked by native people using stone-age weapons or from an ancient human. Chatters sent pieces of the bones to a laboratory for carbon dating, which determined that the age of the skeleton was between 9,200–9,400 years old, making the skeleton one of the oldest, and most complete, ever found in North America.
Once the age of the skeleton was determined, several groups came forward, vying for control of the remains. A group of five Native American tribes in the region, the Umatilla, the Yakama, the Nez Perce, the Wanapum, and the Colville, wanted to accord the remains the same rites given to any Native American, namely a speedy burial. They cited the legal authority of the Native American Graves and Repatriation Act (NAGRA), which requires the return of American Indian remains to tribes. As news of the unique find spread throughout the scientific community, a coalition of eight anthropologists and archaeologists petitioned for their right to study the ancient remains prior to burial. The scientists believed that study of the Kennewick Man could reveal important information about early human migrations into North America. The Native American group believed that any manipulation of the remains would show enormous disrespect to the dead and vehemently opposed scientific investigation of the skeleton, which they called the Ancient One. Because some of the features of the Kennewick Man, such as his height and the shape of his skull, indicated that he might not be of Native American ancestry but rather of European descent, a group of people representing the ancient Norse religion called Asatru also petitioned the court for the right to the remains.
The ensuing legal battle raged for more than nine years. One of the key questions of debate in the courts concerned whether or not the skeleton was subject to NAGRA. NAGRA requires that all Native American remains be returned to the tribe for burial, however it was unclear if the Kennewick man was of Native American ancestry. Eventually the court ruled that some scientific study was required in order to establish the origin of the skeleton and between 1998 and 2000, the Department of the Interior coordinated these studies. A 1999 physical examination of the bones established that the Kennewick Man shared most physical characteristics with people from Southern Asia. In April 2000, samples of bone from the Kennewick Man's skeleton were removed and sent to two different laboratories for DNA testing. Because of the age of the bones, it was impossible to extract sufficient DNA for analysis and the results of the study were inconclusive. After a series of appeals by all sides, in February 2004, a U.S. Federal judge ruled that it was impossible to prove that the Kennewick Man's ancestry was culturally affiliated to any of the Native American tribes in the region and gave scientists the right to go forward with their investigation. In 2005, plans were outlined for study three-phase study involving as many as 23 different scientists.
see also Anthropology; Anthropometry; Mitochondrial DNA analysis; Odontology; Skull.
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"Kennewick Man." World of Forensic Science. . Encyclopedia.com. 18 Nov. 2017 <http://www.encyclopedia.com>.
"Kennewick Man." World of Forensic Science. . Encyclopedia.com. (November 18, 2017). http://www.encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-maps/kennewick-man
"Kennewick Man." World of Forensic Science. . Retrieved November 18, 2017 from Encyclopedia.com: http://www.encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-maps/kennewick-man
Kennewick Man
Kennewick Man
The dead have power to create controversy in the world of the living. One example involves a 9,500 year-old skeleton, one of the best-preserved early human fossils found in North America, whose discovery triggered a bitter debate over who owns America's past.
Found accidentally in the Columbia River of eastern Washington in July 1996, was a nearly complete skeleton that showed traces of a hard life: a fractured elbow, broken ribs, a possible head injury, and a spear wound in the pelvis. The cause of his death is uncertain, as is the issue of whether he was interred or naturally buried. The man's skull, facial characteristics, and teeth are distinct from those of all modern peoples, including American Indians, but most similar to Polynesians and the Japanese Ainu. These differences, which are also seen in other American skeletons older than 9,000 years, raise the possibility that more than one wave of early immigrants peopled America, and that American Indians were not the western hemisphere's first inhabitants.
The skeleton quickly became the center of a political battle pitting conservative Indian tribes and the Native American Identity Movement against the interests of science and the human community at large. Upon announcement of the discovery, leaders of five Pacific Northwest Indian tribes claimed the bones under the Native American Graves Protection and Repatriation Act of 1990 (NAGPRA). The U.S. government, which owned the skeletal remains, agreed to turn them over to the tribes, rebuffing a request by scientists for the opportunity to study this rare find. Eight prominent anthropologists sued to stop reburial, bringing the first test case of the NAGPRA. The case ignited a conflict between religion, the political position of Native American activists, and the public's right to scientific knowledge about the ancient history of its homeland.
On the religious front, the tribes believe that ancestors' remains must be returned to the earth lest their spirits be offended and wreak havoc among the living. Because the tribes' present-day religion holds that they were created near where the skeleton was found, they insist that Kennewick Man must be their ancestor. To deny their religious claims, they say, is to disrespect and weaken their culture.
However, the religious claim is the less important one from the perspective of the Native American Identity Movement, which has as its goal the assertion of tribal rights to land and resources. In the 1970s this movement selected American Indian skeletons kept in museums as an issue of debate, maintaining that placing their skeletons in museum displays compromised their human rights. Kennewick Man and the few other early skeletons are particularly important because they suggest the chilling possibility that Indian people were not the first to occupy the Americas. If the U.S. government acquiesces to the tribes' claim to these most ancient remains, however, it affirms the movement's claim that Indian people were the first occupants of the American continent and supports the tribes' original right to land and resources. The movement is concerned that proof of an earlier people preceding them will weaken its claims.
For the scientific community, the skeleton is a story about the life and death of an ancient American and a window into the history of the human species. Are the most ancient human fossils and artifacts the property of a few or should they be considered the heritage of all peoples? The answer, scientists feel, has global implications for their quest to understand the history of the human species.
See also: Grief and Mourning in Cross-Cultural Perspective; Human Remains
Chatters, James C. Ancient Encounters: Kennewick Man and the First Americans. New York: Simon & Schuster, 2001.
Chatters, James C. "The Recovery and First Analysis of an Early Holocene Human Skeleton from Kennewick Washington." American Antiquity 65, no. 2 (2000):291–316.
Thomas, David H. Skull Wars. New York: Basic Books, 2000.
Preston, Douglas. "The Lost Man." The New Yorker, 19 June 1997, 70–81.
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"Kennewick Man." Macmillan Encyclopedia of Death and Dying. . Encyclopedia.com. 18 Nov. 2017 <http://www.encyclopedia.com>.
"Kennewick Man." Macmillan Encyclopedia of Death and Dying. . Encyclopedia.com. (November 18, 2017). http://www.encyclopedia.com/social-sciences/encyclopedias-almanacs-transcripts-and-maps/kennewick-man
"Kennewick Man." Macmillan Encyclopedia of Death and Dying. . Retrieved November 18, 2017 from Encyclopedia.com: http://www.encyclopedia.com/social-sciences/encyclopedias-almanacs-transcripts-and-maps/kennewick-man


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Results 1 to 5 of 5
Thread: WDYT of this sister set?
July 16th, 2014 02:28 PM #1Senior Member
- Join Date
- Mar 2014
- Posts
- 1,467
July 16th, 2014 02:51 PM #3
July 16th, 2014 02:54 PM #5
I love the second and third names, I love the middle names too. I think both combos flow really well and the sibset goes together well. I don't like Violet James, mainly b/c Violet is nms, but also because sounds like "Violent James" to me, and it doesn't flow like the others do.
Annabel Ray
Olivia Drew
Evangeline James nn Vangie or Evie
Edith James
Estelle James
Elodie James nn Ellie or Edie
Eden James
Evelyn James
Viola James
Valery James
Valentine James
Vivian James* My name is Adelaide. I love to play with names.*Saffron Jane Calliope - Seraphine Mariah Rose - Rhiannon Noelle - Rowan Mazarine - Oswell Constantine - Maceo Alexander - Lucas Balthazar - Israfel Ronin - Paul Constantine - Malcolm Ephraim
July 16th, 2014 04:28 PM #7
Beautiful together!
July 16th, 2014 05:13 PM #9Senior Member
- Join Date
- Jul 2014
- Posts
- 611