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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Cryptophytes are highly compartmentalized organisms, expressing a secondary minimized eukaryotic genome in the nucleomorph and its surrounding remnant cytoplasm, in addition to the cell nucleus, the mitochondrion and the plastid. Because the members of the nucleomorph-encoded proteome may contribute to essential cellular pathways, elucidating nucleomorph-encoded functions is of utmost interest. Unfortunately, cryptophytes are inaccessible for genetic transformations thus far. Therefore the functions of nucleomorph-encoded proteins must be elucidated indirectly by application of methods in genetically accessible organisms.
Orf222, one of the uncharacterized nucleomorph-specific open reading frames of the cryptophyte Guillardia theta, shows homology to slr1649 of Synechocystis sp. PCC 6803. Recently a further homolog from Synechococcus sp. PCC 7002 was characterized to encode a phycocyanin-β155-bilin lyase. Here we show by insertion mutagenesis that the Synechocystis sp. PCC 6803 slr1649-encoded protein also acts as a bilin lyase, and additionally contributes to linker attachment and/or stability of phycobilisomes. Finally, our results indicate that the phycocyanin-β155-bilin lyase of Synechocystis sp. PCC 6803 can be complemented in vivo by the nucleomorph-encoded open reading frame orf222.
Our data show that the loss of phycocyanin-lyase function causes pleiotropic effects in Synechocystis sp. PCC 6803 and indicate that after separating from a common ancestor protein, the phycoerythrin lyase from Guillardia theta has retained its capacity to couple a bilin group to other phycobiliproteins. This is a further, unexpected example of the universality of phycobiliprotein lyases.
Phycobiliproteins are subunits of the major accessory light-harvesting complexes (LHC) of most cyanobacteria and red alga and are present in the thylakoid lumen of cryptophytes as well. Covalently linked to the proteins are chromophore groups, the phycobilins [1,2]. These open tetrapyrrole rings are coupled to conserved cysteine residues via a thioether bond and are necessary for light harvesting and efficient energy flow [3]. Various phycobiliproteins, namely allophycocyanin, phycocyanin, phycoerythrin, phycoerythrocyanin, carry different numbers of bilin groups.
Attachment of bilins to phycobiliproteins is an enzymatically catalyzed reaction, which also occurs spontaneously, but at low efficiency [4]. Several bilin-attaching lyases are described. One of the dimeric enzymes encoded by cpcE and cpcF genes links the chromophore to the phycocyanin α-subunit [4,5]. PecE and pecF genes encode the second known lyase, specific for the phycoerythrocyanin α-subunit [6-8]. Recently Zhao and co-workers discovered that a CpeS-like protein functions as a phycocyanobilin-cysteine-beta84 lyase in Anabaena sp. PCC 7120, which was the first lyase identified for a β-subunit of a phycobiliprotein [9]. Another lyase specific for a β-subunit of a phycobiliprotein was found by Shen et al. [10]. They identified the gene product of cpcT to be a Cys-β153-phycocyanobilin lyase in Synechococcus sp. PCC 7002. Moreover, Zhao et al. reported the Anabaena sp. PCC 7120 CpeS1 as a "near-universal" lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins [11,12].
In most cyanobacteria and red algae phycobiliproteins are organized in multimeric complexes, called phycobilisomes [13-15]. Their antenna structure, located on the cytoplasmic surface of the thylakoid membrane, consists of various linker polypeptides and phycobiliproteins. Each phycobilisome is on its part a multimeric complex, composed of a core and several rod structures. Phycobilisomes can be subdivided according to their structure. The most common type in cyanobacteria, the hemidiscoidal one, consists of a tricylindrical core and six rods.
Allophycocyanin (AP, λmax = 650 nm) forms the core structure, connecting the phycobilisomes to the thylakoid membrane via linker proteins. Rods can be composed of three different phycobiliproteins: phycocyanin (PC, λmax = 617 nm) is located proximal to the core, whereas phycoerythrin (PE, λmax = 560 nm) and phycoerythrocyanin (PEC, λmax = 575 nm) are located distal to the core [16,17]. The phycobilisome rods of each organism differ in their phycobiliprotein composition. Synechocystis sp. PCC 6803 harbors hemidiscoidal phycobilisomes. PC, the only biliprotein in the rod structures in this organism, is composed of α- and β-subunits. These subunits dimerize to heterodimers, assemble to hexameric (αβ)6 discs, and are subsequently coupled to each other, as well as to the AP-core via linker proteins [18,19]. Depending on their location (in core or rods), and their molecular mass, linker proteins are divided into four groups [20,21]. Beside their main function of mediating the assembly and stability of the phycobilisomes, linker proteins also promote energy transfer towards the reaction centres [20].
Guillardia theta is a cryptophyte possessing phycoerythrin as a phycobiliprotein. The β-subunit is encoded on the plastid genome [22], whereas the phycoerythrin α-subunits are encoded by a nuclear-located gene family. In the latter case, the genes encode preproteins containing a tripartite topogenic signal responsible for the translocation across five biological membranes [23]. Because a wide range of genomic data exists from this unicellular phototrophic organism, existing knowledge can be used to reconstruct the biochemistry of these organisms. The elucidation of protein functions encoded by open reading frames in the nucleomorph genome of Guillardia theta is of special interest, as this genome is minimized and should therefore encode only essential proteins. After analyzing the nucleomorph genome data, Orf222 was identified as being homologous to a number of proteins including Slr1649 from Synechocystis sp. PCC 6803 and CpcT from Synechococcus sp. PCC 7002. Because cryptophytes are inaccessible to genetic manipulations, we created a slr1649-loss-of-function strain of Synechocystis sp. PCC 6803 and complemented this strain with the nucleomorph-encoded orf.
The generated Slr1649 loss-of-function mutant generally has characteristics conductive with the description by Shen et al. for a cpcT knock-out in Synechococcus sp. PCC 7002 [10]. Nevertheless, additional effects in the slr1649 knock-out mutant of Synechocystis sp. PCC 6803 were identified in respect to linker proteins within the phycobilisomes of the mutant. Complementation of slr1649 with the nucleomorph-specific orf222 indicated that the cryptophytic protein, although having originated from an organism using phycoerythrin as accessory pigment, attaches a bilin to the position Cys-β155 of phycocyanin in the cyanobacterium.
After analyzing the nucleomorph genome of the cryptophyte Guillardia theta, we identified an open reading frame (orf222) with a high degree of similarity to cyanobacterial genes (Table (Table1),1), that encode soluble proteins possessing a DUF1001 domain. Alignments of the cryptophyte sequence with these cyanobacterial sequences indicated that orf222 should encode an additional transit peptide as shown by a N-terminal extension. Further orfs with homology to orf222 and the cyanobacterial homologs are additionally present in the nuclear genome of red alga [24]. In higher plants, i.e. Arabidopsis thaliana, orfs with some similarity are also present [25]. Even the bacteriophage S-PM2, which infects Synechococcus strains, encodes a homolog of orf222 [26] (Table (Table11).
The number of Orf222 homologues in cyanobacteria varies in several species and does not correlate with the number of phycobiliproteins. Nevertheless, there is a strong tendency to express more than one species of Orf222 homolog in organisms containing multiple types of phycobiliproteins in the rods (Table (Table1).1). Based on amino acid sequence alignments and phylogenetic networks, four monophyletic groups can therefore been assigned (Fig. (Fig.1).1). Two of them resemble CpeT-like proteins (phycoerythrin operon protein); the other two groups harbor members of Slr1649-like type. Neither the Guillardia theta sequence nor any other eukaryotic sequences can be assigned to any one of the four monophyletic groups.
Additionally, clear affiliations of the Gloeobacter violaceus PCC 7421 (glr1182) and Synechococcus sp. PCC 7002 (CpcT) sequences can not be extrapolated. With the exeption of the Prochlorococcus species, at least one member of the Slr1649-like group is present in all cyanobacteria investigated to date. CpeT-like proteins were only detected in cyanobacteria encoding phycoerythrin and/or phycoerythrocyanin. Although the proteins of both groups seem to have the same function, further investigations on the corresponding genes relevant in the genomic context revealed a noticeable difference. Unlike the genes of the cpeT-group, the slr1649-group is by far less conserved in its genomic localization (Table (Table1).1). Except for Nostoc sp. PCC 7120, Anabaena variabilis ATCC 29413 and Trichodesmium erythraeum IMS 101, the localization of the homolog gene is always downstream of cpeS. In few cases it is followed by cpeR. On the other hand, genes for the slr1649-group are rather randomly distributed in the investigated cyanobacterial genomes (Table (Table11).
We used Synechocystis sp. PCC 6803 as a model organism and created first a slr1649 knockout strain (Δslr1649) by inserting a kanamycin resistance cassette into the slr1649 open reading frame via homologous recombination. The generated homozygous knock-out mutant showes identical features described in Shen et al. [10]. Just like the characterized cpcT mutant in Synechococcus sp. PCC 7002, our knock-out mutant contains a decreased level of phycocyanin up to 60% and a resulting pale green phenotype. The knock-out cells produce smaller phycobilisomes, which could be the cause of their different migration behaviour in sucrose density gradients in comparison to wild type phycobilisomes. Furthermore, isolated phycobilisomes showed a red-shifted absorbance maxima and a slightly smaller apparent molecular mass in the β-subunit of phycocyanin on SDS-PAGE (data not shown). After the digestion of purified phycocyanin with formic acid and a phycocyanobilin addition assay, Shen et al. concluded after digestion that the cpcT gene from Synechocccus sp. PCC 7002 encodes a bilin lyase responsible for the attachment of phycocyanobilin to Cys-153 on the β-subunit of phycocyanin [10]. The same is most likely true for the Synechocystis homolog Slr1649 due to the high homology and the similar phenotype between the two knock-out mutants. Thus, Slr1649 is thought to attach a bilin group to the homolog position Cys-155 of β-phycocyanin in Synechocystis sp.PCC 6803.
By isolating and analysing phycobilisomes from our knock-out mutant, we observed one additional feature besides the one already known from the characterization of the Synechococcus sp. PCC 7002 homolog. As shown in Fig. Fig.2A2A two linker polypeptides, CpcC2 and CpcD, encoded by the genes cpcC2 and cpcD respectively, are missing in the isolated phycobilisomes of the mutant but could be identified in the wild type during mass spectrometry analysis. The other linker polypeptides CpcC1 (cpcC1), CpcG1 (cpcG1), ApcC (apcC) and ApcE (apcE) are present in both strains.
Upon further investigations, the absence of the linker proteins was proven not to be a transcriptional effect. In exemplarily reverse transcription experiments for cpcC2, the presence of identical cpcC2 transcripts was confirmed in both the mutant and the wild type strain by sequencing the obtained RT-PCR products (data not shown).
In order to investigate if the nucleomorph-specific reading frame orf222 from the cryptophyte Guillardia theta is able complement the effects of the slr1649 loss-of-function, we integrated this potential gene without its putative transit peptide into the cyanobacterial genome of Synechocystis sp. PCC 6803. This simultaneously affected the reading frame of slr1649 and its cis-acting upstream signals (Fig. (Fig.3A).3A). In the complemented strain, slr1649 is separated from its natural upstream region, generating a promoter-less truncated gene, in which the translational initiator codon and the next two codons are no longer present in the reading frame. The loss of the slr1649 gene product and the complete segregation of the mutation were shown by immunoblot experiments using polyclonal antibodies generated against Slr1649 (Fig. (Fig.3B).3B). Here, cross-reactions of the antibody were shown to be present in the wild type but not in the mutant strain extract. Additional analysis of the complemented strain by RT-PCR and sequence analysis showed that the integrated cryptophytic orf222 is transcribed (data not shown).
Interestingly the phenotype of the complemented strain is similar to that of the wild type strain, as indicated by the greenish color of the culture (data not shown). During sucrose density-gradient separation of isolated phycobilisomes from the complemented strain, we noticed no difference in the migration behaviour of the prominent band in respect to the wild type strain in contrast to the migration behaviour of the knock-out mutant (Fig. (Fig.4).4). This indicates that the size of the phycobilisomes is identical in both strains and could indeed be confirmed by a proteome and mass spectrometry analysis. Here we showed that the missing linker proteins of the slr1649 knock-out strain were present in the complemented strain (Fig. (Fig.2A).2A). Additionally, there was no molecular mass shift in the β-subunit of phycocyanin of the complemented strain on SDS-PAGE visible. Further analyses revealed that the chromophore group, missing at positions Cys-β153 and Cys-β155 in the knock-out mutants of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 respectively, most probably reappeared in the complemented strain, because Zn2+ stainings of phycobilisomes separated by SDS-PAGE showed an equal signal intensity of the phycobiliproteins of the wild type and complemented strain (Fig. (Fig.2B2B).
To clarify this we digested isolated phycobiliproteins with formic acid. In doing so, CpcB is cleaved at a single site while all other phycobiliproteins remain unaffected. The expected sizes for fluorescent fragments are 15.36 kDa with a chromophore group at position Cys-β84 and 2.78 kDa for the fragment with the chromophore group at position Cys-β155. As shown in Figure Figure5,5, these expected fragments were obtained. In addition to a signal at 15.36 kDa, a signal at 2.78 kDa was detected in the lane containing wild type strain protein and protein from the complemented strain but not in the one containing Δslr1649 protein. These data confirmed that the chromophore group, missing in the knock-out mutant, is present in the complementation. This together with the identification of the linker protein spectrum in the complemented strain indicated a wild type phycobilisome structure.
Cryptophytes are important organisms for several reasons. In terms of cell biology, their complex compartmentalization is of major interest, because several plasmas and genomes coexist in these organisms, which can be traced back to either a prokaryote or a eukaryote [27]. One of the hallmarks of cryptophytes is the remnant of a second nucleus, which originated by the reduction of the cell nucleus of an engulfed phototrophic eukaryote by another eukaryotic cell [28]. This compartment, the nucleomorph, is minimized in its coding capacity and expresses – in the case of Guillardia theta – only approximately a tenth of that of the E. coli K12 genome [29]. The reduced coding capacity leads to the impression that the genes are still present in the nucleomorph may encode important functions. Thus, we are interested in addressing the functions of proteins encoded by the nucleomorph. However, due to the lack of a method of transfecting cryptophytes, we are studying homologs of the nucleomorph genes and their encoded proteins in genetically accessible organisms in order to identify the functions of the cryptophytic proteins indirectly. One of the best-studied and genetically accessible cyanobacterium is Synechocystis sp. PCC 6803.
Orf222 is one of the uncharacterized nucleomorph-specific open reading frames, for which homologs are present in many cyanobacteria. Analysis of the contribution of this gene within different organisms indicated that a clear correlation between orf222-homolog genes and phycobiliproteins is present, because at least one orf222 homolog is encoded in all organisms expressing phycobiliproteins, including red alga. Phylogenetic studies demonstrated that homologs of the orf222 gene can be classified into the following four groups (Fig. (Fig.1):1): Slr1649-like a, Slr1649-like b, CpeT-like a and CpeT-like b. Because the method for network construction as well as sampling in our studies is different from that of a recently presented phylogeny [10], it is not surprising that slightly different affiliations are resolved. However, our network corrects erroneous affiliations and indicates uncertainties of the basal grouping. This may be seen in the position of the bacteriophage sequence, which is in the network presented here in the neighbourhood of the bacteria they infect and not in the same branch as the cryptophyte sequence.
Despite the high degree of homology, the members of Slr1649-like and CpeT-like groups differ in the genomic context of the corresponding genes (Table (Table1).1). Members of the CpeT group are predominantly localized in the phycoerythrin associated linker protein operon [30,31] next to the cpeS gene. In some cases, even the cpeR gene is localized directly downstream of cpeT. Because operon structures connect functionally related genes in many cases, CpeT could be an either structurally or functionally moiety of the phycobilisome and it has been shown to be responsible for the attachment of a bilin group to a specific site from β-phycocyanin [10]. It is remarkable that a congruent distribution of members of the Slr1649-groups is not visible, because the genes seem to be localized randomly throughout different genomes. Interestingly, slr1649-homologs exist in some higher plants such as Oryza sativa and Arabidopsis thaliana (Fig. (Fig.1).1). The encoded proteins of these land plants are characterized by a DUF1001 domain as well, but obviously have paralogous functions, since the Arabidopsis thaliana homolog seems to be required for plastid division [25]. It is also suggested to play an important role in cell differentiation and the regulation of the cell division plane in plants [25]. The same could be true for the copy of the bacteriophage S-PM2, but seems to be unlikely since this phage infects different Synechococcus strains and its resource of the homolog may be the result of a selective advantage.
The homozygous knock-out mutant Δslr1649 in Synechocystis sp. PCC 6803 showed features identical to a cpcT knock-out mutant from Synechococcus sp. PCC 7002 described in Shen et al. [10]. Here, the same pale green phenotype and a reduced phycocyanin content, resulting from a missing bilin group in phycobilisomes, was created by knock-out of cpcT, homolog to slr1649 homolog in this cyanobacterium. This indicates that the lyase function of the homologous proteins of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 is comparable.
Nevertheless, we obtained one additional, not described feature in the Synechocystis sp. PCC 6803 knock-out mutant. Two linker proteins, CpcC2 and CpcD, were missing from the phycobilisomes in the knock-out mutant Δslr1649. CpcD is a small linker (10 kDa) located at the distal tip of rods, possibly functioning as a rod terminating factor [32]. The CpcC2 rod linker (30 kDa) connects the most distal located phycocyanin discs [33]. Both genes are located in the phycocyanin operon from which they are co-transcribed with the phycocyanin subunits and the cpcC1 linker gene [33]. A transcriptional effect causing the loss of the linker proteins appears to be very unlikely, because the α-subunit, the β-subunit and CpcC1 linker are present, although the CpcD and the CpcC2 linker are simultaneously absent. This is indicative of our finding that the cpcC2 gene is indeed transcribed in the mutant as indicated by reverse transcription experiments (data not shown). Therefore, the deficit of the two linker proteins in mutant phycobilisomes is a post-transcriptional effect. However, we can not rule out that a decreased stability of phycobilisomes caused by the altered β-phycocyanin may be the reason for the lack of the two linker proteins in our preparations. In any case, the lack of the linker proteins is a molecular marker for the loss of lyase function, which may be interested to be studied in Synechococcus sp. PCC 7002 [10] as well.
Guillardia theta, the cryptophyte on which we are primarily focusing expresses a homolog of slr1649 in association with phycoerythrin. Phycobiliproteins are located in the thylakoid lumen and apparently not organized in phycobilisomes in cryptophytes. Because the cryptophyte Guillardia theta uses phycoerythrin and not phycocyanin as an accessory pigment for photosynthesis, one might not expect that the putative cryptophytic lyase is able to complement the one of Synechocystis sp. PCC 6803. Surprisingly, the complemented strain showed wild type phycobilisomes structures as shown by the correct attachment of chromophore groups and the linker protein spectrum. Thus, Orf222 from the cryptophyte is able to complement the loss-of-function of Slr1649, indicating that the cryptophytic phycoerythrin lyase has still retained the capacity to couple a bilin group to β-phycocyanin, even after the progenitor of both classes of proteins evolved into apparently paralogous ones. However, a pleiotropic function of a biliprotein lyase with a specificity for phycobilin:cysteine-84 was recently shown in vitro for CpeS1 from Anabaena PCC 7120 [11], implicating that a multiplicity of proteins like the cryptophytic phycobilin:cysteine-β155 lyase has the capacity to couple bilins to homologous positions in a variety of phycobiliproteins.
Loss-of function of a bilin lyase leads to a variety of effects in phycobilisome structure. This is already shown for a cpcT mutant in Synechococcus sp. PCC 7002 and could be confirmed by the generation of a slr1649 knock-out mutant in Synechocystis sp. PCC 6803, homolog cpcT. One additional feature, the lack of two distal linker proteins, fits with the already known altered phycobilisome structure and may be the reason for the decreased phycocyanin content in mutant missing the bilin lyase.
Loss of Slr1649 was complemented in vivo by the homolog Orf222, which is encoded by the tiny vestigial nucleus of the eukaryotic endosymbiont from the cryptophyte Guillardia theta. Thus, Orf222 is supposed to be a phycoerythrin-bilin lyase in cryptophytes. Despite having originated from an organism using phycoerythrin as its accessory pigment, the protein still has the capacity to couple a chromophore group to the β-subunit of phycocyanin, indicating the functional universality of bilin lyases on the one hand and demonstrating the importance of nucleomorph-encoded cellular functions on the other.
Synechocystis sp. PCC 6803 strains, wild type, Δslr1649 and the complemented strain, were grown at 30°C in Erlenmeyer flasks containing BG-11 media [34] with gentle swirling under standard light conditions (70 μE) and atmospheric CO2 levels. For growth on plates, BG-11 medium was supplemented with 1% Agar. Plates were incubated under the same conditions as liquid cultures.
Two pairs of primers were used to amplify the flanking regions of the knock-out construct: 1649a_f (5'-GGT TAC TGC TCG AGG CGC ATC A-3') and 1649a_r (5'-GGA CGG CAA GGG ATC CTA TCT GG-3') generate fragment slr1649a, 1649b_f (5'-GGA CGG CAA GGG ATC CTA TCT GG-3') and 1649b_r (5'-CAG AAA TTG CCG CGG CCA ATC TC-3') fragment 1649b. Both were ligated into the pGEM-T vector (Promega, Mannheim) and after verification of the sequence, transferred into the pBluescript II SK (Stratagene, Amsterdam) vector. Escherichia coli strain MRF' XL-1 blue was used as plasmid host for cloning steps.
Using the BamHI restriction site (inserted by the primer 1649a-r and 1649b_f), a kanamycin resistance gene was cloned between the two fragments resulting in plasmid pΔ1649. After transforming into wild type Synechocystis sp. PCC 6803 cells with this plasmid, transformants were selected on BG-11 agar plates supplemented with kanamycin (5 μg/ml starting concentration). Kanamycin resistant clones were transferred to BG-11 liquid media. A homozygous culture was achieved by increasing kanamycin concentrations (50 μg/ml final concentration). Complete knock-out was confirmed via Southern blot analysis.
Nucleotide sequence of orf222 was amplified from Guillardia theta DNA without its putative transit peptide by using the primers 222komp2_f (5'-CAT ATG AAT TAA AAC CAA TCC TTA ATT G -3') and 222komp_r (5'-GTT AAA ATT AAA TGA ATT CTA ATA A-3'). Two pairs of primers were used to amplify the flanking regions: 1649a_f (5'-GGT TAC TGC TCG AGG CGC ATC A-3') and 1649kompa1_r (5'-CAA TAA CTA CAT ATG TCC CAT TCC-3') generated the fragment Compa, which includes the upstream region for slr1649, 1649kompa2_f (5'-TTT ATG TCG AAT TCC ACT GAT C-3') and 1649b_r (5'- GAG ATT GGC CGC GGC AAT TTC TG-3') generated fragment Compb. All three fragments were ligated into the pGEM-T vector (Promega, Mannheim) and after verification of the sequence, transferred into the pBluescript II SK (Stratagene, Amsterdam) vector using different restrictions sides inserted by the primers leading to a precursor construct. By using EcoRI restriction sites, a spectinomycin cassette was cloned between the two fragments Compa/orf222 and Compb resulting in plasmid pComp222 Δslr1649. After transforming the Synechocystis sp. PCC 6803 wild type strain with this plasmid, transformants were selected on BG-11 agar plates supplemented with spectinomycin (5 μg/ml starting concentration). Spectinomycin resistant clones were transferred to BG-11 liquid media. A homozygous culture was achieved by increasing spectinomycin concentrations (30 μg/ml final concentration).
Synechocystis sp. PCC 6803 cells were collected by centrifugation of 5 ml cell culture at 3200 × g. For DNA isolation, the pellet was resuspended in 400 μl TE buffer pH 7.0. After addition of breaking buffer (10% sodium dodecyl sulfat (w/v), 5% sodium lauryl sulfat (w/v)), 200 μl glass beads (0.2 mm diameter) and 400 μl phenol, cells were lysed by vortexing the suspension three times for 10 s. The suspension was then centrifuged at 12 000 × g and the resulting upper phase transferred to a new cup. This sample was treated twice with phenol-chloroform-isoamylalcohol (25:24:1) and centrifuged as before. By adding 1/10 Vol. NaAc pH 4.8 and two Vol. 96% ethanol, the DNA was precipitated for 1 h at -20°C. Afterwards, an additional washing step with 70% ethanol was performed. The pellet was dried and resuspended in H2O.
RNA was isolated from Synechocystis cells with Trizol© (Invitrogen, Karlsruhe) according to the manufacturer protocol. Northern Blot and Southern Blot analysis were performed according to standard protocols (Sambrook). Probes were constructed using the PCR DIG Probe Synthesis Kit (Roche, Mannheim).
To generate an antibody against Slr1649 we used the primers ex1649_f (5'-GGA TCC TTA TGT CCC ATT CCA CTG-3') and ex1649_r (5'-CTC GAG GCT GGC TAA AAA CTA ACT-3') to amplify the slr1649 gene, which was finally cloned in the pGEX-5X-3 vector (GE Healthcare Biosciences). After overexpression and purification of the Slr1649 GST fusion protein, immunization steps were executed by the Eurogentec company (Seraing).
The IgG fraction was purified from serum by protein A sepharose beads (GE Healthcare Biosciences).
Phycobilisome isolation was performed according to Gray et al. [35]. Cells were collected by centrifugation at 5000 rpm for 10 min at room temperature. After an additional washing step with BG-11 media, cells were resuspended in 0.75 M potassium-phosphate buffer pH 7.0 (PPB), containing a protease inhibitors cocktail (PIC, 2 mg/ml Antipain, 5 mg/ml Chymostatin, 2 mg/ml Aprotinin, 5 mg/ml Trypsin-Inhibitor, 2 mg/ml Pepstatin, 5 mg/ml Leupeptin, 1 mg/ml Elastatinal and 2 mg/ml Na2EDTA in HEPES/KOH. Final concentration 200 μg/ml Inhibitor) and afterwards broken by two passes through a French press (Aminco) at 124 MPa. The lysates were incubated with Triton X-100 (2%) for 15 min at room temperature and subsequently centrifuged at 20 000 rpm for 1 h to pellet unbroken cells and membrane debris. The supernatant was immediately loaded on a 10%–40% linear sucrose gradient, solved in PPB and centrifuged at 18 000 rpm for 16 h at 15°C.
Standard SDS-PAGE was performed with an Hoefer SE 250 apparatus (83 mm × 101 mm, 0,75 mm thick) or a custom made system (250 mm × 150 mm and 1,0 mm thick) using the Laemmli buffer system [36]. The polyacrylamide content in the separating gel was a gradient of 10% to 15%. The stacking gel contained 6% polyacrylamide. To achieve a better resolution of polypeptides with masses less than 15 kDa, the SDS-Tricine gel system was used [37]. Staining of gels was generally carried out with Coomassie brilliant blue G-250 dissolved in solution A (2% phosphoric acid v/v, 10% (NH4)2SO4 w/v) and methanol (40:9:1). To visualize the bilin carrying proteins gels were incubated in a 0.2 M ZnSO4 solution [38,39] and highlighted with UV in a transilluminator (Bio-Rad).
Phycobilisomes were precipitated with Methanol/Chloroform [40] and resuspended in cleavage buffer. Cleavage was done according to Piszkiewicz et al. [41]. 30 μg of phycobilisomes were incubated for 16 h at 37°C with 70% formic acid in before adding SDS sample buffer and analysis by Tricine SDS-PAGE on a 17% polyacrylamide gel.
Synechocystis sp. PCC 6803 cells were grown and harvested as described above. The cell pellet was resuspended in TEN100 buffer [42], containing PIC. Cell lysis was performed as described above.
The protein spots were subjected to in-gel trypsin digestion before mass spectrometry analysis as described previously [43]. The peptide mixtures from the tryptic digests were desalted and concentrated using ZipTips™ columns made from the reverse chromatography resins Poros and Oligo R3 (Applied Biosystems). The bound peptides were washed with a solution of 0.5% formic acid and eluted from the column in 1 μl of 33% (v/v) acetonitrile/0.1% trifluoroacetic acid solution saturated with α-cyano-4-hydroxycinnamic (Bruker Daltonics) directly onto a MALDI target plate and air dried before analysis in the mass spectrometer. Mass spectrometry measurement was performed on an Ultraflex-TOF TOF tandem mass spectrometer (Bruker Daltonics). Peptide mass fingerprint spectra were acquired in the reflectron positive mode with a pulsed extraction using approximately 100 laser shots. The spectra were acquired after an external calibration using reference peptides (Peptide mixture II Bruker Daltonics). The acquired spectra were further internally calibrated using trypsin autolysis peaks as internal standards (842.5100, 2211.1046 Da). Monoisotopic masses were assigned and processed using Biotools ™ and FlexAnalysis ™ software (Bruker Daltonics) before submitting them to the Mascot program [44] for searches against the non-redundant NCBI database. The parameters used in the Mascot peptide mass fingerprint searches were as follows: Taxonomy, Synechocystis; search all molecular masses and all isoelectric points; allow up to one missed proteolytic cleavage site and a peptide mass tolerance of 50 ppm. Methionine oxidation was considered as an optional modification and cysteine carbamidomethylation as a fixed modification in all the searches. Matches to Synechocystis proteins were considered unambiguous when the probability score was significant using the Mascot score with a p value < 0.05 and when there was a minimum of five peptides and with a sequence coverage greater than 20%. For each protein the identity was further validated by tandem MS-MS analysis of selected peptides.
Blast search analyses were done by NCBI protein-protein blast [45] (see Additional file 1). This program was also used for conserved domain predictions. Transmembrane domains were predicted by using TMHMM server v. 2.0 [46] and the SOSUI protein prediction server [47]. Genome data from Synechocystis sp. PCC 6803 were obtained from CyanoBase [48]. In silico cleavage was performed by PeptideMass [49].
The 41 sequences were aligned using MUSCLE [50] with 16 iterations. The output format was set to the standard ClustalW format [51]. The alignment contained 307 sites including 191 gapped sites that were excluded from the analysis. From the remaining 116 sites logdet distances were estimated using LDDist 1.3.2 [52]. These distances were used to construct a Neighbor-net splits graph [53] that was visualized with SplitsTree4 [54].
KB, OK and JP performed the experiments except phylogenetic NG and MALDI-TOF analyses JN, UGM initiated the project and was the supervisor. The manuscript, to which all authors contributed, was designed and written by KB, OK and UGM All authors have read and approved of the final version of the manuscript.
Accession numbers. Sequences were obtained from NCBI and the Cyanidioschyzon merolae Genome Project with the exception of cpcT from Synechococcus sp. PCC 7002, which was downloaded from [10].
We thank Andrew Bozarth for comments on the manuscript. This work is supported by the Deutsche Forschungsgemeinschaft (SFB-TR1, TP A7).
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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Steven visits Empire City with Greg and Pearl.
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My post today concerns a comment made by Foreign Affairs Minister John Baird on the latest outbreak of violence between Israel and Gaza. In his speech to a Jewish community gala, Baird refers to "the phoenix-like rising of the modern state of Israel, from a barren desert to the dynamic country we see today." (For the full speech, click here.)
Now, I'm not sure that it's possible to make a comment about Israel/Palestine without offending someone. I'm not sure that it's desirable, either. But that's not the fray I want to walk into today. Instead, I'd like to consider Baird's comments from a standpoint somewhat (but not completely) outside the current conflict. (more…)


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Have you ever had one of those nights that so many thoughts are going through your head you can't keep track of them all? They rush by so fast you can't grip a'one of them greased pigs to hold them still long enough to figure them out.
Of course you have, we all have!
Well that's the night I'm having. That's the night I've been having for the past week. It's put a damper on my ability to sleep and function like a normal person might (whatever normal is, and whatever my 'normal' happens to be.)
As I think to myself, "What do my precious followers want to read from me tonight?" A number of things go through my mind. The ones that slow down enough to glimpse? My penchant for being a Grammar Nazi? NAH. Music? MAYBE. Probably not. WRITING. Maybe! Maybe? Um, the new book I bought (called "The Well-Spoken Thesaurus" by Tom Heehler) but that may just lead me into Grammar Nazi mode and nobody wants me to go there.
We'll go with writing. It has the least amount of "hell no's" attached.
First, I must say, "You Can Do It" By Ice Cube (which just started playing on my new Ipod) is not the appropriate music to read literary quotes with. There we go. Bach.
"Our subject being Poetry, I propose to speak not only of the art in general but also of its species and their respective capacities; of the structure of plot required for a good poem…" -Aristotle, The Poetics.
Along with wonderful suggestions on how to word things not so commonplace, which is helping my writing tremendously, this book also has some damn fine quotes.
Anyway, that's right… writing.
I wish us writers were still referred to as "poets", regardless of whether we write poetry or not. Once upon a time, anyone who took up a pen and parchment was referred to as "the poet" and I believe that term had a certain sort of (pardon the pun) poetical aspects to it. Also, a respectable aspect as well. We may be seen as flighty, or scruffy, artistic and maybe a little odd- but it was well respected. Nowadays, I tell someone I'm a writer, and they say a noncommittal "oh, how nice" and move on. Has it now eluded the common man that writing, no matter what it is that is being written, is no more simple or commonplace than it is to paint a masterpiece of art? After all, writing is nothing but a painting of words, a mural pieced together by those of us who see words for all the fine color and beauty they are. I am both an artist and a poet (in the modern literal and past respective meaning of the word) and I tell you, it feels exactly the same to paint as it does to write. The same places in my body, mind and soul are active- and what, besides these two functions (and music) can you say awakens all three parts of the complete human?
Out of many who shrug it off as more-or-less an "eccentricity" or "laziness" to be overlooked or "tolerated" from me when I say I am a writer, are the ones who assume I am in it for the money. This bunch I like to refer to politely as "ignorant". Everyone has heard of the term "starving artist" Well then, "starving poet" quite applies in most scenarios as well. Furthermore, even if I was somehow "getting rich" from my writing, I'm appalled at the inference that money is the only reason I do it.
"The Utopians wonder how any man should be so much taken with the glaring doubtful luster of a jewel or stone, that can look up to a star, or to the sun himself." Sir Thomas More, Utopia
It is because of these attitudes that I consistently fight the urge to feel lazy and unfit during the times I am writing, or sitting down to write. As we all know, writing takes time, and trying to figure out what to write takes longer. To sit here and do nothing but think, and wonder, and come up with ideas to discard and reexamine, then discard again, looks to others as a waste of time (trust me, I wish I had great amount more time in which to devote to my writing). They say I should just write the whole time I set aside to write, if I should set aside any at all. To me, that's like trying to paint a rainbow without mixing your colors. You can't make a truly believable rainbow with the only three prime colors Red, Yellow and Blue. If I don't contemplate my words thoroughly, then one of two things happens: 1. It comes out poorly and fake. And 2. It doesn't come out at all. Everyone knows that writers can "suffer" from writer's block on a fairly regular basis- I don't need to feel lazy on top of it. It just distracts me from all the magnificent writing I COULD be creating.
"It was only that, having written down the first few fine paragraphs, I could not produce any others- or, to approximate Gertrude Stein's remark about a lesser writer of The Lost Generation- I had the syrup, but it wouldn't pour." William Styron, Sophie's Choice.
I realize times have changed quite phenomenally, even from when I was a little girl, curled up in bed with a book begging my mother to let me finish "just one more chapter" before bed (then trying to see how many chapters I could get in before she came back to "remind" me that I was allotted just "one" more chapter- I can't tell you how many books I finished this way. "I'm almost done with the chapter, I promise!") to nowadays, when the most reading I do on days I don't write includes the horrendous forced short-hand of texts with the even more horrendous awareness that current education is failing this new generation- It's appalling. I'm not saying one has to know how to spell to magnificent proportions, or that short-hand is a bad thing (with 160 word limits on my texts, I am a sad-to-say habitual user of texting short-hand myself) but the attitude looked upon (and down) the written word, the lack of effort put into its productivity and completion, the lack of caring one has pertaining to the way they "sound" when they write is… how should I put this delicately? Borderline stupidity? Ignorance not to be ignored? Ignorance of the worst kind, indeed. Regardless of the technological era and the fact that our children nowadays can name hundreds more video games than they possibly could species of flower or animal or even book titles, people still fail to realize that more than half of communication we do (ESPECIALLY during this technological age, with the internet readily available and texting now easier than dialing a call) is WRITTEN. Why can we not find it in ourselves to learn to use our words properly?
"By profession he is, or has been, a scholar, and scholarship still engages, intermittently, the core of him." J.M Coetzee, Disgrace
I'll admit now, I am little less than screwed if I did not have my computer's spell check (from a publication standpoint, where professionally published novels and their readers are unforgiving of more than a couple spelling errors). But I do not turn it off and I do not choose to ignore it- I learn from it. Every red underlined word I find I MUST understand WHY it is "wrong" (if indeed it is). This creates new learning every time I write. Whether it be that I mistyped a word I know, and how to train myself from making the same mistake again, learning to spell a new word or even an old word I can't for the life of me remember how to spell- it isn't a cheat. It's a learning tool. Did anyone ever refer to a dictionary as a cheat? I use dictionaries, thesauruses and the internet to define words for me all the time, and often learn better ones to use in their stead. In conclusion: Words just fascinate me. Maybe I'm being too harsh on the rest of the world to say they should learn to spell correctly. Maybe I'm sounding pompous. Or maybe, just maybe…
This new "trend" of short-hand texting coupled with the deplorable education system of this nation is driving me crazy. "What" is not "Wut", "Brother" is not "Bruther", "Psycho" is not "Syco" I mean, I realize many schools start out their kindergarteners and first-graders with their words using phonetics and sounds- but then they seem to forget to teach them the rest. English is not a sound-based language, sad to say. The WORST advice I got BY FAR in school was this: "Just sound it out."
Oh boy, I think I've trailed into Grammar Nazi land again. I realize I've dedicated my life to the written word (among other things) but it is not my place to correct others, I realize that. If the urge to correct others drives me this insane, perhaps I should have pursued being a teacher instead (I'll pass).
No, us poets were meant to learn our words and utilize them to the best of our abilities. Not everybody is meant to be a poet. Just like not everyone is meant to be a mathematician. As I am sitting here complaining of the atrocious spelling I find all around me, somewhere out there is a Math Nazi blogging "Why can't they just learn simple division??" Or "She" in my case. There's my own piece of humble pie, served straight from Humbledoore to my ego's mouth-watering appetite.
Chomp Chomp. Nom Nom.
"… And the dead tree gives no shelter, the cricket no relief…" T. S. Eliot, The Waste Land
There is something to be said for the technological age. The fact that I can cart around 1000 (more or less) books in something the size of a paperback novel and read it wherever I want. That I can listen to Bach and Beethoven with headphones as I write and not madden the others in my vicinity. THAT I HAVE THE ABILITY TO WRITE AT ALL. This computer, a modern-day poet's savior. I have written well over 3,000,000 words in my 19 years as a writer (my first published writing was a well-worded book review at the age of nine) I could not even get half that down (or anywhere close) if all I had was an ink pen and parchment. Everything I've written by hand, I've altered and added upon transferring to an electronic device.
But, in my stubbornness, my penchant for the old fashioned, and in yearning for respect as a writer, I still maintain it would be fantastic if us writers were once again referred to as the "poets" of our society.
"Of Life immense in passion, pulse, and power, Cheerful, for the freest action form'd under the laws divine, The Modern Man I sing." Walt Whitman, Leaves of Grass, Book 1 "Inscriptions"
I am by no means laying claim to the theory that my writing is in any way shape or form better or worse than my fellow poets. But I write from the heart, always. Leaving me to leave you with this one last quote, taken from the book but with no explained source:
"If his performance was not electrifying, at least it was believable."
Thank you, and Happy Saint Patrick's day.
* Visit The Legends of Sangue *


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60 5 8
Thu May 18, 2017 8:00 am
I'm hope I'm in the right place. Anyway, sorry if this is a dumb question but I was just wondering, if I were to write a satirical review of a board game, would the Fextralife blog be an appropriate place to post it or no? And if so, should I submit it as just a normal article or as an actual review of the game, or doesn't it matter? Also, what would be the limits on profanity? Thanks.


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If you have a family of growing children or your grocery store funds are a little tight, learning how to save money on groceries can be important. Maybe your funds are tighter in other places which means your grocery store bill needs to shrink a bit but the food needs to increase. Whatever your reason is, this article is here to help you out.
Frugal shoppers know how to save money on groceries and are able to save money at the grocery store in many ways. They may save between $10 and $60 each time they go to the store. They have several things that they do to make this happen. Here are some tips that will help you when you go to the grocery store.
o Grocery Store Ads
One way to save money at the grocery store is to base your meals off of what is on sale at the store. Review the weekly ads for all the stores in the city you shop in and come up with meals that go along with


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Welcome to the newest phase of Cypress Meadows in Broussard where local builder CA Homes is once again building exceptional quality homes! With over 30 years of experience in home building, CA Homes are trusted and experienced in new home construction, providing exceptional service and unparalleled value. 209 Windy Feather is an absolutely beautiful new construction home and there is just so much to love about this home! The kitchen features stainless steel appliances, custom cabinets, large island with granite counters and a separate breakfast area. No carpet in this home - just beautiful tile and wood throughout. The living room is large with gas fireplace and beautiful coffered ceilings.
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Free shipping*For Orders above Rs.500*
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Kenny Delights Pitted Prunes, 170gm0 out of 5 Stars, 0 reviews
Brand: Kenny Delights
Availability: Out Of Stock
Weight: 0.17 kgPrice: Rs.200
Free Shipping for orders above Rs.500 (Mumbai,Pune) & Rs.999 (Rest of India)
Pitted Prunes are dried plums which have deep sweet taste and sticky chewy texture. Kenny Delight Pitted Prunes are nutrient-rich dried fruits which have a multitude of health benefits. These are snack time favorite of health-conscious folks and can even be added to bakery items and cereals.
- Natural laxative
- Rich source of fiber
- Antioxidant source
- Contains high level of phenols
- Contains Vitamin C
- Good source of potassium and calcium.
Benefits of Kenny Delight Pitted Prunes:
- Higher fiber content helps regularize bowel movements and maintain healthy digestive system.
- Antioxidants reduce cellular damage caused by free radicals and boosts immunity.
- Potassium aids in muscle contraction and maintains healthy blood pressure.
- Vitamin C aids in absorption of iron which help prevent anemia caused by iron deficiency.
- Improves mental alertness.
- Protects heart from chronic diseases.
- Keeps blood sugar stable.
- Prevents overeating and may help in weight loss.
- Good for eyes, skin, and hair.
How to use:
Include Kenny Delight Pitted Prunes in your diet daily or add them to desserts , bakery product sand salads.
Pitted prunes.
Side effects:
Excessive intake of this product can lead to a spike in blood sugar levels, frequent bowel movements, gas and bloating.
Information about the brand:
Kenny Delights aims at bringing high-quality fruits, super-powered seeds and vegetables with high nutritional value to the customers so that they can enjoy a healthy life. They make sure that the food products are preserved using advanced technology so that nutritional value is not affected and they can successfully be stored for a longer time period. With Kenny Delights, the customers can relish premium quality food products and stay healthy.Related Products (8)
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- Snacks


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Dr. Joseph S. Covert, 89, of Zanesville, died Sunday, February 6, 2011 at the Justin T. Rogers Hospice Care Center in Akron. Dr. Covert was born in Zanesville, May 2, 1921, the son of the late Chester L. and Margaret Hormuth Covert. He graduated from Lash High School, then from the Illinois College of Optometry in March 1943. Immediately following graduation, he was enlisted in the United States Army Medical Corps where he was instrumental in establishing the eye clinic at Ft. Moultrie near Charleston, S.C. In 1995, Dr. Covert was one of 12 optometrists of the World War II era honored for distinguished service in the United States Army Medical Corps. Upon his discharge in 1946, he returned home to Zanesville where he entered the private practice of optometry until his retirement in 1992. During his career, he was instrumental in establishing optometry as a clinical entity. He served two terms on the Ohio State Board of Examiners, the second term as President. He also served on National and International Boards of Optometry, the Executive Board of the Ohio Optometric Association, and the Legal and Legislative Council of the American Optometric Association.
He was preceded in death by his wife Gertrude, whom he married on Valentine's Day 1943.
He is survived by their children, Stephen, Kathleen, Elizabeth Alberto and Chester James; ten grandchildren, Joseph Covert, Karen Covert, Joshua Haugh, Allison Moten, Marianne Haugh, Carrie Covert, Jocelyn Schultz, Emily Covert, Samuel Covert and Chad Covert; and five great grandchildren.
In addition to his parents and wife, he was also preceded in death by sisters, Jane Ross and Vivian Householder.
Friends and family may call from 6-8 p.m. Thursday, February 10, 2011 and 11:00 a.m. to 1:00 p.m. Friday, February 11, 2011 at the Bolin-Dierkes Funeral Home, 1271 Blue Ave., Zanesville. Funeral services will be held at 1:00 p.m. Friday, February 11, 2011 at the funeral home with Rev. Doris Flynn officiating. Dr. Covert will be laid to rest beside his beloved Gertrude in Williams Cemetery, Zanesville. The United States Army in conjunction with VFW George Selsam Post #1058 will hold full military rites at the graveside.
In lieu of flowers, memorial contributions may be made to the Spina Bifida Association, 4590 MacArthur Blvd. NW, Suite #250, Washington, DC 20007.
To send a note of condolence, a sympathy card or comfort food to the Covert family: www.bolin-dierkesfuneralhome.com


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a small radio receiver fitted with a buzzer to alert a person to telephone their home, office, etc, to receive a message
Tagged: r
Read Also:
- Radiopathology
radiopathology ra·di·o·pa·thol·o·gy (rā'dē-ō-pā-thŏl'ə-jē) n. A branch of radiology or pathology dealing with effects of radioactive substances on cells and tissues.
- Radiopelvimetry
radiopelvimetry ra·di·o·pel·vim·e·try (rā'dē-ō-pěl-vĭm'ĭ-trē) n. Measurement of the pelvis by radiography.
- Radiophare
[rey-dee-oh-fair] /ˈreɪ di oʊˌfɛər/ noun 1. a radiotelegraphic station used by vessels to determine their positions; radio beacon.
- Radiopharmaceutical
[rey-dee-oh-fahr-muh-soo-ti-kuh l] /ˌreɪ di oʊˌfɑr məˈsu tɪ kəl/ noun, Pharmacology. 1. any of a number of radioactive drugs used diagnostically or therapeutically. radiopharmaceutical ra·di·o·phar·ma·ceu·ti·cal (rā'dē-ō-fär'mə-sōō'tĭ-kəl) n. A radioactive compound used in diagnostiis or therapy. ra'di·o·phar'ma·ceu'ti·cal adj.


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By Brian L. Willison, Chair
Wisconsin Law Enforcement Memorial, Inc.
The Wisconsin Concerns of Police Survivors (WI COPS) and Wisconsin Law Enforcement Memorial, Inc. (WLEM), law enforcement charity organizations, have combined with Charter Communications and a statewide bowling group, Community Entertainment Centers, to establish a month-long bowling tournament during weekends in June of 2005. The bowling tournament will be held in seven different bowling centers located throughout Wisconsin to reduce travel time for all of those who participate. The bowling centers, tournament dates and times, and contact information is listed below. We will be raising needed money for WI COPS and WLEM from different sources. If we are successful and generate officer and friend support, this will become an annual event and will be the primary funding source for both charities.
Our 8-Pin Bowling Tournament Overview
This fun style 8-pin bowling tournament is designed to appeal to the average and not-so-average bowler to broaden participation among both law enforcement and our community supporters. This is a team-style event – four adult men, women or a mixed team – costing $25.00 per team member. Twelve dollars from each person's tournament entry goes directly to the WI COPS and WLEM support funds. The remainder of the entry cost goes to prize money and bowling expenses. In 8-pin bowling, leaving two or less pins on the first ball counts as a strike. You do not have to be a league bowler or even a good bowler to score well in this format! Three games are bowled by each team member. Bowling center staff will be on hand to get us all started with computer scoring. Teams can win prize money and other donated prizes during the event. Following each day's tournament, Pepsi Bottling Company will sponsor two hours of free glow-in-the-dark cyber bowling for family members who accompany tournament bowlers. Bumper bowling will also be available at this time for smaller family members. We want all who participate or come to watch to have fun!
Your Help!
We are asking that active and retired officers and supporters of law enforcement come together with fellow officers or friends to form a 4-person team and bowl in this event. If just one active and one retired officer from each state department formed a team and bowled, this tournament would be a dramatic success! Some departments are planning a motorcycle ride to a bowling center to make it a fun day-long trip. All of the bowling centers have quality house bowling balls available to make this possible. No matter how you get there or how talented you are as a bowler, please help WI COPS and WLEM by participating in this event. If we can generate enough participation and support, this tournament and corporate sponsorship will become an annual event. Entry forms can be found by visiting www.bowlforwicops.com or our WLEM web site at www.WLEM.com.
Bowling shifts will take place at noon and 3:00 p.m. every Saturday and Sunday in June, and at 9:00 a.m. on Saturdays, June 11 and 18, at the following locations:
Participating Community Entertainment Center Bowling Centers
|Spartan Bowl
4711 Farwell St.
McFarland, WI 53558
(608) 838-8014
Tournament Director: Matt Chizek
Wagner's Lanes
North Star Lanes
Thunderbird Lanes
|Classic Lanes
7501 S. Howell Ave.
Oak Creek, WI 53154
(414) 764-1120
Tournament Director: Al Passehl
Lakeview Lanes
Pioneer Lanes


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Michele FulkersonUnion, Missouri, US
I am new at this, I just need a chance to show you that I will give 100% and get the job done for you.
English (North American), English
Child, Middle Aged, Senior, Teen, Young Adult
Special Skills
Audiobooks, Business, Cartoons, Documentaries, Educational, Internet, Movie Trailers, Podcasting, Radio, Telephone, Television, Videogames
Turnaround Time
ASAP, I will definitely give 24 hours unless requested earlier
Service Description
I am a dedicated into anything I do and will give everything to make it perfect for you.
Payment Terms
Hire Michele as a voice actor. Payment is made through Voices.com SurePay, an escrow service that guarantees your work is done to your satisfaction. Send payment securely by Visa, Mastercard or PayPal through Voices.com then download your files from the website.
Watch a video to see how to make a payment
Next Steps
To contact Michele Fulkerson about your project, request a quote today. We'll walk you through the process step-by-step. It's fast and easy.
This talent has not uploaded any demos yet.
Hire Michele Fulkerson as a voice actor. Working with Michele Fulkerson is fast and easy. We'll walk you through the process step-by-step.
Request a quote for your project today.


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REINE ANGEVINE was odds-on for her first three starts but won just one, a bumper at Killarney in August. She looked an improved performer when beating a big field over the C&D last month and with her allowance coming into play may have too much basic speed for Rogue Angel. Mouse Morris' point-to-point winner is a chaser in the making and didn't impress everyone at Galway last time though he'll love the heavy ground. Blacklough has battled on to land a Killarney bumper and a maiden hurdle at Fairyhouse and those qualities should stand him in good stead in these testing conditions. The remainder will all need to find improvement to get involved.
Blacklough (IRE) 10-1 (11-9) Tracked leaders, improved to dispute 2nd 2 out, ridden to lead narrowly before last, stayed on well run-in, won at Fairyhouse 2m mdn hdl sft beating Akatara (10-4) by nk, 14 ran.
Rogue Angel (IRE) 1-6fav (11-7) Tracked leaders in 3rd, improved to challenge 2 out, soon ridden to lead, clear last, easily, won at Galway 2m 4yo mdn hdl in Oct beating Up Sluggarh (11-0) by 8l, 11 ran.
Apache Gold (IRE) 8-1 (8-11) Chased leaders, 5th halfway, ridden in 6th 2f out, no impression in 5th 1f out, kept on same pace, 5th of 14, 3 1/2l behind The Reek (8-9) at Dundalk 1m hcp 47-65 pol (flat).
Bad Cigar (IRE) 25-1 (10-11) Soon chased leaders, a bit slow 4 out, soon no impression, not fluent 2 out and kept on same pace, 10th of 14, 37l behind Rawnaq (11-9) at Bellewstown 2m 1f hcp hdl 80-102 in Aug.
Fiveinthemorn (IRE) 33-1 (11-4) Mid-division, 6th 3 out, soon weakened, 11th of 14, 18l behind Blacklough (11-9) at Fairyhouse 2m mdn hdl sft.
Paddy Wicklow (IRE) Statue Of Liberty gelding out of Pregonda.
Reine Angevine (FR) 4-1 (11-1) Tracked leaders, moderate 3rd after halfway, took closer order 3 out, led travelling well approaching 2 out, soon drew clear, mistake last, eased run-in, impressive, won at Gowran Park 2m 4yo mdn hdl gd in Oct beating Dumbarton (11-8) by 10l, 20 ran.
Cheap As Chips (IRE) 50-1 (10-6) Towards rear for most, never a factor, 11th of 17, 46l behind The Boys Dont Know (10-13) at Thurles 2m mares mdn hdl.


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I know the official line about Osama Bin laden. But too many things just dont add up. What are other possibilities?
sort by best latest
For sure - the cover-up was the work of Cheney and company. George Bush was the president of the United States! It is under this watch that America refused to investigate the "supposedly terrorist attack". Well George Bush and company should be tried for obstructing justice and they should be punished for that treason. Nobody can prove to me that those towers came down as the news and government want us to believe. But as in the Kennedy investigation. MLK, etc. there will no investigation. The real investigations are on TV. Perry Mason, etc.
We will never know how 911 really went down because of treason by the president and vice-president. But too many people are hung up on the capitalism that ain't working. It's slavery. The two party system makes sure we have two lawyers for every election. Lawyers refuse to run courtrooms properly. America is the most imprisoned nation on the face of the Earth. Our children are sent to war every few years. If this is "the plan" from our so called leaders - we are in hell! THIS is their plan? Nothing else like peace is fathomable? Make no mistake- the people "in charge" of us are selfish evil children. God bless America. It will take God to rescue America because the people are sheep.
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It is real travesty if justice in this world that Muslims are blamed for something they had nothing to do with. The 911 was a plot hatched by the US power brokers and Zionist machine of USA at the time. Too many scientists, architects, explosives experts & engineers have already concluded that this was not pulled off by some some group of Muslim fundamentalists that actually don't even know their own religion and have to write very stupid letters to each telling them that they are Muslims and they need to do xyz before carrying out the activity.
The funny thing is that they find one of the so called Muslim terrorists passport some 200-300 yards away from the twin towers, apparently according to expert opinion at the time it had dropped out of the plane when it exploded, to believe that you'd either have to be confined to the mental hospital or be a idiot that does not question the fodder that the media and US government feeds people. You may want to check out my hub http://hubpages.com/t/1d1ef6
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America made the 9/11.
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This is Set Two of 34DD Erica Campbell's big photo debut and the first set we ever shot with her and, as always, Erica is cute as ever and always photogenic. Just don't ask about the green-logo on the t-shirt though... we don't know what we were thinking, either. ;-)
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This pattern is an Instant Download file for a soft, stretchy newborn knit wrap / layering blanket.
***Note: This is a pattern and NOT a physical item.
This pattern makes a beautiful stretchy and soft cable knit blanket that can be used as a wrap or for layering/stuffing baskets in newborn photography.
Materials: You will need 8.0 mm straight knitting needles and worsted weight yarn.
You have my permission to sell any items that you create with my patterns, but they are copyrighted so please do NOT copy, share, adjust, or resell this pattern as your own. As well, the images in my patterns are NOT to be used as your own if you are selling the items that you make with the patterns. Please use your own images of the actual items that you make.
Your files will be available to download once payment is confirmed.
I do not offer refunds on my patterns due to their electronic nature, therefore please read all listing descriptions. Thank you!!


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Forman's treatment is another matter entirely - infinitely more subtle and, using the intrinsic bias of film, far more naturalistic. [18 Nov 1989]
The Frears version is cerebral and claustrophobic, an exercise in sexual mindplay. show more
The film is rapturously beautiful, enticing us into a lush, aristocratic world. show more


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OBEY Shasta 5-Panel Cap - Black
Adjustable strapback closure | Leather look badge logo on front
Available in one size fits most!
Formed in 2001, OBEY clothing is a new art medium for renowned artist Shepard Fairey. Influenced by classic military design, workwear basics and the cultural movements and elements Fairey is known for, this fashion line was an instant hit and has become the go-to clothing brand for the skate, punk-rock and street subcultures. OBEY clothing challenges you to think about your environment and question the world: the medium is the message.
- OBEY accessories
- For men
- Style: Shasta 5-Panel Cap
- Colour: Black
- Adjustable strapback closure
- Leather look badge logo on front
- Contrast flat peak
- Materials: 40% polyester, 39% wool, 12% acrylic, 9% nylon
- Country of origin: Vietnam


text:
While wearing a safety glasses, Samantha Monroe concentrated as she operated machinery inside a shop at Sampson Community College.
As she moved around the Occupational Building, a crowd observed her operate a FMS-200 system, which is used to train students in industrial automation. Along with other students, she was happy to show how it works during an open house. Thanks to a generous grant from Duke Energy, the system is giving Monroe experience for her future goals in the industrial field or mechanics.
"It's an hands-on situation like in the real world and there's always a solution for something," she said.
Monroe, a dual-enrollment student from Clinton High School, takes classes at the college in the afternoon.
"I like working with technology because I like working with my hands," Monroe said. "It's always different — nothing is the same. If something messes up, you have to find a solution."
The machine assembles a turning mechanism, which moves large industrial objects. It includes an aluminum body, a bearing of a shaft, cover and screws. Instructor Durwood King said the automatic assembly process involve hydraulics, pneumatics, programmable controllers to produce a final product. Through the program, the students learn how to solve problems for automatic systems.
"They're learning the technology of how all this stuff ties together and runs automatically like it does in plants, like Smithfield, Butterball and Schindler," King said. "The process may not be the same of what they're doing, but a lot of devices that we're using are similar."
Duke Energy supported the program by giving a $250,000 grant to SCC for the Industrial Systems Technology Program. John Elliott, Duke Energy's director of government and community relations, presented the check in January and was present Thursday during the open house.
"I'm very thankful that they put money in it because that has helped us to get on another whole level on the training and preparing people," King said.
Elliott said it was exciting to see a vision of preparing students for jobs in the community with the technology.
"That's when it all comes together," Elliott said. "I can't enough of the faculty and staff that had the vision for programs like this for students to be ready for the workforce."
SCC President Paul Hutchins said he's been looking forward to the open house event for several months and thanked everyone who made it possible.
"A community college is all about preparing men and women for opportunities that lie ahead," Hutchins said. "For community colleges, it's vital that we train on state-of-the-art equipment."
Darrell Matthews, division chair for Business and Occupational Technologies Programs, said it was a good chance to thank Duke Energy and other supporters.
"To have something like this is unique, but to have the latest, greatest technology right here in Sampson County training the students who will work for our local industries, that's even better," Matthews said. "It's a win-win for everybody. It wouldn't be possible without Duke Energy."
Kate Brown, director of customized training and workforce development programs for SCC, said she was proud to be recipient of the grant.
"It's an added value for the business industry," Brown said. "We have worked productively and we've been committed to working with our business industries and creating more efficiency."
The Industrial Technology program is designed to prepare students to service, maintain, repair or install equipment. Some of the technical skills taught include reading blueprints, welding working hydraulics. Another purpose is to prepare an individual for employment in a certain occupation. The program takes about one year or less to finish.
SCC offers certificate programs for agricultural business maintenance, commercial building maintenance, electrical, industrial maintenance, machine operations and maintenance operations. Jared Hudson, is one of many students enrolled in the program. He also participated in the demonstration. In the future, he would like to be in the wiring and maintenance for hog farms.
"I really enjoy it," Hudson said. "It's pretty awesome to learn."
Reach Chase Jordan at 910-249-4617. Follow us on Twitter at @SampsonInd and like us on Facebook.


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|.1 lbs
Pick Pocket by Berndt Woods
For over 35 years BERNDT has meant the finest name in woods, design and quality. We supply birdseye maple and tiger maple to the world's finest guitar builders, artists and luxury brands. We now produce the finest drumsticks, chopsticks, pickpockets, trays, furniture, wall paneling, and wall art from these woods.
We can emboss any name/logo on the interior of the pickpocket. Artwork must fit into a 3/4 inch circle.
Out of stock


text: Hello the net, I finally got the new computer assembled and working, the bad power supply slowed me down. The new computer is a 2.4 G pentium 4 with HT, 800 Mhz FSB, 512 M RAM per channel Intel D865PERL motherboard and a 120G serial ATA hardrive, SATA I have on hand KNOPPIX V3.3 on a CD and I also have SUSE 9.0 pro. The KNOPPIX loads (mostly) fine onto a RAMDISK and I am figuring how to get it onto the hard disk. KNOPPIX does not properly auto recognize the AC'97 sound card , but I have a soundblaster PCI card I will try. I know that I will have to add the ALSA drivers for the DELTA 44 card. SUSE stalls on loading without an error message, just hangs up. (locks up) last line on the screen is: hde: attached ide-disk driver hde is the SATA hard disk which was previously partitioned and formatted in FAT32 can not get to YAST. Questions: 1. is there any way I can single step the installation ? The pause key has no effect. 2. Any known problems with SUSE and SATA hard drives ? I am slowly creeping up the learning curve. Stan, WA1ECF zzzzzLINRADDARNIL


text:
CLA summit will explore how landowners can help solve rural housing crisis
An organisation representing country landowners and businesses is holding a summit designed for the private rural housing sector next month.
The CLA says it has gathered a high profile mix of speakers for the event taking place on 5 July, including politicians, policy makers and industry leaders, at what is said to be the first such housing summit of its kind.
Government chief planner Steve Quartermain and shadow housing secretary John Healey MP are among those billed to speak at the Strong Foundation event. The debate is intended to help solve the acute shortage of housing in rural areas and is aimed at landowners who own and rent out properties or who are seeking to develop new homes in rural areas.
"Landowners play an important economic and social role within rural communities and have a big role to play in helping to provide more new homes of every type and tenure," said Ross Murray, the CLA's president.
"The summit will look at the role of landowners in providing homes in their communities across England. It will explore the future of the rural rental market and the role of the planning system in delivering thriving and sustainable communities.
"We shall be highlighting how the right support for private investment in the rural housing sector can help to encourage rural businesses to deliver much needed homes for the next generation to live and work in our countryside."
New research about rural landowning businesses and their contribution to rural housing needs will be unveiled at the summit, the CLA said.
Confirmed speakers at the summit are: Steve Quartermain CBE, chief executive of the Planning Inspectorate; shadow secretary of state for housing John Healey; Lord Best OBE DL, crossbench peer; Sue Chalkley OBE, chief executive of Hastoe Housing Association; Ben Bolgar, senior director at the Prince's Foundation for Building Community.
Themes and panel sessions include the rural housing challenge, planning, private rented sector housing that works for rural areas and a debate on the Green Belt.


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|10 Oct 2011
thanks for the tip ill write a note about it since it will make it easier for the person repairing it. just hope they do fix it and not just return it back un touched...
|My System Specs
|10 Nov 2011
well its been while but after sending the laptop to HP , i received it after a week and so far its working ok.
the problem that they said was that there was a problem with the SDRAM(Synchronous Dynamic Random Access Memory).so yeah you guys can closed this post and have it solved
|My System Specs
|10 Nov 2011
Microsoft Windows 7 Home Premium 64-bit Service Pack 1
Glad to hear that you are OK now.
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text: With over one hundred years of experience, our attorneys have seen it all. We have experience with just about every aspect of the law and we are willing to listen, for free, to every potential client's situation. If you do not see your issue listed here, please contact us for more information. If we are unable to personally take your case, then we will put you in touch -free of charge- with an experienced attorney who can help you.


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Don't Lose it all, Time to get dataMonday, January 14, 2013
Do you still remember "The Long Distance Relationship" post?
Amy has been facing her frustrations badly because Nick hasn't been replying. These are obviously the consequences of being in a long distance relationship – but of course they're both aware of that. However, in this day of age, we have DATA – so where did they go wrong? Now that Nick has been away for sometime, is Amy moving on? But whose ring is that?
"This is intense peeps! Is that Amy with Joe? What about Nick? Wait till the story unfolds on 18 January 2013. http://bit.ly/dontLoseItAll #DontLoseItAll @xpaxsays"


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E.L.F. Cosmetics, Eyebrow Kit, Gel & Powder, Ash, 0.123 oz (3.5 g)
Quantity Discount
Buy 3+Save 5%
- You earn loyalty credit for every order you place at iHerb!
- Loyalty credit is equal to 5% of the value of your order after discounts and credits applied, excluding shipping charges.
- Your loyalty credit will be applied towards your next order as soon as your current order has shipped.
- Your loyalty credits are valid for up to 90 days from the date of your last purchase.
E.L.F. Cosmetics, Eyebrow Kit, Gel & Powder, Ash, 0.123 oz (3.5 g)
Expiration Date:?01/2020Expiration Date
The length of time for the expiration date or "best used before" date depends on the type of product, as well as the brand.
Perishable items (such as flax oils or certain probiotics) generally have shorter expiration dates. Although our warehouse is fully air-conditioned, these more fragile items are put in cold storage (freezer or refrigeration unit) for maximum freshness.
Our receiving department does its best to verify and then enter the correct expiration dates for all incoming products. However, discrepancies do occur from time to time. This being said, the exceptionally high turnover at iHerb ensures that our inventory is among the freshest in the industry.
Shipping Weight:?0.12 lbsSwitch to Metric units
- Product Code: ELF-81304
- UPC Code: 609332813045
- Package Quantity: 0.123 oz
0.6 x 2.9 x 2.6 in, 0.1 lbsSwitch to Metric units
- Madre Labs, Argan Oil Hand Cream with Marula & Coconut Oils plus Shea Butter, Soothing and Unscented, 2.5 oz (71 g)
- Save $2.00! Normal price: $5.00
Featured Products
- New
- Mirror and Dual Brush Applicator Included
- Become Your Own Makeup Artist
Creates brows that are fuller, thicker, and more defined
Shapes and sets your eyebrows
Gives lasting color definition for a beautiful natural look
Suggested Use
Directions: Use the angled brush to apply the pigmented brow gel to define and shape the brows. Set the color in place with the brow powder using the blending brush for a natural finished look.
Other Ingredients
Eyebrow Gel Ingredients: Net Wt. 0.042 oz (1.2 g)
Ethylhexyl palmitate, hydrogenated castor oil, synthetic fluorphlogopite, copernicia cerifera (carnauba) wax, paraffinum liquidum (mineral oil), polyisobutene, ceresin, bis-diglyceryl polyacyladipate-2, microcrystalline wax (cera microcristallina). May Contain: Titanium dioxide (Cl 77891), iron oxides (Cl 77491, Cl 77492, Cl 77499), manganese violet (Cl 77742)
Eyebrow Pointer Ingredients: Net Wt 0.081 oz (2.3 g)
Talc, mica, hydrogenated polyisobutene, caprylic/capric triglyceride, ethylhexyl palmitate, sodium dehydroacetate. May Contain: Titanium dioxide (Cl 77891), iron oxides (Cl 77491, Cl 77492, Cl 77499)
For external use only. Discontinue use if signs o irritation appear. Keep out of reach of children.
No animal testing
Works very well
Verified Purchase
Works very well
I only use the darker brown, as the lighter one makes the color a little to light for me. I have medium/dark blond hair. The color might feel just a little bit green-ish sometimes, but overall I am happy with this product and will probably continue to use it.
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Nice Product
Posted by iHerb Customer on Oct 31, 2017 | Verified Purchase
It gives your brows this natural gel look :)
Great Price and Colour
Posted by iHerb Customer on Oct 22, 2017 | Verified Purchase
I've finally found an eyebrow set that perfectly matches my natural light colour (ashy blonde). Great price too.
Good value! Get 5 - % - off with ref 533 (without s.p.a.ce!)
Posted by iHerb Customer on Oct 22, 2017 | Verified Purchase
First off, the color is much darker in real life than what it appears on the picture. But it works well for me as a brunette, and I find it smooth and creamy. The pomade has a little slip and isn't as dry as the Anastasia pomade, so if you're into that this one is a miss. I like that they include a brush and a mirror, for on the go application and touch-ups. And you will have to touch up the pomade if you don't make sure to set it properly with the powder. About the brush; it's okay for on-the-go use, but I prefer using a more precise brush with a longer handle. The short handle can make the pressure you apply too hard. The end of the brush intended for powder is no good. Too bushy and unprecise. All in all it's good value for the low price. Make sure you like my review if you found it helpful, and visit my reviews page for more reviews. :-)
Nice and subtle
Posted by iHerb Customer on Aug 27, 2017 | Verified Purchase
Very nice product. Most eyebrow colour has too much red for me. This is perfect.
The Color Is Too Light
Posted by iHerb Customer on Aug 25, 2017 | Verified Purchase
The color is too light that I can't see the difference


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Item description for Gimme an "A" (Slam Dunk Series) by Tess Eileen Kindig & Eileen Kindig...While trying to improve his failing math grade in order to stay on the basketball team, Mickey learns a lesson about forgiveness.
Promise Angels is dedicated to bringing you great books at great prices. Whether you read for entertainment, to learn, or for literacy - you will find what you want at promiseangels.com!
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One of our favorite emerging wedding trends is the wedding movie trailer. We just can't get enough of these "film shorts"!
Rather than a movie preview, a wedding movie trailer is a brief, artsy recap of the sights and sounds of your wedding–after the fact. We think these 1 to 3 minute films are perfect for sharing on your Facebook page when you just can't wait to share it with the world.
Sure, your full length wedding video will come, but we bet this is the one you'll watch over and over again.
Check out this extraordinary wedding at The Westin Kierland Resort & Spa. The trailer captures the elements of two ceremonies–one Indian, one American–celebrated by Amanda and John and their families. The groom even comes in on a white horse!
Thanks to Taryn and Jeff at Serendipity Video for sharing this slice of the sublime with us.Share on Facebook


text:
Engine Prototype #4 was built as a siege weapon to break the standstill while trying to breach the Elven city of Cullebra. It's new offensive system dubbed the Arcane Mortar , is viewed as one of the most destructive forces in all Terescence.
Why rum all gone?
Tell me what you eat and I'll tell you what you are…
Used to this day, it is used when a person knows they are out matched but they need anything they can to help rid themselves of their foes.
Found at the best cafes, and commanding the highest prices…
E'Scatonia's demon-wrought strongbox held her favorite things.
The merchant grinned "Dear sir, I noticed you had your eye on that charm. Might I interest you in a little bit of its history? Of course I would. I will have you know, that charm has seen its way through many an adventure, and saved the lives of many of the adventurers therein. Legend tells that it was crafted centuries ago by Yurid Norcral, the greated Dwarven runesmith to ever live. Later it was said to have been enchanted by ancient Elven magics which have since been lost to the ages. Quite a rare find."
"Let me to battle, Brother. I shall sweep thine enemies as the wind, and thou shalt have vict'ry." -St. Vedast's revelation to Hural
Winner of The Shards of the Storm Quest.
A goblet created with the pommel of the sword of storms.
The Sextant of the Soul is a potent magical artifact that can only be found within the dreamscape.
Misery, trouble and general bad luck will dog all who drink from the seemingly wonderful Goblet of Sin…
With the application of kinetic energy, this shard radiates the aura of worry, fear, and doubt that the fiercest storms bring.
Falconry and Hawking has been important skill in a variety of places through out history. It is the sport of nobles and nomads, and many in between. In some places and times, Raptors are bonded to people, using their magic connection to everyones benefit. It is only natural that magical items connected to raptors and Hawking came into existence.
A shadow shrine is an elegant solution to a complicated problem for those that are Godsworn.
Yes my child, the pig can see the winds, and will tell you of the storms.
This is a Quest Submission for the Shards of the Storm Quest.
The shards of Typhoon and Monsoon were recognized by the Wind and Sky, and so the lands beneath them permitted them to exist, and thus remembered, so they continue to exist.
But what of the shards themselves? When the hands of mortal interfere with the Gods, woe to thee who believes them worthy.
The Rod of Arcane Power is saught after by wizards everywhere as a source of limitless power. The Rod of Arcane Might is an item that complements Siren no Orakio's Item submission "Shards of the Storm". This is a quest submission.
Archaeologists have long wondered about the origins of "cup and ring-marked stones", the mysterious rocks found on deserted tracts of moorland bearing markings resembling small shallow pits with concentric rings and dating to the Bronze-age.
This strange tobacco mixture that natives of Ankorill use
for both traditional ceremonies and everyday events.
A simple net made of strong jute,this otherwise innocous looking object when combined with the amazing Ahal paste used by the tribsemen of the Powi,can be a serious threat to any warrior who finds himself menaced with this traditional Powi wepaon.
Elves use nature as strength and borrow from the trees to keep them living. An elf out of the forest for more than a day begins to die and within 3 days is dead without borrowing the strength of nature.


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Cartier Santos W20057C4
This Cartier has undergone a thorough inspection of accuracy, functionality and condition at our Cartier certified service centre to determine the level of reconditioning required to meet our strict standards. It has also been checked against our records to ensure authenticity.
Product Description
|Approx. 19 years
|Product code
|Case size
|Case material
|Steel & Yellow Gold
|Bracelet material
|Steel & Yellow Gold
|Dial type
|Silver Roman Numeral
|Water resistance
|30 metres
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text:
Just booked last minute trip to NY. Arriving Friday, Jan 8 in time for lunch and leaving Sunday around 330p.
Plan to go to theater either Friday or Saturday night but will be 90 minute, no intermission so could do a late dinner (Bernard-Jacobs Theater).
Food is the key. Prefer smaller, intimate neighborhood restaurants but open to anything. Don't like ornate, heavy dining rooms and don't really want a 3-4 hour tasting menu. However, price is not an issue. Like all cuisines, especially Italian. Prefer lighter over heavy. Desserts are not an interest at all but like a nice wine or saki list. Tried to get into Lupe on recommendation and may do for lunch though dinner looks doubtful. Some other recommendations from friends were Chiyona, Cafe Cluny, Momofuka Noodle.
Coming from Atlanta where some of our favorites are Shaun's, Rathbuns, Sotto Sotto, Holeman and Finch.
I did review several recent posts but am having a hard time getting a handle on the best plan and would appreciate any advice.


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You'll need that inner voice of scorn for later, when you're revising - but while you're working on a first draft, you have to drown it out, possibly with loud Finnish death metal.
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