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Molecular genetic and chemotaxonomic characterization of the terrestrial cyanobacterium Nostoc commune and its neighboring species
The phylogeny of the terrestrial cyanobacterium Nostoc commune and its neighboring Nostoc species was studied using molecular genetic and chemotaxonomic approaches. At least eight genotypes of N. commune were characterized by the differences among 16S rRNA gene sequences and the petH gene encoding ferredoxin-NADP+ oxidoreductase and by random amplified polymorphic DNA analysis. The genotypes of N. commune were distributed in Japan without regional specificity. The nrtP gene encoding NrtP-type nitrate/nitrite permease was widely distributed in the genus Nostoc, suggesting that the occurrence of the nrtP gene can be one of the characteristic features that separate cyanobacteria into two groups. The wspA gene encoding a 36-kDa water stress protein was only found in N. commune and Nostoc verrucosum, suggesting that these Nostoc species that form massive colonies with extracellular polysaccharides can be exclusively characterized by the occurrence of the wspA gene. Fifteen species of Nostoc and Anabaena were investigated by comparing their carotenoid composition. Three groups with distinct patterns of carotenoids were related to the phylogenic tree constructed on the basis of 16S rRNA sequences. Nostoc commune and Nostoc punctiforme were clustered in one monophyletic group and characterized by the occurrence of nostoxanthin, canthaxanthin, and myxol glycosides.
- Anhydrobiosis
- carotenoid
- ferredoxin-NADP+ oxidoreductase
- nitrate/nitrite transporter
- random amplified polymorphic DNA
- water stress protein
Cyanobacteria are photoautotrophic prokaryotes that perform oxygen-evolving photosynthesis with water as the primary electron donor. Requiring only light, water, carbon dioxide, and inorganic salts, cyanobacteria have very simple nutrient requirements that allow these organisms to occupy highly diverse ecological niches. Cyanobacteria are found in almost all terrestrial, freshwater, and marine habitats. Some cyanobacteria form symbiotic associations with hosts, such as fungi (lichens), bryophytes, cycads, mosses, fern (Azolla), and one angiosperm, Gunnera (Rai et al., 2000). Interestingly, most of the cyanobacterial photobionts in lichens belong to the genus Nostoc (Rikkinen et al., 2002).
The terrestrial cyanobacterium Nostoc commune is a well-known species of the genus Nostoc, and N. commune may be a cosmopolitan species that is adapted to terrestrial environments (Potts, 2000). In natural habitats, N. commune forms nonspherical, irregularly spreading, macroscopic colonies that consist of trichomes of cells and extracellular matrix (Potts, 2000). Nostoc commune is very unique because of its extreme desiccation tolerance; desiccated colonies have no metabolic activity and sustain the capacity for cell growth for over 100 years (Lipman, 1941; Cameron, 1962). This phenomenon is termed 'anhydrobiosis' (Crowe et al., 1998; Clegg, 2001; Potts, 2001; Crowe, 2002). Nostoc commune is considered an anhydrobiotic microorganism that has oxygenic photosynthetic capabilities and does not differentiate into akinetes (spores) (Potts, 2000, 2001). The mechanism of extreme desiccation tolerance by this species is thought to involve multiple processes that include extracellular polysaccharide (EPS) production, compatible solute accumulation, regulation of photosynthesis, and protection from UV radiation. Removal of EPS in N. commune causes a significant reduction in the stress tolerance of photosynthesis during desiccation (Tamaru et al., 2005). The cessation of photosynthesis and trehalose accumulation occurs as an acclimatizing response to matric water stress (desiccation) and osmotic water stress (high salt concentration) (Sakamoto et al., 2009; Yoshida & Sakamoto, 2009). With UV-absorbing pigments of mycosporine-like amino acids and scytonemin (Wright et al., 2005; Sakamoto et al., 2011), N. commune may be able to adapt to terrestrial environments with high levels of solar radiation.
To investigate a unique mechanism responsible for the acquisition of the extreme desiccation tolerance of N. commune, a taxonomic study of this organism is necessary. In this study, we investigated the phylogeny of N. commune within the genus Nostoc using a molecular phylogenic approach that uses genetic techniques and a chemotaxonomic approach that uses carotenoid patterns to characterize N. commune and its neighboring Nostoc species.
The 16S rRNA gene is the most highly studied gene in phylogenic analysis and is useful in determining the identification of cyanobacterial species (Rasmussen & Svenning, 2001; Svenning et al., 2005; Rehakova et al., 2007). Ferredoxin-NADP+ oxidoreductase (FNR) catalyzes electron transfer from the reduced ferredoxin to NADP+ to produce NADPH in the final step of photosynthetic electron transfer. The cyanobacterial FNR contains a unique N-terminal domain similar to the 9-kDa phycocyanin-associated linker polypeptide CpcD (Nakajima et al., 2002; Morsy et al., 2008a). Thus, the petH gene that encodes cyanobacterial FNR was used for the phylogenic study, serving as a specific and characteristic gene of cyanobacteria. Two distinct types of nitrate/nitrite transporters (NRT) have been identified in cyanobacteria thus far. An ATP-binding cassette (ABC)-type NRT, in which NrtA functions as a high-affinity nitrate/nitrite-binding protein, occurs in freshwater strains (Omata, 1995), whereas marine cyanobacterial strains have an NrtP-type permease (Sakamoto et al., 1999; Wang et al., 2000). Because the freshwater cyanobacterium Nostoc punctiforme has the NrtP-type permease (Meeks et al., 2001; Aichi et al., 2006), the attribution of the types of NRT to the habitats of cyanobacteria is questioned. Hence, the distribution of the types of NRT was studied. The groEL gene encoding chaperonin 60 is transcribed at similar levels in both hydrated and desiccated cells of N. punctiforme (Yoshida & Sakamoto, 2009). Thus, the groEL gene was studied as a constitutively expressing gene. The wspA gene encodes a 36-kDa water stress protein in the extracellular matrix of N. commune (Wright et al., 2005; Morsy et al., 2008b; Sakamoto et al., 2011). There is no homologous gene to the wspA gene in the genome of N. punctiforme ATCC 29133 (Meeks et al., 2001), although N. punctiforme is believed to be closely related to N. commune. In our recent study, the wspA gene was found in the aquatic cyanobacterium Nostoc verrucosum (Sakamoto et al., 2011). Thus, the occurrence of this gene was studied in N. commune and its neighboring species. Carotenoids in cyanobacteria have two main functions: as accessory pigments for photosynthesis and in protection against photooxidative damage (Hirschberg & Chamovitz, 1994). Cyanobacteria synthesize unique ketocarotenoids and glycosyl carotenoids that are not found in higher plants (Takaichi et al., 2005). Thus, carotenoid patterns were studied in the genus Nostoc as a chemotaxonomic marker.
Materials and methods
Organisms and culture conditions
Nostoc commune colonies growing naturally in the field were collected in Japan, and samples from China and France were kindly provided for this study. Nostoc commune strain IAM M-13 (NIES-24), Nostoc muscorum strain IAM M-14, N. punctiforme strain IAM M-15 (NIES-2108), Nostoc linckia strain IAM M-30 (NIES-28), and Nostoc carneum strain IAM M-35 (NIES-2107) were retrieved as cultivated samples from the Institute of Molecular Biosciences at the University of Tokyo. Antarctic strains of Nostoc cf. commune SO-42, SO-36, KG-54, and KG-102 were provided by Dr S. Ohtani at Shimane University. Nostoc sp. strain PCC 7120 (also known as Anabaena sp. strain PCC 7120) was provided by Dr A. Higo at Saitama University. The domestic strains of Nostoc sp. KU001 and N. commune KU002 were isolated from the Kakuma Campus at Kanazawa University, and they were purified by streaking on agar plates. These cyanobacterial strains originally isolated by the authors will be available upon request. Cells were grown at 25–30 °C under constant illumination from fluorescent lamps (5–10 μmol m−2 s−1) on modified BG110 liquid medium (without a nitrogen source) with shaking for aeration or BG110 solid medium with 1.5% agar supplemented with a vitamin mix to give final concentrations of 1 μg L−1 of biotin, 2 mg L−1 of thiamin, and 1 μg L−1 of cyanocobalamin (Castenholz, 1988). These culture media were buffered with 20 mM HEPES-NaOH (pH 7.5). For culturing N. carneum strain IAM M-35, 1.5 g L−1 of NaNO3 was added as a nitrogen source. These cyanobacterial cultures were unialgal but not completely bacteria-free.
PCR direct sequencing and phylogenic analysis
The DNA fragments containing the 16S rRNA gene, the petH gene encoding FNR, the nrtP gene encoding nitrate/nitrite permease, and the groESL gene encoding chaperonin 60 were amplified by PCR, and their nucleotide sequences were determined. The field samples of N. commune and cultivated cyanobacterial cells were lyophilized until dry and ground to a powder. A genomic DNA template was extracted from approximately 15 mg of the powder using the DNA Plant Mini Kit (QIAGEN) according to the manufacturer's instructions. The gene-specific primers for the 1.4-kb 16S rRNA gene, the 1.9-kb petH gene, the 1.3-kb nrtP gene, the 2-kb groESL gene, and the 1.7-kb groEL gene (Table S1) were designed and synthesized based on the nucleotide sequence of the N. punctiforme strain ATCC 29133 genome (Meeks et al., 2001). The reaction mixture contained 5 μL of 10× PCR buffer (100 mM Tris–HCl pH 8.3, 500 mM KCl, and 15 mM MgCl), 4 μL of 2.5 mM dNTP mixture, 50 pmol of a forward primer, 50 pmol of a reverse primer, 1.25 U of Taq polymerase (R001A; TaKaRa, Kyoto, Japan), and 5–20 ng of the genomic DNA template in a total volume of 50 μL. PCR was performed under the following conditions: initial denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 30 s with a final extension step at 72 °C for 5 min and final soak step at 4 °C. The PCR product was purified using a QIAquick PCR Purification Kit (QIAGEN) to remove excess PCR primers. Cycle sequencing reactions were performed to determine the nucleotide sequence by 3′ dye-labeled dideoxynucleotide triphosphates (BIG DYE terminators; Applied Biosystems company) using the internal specific primers and the template DNA amplified by PCR and run on an ABI PRISM377 DNA Sequencer (Perkin-Elmer ABI, Foster City, CA). The nucleotide sequences determined in this study have been submitted to GenBank/EMBL/DDBJ with the accession numbers listed in Table S2. The accession numbers of the 16S rRNA gene sequences obtained from the GenBank/EMBL/DDBJ database are listed in Table S3. The phylogenic analysis was performed using phylip (Phylogeny Interference Package) version 3.6 (Felsenstein, 2004). The sequence data were aligned using clustal x version 1.83 and corrected manually using MacClade version 4.0. The distance matrix was computed using Restdist program on the Kimura-2-parameter model, and a phylogenic tree was constructed using Neighbor program. Bootstrap test was performed using Seqboot program.
PCR–restriction fragment length polymorphisms (RFLP) analysis
To characterize the genotypes of the field-isolated natural colonies of N. commune, PCR–RFLP analysis was performed using 16S rRNA and petH genes (Table S4). The 1.4-kb DNA fragment of the 16S rRNA gene was amplified from the genomic DNA template by PCR using the specific primers (Table S1) and digested by the restriction enzymes Ase I, Bsm I, and Hpa I, respectively. The 1.8-kb DNA fragment of the petH gene was amplified by PCR and digested by Sau3A I. These digestion products were separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. The DNA fragment patterns were compared with those from the known genotypes. Four distinct types designated A, B, C, and D could be recognized by this analysis (Table S4).
Random amplified polymorphic DNA (RAPD) analysis
Three oligonucleotide primers, 5′-CCA(A/G)TCCCCA(A/G)TCCCC-3′ containing short tandem repeat repetitive sequences (STRR; Mazel et al., 1990), 5′-GCGATCGCACC-3′ (Bhaya et al., 2000), and 5′-TAAACCTTTAGGCGATCGCG-3′ containing highly iterated palindrome sequences (HIP1; Gupta et al., 1993), were used as a single primer for the random amplification of the genomic DNA template. The reaction mixture contained 5 μL of 10× PCR buffer (100 mM Tris–HCl pH 8.3, 500 mM KCl, and 15 mM MgCl), 4 μL of 2.5 mM dNTP mixture, 100 pmol of a RAPD primer, 1.25 U of Taq polymerase (R001A; TaKaRa), and 5–20 ng of the genomic DNA template in a total volume of 50 μL. The PCRs were performed using the following PCR profile: an initial denaturation at 95 °C for 6 min, followed by 30 cycles with denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, and extension at 65 °C for 5 min with a final extension step at 65 °C for 16 min and final soak step at 4 °C (Mazel et al., 1990). The amplified DNA fragments were separated by electrophoresis on a 2% agarose gel and stained with ethidium bromide.
PCR amplification of the wspA gene
A 0.8-kb DNA fragment containing the wspA gene was amplified by PCR from the genomic DNA templates as previously described (Sakamoto et al., 2011). The degenerate primers used a forward primer (23-mer with 96-fold degeneracy) 5′-TA(T/C) GGI TA(T/C) ACI AT(T/C/A) GGI GA(A/G) GA-3′ derived from the amino acid sequence YGYTIGED and a reverse primer (23-mer with 96-fold degeneracy) 5′-TC (T/C)TG (A/G)TA ICC IGT (A/G/T)AT (T/C)TC (A/G)TA-3′ derived from the amino acid sequence YEITGYQD. PCR was performed under the following conditions: initial denaturation at 94 °C for 1 min, followed by 40 cycles of 94 °C for 1 min, 50 °C for 2 min, and 72 °C for 3 min with a final extension step at 72 °C for 5 min. The amplified DNA fragments were separated by electrophoresis on a 1.2% agarose gel and stained with ethidium bromide. The nucleotide sequences of the wspA genes from N. commune and N. verrucosum have been submitted to GenBank/EMBL/DDBJ with the accession numbers listed in Table S2.
Southern hybridization analysis
Genomic DNA (2 μg) was digested by Hind III and EcoR I, separated by electrophoresis on a 0.8% agarose gel, and transferred onto a nylon membrane (Hynbond N+; GE Healthcare UK Ltd.). For the digestion of the genomic DNA from Nostoc sp. KU001, Hind III and BamH I were also used. The 1.4-kb nrtA probe was amplified by PCR using the specific primers (Table S1) and the genomic DNA template of Nostoc sp. strain PCC 7120. The 1.3-kb nrtP probe was amplified by PCR using the specific primers (Table S1) and the genomic DNA template of N. commune strain KU002. The 0.7-kb wspA probe was amplified by PCR using the specific primers (Table S1) and the genomic DNA template of N. commune strain KU002. The DNA fragments were labeled using AlkPhos Direct Labelling Module (GE Healthcare), and hybridizations with these probes were preformed in AlkPhos Direct Hybridization Buffer (GE Healthcare). After excess probes were washed, the hybridization signals were detected by a chemiluminescence detection system (CDP-Star Detection Reagent, GE Healthcare) and visualized with a luminoimage analyzer (LAS-3000; FUJIFILM).
Identification and composition of carotenoids
Colonies of N. verrucosum growing naturally in the stream were collected from Shishiku Park, Hakusan-shi, Ishikawa, Japan (36°26′8″N, 136°38′37″E) (Sakamoto et al., 2011). Colonies of N. commune growing naturally in the field were collected, washed with tap water to remove soil, air-dried, and stored at room temperature. Cells of N. muscorum strain IAM M-14, N. linckia strain IAM M-30 (NIES-28), and Nostoc sp. strain KU001 were grown diazotrophically for 4 weeks at 25 °C under constant illumination from fluorescent lamps (5–10 μmol m−2 s−1) on modified BG110 liquid medium (without a nitrogen source) buffered with 20 mM HEPES-NaOH (pH 7.5) with shaking for aeration. Cells of N. carneum strain IAM M-35 were grown for 2 weeks on BG11 liquid medium containing 1.5 g L−1 of NaNO3 as a nitrogen source otherwise under identical conditions. The light intensity was measured using a photometer (model LI-185B; LI-COR, Lincoln, NE).
Pigments were extracted from wet cells with acetone/methanol (7 : 2, v/v) and analyzed by the HPLC system equipped with a μBondapak C18 column (8 × 100 mm, RCM-type; Waters, Milford, MA) and an MCPD-3600 photodiode array detector (Otsuka Electronics; Osaka, Japan). After elution with methanol/water (9 : 1, v/v) for 20 min at a flow rate of 2.0 mL min−1, the mobile phase was changed to 100% methanol. For quantitative analysis, the molar extinction coefficient in the HPLC eluent at the maximum wavelengths of each carotenoid was assumed to be the same (Takaichi et al., 2001; Iwai et al., 2008).
For the identification of the carotenoids, their absorption spectra and their specific retention times on HPLC were compared with N. commune strain IAM M-13 (NIES-24) (Takaichi et al., 2009). For the identification of the glycoside moieties of myxol glycosides, they were purified using silica gel TLC and the HPLC system as described earlier. Their relative molecular masses were measured by FD-MS using an M-2500 double-focusing gas chromatograph-mass spectrometer (Hitachi, Japan) equipped with a field-desorption apparatus. The proton nuclear magnetic resonance (1H-NMR) (500-MHz) spectra of carotenoids in CDCl3 at 24 °C were measured using the UNITY INOVA-500 system (Varian).
Results and discussion
Genetic diversity of Nostoc commune and phylogenic analysis
The 1.4-kb DNA fragment of the 16S rRNA gene was amplified and sequenced using 34 samples of field-isolated, naturally grown colonies of N. commune collected in Japan. In total, eight distinct sequences of the 16S rRNA gene were found. These nucleotide sequences obtained from the field samples of N. commune were > 96% identical to each other. These sequences were also highly similar to the 16S rRNA gene sequences obtained from N. commune strain IAM M-13 and the Antarctic strains of N. cf. commune SO-42, SO-36, KG-54, and KG-102 with a sequence identity of > 96%. These data confirmed a high level of genetic similarity among all field samples of N. commune. The 1.8-kb DNA fragment of the petH gene encoding FNR was amplified and sequenced, and seven distinct sequences of the petH gene were found in the 26 field samples of N. commune examined. The deduced amino acid sequences of PetH were highly similar to each other with a sequence identity of > 91%. These PetH sequences in N. commune exhibited < 84% sequence identity to other cyanobacterial PetH sequences obtained from the GenBank database, suggesting that N. commune can be recognized as a discriminative species by the PetH sequence. When the identical 16S rRNA gene sequence was obtained, an identical or very similar sequence of the petH gene was found, indicating that these genotypes are consistently recognizable by either the 16S rRNA gene sequence or the petH gene. Thus, these genotypes were designated A1, A2, B1, B2, C1, C2, D1, and D2 and characterized further.
By comparing the nucleotide sequences of 16S rRNA and the petH gene obtained from the genotypes of N. commune, RFLP in these genes were consequently found (Table S4). These RFLPs allowed us to characterize the four distinct genotypes, designated A, B, C, and D, of the field-isolated natural colonies of N. commune by PCR–RFLP analysis (Table S4). In total, more than 45 colonies were examined by the 16S rRNA gene sequence and PCR–RFLP analysis, and all of the colonies were recognizable as N. commune with the characteristics of this organism as previously described (Rehakova et al., 2007). Colonies of N. commune were found throughout Japan, and there was no specific regional distribution of these genotypes (Supporting Information, Fig. S1). Moreover, three genotypes were found in a small area at the Kakuma Campus of Kanazawa University, and occasionally, two distinct genotypes were mixed together in a small fragment of N. commune colonies (data not shown). It is noteworthy that these genotypes of N. commune were hardly distinguishable morphologically, and their genetic differences were not great enough to recognize these genotypes as distinct species.
The 16S rRNA gene phylogenic tree of N. commune and its neighboring species of Nostoc was constructed by the neighbor-joining method (Fig. ⇓). Anabaena cylindrica strain PCC 7122 was included as an outgroup to root the tree. All of the genotypes of field-isolated, naturally grown N. commune were clustered in a monophyletic group with other strains of N. commune, Nostoc flagelliforme, and N. punctiforme. This clade including N. commune and N. punctiforme was significantly exclusive from other Nostoc species. Symbiotic Nostoc strains that are thought to belong to N. punctiforme were also included in this clade when included in the phylogenic analyses (data not shown). This analysis is in agreement with previous studies of the phylogeny of symbiotic Nostoc strains (Svenning et al., 2005). The phylogenic trees constructed using the petH, nrtP, and groEL genes were essentially the same as the trees constructed using the 16S rRNA gene (data not shown), confirming the result of the 16S rRNA gene analysis (Fig. ⇓). These results suggest that N. commune can be considered a synonym of N. punctiforme, and N. commune and N. punctiforme, including symbiotic Nostoc strains, are closely related, although their habitats are superficially different. Nostoc commune forms free-living macroscopic colonies in terrestrial environments, and N. punctiforme is found as a phycobiont in the root of cycads (Rehakova et al., 2007).
RAPD analysis using the primers derived from cyanobacterial repetitive sequences of STRR (Mazel et al., 1990) and HIP1 (Gupta et al., 1993) is a powerful tool to study the genetic diversity of cyanobacterial strains. Because the DNA profile generated by random amplification using cyanobacterial-specific repetitive sequences resolves the small differences in the entire genome in an enhanced manner, this analysis is one of the most sensitive methods for distinguishing among closely related strains (Prabina et al., 2005). The eight genotypes of N. commune recognized by the 16S rRNA gene were analyzed by RAPD (Fig. ⇓). Each primer produced a distinct set of DNA fragments for each of the eight genotypes of N. commune (Fig. ⇓a,c,d). When seven samples of the type A1 (characterized by the laboratory culture strain of N. commune KU002) from distant geographic locations were examined, all of the DNA profiles obtained were identical to each other (Fig. ⇓b). The band patterns shown in Fig. ⇓a were converted into binary data to construct a dendrogram (Fig. S2). The dendrogram graphically represents the differences among the genotypes according to their band patterns. These results indicate that the genotypes of N. commune can be distinguished by the DNA profiles specific to genotype using this technique.
In this study, the genetic diversity of the cosmopolitan terrestrial cyanobacterium N. commune was analyzed, and we designated eight genotypes of N. commune in Japan based on the nucleotide sequences of the 16S rRNA gene. The eight genotypes of N. commune are genetically similar, and the morphological features of the macroscopic colonies and microscopic trichomes are almost indistinguishable. These genotypes are easily recognized by PCR direct sequencing of the 16S rRNA gene and PCR–RFLP or RAPD analysis as described earlier. Our results support the results of other studies that found that N. commune, N. punctiforme, and symbiotic Nostoc strains are clustered in a monophyletic group and originate from a common ancestor (Rikkinen et al., 2002; Svenning et al., 2005; Rehakova et al., 2007). The aquatic cyanobacterium N. verrucosum forms visible colonies and retains a large amount of EPS. The appearance of N. verrucosum colonies is superficially similar to that of N. commune, but N. verrucosum is sensitive to desiccation (Sakamoto et al., 2011). The 16S rRNA gene sequence of N. verrucosum strain KU005 showed 95% identity to the sequence of N. commune strain KU002, suggesting that these strains are genetically similar but different species of cyanobacteria. Nostoc verrucosum may be the origin of the terrestrial cyanobacterium because of the morphological resemblance. However, we could not precisely resolve the phylogeny of N. verrucosum within the genus Nostoc by phylogenic analysis using the 16S rRNA gene sequence (data not shown).
Nitrate/nitrite transporters in Nostoc
Two distinct types of NRT, an ABC-type NRT in freshwater strains (Omata, 1995) and an NrtP-type permease in marine strains (Sakamoto et al., 1999; Wang et al., 2000), have been identified in cyanobacteria. The nrtA gene encodes a high-affinity nitrate/nitrite-binding protein that is a component of the ABC-type NRT (Omata, 1995). The distribution of these types of NRT in the genus Nostoc was characterized. Figure ⇓ shows the results of Southern hybridization using nrtA (Fig. ⇓a) and nrtP (Fig. ⇓b) gene probes. The signals hybridizing with the nrtA gene probe were detected in Nostoc sp. strain KU001, in N. carneum strain IAM M-35, and in the positive control Nostoc sp. strain PCC 7120 (Fig. ⇓a, lanes 1–4). The signals hybridizing with the nrtP gene probe were detected in N. carneum strain IAM M-35, N. linckia strain IAM M-30, N. commune strain KU002, and N. commune strain IAM M-13 (Fig. ⇓b, lanes 4–7). According to these results, the nrtP gene was isolated by PCR using the gene-specific primers designed from the nucleotide sequence of the N. punctiforme strain ATCC 29133 genome (Meeks et al., 2001). As summarized in Table S2, the nrtP genes from 15 strains of the genus Nostoc were PCR-amplified and sequenced, indicating that the nrtP gene is widely distributed in the genus Nostoc. Because the signals hybridizing with the nrtA probe were also detected in N. carneum strain IAM M-35, we tried to isolate the nrtA gene by PCR using the primers designed from the nucleotide sequence of the nrtA gene of Nostoc sp. strain PCC 7120. However, the nrtA gene could not be isolated from N. carneum strain IAM M-35 by this approach (data not shown). Thus, future studies need to determine whether N. carneum is an intermediate species having two distinct nitrate/nitrite transport systems; N. carneum clearly has the nrtP gene (Table S2).
The distribution of the nrtP gene in the genus Nostoc was related to the phylogenic tree (Fig. ⇑), but the habitats of cyanobacteria were unrelated to the type of NRT. The nrtP genes were found in all of the marine, freshwater, and terrestrial cyanobacteria. These results suggest that the occurrence of the nrtP gene can be one of the characteristic features that separate cyanobacteria into two groups. Future studies need to examine how these two systems of NRT were originated and selected in cyanobacteria.
Occurrence of the wspA gene in Nostoc
To examine the distribution of the wspA gene in the genus Nostoc, the wspA gene was amplified by PCR using the degenerated oligo DNA primers that were designed according to the deduced amino acid sequence of WspA (Fig. ⇓). The 0.8-kb PCR product of the wspA gene was obtained from the genomic DNA template of N. commune strain KU002 (Fig. ⇓, lane 1), N. verrucosum strain KU005 (Fig. ⇓, lane 2), and the field-isolated, naturally grown N. verrucosum (Fig. ⇓, lane 3). The wspA gene of N. commune strain KU002 encoded a 333-amino-acid polypeptide with 77% sequence identity to WspA of N. commune strain DRH1, and the wspA gene of N. verrucosum strain KU005 encoded a 330-amino-acid polypeptide with 73% sequence identity to the WspA of N. commune KU002 (Sakamoto et al., 2011). No DNA fragment was obtained in the other 12 strains of Nostoc tested, including strains closely related to N. commune (Fig. ⇓, lanes 4–15). Using the 0.7-kb probe of the wspA gene from N. commune strain KU002, Southern hybridization analysis was performed to confirm the result of the PCR analysis using the degenerated oligo DNA primers, and hybridization signals were detected in N. commune strain KU002 and N. verrucosum but not in the other cyanobacteria tested (data not shown). These results suggest that the wspA gene exclusively occurs in two Nostoc species, N. commune and N. verrucosum, and these species form massive colonies with a large amount of extracellular matrix in natural habitats. WspA is a major extracellular matrix protein with β-galactosidase activity (Morsy et al., 2008b) and is assumed to maintain the structure and/or functions of the extracellular matrix (Wright et al., 2005). The primary structure of WspA shows no similarity to the known proteins in the database and is thought to be a unique protein in N. commune. It is noteworthy that several N. commune strains lack the wspA gene (Fig. ⇓). This distribution of the wspA gene raises new questions surrounding the origin of this gene and genetic relationships between N. commune and N. verrucosum.
Identification and composition of carotenoids in Nostoc
β-Carotene, its hydroxyl derivatives of zeaxanthin, caloxanthin, and nostoxanthin, and its keto derivatives of echinenone and canthaxanthin were identified based on the compatible absorption spectra and the specific retention times on HPLC of N. commune strain IAM M-13 (NIES-24) (Takaichi et al., 2009). Two carotenoid glycosides were purified from N. verrucosum and were identified as myxol 2′-fucoside and 4-ketomyxol 2′-fucoside based on their relative molecular masses of 730 and 744, respectively, and 1H-NMR spectra (Takaichi et al., 2005). Nostoc cameum IAM M-35 and Nostoc sp. strain KU001 contained myxol methylpentoside that was identified based on a relative molecular mass of 730, which was thought to be fucoside, rhamnoside, or quinovoside. Nostoc punctiforme strain IAM M-15 contained myxol methylpentoside and 2-hydroxymyxol methylpentoside that were identified based on the presence of nostoxanthin. Minor components in Nostoc species lacking nostoxanthin were thought to be 4-hydroxymyxol methylpentoside. Of note, 2,2′-β-hydroxylase, CrtG, catalyzes the reactions of zeaxanthin to nostoxanthin and myxol to 2-hydroxymyxol (Iwai et al., 2008).
Carotenoid compositions in N. commune and its neighboring Nostoc and Anabaena species are summarized in Table ⇓. The most common carotenoids in cyanobacteria are β-carotene, zeaxanthin, the ketocarotenoid echinenone, and the myxol glycosides (Takaichi & Mochimaru, 2007). In all of the cyanobacterial species examined, β-carotene was found to be a major component, and the ketocarotenoids of echinenone and canthaxanthin were also present. Three groups of distinct carotenoid patterns were found. Group 1 was characterized by the absence of hydroxylated molecular species, such as β-cryptoxanthin, zeaxanthin, caloxanthin, and nostoxanthin. Group 2 was characterized by the absence of myxol glycosides. Nostoc commune was found to belong to Group 3 and was characterized by the presence of hydroxylated carotenoids and myxol glycosides (Table ⇓). This carotenoid composition is in agreement with a previous report of the pigment analysis in N. commune (Potts et al., 1987), in which β-carotene and echinenone predominate. These three groups designated by carotenoid composition were strongly related to the phylogenic tree constructed on the basis of 16S rRNA gene sequences. Each group was discriminated from the others and recognized as a separated clade (Fig. ⇑), indicating that this chemotaxonomic approach using the carotenoid patterns is a valid approach and is useful for distinguishing Nostoc strains.
Data from a single experiment are shown but the occurrence of the carotenoids was confirmed at least twice.
β-caro, β-carotene; β-cryp, β-cryptoxanthin; Zea, Zeaxanthin; Calo, Caloxanthin; Nosto, Nostoxanthin; Echine, Echinenone; Cantha, Canthaxanthin; 3′-OH-E, 3′-OH-Echinenone; M-G, Myxol glycoside; KM-G, 4-Ketomyxol glycoside; 2OH-M-G, 2-hydroxymyxol glycoside; 4OH-M-G, 4-hydroxymyxol glycoside.
↵Takaichi et al. (2005),
↵Takaichi et al. (2006),
↵Takaichi et al. (2009),
↵free (not glycoside),
↵methyl pentoside,
↵these might be 4OH-M-G due to lack of nostoxanthin.
As a chemotaxonomic tool to characterize Nostoc species, fatty acid profiling has been reported (Liu et al., 2003; Temina et al., 2007). Because a quantitative and statistical analysis is required for fatty acid profiling, this approach may be complex and it is difficult to avoid the fluctuation in fatty acid composition that depends on the growth conditions. Compared with the previously reported fatty acid profiling, the differences in the carotenoid patterns found by this study can be supported by the presence and/or absence of the specific genes involved in the carotenogenesis pathways. These comparative studies of the genes involved in the carotenogenesis pathways in the genus Nostoc are important to confirm the validation of this chemotaxonomic tool. This confirmation remains to be directly demonstrated in future studies.
Concluding remarks
The taxonomic characters for the genus Nostoc are limited, and these characters are hardly distinguishable morphologically. Thus, molecular genetic and chemotaxonomic tools are useful and convenient for the characterization of Nostoc species. The terrestrial cyanobacterium N. commune can be discriminated from the other Nostoc species by a combination of the presence of genes related to the structure and functions of the extracellular matrix, such as the wspA gene, and the presence of the specific carotenogenesis pathways for both hydroxylated carotenoids and myxol glycosides. To elucidate the mechanisms of terrestrial cyanobacteria adaptation to the extreme environments and expansion of their cosmopolitan distribution, the freshwater Nostoc species that is genetically closest to N. commune must be determined. Further studies are required to identify the characteristic genes attributive to the unique habit of anhydrobiosis of the terrestrial cyanobacterium N. commune.
Supporting Information
Additional Supporting Information may be found in the online version of the article:
Fig. S1. Sampling locality of field-isolated, naturally grown colonies of Nostoc commune in Japan.
Fig. S2. Cluster analysis using RAPD patters in the 8 genotypes of N. commune.
Table S1. PCR primers.
Table S2. Accession numbers for the nucleotide sequences of 16S rRNA gene, petH, groESL, nrtP, and wspA.
Table S3. Accession numbers of 16S rRNA gene sequences retrieved from the EMBL/GenBank/DDBJ database.
Table S4. PCR–RFLP analysis.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
The authors thank Dr T. Maoka (Research Institute for Production Development) for the NMR analysis of certain myxol glycosides and Y. Tamaru (Kanazawa University), F. M. Morsy (Kanazawa University), Y. Andoh (Kanazawa University), E. Hiratani (Kanazawa University), and S. Matsumura (Kanazawa University) for their technical assistance. This work was supported by Japan Society for the Promotion of Science (KAKENHI 22510227), Suntory Institute for Bioorganic Research (SUNBOR Grant), Micro Algae Corporation, and HABA Laboratories, Inc.
Editor: Riks Laanbroek
- © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved


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7 June 2016 – Eight out of ten 18-year-olds believe young people are in danger of being sexually abused or taken advantage of online, and more than five out of 10 think friends participate in risky behaviours while using the internet, a new Unicef study shows.
Perils and Possibilities: Growing up online is based on an international opinion poll of more than 10,000 18-year-olds from 25 countries, revealing young people's perspectives on the risks they face growing up in an increasingly connected world.
"The internet and mobile phones have revolutionized young people's access to information, but the poll findings show just how real the risk of online abuse is for girls and boys," said Unicef's Associate Director of Child Protection, Cornelius Williams. "Globally, one in three internet users is a child. Today's findings provide important insights from young people themselves. Unicef aims to amplify adolescents' voices to help address online violence, exploitation and abuse, and make sure that children can take full advantage of the benefits the internet and mobile phones offer."
The new report finds that adolescents appear confident with their own ability to stay safe, with nearly 90 per cent of interviewees believing they can avoid online dangers. Approximately six out of 10 said meeting new people online is either somewhat or very important to them, but only 36 per cent strongly believe they can tell when people are lying about who they are online.
More than two-thirds of girls, 67 per cent strongly agree they would be worried if they received sexual comments or requests over the internet, this compares to 47 per cent of boys. When online threats do occur, more adolescents turn to friends than parents or teachers, but less than half strongly agree they know how to help a friend facing an online risk.
In the UK, 88 per cent of 18 year olds surveyed agree that children and adolescents are in danger of being sexually abused or taken advantage of online and would turn to their friends if they felt threatened or unsafe online. In comparison, 70 per cent would talk to their parents or caregiver if they perceived a threat online while less than half, 42 per cent, would talk to a teacher.
ChildLine, run by the NSPCC, is a free platform that provides advice on a range of issues including online risks for young people in the UK. Through message boards, the ChildLine website and their YouTube channel, children and young people can support each other if they feel unsafe online.
Putting young people front and centre as messengers and advocates to keep themselves safe online, is key to Unicef's global #ReplyforAll campaign, part of its EndViolence Against Children initiative. Though #ReplyforAll,children and adolescents worldwide will be asked to give their advice on the best ways to respond to online violence or risks and to raise awareness among friends through social media. This work has been supported by the WePROTECT Global Alliance, which is dedicated to ending the sexual exploitation of children online through national and global action.
Unicef, together with the WePROTECT Global Alliance, is calling on national governments to establish coordinated responses between criminal justice systems including law enforcement, and child welfare, education, health and the Information Communication Technology (ICT) sectors, as well as civil society, to better protect children from online sexual abuse and exploitation.
"When young people, governments, families, the ICT sector and communities work together, we are more likely to find the best ways to respond to online sexual abuse and exploitation, and send a strong message that confronting and ending violence against children online – indeed anywhere – is all of our business," said Williams.
Download broadcast quality photos and video.
Perils and Possibilities: Growing up online.
About the WePROTECT Global Alliance
The WePROTECT Global Alliance is dedicated to ending the sexual exploitation of children online through national and global action. Its vision is to identify and safeguard more victims, apprehend more perpetrators and create and internet free from this crime. The WeProtect Global Alliance is comprised of governments, companies and civil society organizations signed up to the commitments made at the WePROTECT Children Online summits in London (2014) and Abu Dhabi (2015) and the members of the Global Alliance Against Child Sexual Abuse Online.
About Unicef
Unicef is the world's leading organisation for children, promoting the rights and wellbeing of every child, in everything we do. Together with our partners, we work in 190 countries and territories to translate that commitment into practical action, focusing special effort on reaching the most vulnerable and excluded children, to the benefit of all children, everywhere.
Unicef UK raises funds to protect children in danger, transform their lives and build a safer world for tomorrow's children. As a registered charity we raise funds through donations from individuals, organisations and companies and we lobby and campaign to keep children safe. Unicef UK also runs programmes in schools, hospitals and with local authorities in the UK. For more information please visit unicef.org.uk
About ChildLine
ChildLine can be reached by calling 0800 1111
For further information, please contact:Lilly Carlisle, Unicef UK, 02073756212, firstname.lastname@example.com
Unicef UK Press Office, 0207 375 6030, email@example.com
UNICEF IS FUNDED ENTIRELY BY VOLUNTARY CONTRIBUTIONS.WE RECEIVE NO FUNDING FROM THE UN BUDGET© Unicef UK. The United Kingdom Committee for UNICEF Registered office: 30a Great Sutton Street, London EC1V 0DURegistered charity 1072612 (England and Wales) and SC043677 (Scotland). Registered company limited by guarantee 3663181 (England and Wales). All images © Unicef except where noted.

subset: commoncrawl


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Have you visited Dr. Rajesh Shetty? Rate you experience below.
Ophthalmologists in Jacksonville, FL
Dr. Rajesh Shetty specializes in ophthalmology in Jacksonville, Florida.
Dr. Rajesh Shetty has been compared 131 times. Details of Dr. Shetty's 20 years experience as an MD, his hospital affiliation and education at Indiana University are available on your free doctor report.
Dr. Rajesh Shetty has an average rating of 5 stars (out of 5) with a rating of Excellent based on 5 reviews.
Have you visited Dr. Rajesh Shetty? Rate you experience below.
Dr. Rajesh Shetty has been identified as specializing in Ophthalmology.
He obtained board certification from The American Board of Ophthalmology for Ophthalmology.
Insurance plans and networks accepted by a doctor are not necessarily universal across all locations at which the doctor practices. This can be affected by many factors such as different group practices, hospital affiliations and working arrangements at those locations. Checking that the doctor you are visiting and the facility they are at are in-network providers for your insurance plan can save you money and headaches later from surprise medical bills.
Dr. Rajesh Shetty accepts the following plans at Florida Eye Specialists
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Dr. Shetty is affiliated with 1 hospital:
Dr. Shetty attended medical school at Indiana University School of Medicine and graduated in 1996 having 20 years experience.
While there is no proven correlation between the quality of one's educational institution and the quality of one's professional competence, it is often an indicator. Medical schools have various levels of selectivity (some are harder to get into than others), and both the quality of their clinical training and level of research are measured. Additional medical training in both residency and fellowships and the facilities they are conducted at also provide a history of the doctors' level of training and experience.
Patients may also know Dr. Rajesh Shetty MD as:
2 Shircliff Way, Jacksonville, FL 32204
1034 Riverside Ave, Jacksonville, FL 32204
Jacksonville Eye Center, 2001 College St, Jacksonville, FL 32204
University of Florida College of Medicine, 580 W 8th St, Jacksonville, FL 32209
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This video says about itself:
Cute Lion Cubs Only 6 Weeks Old! – This Wild Life – BBC
1 September 2016
In Samburu, Northern Kenya there are three prides of lions that have a new generation of cubs.
This video says about itself:
West African lion – Video Learning – WizScience.com
24 September 2015
The "West African lion" , also known as the "Senegal lion", is a lion subspecies native to western Africa. Results of genetic research indicate that the Western and Central African lions form a different clade of lions and are perhaps more related to Asian lions than to lions from southern or eastern Africa. The genetic distinctiveness is particular of interest, since lions are regionally endangered in western Africa. With a total population of perhaps less than 1,000 individuals in all of West and Central Africa and no captive population, the West African lion is one of the most endangered subspecies of big cats.
Lions from western and central Africa are believed to be smaller than lions from southern Africa. It is also suggested that they have smaller manes, live in smaller groups, and they may also differ in the shape of their skull.
In the Pendjari National Park area, which is within the range of the West African lion, almost all males are maneless or have very weak manes.
The West African lion is distributed in western Africa south of the Sahara from Senegal in the west to the Central African Republic in the east. Another subspecies or North East Congo lion is traditionally described from northeastern Democratic Republic of the Congo.
Lions are rare in western Africa and may be critically endangered in this region. In 2004 there were probably only 450-1,300 lions left in West Africa. In addition, there were about 550-1,550 in Central Africa. In both regions, the area inhabited by lions has been reduced until 2004 to less than 15% of the historic range.
From Leiden University in the Netherlands:
Lions in West and Central Africa apparently unique
10 August 2016
Lions in West and Central Africa form a unique group, only distantly related to lions in East and Southern Africa. Biologists at Leiden University confirm this in an article published in Scientific Reports.
Genetic data
In this study, the researchers gathered a genetic dataset of lion populations covering a total of 22 countries. This included samples from each remaining lion population in West and Central Africa, a region where lions and other wildlife are rapidly declining as a consequence of the increasing human population. The researchers managed to gather all the information by teaming up with other people in the field and local conservationists.
300,000 years ago
Based on the genetic data, it was estimated that the split between the two major groups that can be identified in the lion must have occurred 300,000 years ago. To explain what happened in their evolution, the researchers made a reconstruction of African climatological history. It seems that periodic expansions of the rain forest and the desert drove lions into isolated pockets of suitable habitat, where the different genetic lineages originated that can still be observed today.
Other mammals
This influenced not only the patterns we observe in the lion, but also in other large mammals such as giraffe, buffalo, hartebeest, cheetah and spotted hyena. A general pattern is emerging that shows that many large African savannah mammals show very similar arrangements, with unique lineages in West and Central Africa.
Reason for concern
The strong declines in wildlife populations in large parts of West and Central Africa are therefore a reason for major concern. The fact that this region seems to harbour a lot of unique genetic lineages makes conservation in the area extremely important. A delegation from Leiden University will participate in the IUCN World Conservation Congress in September 2016, and will lead a Side Event that aims to establish a Species Action Plan for West and Central Africa. The researchers hope that this will facilitate coordination and funding of projects in the region.
This video says about itself:
Lion Facts for World Lion Day 2016
Aug. 10th is World Lion Day. How much do you know about these awesome big cats? Watch & share!
Art by Ken Gitau
From National Geographic:
Today is World Lion Day. Give a roar for the big cats you've helped save!
It's a special day to celebrate lions and to recognize their critical role in helping nature to thrive.
But it's also the day to remember Cecil, the lion killed by an American trophy hunter, and so many other lions now at risk. With lion and other big cat populations declining worldwide due to hunting, conflict, habitat loss, and other threats, it's urgent that we take action for all big cats in the wild now!
Please celebrate World Lion Day and help give threatened big cats a future by donating to the National Geographic Society's Big Cats Initiative today.
To show how your generous support can make a difference, I'd like to share updates from two innovative, community-based programs that are saving lions right now.
Shivani Bhalla works in northern Kenya. She hires young Samburu warriors to be peaceful ambassadors for lions, and reduces conflicts between humans and big cats through her Warrior Watch program. They've already prevented the killing of at least 35 lions! And now, thanks to the funding provided by National Geographic Society supporters like you, she's training more warriors to join her efforts.
Amy Dickman is helping farmers and families in Tanzania prevent conflict with lions by using proven techniques like reinforced bomas (fences) and guard dogs to better protect livestock. She started with five villages, but now she will be expanding to 15 with your help. Your generous donation to the Society could prevent the deaths of up to 1,000 lions and 200 cheetahs!
The impact of these projects is powerful and proven. They show how, with your generous support, the Society funds innovative ideas that empower our explorers to test, improve, and expand effective conservation programs.
But to make a larger difference more funding is needed now to invest in the solutions that save lions and other big cats. Today, I am asking for YOUR support.
Please help the National Geographic Society invest in more innovative programs to protect lions and other threatened wildlife today.
As you think about what World Lion Day means to you, remember that lions aren't only amazing and majestic creatures on their own . . .
They are also a vital part of a larger habitat that includes many other animals. When lions thrive, the whole ecosystem is healthier. When lions and other big cats decline, nature is thrown out of balance.
Shivani Bhalla and Amy Dickman are just two of the brilliant conservationists working with communities to help save lions. The impact of their work is spreading!
And so is the impact of our generous supporters. In the last six years, Society donors have helped us support 88 projects in the field in 27 countries.
Now it's time to do even more together. Because with lions and other big cats facing increasing risks, we must intensify our efforts and expand proven programs.
You can help. Please celebrate World Lion Day right now by joining with the National Geographic Society to fund even more innovative, on-the-ground solutions where they are needed most.
Only by working together can we give lions and other threatened wildlife a future. We need your help.
Thank you!
Sarah Stallings
Director, Annual Giving
This video says about itself:
14 June 2016
We all were anticipating a fairly easy catch by these 2 hunting lions. But this turned out to be a hilarious fail by them!🙂
Taken on a safari in South Africa.
Video by: Natasha
This video from South Africa says about itself:
Lions Bring Down Buffalo | Absolutely Crazy Ending!
23 May 2016
We have had the honor of hundreds of people from around the world sending in their incredible videos for us to share, and each is unique. This one however is one that is just extremely special and plays on every emotion!
Incredible too see how two lionesses from one pride chase the herd into two males from a different pride. The 4 of them are happy to share the meal, however, "the enemy of my enemy is my friend" happened to the buffalo when the lioness from the male's pride comes in and a huge fight breaks out, leaving the buffalo a great opportunity to escape without even a limp.
Taken in the Mala Mala Private Game Reserve in the Greater Kruger National Park in South Africa.
Video by: Ranger Mike
This video from South Africa says about itself:
Lions Chase Buffaloes Into Tourists' Car
14 May 2016
Scary video showing a group of tourists being at the wrong place at the wrong time as 2 male lions take their chance at buffaloes, chasing the herd right into a spectating vehicle.
Thank goodness the people in the car are doing well and weren't hurt.
Taken on the H1-4 near Olifants in the Kruger National Park in South Africa.
Video by: Penny Osborne
This video from South Africa says about itself:
Roaring rescued lions free at last
1 May 2016
Thirty-three abused circus lions arrived at OR Tambo Airport from Peru and Colombia, en route to their new home in Limpopo.
Animal Defenders International co-founder Tim Phillips describes the difficulties of rescuing the 33 lions who were scattered around in 12 different illegal circuses.
For more about this story click here.
Also related to the arrival of the lions – read here.
This video from South Africa says about itself:
Rescued Circus Lions Given Freedom In South Africa
2 May 2016
Thirty-three lions rescued from South American circuses landed in South Africa. Tasting their new freedom, the lions were released into a bush sanctuary for big cats.
Animal Defenders International said it rescued 24 of the lions in surprise raids on circuses in Peru.
In the statement released by the organization, the company said, "Nine were voluntarily surrendered by a circus in Colombia. Almost all of the rescued lions have been mutilated to remove their claws, one has lost an eye, another is almost blind, and many have smashed and broken teeth so would not survive in the wild," the organization.
The president of Animal Defenders International said it was their largest airlift in history.


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THOUSAND OAKS, Calif. – There's an intriguing conundrum in professional golf. The better a player is, the more he's criticized when he doesn't have his best stuff. Tiger Woods could tell you a thing or two about what he thinks of that. Nobody cares if a journeyman misses the cut, but when an elite talent "only" finishes in a share of eighth place? Sound the alarms...
Rickie Fowler Plagued by Back Problems Since U.S. Open
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craft beer
The Right Way to Pair Beer and Food
There are two drinks we care about on this week's podcast episode: beer and coffee. It's no surprise that our editor in chief has *a lot* of thoughts on the matter.
10 More Beers that *Should* Be Renamed 'America'
These locally-brewed, innovative beers deserve to make the All-American squad.
Why the World Needs More Crowlers—Wait, What's a Crowler?
What is a Crowler? For those who love beer, it's about to change the game.
Crush These 14 Canned Craft Beers on Super Bowl Sunday
For today's craft brewers, it's all about aluminum (and cool graphics).
Boozy Root Beer is Taking the Country By Storm
How Not Your Father's Root Beer came to dominate the hard root beer market, and why people are clamoring for the boozy, nostalgic soda pop.


text:
Take-Two Launches Sports Label
2K Games internal development studios will be Visual Concepts (ESPN NFL 2K5, ESPN NBA 2K5, ESPN Major League Baseball), Kush Games (ESPN NHL 2K5), Indie Built (Top Spin, Amped, and Links) and Venom Games (Rocky and Rocky Legends) along with PopTop Software and Frog City Software. It will also incorporate the Take-Two licensing team. Some titles planned as Global Star releases could be the first to bear the brand on store shelves.
"Consistent with our strategy to diversify Take-Two's product portfolio, the team at 2K Games is rapidly building a sweeping lineup of high-profile licensed and proprietary titles for console, PC and handheld platforms," said Take-Two President Paul Eibler. "From high-profile Hollywood licenses to PC-only titles to sports titles, 2K Games will be defined by exceptional games for all genres and platforms," he added. That last part explains the inclusion of PopTop and Frog City - known best for strategy titles Railroad Tycoon 3 and Tropico 2 respectively - under the new label.


text:
The third version of Google's much-maligned Penguin algorithm was released back at the end of 2014. Since then, we have enjoyed relatively stable search results, at least where links are concerned. That all changed a few weeks ago, when the fourth version was released, shaking things up once more.
As a quick review, what actually is the Penguin algorithm? Penguin is a set of filters and changes to how Google ranks sites, based entirely on links. While we don't think of penguins as predators in nature, Google's version of the animal seeks out cases where links have been abused to rank a site higher than it deserves.
Before the first set of Penguin updates hit, links were either valuable or not. If a link you had pointing at your site was considered spammy by Google, they devalued it. It didn't hurt your site beyond the loss of the value of that link, and it was easy enough to simply create a few more links to counteract that loss.
Penguin added a penalty on top of that. When a link was discovered and labeled spammy, Google would essentially make that link toxic to your site. Instead of simply being valueless, it would actively hurt your site until it was either remove…


text:
Thirty Frames a Second
Simple MindsBuy this song
Today I saw a film going backwards Thirty Frames A Second Was a man I recognized Say who you are I lost my job Security Self confidence Bank account Identity The league of youth Coming backward at me Thirty Frames a second Say who you are Go back to father Father where's my food? You food is on the table That can't be food It's dirt Say who you are Look at my skin Look at my face Look at my arms Getting younger Going backwards Thirty Frames A Second Say who you are I crash my car Collapse in town Young immigrants And legionnaires Fighting back back backwards Thirty Frames A Second Say who you are Some friends of mine I thought were dead A coming back "What are you doing coming back Jack" Say who you are Feel I'm going backwards Thirty Frames A Second
Written by: CHARLES BURCHILL, DEREK FORBES, JAMES KERR, BRIAN MC GEE, NORMAN MICHAEL MC NEIL
Lyrics © Sony/ATV Music Publishing LLC, Warner/Chappell Music, Inc., BMG RIGHTS MANAGEMENT US, LLC
Lyrics Licensed & Provided by LyricFind
Discuss the Thirty Frames a Second Lyrics with the community:
Use the citation below to add these lyrics to your bibliography:
"Thirty Frames a Second Lyrics." Lyrics.com. STANDS4 LLC, 2016. Web. 22 Oct. 2016. <http://www.lyrics.com/lyric/5042155>.


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Special Needs Children event on October 23, 2016
Become a T-Bird spirit leader for a day!
The T-Bird organization is proud to offer kids with special needs an opportunity to experience being part of cheerleading team at no cost for the families. On October 23rd, registered participants will experience cheering on the sideline during the Midget football game and they will perform a halftime routine with the Midget cheerleaders!
For more information and registration:


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Today, I learned that my new parakeet hates her reflection, and will screech loudly day and night unless I take the mirror out. My other parakeet loves the mirror and constantly cries out when I remove it. I can't win. FML
by bird / 01/08/2014 at 3:27pm / United States (Michigan) / Animals
Today, I'm now able to put my acne cream on my face without having to look in the mirror, because I've memorized the crater and trench-filled war zone that is my acne-riddled face. FML
by Anonymous / 01/04/2014 at 1:38pm / United States (Michigan) / Health
Today, I lost my virginity. We did it on the floor in my step-sister's room, and the entire time he kept pushing my head into the carpet. I lost my V-card but gained rug burn on my face that looks like a fatal disease. FML
by Anonymous / 01/04/2014 at 1:10am / United States (Michigan) / Intimacy
Today, a customer started a conversation by telling me how smart he'd heard I am, and finished it by explaining his theory that only smart people commit suicide. He then gave me a knowing look and said, "Just something I thought you should think about," and left without buying anything. FML
by Okay_Then / 01/01/2014 at 6:49pm / United States (Michigan) / Work
Today, my wife got so drunk she kissed another guy when the ball dropped. FML
by dantko / 01/01/2014 at 12:04am / United States (Michigan) / Love
Today, after being married for 20 years, I found out that my wife has accounts on multiple dating sites, "just in case." FML
by Anonymous / 12/28/2013 at 12:10am / United States (Michigan) / Love
Today, I had sex with my boyfriend for the first time, after he repeatedly told me not to worry about bleeding, and reassuring me that he'd take care of me. He passed out halfway through. FML
by JoshuasGirl / 12/23/2013 at 2:13pm / United States (Michigan) / Intimacy
Today, I did the "walk of shame" sixteen blocks. It wouldn't have been so bad if the sidewalks and streets weren't completely covered in ice. Somewhere along the way I lost what little dignity I had left, along with my left shoe. FML
by Anonymous / 12/21/2013 at 6:29pm / United States (Michigan) / Intimacy
Today, I finally got the courage to tell the girl I like how I feel. She instantly burst out laughing and said "A crush? Dude, what are you, 12?! Hahaha!" FML
by um...no? i don't think so anyway / 12/15/2013 at 2:16pm / United States (Michigan) / Love
Today, I received an unexpected package from my mother. It contained my cat's ashes. Apparently he died 3 weeks ago and she thought this was the best way to tell me. FML
by Cat Ashes / 12/05/2013 at 12:18am / United States (Michigan) / Animals
Today, after having my sinuses draining for a couple days, my ma stopped by for a surprise visit. Upon discovering the trash can full of used tissues, she called my pastor grandfather to talk to me about the chronic masturbation problem I don't have, but that everyone now thinks I have. FML
by Thors_Hammer9999 / 11/23/2013 at 1:05am / United States (Michigan) / Miscellaneous
Today, I tried to put the little girl I babysit in timeout for lying to me. I was about to sit her down when she made a mad dash for the basement stairs, slipped on the wood floor, and fell down them. She told her parents and everyone at the ER that I had thrown her down the stairs. FML
by little_star78 / 11/13/2013 at 6:08am / United States (Michigan) / Kids
Today, while taking an order over the phone for the customer at work, I began to hear slight moans. The moans gradually became faster and louder, until climax was achieved and I realized I was being used for phone sex. FML
by long day / 11/12/2013 at 3:29pm / United States (Michigan) / Work


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Quick rebound on Thomas Jockin lovely Line Slab. While doing this I realized how challenging this kind of style can be.
I only played with the way the slabs are dealt with. I felt like opening the slabs would lighten up the design, make it a bit simpler while bringing some "fanciness" to it. Doing this, I had to close the left top slab of the T for consistency, and keep the slab of the a closed. I know it might seam weird, but if you respect the ductus, this actually make sense… I think :)
For the serif, I used the same system and brought the slabs back to the h and the m, to see if it works.
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Hello everyone, the name's Brandon and I live in northern Jersey. I have been raising my two hens for 3 months now and would like to show you all my setup. I have made a quick video earlier today and was hoping some of you can check it out for suggestions/comments. I am fairly new at the backyard chicken thing but I have much enthusiasm for the hobby. Thank you all very kindly. The link is below to the YouTube video I uploaded.
Quick background: I bought these two beautiful hens at a hatchery in Quakertown PA (Moyer's Chicks I believe). They hatched the same day I picked them up. They are "Red Rocks" which I believe are the cross between a Rhode Island Red x White Rock chicken. I also think I may have misspoke in the video where I said "White Rocks" which is clearly not the case. My apologies in advance. I got them Sept. 30 2016 and have raised them in a makeshift brooder I had until early November where I migrated them to the coop I managed to buy/build/modify in my backyard (which you will see in the video). They are very social/friendly and seem to be very healthy so far and have had no problems. I have done extensive research especially on this website's forums and Learning Center. I just want to join the forum in a friendly manner and hope to make chicken buds (as corny as that sounds). Thank you all. I am also new to this forum so sorry for posting this in an inappropriate section of the forums.
Edited by JerseyChickhens - 12/5/15 at 4:26pm


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This page contains Cheats for Nintendogs + Cats: Golden Retriever And New Friends organized by sections for 3DS. This game has "Simulation Virtual" as genre, made by Nintendo, released on Mar 27, 2011. If you can't find a hint or secret in our list, then please check this page periodically for the latest updates.
- Developer: Nintendo
- Publisher: Nintendo
- Genre: Simulation Virtual
- Release: Mar 27, 2011
- ESRB: Everyone
If you know cheat codes, secrets, hints, glitches or other level guides for this game that can help others leveling up, then please Submit your Cheats and share your insights and experience with other gamers.
How do you shower? I really need to clean my dog, lady, and she is so dirty!...
Posted on: 02-17-2016
I dont understand how to make the dogs cuddle everyone keep saying try to make them cuddle with the Frendly Whiff, Flower Waltz or the Nighttime Song. I dont have the Fre...
Posted on: 11-30-2014
Is there an easier way to get more money and, or rare items example ( lucky collar )...
Posted on: 01-14-2014
How do you view your trainer points please if any one knows please tell me!...
Posted on: 11-23-2013
If you get far in the game, at a BARC shop (it's spelled BARC, not bark or barck), they will sell a gold bar. It is so expensive! besides, i don't even know what it does....
Posted on: 07-20-2013
I don't know where the trainer points are in this game. Please tell me where they are!...
Posted on: 07-20-2013
In the first nintendogs, when you bought a house design, the next time you wanted it, it was free. In the new nintendogs, they make you pay for it all over again! If anyo...
Posted on: 07-19-2013
Stuck in this game? Set a specific, clearly defined question and let other gamers answer your question or view answers to previously asked questions. If you think you are an expert then please try to help others with their questions.
Nintendogs + Cats: Golden Retriever And New Friends - Trailer
- Aug 25, 2014, Views: 129
Nintendogs + Cats: Golden Retriever And New Friends - Gameplay #2
- Aug 25, 2014, Views: 68
Nintendogs + Cats: Golden Retriever And New Friends - Gameplay #2
- Aug 25, 2014, Views: 80
- The Journey: Bob's Story, (PC)
- Call Of Duty: Infinite Warfare, (PS4)
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- Cartoon Network: Battle Crashers, (3DS)
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Product DescriptionBaby Born: If your dolly s feeling poorly then give him/her a night on the Baby Born Doctor s Bed. It comes with lots of accessories including a blanket, pillow and medical patient chart. The doctor s bed also has four wheels so you can gently push it along without disturbing your dolly. It s the perfect place to make a full recovery! A great gift for Baby Born fans aged three and up. Please note: doll is not included.
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Join Janine Smith for an in-depth discussion in this video Adding captions and notes, part of Photoshop Elements 9: Scanning and Restoring Photos.
- Overview
- Transcript
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An important part of photo restoration that often gets overlooked by the family…historian is the inclusion of historical data.…The little details of who's in the picture and what's happening that will get…lost down the road and nobody knows who that is.…How do you attach historical data to a photograph?…We use to type out the information and make a copy of it with the photo to put…in our records or heaven forbid we'd write on the back of the photograph itself.…But with the millions of electronic copies of photos being passed around the…Internet today that no longer works very well.…
You can however attach information to a photo electronically in the form of…metadata, or as it's known in the Elements organizer, captions and notes.…Still in the Organizer workspace, we will select this photo and go up to Window…and Properties and here you have a space to make all your notes and a caption.…Then you Caption you want a good description of what's going on in the photo? Who it is?…So we will put Nana and this is her graduation photo, so Nana's graduation.…
- Determining equipment needs
- Scanning negatives, slides, and film
- Importing photos in Photoshop Elements
- Adding captions, keywords, and Smart Tags
- Adjusting contrast
- Fixing fading with Threshold
- Making automatic fixes with guided edit
- Removing dust, spots, and texture with the healing tools
- Repairing rips and tears
- Sharing restored images


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Killesberg Tower Chilis Denial Behind the Train Station at night Impatient? People of London People of London - the blue bag Silhouette Selfportrait In The Eye People of London Konfi Lukas (color) The Wall People of London People of London London Tube - Downstairs (bw) Red House London Tube - Downstairs (bw) The Door Stupid Hotel Games: Part Four: The Mirror The Bow (Panty) The Bow (Bra) Portrait of a Killer On The Woodblock Done. Killing Time Don't mess with me buddy Wanna meet her in the forrest at night? She's gonna take you Wanna meet her in the forrest at night? Dan Dan & G Dan Portrait Anna Jump Anna Jump Throw Wind Path Desert Rose Jump Jump Queen of the castle hey you The cigarette The crop Late Night Cigarette Late Night Cigarette (vintage version)
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Should I Divorce? 11 Questions To Answer Before You Decide to Stay or Go
by Jennifer Jessica
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Victim of adultery, abuse, or just fed up with your present marriage and life ?
If you're contemplating a divorce, then you're already aware that you are considering making one of the most significant decisions of your life. There are so many issues to evaluate: finances, children, compatibility etc. You may feel like throwing in the towel one day, yet feel like you want to persevere in your marriage the next. There's a good chance you're losing sleep over this decision. You may even feel like you're going crazy.
We're surrounded by examples of marriage, both discouraging and inspirational. On the one hand, we have the example of the infamous 72-day marriage of Kim Kardashian and Kris Humphries. On the other hand, we have the inspirational example of Dana and Christopher Reeve. Christopher (most famous for his role as Superman) married Dana in 1992, but was paralyzed in an equestrian accident in 1995. Dana cared for Christopher until he died in 2004, and Dana passed away soon after his death.
Was Kim right to give up on her marriage after 72 days? Was Dana robbed of a rich life with a fully functional partner, or did she make the right decision? The decision to remain married or to divorce is a highly personal, and subjective decision. No one but Kim knows if she should have left, and no one but Dana knows if she made the right decision when she chose to stay.
In a national survey divorced Americans were asked if they wished they had worked harder to save their marriage, and if they wished their ex had worked harder to save the marriage. A surprising 62% of the divorced Americans surveyed said they wished their ex had worked harder to save the marriage, and only 33% of the people surveyed answered no to both questions. Several other studies have come to similar conclusions: that the majority of divorced Americans wish that either they, or their ex, had worked harder at the marriage before deciding to divorce.
Eleven Questions to Ask Yourself Before You Get a Divorce
Your decision to work on your marriage or to get a divorce is an emotionally charged decision. The following eleven questions address the most important pillars of marriage and the issues couples consider the most important factors necessary for a successful union. As we address each topic, we encourage you to ask yourself the questions presented, and to evaluate your answers. Are there ways to constructively deal with the problem, or is the relationship beyond repair?
Find out now !
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by on October 25, 2016
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Hotels in Castelrotto, Italy
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- Most bookedChalet Tianes
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"Unbelievable gem high in the Seiser Alm. Gorgeous, beautifully appointed room with stunning views from the balcony. Spa and pool area were world-class. And then there was the food. Half board: amazing breakfast and dinner. We travel frequently and this has to be in our top three favorites o..."
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"My room had an enclosed balcony with an incredible view of Seiser Alm!!"
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"You will find the rooms, the food, the spa, the service, the gardens and view spectacular. This is a spectacular hotel. Go!"
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"All of the staff at the Plaza, were great. But Robert, seemed to be everywhere, performing any task, all the time. He easily shifted to cater to each of his client's language, customs and needs, and always with a big smile. And Barbara couldn't have been any kinder, nor more helpful to us. At man..."
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If you are looking for Castelrotto hotels in a particular area, you can use the search feature at the top of the page to search by neighborhood, landmark, address or zip code, or you can search by using the built-in map feature. You can also browse hotels in Castelrotto by type, amenities, and star-rating so that you can find the best hotel. Currently, top rated hotels in Castelrotto are:
- Hotel Saltria - true alpine living
- Hotel Arvina
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The location of your Castelrotto hotel is important. If you are traveling to Castelrotto, &StateorCountry for business, you may want to consider getting a hotel close to the airport or near your meeting venue. By doing so, you can really cut down on transportation costs and time. You may also want to book a hotel that offers free wifi just in case you need to get some work done.
If you are traveling for vacation, figure out which attractions or landmarks you want to see and book a hotel that is within close proximity. Although hotels near attractions might cost a little more, you may find that spending those extra few dollars is worth the convenience. You can even find hotels that align with your interests. For example, if you are looking for a relaxing vacation, a spa hotel may be the perfect choice for you.
You'll also want to determine which type of accommodations will suit your needs before booking a hotel in Castelrotto. If you're traveling with the kiddos, a family friendly hotel will offer larger rooms and more sleeping arrangements. For those traveling for an extended period of time, booking a hotel suite with a kitchenette is a smart move and will feel a little more like home. If you are budget conscience and just need a place to rest your head, a hostel or motel may be all you need.
If you are looking to score the best discounts and deals, use the filter to sort by price from low to high. You can book a hotel in Castelrotto for as low as $71 per night. If you are looking to cut down on expenses, look for hotels that include free breakfast and parking. Some hotels even offer free shuttle service to and from nearby places. You can also sort by star rating, proximity, and guest rating.
Before you book, check out user reviews. Hotels.com has 92 reviews in Castelrotto. We show reviews from Expedia and TripAdvisor so you know that you are reading genuine, reliable reviews. Be sure to download the Hotels.com app so you can book your reservations on the go, view amenities, and save the hotels you want to view and compare later.


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Home > Web Development::Site Administration > Force 1 DomainCheck 1.0
Force 1 DomainCheck 1.0
F1 DomainCheck for Windows, see if a domain is available.Company: Force 1 Network
Platform: WinXP,WinVista,WinVista x64,Win7 x32,Win7 x64
Size: 1381 KB
Price: USD $0.00
Release Date: 2011-06-14
Screenshot URL: http://force-1.net/dwn/domaincheck.jpg
Category: Web Development::Site Administration
Force 1 DomainCheck will let you to check the availability of domains immediately. Search, look up, purchase, register, transfer and/or monitor domain names, internet Web addresses, or URLs. You simply type your domain, and the program will automatically inquire your input.
Related keywords:
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Force 1 DomainCheck Related Titles:GrafX Website Studio 2.5.11 - Responsive web design makes your website flexible to your tablets or mobile.
SEO SpyGlass Enterprise 6.16.7 - SEO SpyGlass Enterprise: powerhouse Google competition analyzer for SEO pros.
SEO SpyGlass Enterprise 6.16.7 - Matchless backlink checking tool analyzing up to 100,000 links per search
WebSite Auditor Enterprise 4.14.7 - An SEO tool that makes on-page SEO easy (optimizes site structure AND content!)
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SEO PowerSuite Enterprise 55.6 - SEO PowerSuite offers fullest professional SEO management of a website.
SEO PowerSuite Enterprise 55.6 - A one-stop-shop SEO toolset for rank tracking, backlink analysis & SEO reporting
SEO SpyGlass Professional 6.15.6 - Exceptional backlink analyzer and competition researcher for SEO experts.
SEO SpyGlass Professional 6.15.6 - Backlinks in one click with SEO SpyGlass backlink checker by Link-Assistant.Com
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SEO PowerSuite 54.3 - The ultimate collection of rocking SEO tools for effective optimization.
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"Thanks Tracey, Lexi loved you and would be very happy to see you again soon. we will be in touch..."
"We just had Yvette stay at our house for 4 nights and 5 days looking after our 2 furry kids Jack..."
"Amy is a lovely, caring girl who developed a lovely bond with my pets who were very happy on my..."
"Cait was fantastic, we came home to very happy pets and clean house. The meet and greet was..."
"Ben was Excellent. He treated our boys like they were his own & in top of being well cared for they ..."
"Hannah was great, dogs were happy All good. "
"Lovely sitter with great space for the dogs. Thanks Ailsa!"
"Dogs happy and contented. Very polite, respectful, house left extremely tidy. Would like a few..."
"Picked Jac up yesterday. She was very happy. She seems to have had a great holiday playing with..."
Pet sitting in Pakenham . Pet boarding alternative to kennels in Pakenham . Find a trusted pet sitter in Pakenham . We verify our pet sitters and the stay of your dog or cat is insured.


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Dazadi Coupons + cashback
Add more entertainment and fun to your home courtesy of Dazadi. It is the online destination that offers the largest selection of topnotch sporting goods, game rooms, trampolines, swimming pool, arcade games, and so much more. Unlike other shops, all of their products are offered at discounted prices all year round so you can get whatever you want. More information available at dazadi.com
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Main Content
Let your young fan celebrate his New York Giants fandom with this Odell Beckham Jr Name & Number T-shirt. It features New York Giants graphics along with his favorite player's name and number on the back. This stylish New York Giants tee also boasts a rib-knit collar.
- Material: 100% Cotton
- Rib-knit collar
- Tagless collar
- Crew neck
- Short sleeve
- Screen print graphics
- Machine wash
- Officially licensed
- Imported
- Brand: Outerstuff
Giants Customers also liked:
Special event items are produced by manufacturers only after the outcome of a game or event. These are advanced sale items and will ship immediately after they are received in our warehouse.
Manufacturer direct items are shipped directly from the manufacturer. These items are not available for international or expedited shipping.
Customized items can be personalized with options such as your name, your favorite number, and/or designs. Some options may be limited by league rules.


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24 Thanksgiving Dinner Recipes and Fall Craft Projects
Bonus: Get our newsletter & special offers for free. We will not share or sell your email address. View our Privacy Policy
Leafy Autumn Wreath
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Click here to view your Craft Projects.
This leafy autumn wreath from The New Image Group is an autumn or Thanksgiving craft that your kids can help you make. It will look great on the wall or hanging on a door to welcome in the rich colors of autumn.
- Scissors
- Fabric Glue
- 12 x 18 Red EZ Felt Sheet
- 12 x 18 Yellow EZ Felt Sheet
- 12 x 18 Tangerine EZ Felt Sheet
- 12 x 18 Cocoa EZ Felt
- 36" String of Jute/Twine
- Pattern
Cut out 1 Each of Leaves on all 12 x 18 Sheets of EZ Felt.
You should have 4 Oak Leaves, 4 Maple Leaves, and 4 Standard Leaves
Begin arranging how you would like your wreath to look. You can follow the picture above or create your own. You will need to overlap the figures. Our wreath is about 18" in diameter.
Begin gluing pieces in place with fabric glue. Let dry.
Once glue has dried, glue Jute/Twin to the back of the wreath.
- Hang on door.
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- 268 Thanksgiving Crafts for Kids and Adults
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- "9 Thanksgiving Decorations to Make" eBook
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- Homemade Halloween Decorations
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- 11 DIY Christmas Decorations and Gift Ideas
- 11 Teacher-Approved Crafts for School
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The Hospice of Southern Illinois Board of Directors is also steadfastly dedicated to its mission of enhancing the quality of life for individuals and their loved ones touched by a terminal illness. The Board is composed of professionals from across the Hospice of Southern Illinois service area who serve on a volunteer basis.
|Carol Mestemacher, Chairperson
Karen Kunsemiller, Vice Chairperson
Matthew Waters, Treasurer
Jennifer Barbieri, Secretary
Carolin King Harvey, Member-At-Large
Abel Anderson
Laura Beckman
Lareva Bradley
Rex Budde
|Cheryl Creasy
Sharon Frederking
Anthony Garavalia
Kay Mueggenburg
Catherine Newbold
David Olson
Dean Salvatore
Sandy Wagner


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Amenities + Details
Handicap Accessible
Liability Insurance
On-Site Accommodations
Outdoor - Covered
Reception Area
For more details about amenities, please message the Venue.
$ – Inexpensive
Ballroom, Hotel, Restaurant, Tented
Food + Catering, Planning, Service Staff
Contact Info
Perfect Wedding DayReviewed on 8/25/2011 Jacqueline V
Kathleen and her staff were excellent and we were thrilled with everything! From our beach ceremony to the outdoor tented reception! They set it all up and provided amazing food, service, value, and flexibility with all of our needs.
Water doesn't stop usReviewed on 7/16/2011 Kelly C
This resort is located less than a mile from Goose Rocks Beach but was secluded with beautiful gardens surrounding the property. We had wanted an outdoor tented wedding with all of our guests staying at the same property. Kathleen and Peter who run the resort made us feel at... Read More home and were available for whatever we needed. Unfortunately our vision of an outdoor ceremony and tented wedding was made impossible by the rain. With Kathleen and Peter's help we were able to have everything under the tent (they provided umbrellas for the walk to the tent) which had clear panels so you can see the rain drops falling. It was wonderful!!! Even during the planning process Kathleen and Peter were quick to make suggestions and able to accommodate our vision. We are very thankful and had a great day!! Not even the rain could stop the party!! If you want an at home feel with an intimate wedding, this is the place to go!
Loved itReviewed on 7/12/2011 sally m
My ceremony was by the pond. Gorgeous, intimate location. We had the reception under a tent and it was just perfect!


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Saturday Markets London
Alice's marks the beginning of the Portobello Road market in London. Saturdays was antiques. Best poking around ever. <3
Portobello Road Market in Notting Hill is one of the most famous street markets in the world. The Saturday antiques market is the most popular but there is a street market six days a week. Portobello Road is lined with well-established shops and is not the average 'High Street' as most are independent stores. There has been a market on this street since around 1870. As well as the antique stalls there are a whole host of arcades, galleries, shops and cafes.
Shop in style. More
London, Greenwich & Blackheat, Greenwich Market, Entrance
London flower market love. flowers. churchill. flowers. fleur. flores. inspiration. inspirational.
Borough Market, Southwark, London - the best market of all out of the many I have seen.
15 Secret Places to Discover in London | Sunday Chapter | Bloglovin'
Christmas markets, festive jumpers, the trees to see, ice rinks and a Christmas lights tour! All you need to get into the festive spirit.
Christmas Markets in London ~ A great list of all the Christmas Markets in London and info on how to get to them.
Add these 20 things to do in London to your next vacation itinerary ranging from shopping to best afternoon teas to Christmas markets! #LondonMoments


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DNA Project Web Video: Cape Town Station Activation
DNA Project and creative agency FoxP2 Cape Town have launched this web video raising our knowledge about the crime investigation.
Thousands of criminals escape conviction due to a disturbed crime scene evidence in South Africa only. Therefore the DNA Project presented an interactive campaign: they made a 6x8m mosaic out of small white stones. The mugshot of a criminal was sprayed on these tiny stones. It was placed at the metro station. When people walked over, the installation faded away, leaving just a white dusty ground. It expresses the interference of ignorant people with a crime scene.
Watch the video and tell us if you liked it.
Advertising Agency: FoxP2 Cape Town, South Africa
Executive Creative Director: Justin Gomes
Art Directors: Reijer Van Der Vlugt, Brendan Hoffmann
Copywriters: Justin Osburn, Kenneth Van Reenen
Installation built by: More Design
Video shot and edited by: NiceOneSteve


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This long elegant maxi dress features a strapless top with padded cups. Floral print through out, tonal stitching. 56 inch measured from shoulders to finished hem. Polyester/spandex. Imported.
You're reviewing: MINT FLORAL PRINT STRAPLESS LONG MAXI DRESS
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