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Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in mediating biotic and abiotic stress responses. In plants, MAPKs are classified into four major groups (A-D) according to their sequence homology and conserved phosphorylation motifs. Members of group A and B have been extensively characterized, but little information on the group D MAPKs has been reported.
In this study, we isolated and characterised GhMPK16, the first group D MAPK gene found in cotton. Southern blot analysis suggests GhMPK16 is single copy in the cotton genome, and RNA blot analysis indicates that GhMPK16 transcripts accumulate following pathogen infection and treatment with multiple defense-related signal molecules. The analysis of the promoter region of GhMPK16 revealed a group of putative cis-acting elements related to stress responses. Subcellular localization analysis suggests that GhMPK16 acts in the nucleus. Transgenic Arabidopsis overexpressing GhMPK16 displayed significant resistance to fungi (Colletotrichum nicotianae and Alternaria alternata) and bacteria (Pseudomonas solanacearum) pathogen, and the transcripts of pathogen-related (PR) genes were more rapidly and strongly induced in the transgenic plants. Furthermore, transgenic Arabidopsis showed reduced drought tolerance and rapid H2O2 accumulation.
These results suggest that GhMPK16 might be involved in multiple signal transduction pathways, including biotic and abiotic stress signaling pathways.
Cotton (Gossypium hirsutum); GhMPK16; Pathogen resistance; Drought sensitivityBackground
Stresses such as drought, high salinity and fungal infections constitute a major limitation to crop productivity. Plants have developed sophisticated defense mechanisms to deal with diverse unfavorable environmental factors. The mitogen-activated protein kinase (MAPK) cascades are conserved pathways through which extracellular stimuli are transduced into intracellular responses in all eukaryotes [1,2]. Plant MAPK cascades have been shown to regulate a number of essential biological processes, including growth, development and stress responses [3].
MAPK cascades are composed of three interlinked protein kinases: MAPKK kinases (MAPKKKs or MAP3Ks), MAPK kinases (MAPKKs, MAP2Ks or MEKs) and MAPKs. MAPKs are the terminal components in this cascade, and they are regulated by the dual phosphorylation of the conserved T-X-Y motif located in the activation loop by upstream kinases (MAPKKs). There are 20 MAPK genes identified in Arabidopsis, and a similar repertoire of genes have been found in other plants, such as rice (Oryza sativa), poplar (Populus sp.) and grapevine (Vitis vinifera) [3-5]. The MAPKs can be categorised into four major groups (A, B, C, and D) based on the phylogenetic analyzes of amino acid sequences and phosphorylation motifs (TEY and TDY). The TEY subtype can be classified into three groups (A, B and C), whereas the TDY subtype is found in the more distant group D [4,5].
In Arabidopsis, three particular MAPKs in groups A and B (MPK3, MPK4 and MPK6) have been extensively studied. Both biochemical and genetic analyzes have been performed for each of these isoforms, which appear to work in multiple signaling pathways and play crucial roles in many distinct processes ranging from stress responses to developmental processes [3]. Information about group C MAPKs has recently emerged. Three members of group C, MPK1 and MPK2 in Arabidopsis and PsMPK2 in pea (Pisum sativum L.), are transcriptionally induced by a variety of stresses [6,7]. More recently, Arabidopsis group C MAPKs, including MPK1, MPK2, MPK7 and MPK14, were reported to be activated by MKK3, and MKK3-MPK7 participates in pathogen signaling [8]. Cotton GhMPK7 may play a role in pathogen resistance, plant growth and development [9].
Based on the phylogenetic analysis and pairwise comparison of Arabidopsis and rice MAPKs, it has been proposed that the rice genome contains more MAPKs with a TDY phosphorylation motif (11 members) than with a TEY motif (6 members). In contrast, the Arabidopsis genome contains more MAPKs with a TEY motif (12 members) than with a TDY motif (8 members) [10]. Detailed functional data about TDY MAPKs was first obtained from a monocot plant. Overexpression of OsBWMK1 (also known as OsMPK12) in tobacco resulted in constitutive PR gene expression and enhanced resistance to fungal and bacterial infections [11]. In maize, ZmMPK6 is able to interact with a 14-3-3 protein, and these data represent the first evidence of the possible involvement of 14-3-3 proteins in the regulation of MAPK cascades in plants [12]. More recently, Arabidopsis MPK9 (a group D MAPK) and MPK12 (a group B MAPK) were found to be preferentially expressed in guard cells, share functional redundancy and function as positive regulators downstream of reactive oxygen species (ROS) in guard cell abscisic acid (ABA) signaling [13]. Moreover, Arabidopsis MPK18 helps to mediate cortical microtubule functions in plant cells [14].
Cotton (Gossypium hirsutum) is one of the most important fibre and oil crops, and its growth and yield are severely impaired in various biotic/abiotic stress conditions. The biological significance of cotton group D MAPKs has not yet been described. In this study, a cDNA clone, GhMPK16, encoding a putative group D MAPK gene was isolated and characterised. Our results indicate that the expression of GhMPK16 is induced by chemical and biological signals. Ectopic expression of GhMPK16 in Arabidopsis results in enhanced disease resistance against fungi and bacteria pathogen. Moreover, GhMPK16 transgenic plants were obviously more drought-sensitive than control plants. We deduced that GhMPK16 may play important roles in regulating pathogen resistance and drought signaling.
Cloning and characterisation of the full-length GhMPK16 cDNA
Based on the conserved region of plant group D MAPK genes, the degenerate primers MP1 and MP2 were designed and synthesized to clone the internal conserved region of MAPKs from cotton, and a putative MAPK fragment (476 bp) was cloned. Next, rapid amplification of cDNA ends-PCR (RACE-PCR) was performed, and the full length sequence was retrieved. A 3'-end fragment of about 1500 bp and a 5'-end fragment of about 360 bp were obtained with the specific primers. The full-length cDNA sequence was PCR amplified using the gene-specific primers (FL1 and FL2), and it showed a high degree of homology to group D MAPKs especially Arabidopsis AtMPK16. According to the nomenclature for plant MAPKs, the novel cotton MAPK gene was named GhMPK16 [4,5], and is the first group D MAPK identified in cotton. The full-length cDNA sequence of GhMPK16 (GenBank accession number: FJ966889) was 2030 bp with a 78 bp 5' untranslated region (UTR) and a 287 bp 3' UTR. The cDNA contains a 1665 bp open reading frame (ORF) that is predicted to encode a protein of 554 amino acids with a predicted molecular mass of 63.18 kDa and an isoelectric point (pI) of 8.61.
A BLAST search (http://www.ncbi.nlm.nih.gov/BLAST webcite) and multi-alignment analysis revealed that GhMPK16 is highly related to other group D MAPKs, sharing a homology of 81.48% to AtMPK16, 72.18% to TaMAPK2, 74.56% to OsMPK16-1 and 76.02% to ZmMPK6 (Figure 1A). Moreover, as with other known plant group D MAPKs, GhMPK16 has the same family signature of 11 conserved subdomains, an activation loop (T-loop), a phosphorylation motif (TDY motif) in the T-loop, and an extended C-terminal region relative to groups A, B and C. Additionally, GhMPK16 also lacks a C-terminal CD domain, a feature conserved in MAPKs belonging to the other groups (A, B, C) [4].
Figure 1. Sequence analysis of GhMPK16. (A) Alignment of the deduced GhMPK16 protein sequence with other known plant group D MAPKs. Amino acid sequences used in the analysis are from Arabidopsis thaliana (AtMPK16, NP_197402), Triticum aestivum (TaMAPK2, ABC54585), Zea mays (ZmMPK6, NP_001105238), Oryza sativa (OsMPK16-1, ACF06191), Gossypium hirsutum (GhMPK16, FJ966888). These sequences were aligned using the DNAman 6.0 program. Identical and similar amino acids were shaded in black and gray, respectively. The protein kinase subdomains are indicated by Roman numerals (I to XI) over the sequences. The activation-loop is boxed, and the phosphorylation motif (TDY) is marked by an arrow. (B) The phylogenetic relationships of GhMPK16 and other plant MAPKs. Numbers above or below the branches indicate bootstrap values (>50%) from 1,000 replicates. The amino acid sequences of MAPKs used for construction of the tree are deposited to the GenBank database under the following accession numbers: AtMPK3 (NP_190150); NtWIPK (BAA09600); PsMAPK3 (AAF73236); OsMPK5 (AAL87689); AtMPK6 (NP_181907); NtSIPK (AAB58396); NtNTF6 (CAA58760); AtMPK5 (NP_567378); AtMPK4 (NP_192046); OsMPK6 (NP_922504); AtMPK7 (NP_179409); OsMPK3 (ABH01189); NtNTF3 (CAA49592); AtMPK1 (NP_172492); GhMAPK (ABA00652); AtMPK18 (NP_175756); OsMPK8 (CAD54742); AtMPK17 (NP_001030941); AtMPK15 (NP_565070); AtMPK9 (NP_566595); OsMPK12 (AAD52659); AtMPK16 (NP_197402); GhMPK16 (FJ966889); OsMPK15 (ACD76441); ZmMPK6 (NP_001105238); OsMPK16-1 (ACF06191); TaMAPK2 (ABC54585). At, Arabidopsis thaliana; Nt, Nicotiana tobacum; Ps, Pisum sativum; Ta, Triticum aestivum; Zm, Zea mays; Os, Oryza sativa; Gh, Gossypium hirsutum. The Ser/Thr kinase domain is indicated as gray and contains part of the activation-loop motif; the CD domain required for MAPK docking is indicated in black. GhMPK16 is boxed in the diagram and classified as a member of the TDY group.
To reveal the evolutionary relationship of GhMPK16 with other MAPKs from various plant species, we constructed a phylogenetic tree using amino acid sequences derived from the GenBank database (Figure 1B). Our results indicate that GhMPK16 has high similarity with AtMPK16 and a close genetic relationship with many monocot (rice, corn, and wheat) group D MAPKs, such as OsMPK16-1, OsMPK15, ZmMPK6 and TaMAPK2, which suggests that these genes may have a similar function across species including dicotyledons and monocotyledons.
Genomic structure and Southern blot analysis of GhMPK16
To isolate a genomic GhMPK16 clone, two pairs of specific primers, FL1/Z1 and FL2/Z2, were designed based on the GhMPK16 cDNA sequence, and the cotton genomic DNA was used as the template to generate two PCR fragments. These two fragments were further linked together through their overlapping region and the full-length GhMPK16 genomic sequence of 5520 bp (GenBank accession number: FJ966896) was deduced. A comparison between the GhMPK16 genomic and cDNA sequences indicated that ten introns were present in the gene (Additional file 1: supplementary Figure S1). Interestingly, exon 8 was only 5 bp, intron 7 did not contain a 3'-splice acceptor AG signal, and intron 8 did not contain the conserved 5'-splice donor GT signal. A comparative analysis of the homologous genes from Arabidopsis thaliana, Oryza sativa and Vitis vinifera was performed (Additional file 1: supplementary Figure S1). This analysis indicated that the size of the exons was conserved, but the length of the introns for each member was different. To date, the most noticeable difference in GhMPK16 was the number of exons. GhMPK16 has 11 exons, and the others have 10.
Additional file 1. Figure S1 Schematic representation of the genome structure of GhMPK16. Length of the exons and introns (A) Arabidopsis thaliana (AtMPK16), (B) Gossypium hirsutum (GhMPK16), (C) Oryza sativa (Os11g0271100), and (D) Vitis vinifera (Vitis vinifera hypothetical protein LOC100246022) are indicated according to the scale below. The exons and introns are highlighted with gray and white bars, respectively. The start codons (ATG) are indicated by (▼), and the stop codons are marked by (*).
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Southern blots were used to investigate the genomic organization of the GhMPK16 gene. Genomic DNA was completely digested with EcoR V, Xba I, Hind III and EcoR I and hybridised to the 3' partial sequence of GhMPK16, which contains no restriction sites of EcoR V, Hind III and EcoR I and only one Xba I site presents in the probe region. As shown in Figure 2, only one band was observed for the EcoR V, Hind III and EcoR I digestions, and two bands were observed for the Xba I digestion. These results imply that there is a single GhMPK16 gene in the cotton genome.
Figure 2. Southern blot analysis for GhMPK16 in the cotton genome. Genomic DNA (30 mg/sample) was digested with EcoR V, Xba I, Hind III and EcoR I respectively, followed by hybridization with the partial α-32P -labeled genomic GhMPK16 fragment. Molecular weight marker is shown on the right.
Subcellular localization of GhMPK16
To reveal the cellular localization of GhMPK16, a reporter gene encoding GFP was fused to GhMPK16 and placed under the control of the CaMV35S promoter, and immunoblot analysis indicated that GhMPK16::GFP was an integrated fusion protein (Additional file 2: supplementary Figure S2). The biolistic transformation system was used for a transient assay in onion epidermal cells. The nuclear localization of GFP-conjugated GhMPK16 was confirmed in individual transgenic cells by GFP fluorescence, using DAPI staining to detect the nuclei and interference contrast images to detect whole-cell structures. As shown in Figure 3B, the 35S-GhMPK16::GFP construct localized to the nucleus, and the 35S-GFP control construct showed GFP signals in both the cytoplasm and the nucleus. In addition, a program that predicts the subcellular localization of proteins (http://www.bioinfo.tsinghua.edu.cn/SubLoc/ webcite) predicted that GhMPK16 is localized in the nucleus with an expected accuracy of nearly 74%. These results indicate that the GhMPK16 protein is likely localized in the nucleus.
Additional file 2. Figure S2. Analysis of the integrity of GhMPK16::GFP fusion protein. (A) Transient expression of 35S-GFP and 35S-GhMPK16::GFP in N. benthamiana cells. Bar = 10 μm. (B) Immunoblot analysis of GhMPK16::GFP fusion protein. 4-week-old N. benthamiana plants were chosen, and proteins were isolated from wild-type and transgenic plant leaves. Each lane was loaded with a total of 50 μg protein.
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Figure 3. Subcellular localization of the GhMPK16 protein in onion epidermal cells. (A) Schematic diagram of the 35S-GhMPK16::GFP and 35S-GFP fusion construct. (B) Transient expression of 35S-GFP and 35S-GhMPK16::GFP in onion epidermal cells. Cells were analyzed by laser confocal microscopy 12 h after particle bombardment. The nuclei of the onion cells were visualised by DAPI staining. Bar = 20 μm.
Expression pattern of GhMPK16 under diverse abiotic and biotic stresses in cotton
To determine whether GhMPK16 expression is triggered by abiotic stresses, cotton seedlings were exposed to low and high temperatures, mannitol, NaCl and wounding treatments. GhMPK16 showed a slightly response to low and high temperatures (Figure 4A, B, C). GhMPK16 transcripts accumulated within 6 h following mannitol treatment, and the induction was still present 12 h after treatment (Figure 4D). As shown in Figure 4E, GhMPK16 expression increased and reached the maximum at 8 h after the NaCl treatment. Wounding rapidly and transiently enhanced the expression level of GhMPK16 (Figure 4F). Taken together, the expression profiles indicate that GhMPK16 is induced by various abiotic stresses.
Figure 4. Expression profiles of GhMPK16 under abiotic/biotic stresses. RNA blots were performed with total RNA extracted from leaves at the indicated times, and treated with low temperature (12°C) (A), high temperature (37°C) (B), normal temperature (25°C) (C), 200 mM mannitol (D), 200 mM NaCl (E), wounding (F), X. campestris pv. Malvacearum (G), C. gossypii (H), F. oxysporum f. sp. Vasinfectum (I), mock (J), 10 mM SA (K), 100 μM ABA (L), 100 μM MeJA (M), or ethanol (N). Ethidium bromide-stained rRNA was used as the loading control.
To explore the roles of GhMPK16 in plant biotic stresses, cotton seedlings were inoculated with pathogens, including Xanthomonas campestris pv. malvacearum (X. campestris pv. malvacearum), Colletotrichum gossypii (C. gossypii) and Fusarium oxysporum f. sp. vasinfectum (F. oxysporum f. sp. vasinfectum). As shown in Figure 4 (G, H, I), infection by all of these pathogens elevated the transcription level of GhMPK16, although the induction kinetic was variable. There was no significant change in GhMPK16 transcript level without pathogen treatment during 7 days (Figure 4J). These results indicate that GhMPK16 may be intimately involved in the plant pathogen defense response.
Additionally, we examined the response of GhMPK16 to exogenously applied salicylic acid (SA), methyl jasmonate (MeJA) and ABA, which are plant signaling molecules involved in plant defense signaling pathways. The expression of GhMPK16 was induced by all of these signal molecules (Figure 4K, L, M). Furthermore, the solvent control (Ethanol) did not significantly induce the expression of GhMPK16 (Figure 4N)
GhMPK16 promoter analysis
Inverse-PCR (I-PCR) was used to obtain a 785 bp fragment of the 5' flanking region upstream of the transcriptional start site, as determined by the GhMPK16 cDNA sequence. In order to find cis-acting elements, the PlantCARE databases were analyzed. Sequence analysis revealed that the GhMPK16 promoter contains TATA and CAAT motifs located at nucleotides -38 and -64 relative to the transcriptional start site, respectively, which is characteristic of eukaryotic gene promoters. As shown in Figure 5, the 785 bp promoter region contains several motifs probably related to pathogen and drought signals, such as a Box-W1 element (fungal elicitor responsive element), an ERE element (ethylene-responsive element), two TCA-element (cis-acting SA-responsive element) and a MBS element (MYB binding site involved in drought-inducibility). This suggests that these putative cis-acting elements are responsible for enhanced expression of GhMPK16 during stress conditions.
Figure 5. Nucleotide sequence of the promoter region of GhMPK16. The predicted transcription initiation site is indicated (+1, A). The start codon is marked with an asterisk, and the putative core promoter consensus sequences (TATA-box and CAAT-box) are highlighted in grey. The putative cis-acting elements are indicated by boxes and their corresponding names are given above each element. Arrows indicate the direction of the cis-element. Box-W1 is a fungal elicitor responsive element, ERE is an ethylene-responsive element, the MBS binding site is involved in gene induction in response to drought-inducibility and the TCA-element is a cis-acting element involved in SA-responsive.
Enhanced resistance of GhMPK16 transgenic plants to pathogenic infections
In order to further explore the function of GhMPK16 in plant defense, the coding sequence of GhMPK16 was cloned into the plant binary vector pBI121 and transformed into Arabidopsis. A total of 32 independent transgenic lines were obtained by kanamycin-resistance selection and confirmed by PCR (data not shown). RNA blot analysis was performed to determine the transgenic expression levels in 7 randomly selected lines (Figure 6A). Two representative lines (#2 and #5) exhibiting different expression levels were selected and their T3 transgenic plants used to evaluate disease resistance.
Figure 6. Enhanced resistance of GhMPK16-overexpressing Arabidopsis to P. solanacearum infection. (A) RNA blot analysis of GhMPK16 expression in T1 transgenic plants. (B) Disease symptoms of wild-type (WT) and transgenic plants (#2 and #5) at 6 d post-inoculation with P. solanacearum (105 cfu/ml). Magnified images are shown in the lower photographs. (C) Growth of P. solanacearum in wild-type and transgenic plants (#2 and #5) after infecting the plants using the dipping method. Data are the mean ± SE (n = 6) from three independent experiments. Different letters at 3- or 6-d data points indicate significant differences with P < 0.05 according to Duncan's multiple range test. (D) RT-PCR analysis of the expression of Arabidopsis AtPR1, AtPR4 and AtPDF1.2 genes in wild-type and transgenic plants (#2 and #5) inoculated with P. solanacearum. Arabidopsis elongation factor EF-1α was used as an internal control.
To analyze bacterial resistance responses in wild-type and GhMPK16-overexpressing plants, 3-week-old plants were inoculated with P. solanacearum and monitored daily for the appearance of typical disease symptoms. P. solanacearum caused more severe chlorotic symptoms in the wild-type plants compared to the transgenic plants 6 d after inoculation (Figure 6B). As shown in Figure 6C, the bacterial titres in the inoculated leaves of the transgenic plants were significantly reduced as compared to those in wild-type plants at 3- and 6-day post-inoculation, which is consistent with the observed symptoms. The rate of bacterial growth in the inoculated leaves was lower for transgenic line #2 than for transgenic line #5. These results indicate that GhMPK16 can enhance resistance to P. solanacearum in a dose-dependent manner in transgenic plants. In order to better understand the role of GhMPK16 in disease resistance in transgenic plants, three marker genes associated with pathogen responses (AtPR1, AtPR4, and AtPDF1.2) were selected, and the transcript levels of those genes were analyzed by reverse transcription-PCR (RT-PCR). The expression of the three marker genes, especially AtPR4 and AtPDF1.2, was more rapidly and strongly induced by the P. solanacearum infection in transgenic plants than in wild-type plants (Figure 6D). These results demonstrate that the overexpression of GhMPK16 activates defense-related gene expression when plants are challenged with P. solanacearum.
As shown in Figure 7, GhMPK16-overexpressing plants were evaluated for their resistance to fungal pathogens (C. nicotianae and A. alternate). Detached leaves were inoculation with C. nicotianae and A. alternate, and after 5 days, the lesions in the wild-type leaves were larger than those in the transgenic leaves, and line #2, which displayed the highest level of GhMPK16 expression, exhibited the least severe disease symptoms. To quantify the lesions, the diameters of the disease spots were measured, and the data were consistent with the visual observations. These results indicate that GhMPK16 can enhance resistance to infections by pathogenic fungi in transgenic plants. After inoculating whole plants with C. nicotianae and A. alternate, the expression of AtPR1, AtPR4 and AtPDF1.2 was observed at 3- and 6-day post-infection in both the wild-type and transgenic plants, and the expression patterns of the three marker genes were similar to those observed with the P. solanacearum challenge (data not shown).
Figure 7. Inhibition of fungal pathogen infections in GhMPK16-overexpressing Arabidopsis lines. (A) Disease symptoms of leaves from wild-type (WT) and transgenic plants (#2 and #5) 5 d after inoculation with C. nicotianae (106 conidia/ml). (B) The diameter (mm) of the lesions was measured 5 d after inoculation with C. nicotianae. Data are the mean ± SE (n = 6) from three independent experiments. Different letters indicate significant differences with P < 0.05 according to Duncan's multiple range test. (C) Disease symptoms of the leaves from wild-type (WT) and transgenic plants (#2 and #5) 5 d after inoculation with A. alternate (106 conidia/ml). (D) The diameter (mm) of the lesions was measured 5 d after inoculation with A. alternate. Data are the mean ± SE (n = 6) from three independent experiments. Different letters indicate significant differences with a P < 0.05 according to Duncan's multiple range test.
Reduced drought tolerance and rapid H2O2 accumulation in GhMPK16-overexpressing plants
Compared with wild-type plants, the GhMPK16-overexpressing plants exhibited obvious drought sensitivity. As shown in Figure 8A, the leaves began to turn yellow and curl in both the 3-week-old wild-type and transgenic plants after a 15-day drought treatment. Additionally, drought treatment led to more serious damage in the transgenic plants than in the wild-type plants. Under normal conditions, no overt morphological differences were observed between wild-type and transgenic plants (data not shown). We tested the seed germination capacity on 1/2 MS agar medium with 50 mM mannitol to mimic drought conditions and found that the germination percentage was higher for wild-type plants than for transgenic plants (Figure 8B, C). The root lengths of both wild-type and transgenic plants were used as indicators of drought stress tolerance. Root growth was inhibited by mannitol treatment to a greater extent in transgenic seedlings than in wild-type seedlings (Figure 8D, E). These results suggest that the overexpression of GhMPK16 in Arabidopsis results in reduced drought tolerance.
Figure 8. Drought stress analysis of GhMPK16-overexpressing Arabidopsis. (A) Phenotypes of wild-type (WT) and transgenic plants (#2 and #5) plants withheld from water for 15 d at the vegetative stage. (B) Seed germination of wild-type (WT) and transgenic plants (#2 and #5) after 4 days on 1/2 MS agar medium containing 50 mM mannitol. (C) Germination percentage of seeds on 1/2 MS agar medium containing 50 mM mannitol. Germination was defined as the emergence of the root radical and was scored daily. Data are the mean ± SE (n = 4) from three independent experiments. (D) Growth of wild-type (WT) and transgenic plant (#2 and #5) seedlings after 100 mM mannitol treatment. Photos were taken 10 d after mannitol treatment. (E) Root length of wild-type (WT) and transgenic plant (#2 and #5) seedlings after 100 mM mannitol treatment for 12 d. Data are the mean ± SE (n = 6) from three independent experiments. Different letters indicate significantly different at P < 0.05 according to Duncan's multiple range test.
One of the important responses of plants under osmotic stress is the accumulation of reactive oxygen species (ROS). Among the different ROS, only H2O2 can cross plant membranes and thus directly function in cell-to-cell signaling. Therefore, we examined endogenous H2O2 accumulation in wild-type and transgenic plants in response to mannitol treatment. Mannitol (200 mM) was used to treat one-week-old seedlings, and the leaves were collected after 0, 1, 3 and 6 h. Staining with 3,3'-diaminobenzidine (DAB) revealed different levels of H2O2 production in the leaves of wild-type and transgenic plants after osmotic stress treatment. Specifically, H2O2 accumulated in transgenic plants at a significantly faster speed than that in wild-type plants (Figure 9). These results suggest that the overexpression of GhMPK16 leads to the rapid production of ROS or ineffective scavenge of excess ROS after osmotic stress.
Figure 9. DAB coloration assay of H2O2 accumulation in wild-type (WT) andGhMPK16-overexpressing transgenic plants after 200 mM mannitol treatment. Mannitol was used to treat one-week-old plants for 0, 1, 3 or 6 h.
Discussion and Conclusion
In this study, we isolated and characterised GhMPK16, a gene whose product belongs to the family of group D MAPKs. GhMPK16 is the first characterized MAPK gene from cotton belonging to the group D MAPKs. The corresponding protein is characterized by the presence of a TDY activation motif in its T-loop, the lack of a CD domain and an extended C-terminal region compared to the TEY subtypes of MAPKs (Figure 1A). These structural differences suggest that group D MAPKs may function differently from other MAPKs.
The subcellular localization of group D MAPKs has not been established. OsBWMK1, a group D MAPK from rice, is found in the nucleus and mediates pathogenesis-related gene expression by activating the OsEREBP1 transcription factor [11]. Intriguingly, the alternative splicing of OsBWMK1 generates three different transcriptional variants that produce proteins with different subcellular localizations [15]. In Arabidopsis, AtMPK18 is located in the cytoplasm [14]. Analysis of the subcellular localization of a GFP-tagged GhMPK16 protein in transiently transformed onion epidermal cells revealed that GhMPK16 is likely localized in the nucleus (Figure 3). Therefore, we speculated that GhMPK16 may act as a transcriptional activator by activating the expression of a set of target genes in the nucleus.
Gene expression patterns are usually an indicator of gene function. Notably, GhMPK16 can be induced by various pathogens, including X. campestris pv. malvacearum, R.solani and C. gossypii. RNA blot analyzes revealed that the expression of GhMPK16 could be induced by signal molecules such as SA, MeJA, and ABA (Figure 4). These results imply that GhMPK16 may play a role in plant defense responses and in the regulation of certain components of multiple stress-signaling pathways. Consistent with this hypothesis, sequence analysis of the GhMPK16 promoter revealed several motifs, such as Box-W1 element, ERE element and TCA-element, related to motifs with important roles in defense signaling.
Recently, a variety of MAPK genes have been identified and subsequently explored by both genetic and biochemical approaches. Increasing evidence has shown that the MAPK cascade is an important pathway in pathogen-triggered signal transduction [16,17]. Signal molecules such as SA, jasmonic acid (JA) and ethylene (ET), regulate distinct sets of pathogenesis-related (PR) genes in different pathogen defense pathways. SA and MeJA are involved in two major defense signaling pathways, the SA-dependent and JA/ET-dependent defense mechanisms, which act against different types of pathogens [18]. In Arabidopsis, AtMPK4 responds to the balance between SA and MeJA through the EDS1/PAD4 module and regulates the SA and JA/ET-related defense responses [19]. When GhMPK16 is ectopically expressed in Arabidopsis, the transgenic Arabidopsis show enhanced disease resistance against bacteria (P. solanacearum) and fungi (C. nicotianae and A. alternate) (Figure 6, 7). PR1 is known as a marker gene for the SA signaling pathway, and AtPR4 and AtPDF1.2 are markers for the JA/ET signaling pathway. Along with the enhanced disease resistance in GhMPK16-overexpressing plants, the expression of the examined defense-related genes AtPR1, AtPR4, and AtPDF1.2 was elevated. Thus, it is reasonable to speculate that GhMPK16 may be involved in the crosstalk between the SA- and JA/ET- mediated pathogen defense pathways.
GhMPK16 showed a response to various abiotic stresses, such as low or high temperatures, mannitol and NaCl (Figure 4). Interestingly, transgenic plants overexpressing GhMPK16 exhibited obvious drought sensitivity (Figure 8). GhMPK16 overexpression resulted in a significant reduction in both germination percentage and root growth after mannitol treatment, and seedling growth was more severely inhibited by drought stress in GhMPK16-overexpressing plants than in wild-type plants. ABA responds to the environmental signals to protect plants from abiotic stresses, such as cold, drought and salt stresses [20,21]. The expression of GhMPK16 was strongly induced by exogenous ABA and the GhMPK16-overexpressing plants were sensitive to drought tolerance comparing with wild-type Arabidopsis, suggesting that GhMPK16 may be involved in ABA signaling and caused negative function for drought tolerance. DAB staining revealed that H2O2 rapidly accumulation in GhMPK16-overexpressing transgenic plants after drought treatment (Figure 9). Excessive ROS can injure plants. GhMPK16 accelerates the accumulation of H2O2, and this may be the reason that drought causes more serious damage in the transgenic plants. In Arabidopsis, MPK9 and MPK12 function downstream of ROS to regulate guard cell ABA signaling positively [13]. Guard cell-specific inhibition of Arabidopsis MPK3 expression causes abnormal stomatal responses to ABA and H2O2 [22]. More physiological, biochemical and molecular experiments are needed to elucidate the mechanism of GhMPK16 in response to drought stress.
On the basis of these observations, we propose that GhMPK16 functions in at least two signaling pathways, one that responds to pathogens and another that is involved in drought stress. GhMPK16 may serve as a point for crosstalk between biotic and abiotic stress response signaling. Further studies on the function and regulation of GhMPK16 are required.
Plant materials and treatments
The cotton cultivar Gossypium hirsutum L. cv Lumian 22 was used for all of the experiments. Germinated seedlings were grown by aquaculture in tissue culture pots (sterile water) under greenhouse conditions at 25°C with a 16 h light/8 h dark cycle. The following treatments were performed on seven-day-old cotton seedlings: 10 mM Salicylic acid (SA), 100 μM methyl jasmonate (MeJA) (ethanol as a solvent control) or 100 μM abscisic acid (ABA) was sprayed onto leaves of cotton seedlings; seedling roots were placed in 200 mM NaCl and 200 mM mannitol solutions for salt and drought stresses, respectively; seedling leaves were cut with scissors for wound treatment; seedlings were placed in a growth chamber at 37°C or 12°C for the high and low temperature treatments, respectively; roots were dipped into suspensions of the bacterial pathogen Xanthomonas campestris pv. malvacearum (X. campestris pv. malvacearum) (OD = 0.001) to cause a bacterial infection and the fungal pathogens Fusarium oxysporum f. sp. vasinfectum (F. oxysporum f. sp. vasinfectum) and Colletotrichum gossypii (C. gossypii) conidial suspensions (105 conidia/ml) in 1% glucose were dropped onto plant leaves to cause the fungal infections. The plants treated with 1% glucose were considered as mock. The leaves, roots and stems were harvested at the appropriate time points as indicated and frozen in liquid nitrogen for further analyzes.
Arabidopsis (ecotype Columbia, Col-0) and transgenic Arabidopsis seeds were planted directly in soil or transplanted after germinating on 1/2 MS agar medium and grown in a chamber under SD (8 h light/16 h dark) or LD (16 h light/8 h dark) conditions at 23°C. All seeds were treated at 4°C for 2 d before being transferred to a growth chamber. Arabidopsis were grown in SD conditions for 2-week and then transferred to LD conditions. 3-week-old Arabidopsis plants were used for the disease resistance assays. The bacterial pathogen Pseudomonas solanacearum (P. solanacearum) was cultivated in King's B medium at 30°C for 2 days, and the bacterial cells were resuspended in 10 mM MgCl2 to a concentration of 105 colony-forming units/ml (cfu/ml). Plants were infected using the dipping method, and bacterial titres were assessed by plating a dilution series of leaves ground in 10 mM MgCl2 on King's B medium as described by Liu et al. [23]. Infected leaves were sampled immediately and at 3- and 6-d after inoculation. To evaluate resistance against the fungal pathogens Colletotrichum nicotianae (C. nicotianae) and Alternaria alternate (A. alternate), two methods were used: the detached leaf inoculation assay and the whole plant inoculation assay. A spore suspension (106 conidia/ml) was prepared in 1% glucose. In the detached leaf inoculation assay, leaves were inoculated with a 10 μl droplet of conidial suspension. In the whole plant inoculation assay, a vaporiser was used to inoculate the plants with the conidial suspension. The inoculated leaves and plants were kept in a moist chamber under dark conditions at 25°C for 48 h and then incubated under LD conditions at 25°C.
For the drought stress tolerance analysis, Arabidopsis were grown in SD conditions for 2-week and then transferred to LD conditions. For 15 d, 3-week-old Arabidopsis plants were completely withheld from water. Additionally, Arabidopsis seeds were germinated in 1/2 MS agar medium or 1/2 MS agar medium supplemented with 50 mM mannitol. Analysis of the growth of transgenic Arabidopsis after mannitol treatment, Arabidopsis seeds germinated in 1/2 MS agar medium and incubated for 3 d. The seedlings were then carefully transferred into 1/2 MS agar medium supplemented with 100 mM mannitol, and root lengths were measured over the next 10 d.
Cloning of the GhMPK16 gene
Total RNA was extracted from cotton seedling leaves using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The full-length GhMPK16 cDNA was amplified by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends-PCR (RACE-PCR).
The first cDNA strand was synthesised using approximately 2 μg of total RNA, the Oligo(dT)18 adaptor primer and M-MLV reverse transcriptase (Promega, Madison, WI, USA) for 1 h at 42°C. To clone the internal conserved fragment, primers MP1 and MP2 were designed and synthesised based on the conserved amino acid and nucleotide sequences of the plant group D MAPK genes. Cloning was performed as described previously [24], and the primer sequences are provided in Table 1.
Table 1. The primers used in this study
Amino acid and nucleotide sequences of other plant MAPK genes were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/Genbank webcite). Amino acid sequence alignments were performed using the DNAman 6.0 program. The phylogenetic tree was constructed by the Neighbour-Joining method using MEGA 4.
Amplification of the GhMPK16 genomic sequence
Total genomic DNA was extracted from cotton seedlings using a modified cetyl-trimethyl-ammonium bromide (CTAB) method [25]. The genomic DNA of cotton was isolated to clone the DNA sequence and the 5'-flanking region of GhMPK16.
Two genomic DNA fragments were PCR amplified with primers designed according to the GhMPK16 cDNA sequence. The first fragment was amplified with primers FL1 and Z1, and the second fragment was amplified with primers FL2 and Z2.
The promoter region was cloned using the inverse-PCR (I-PCR) method. Five restriction endonucleases (Bam I, Bgl II, Dra I, Hind III and Xba I) were used to digest the genomic DNA. Using T4 DNA ligase (TaKaRa, Dalian, China), the DNA fragments were self-ligated to form circles that were used as the template to amplify the GhMPK16 promoter region. The 5' flanking region of GhMPK16 was obtained from the template digested with Dra I. The exterior primers Wup1 and Wdra1 and the interior primers Wup2 and Wdra2 were used for the first and second rounds of PCR, respectively. The sequences of the primers used in this study are provided in Table 1. The PlantCARE program (http://bioinformatics.psb.ugent.be/webtools/plantcare/html webcite) was used to analyze the GhMPK16 promoter sequences, which used default parameters.
Subcellular localization and Histochemical Analysis
To observe the cellular localization of GhMPK16, the GhMPK16::GFP fusion construct was prepared. The GhMPK16 coding region was amplified with primers Wbd-1 and W-GFP, containing an Xba I site upstream and an Xho I site downstream of the deleted stop codon, respectively. The resulting fragment was inserted into the Xba I/Xho I site of the binary vector pBI121-GFP, and the fragment was fused to GFP and placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Particle bombardment was performed according to the manufacturer's instructions (PDS-1000, Bio-Rad Laboratories, Hercules, CA), using gold particles (1.0 μl) and 1350 psi helium pressure. After bombardment, tissues were incubated on MS agar medium under dark conditions at 23°C for 12 h. Nuclei were stained with 100 μg/ml of 4',6-diamidino-2-phenylindole (DAPI) (Solarbio, Beijing, China) in phosphate-buffered saline for 10 min. GFP fluorescence and DAPI fluorescence were examined with a laser scanning microscope (LSM 510 META, ZEISS, Germany) using excitation wavelengths of 488 nm and 350 nm, respectively.
Immunoblot analysis
The GhMPK16::GFP recombinant plasmid was introduced into the Agrobacterium tumefaciens strain GV3101. The A. tumefaciens strains were grown overnight in YEB media. Pellet and resuspended cells was performed as described by Guo [26]. Resuspended cells were infiltrated into leaves of 4-week-old Nicotiana benthamiana plants as described previously [27,28]. Epidermal cell layers of plant leaves were assayed for fluorescence with a fluorescence microscope (BX51, model 7.3; Olympus) 3 d after injection. Total plant proteins were extracted as Silverstone described [29]. The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and analyzed on immunoblots using a 1000-fold dilution of anti-GFP polyclonal antibodies (Beyotime, Haimen, China) and a 5000-fold dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Beyotime). The signals were detected by chemiluminescence.
Southern blot hybridisation
Genomic DNA (30 μg/sample) was digested with EcoR V, EcoR I, Xba I or Hind III then separated on 0.8% agarose gels by electrophoresis and transferred onto a Hybond-N+ Nylon membrane (Amersham, Pharmacia, UK). A gene-specific dCTP-[α-32P]-labelled probe was synthesised with the GhMPK16 fragment (primers FL1 and 5P2) using the Primer-a-Gene® Labeling System (Promega, Madison, WI, USA) according to the manufacturer's instruction. The hybridisation was carried out for 24 h at 42°C. After the hybridisation, the blots were washed two times with 2 × SSC, 0.1% SDS and three times with 0.2 × SSC, 0.1% SDS for 10 min each at 42°C, and the radioactive signal was visualised using a FLA-7000 phosphorimager (FUJIFILM).
RNA blot hybridisation and semi-quantitative RT-PCR analyzes
Total RNA was isolated from cotton seedling leaves using the method described above. Total RNA (15 μg) from each sample was separated on a 1.0% agarose-formaldehyde gel and transferred to Hybond-N+ Nylon membranes (Amersham, Pharmacia, UK). Gene-specific dCTP-[α-32P]-labelled probe was synthesis (primers FL2 and 5P3) and RNA blot hybridisation were performed using the methods described for the Southern blot hybridisation.
To analyze gene expression in transgenic plants by RT-PCR, total RNA was isolated from wild-type and transgenic plants using the method described above for gene cloning. Amplifications were performed at 94°C for 5 min, followed by 25-30 cycles of amplification (94°C for 50 s, 50°C for 50 s, and 72°C for 40 s). The PCR products were separated on a 1.8% agarose gel and visualised after ethidium bromide staining. To ensure that equal cDNA amounts were used in each reaction, EF-1α was used as a loading control. The gene-specific primers used in RT-PCR analyzes are listed in Table 1.
Vector construction and plant transformation
The GhMPK16 coding region was amplified using primers Wbd-1 and Wbd-2 (Table 1). The amplified fragment was then subcloned into the binary vector pBI121 under the control of the CaMV 35S promoter. The recombinant plasmid was introduced into the A. tumefaciens strain GV3101. Arabidopsis was transformed using the floral dip method [30]. Transformants were selected on 1/2 MS agar medium containing 30 μg/ml of kanamycin. The transgenic T3 lines were used in the experiments.
Histochemical detection of H2O2
The substrate, 3,3'-diaminobenzidine (DAB), was used to visually detect H2O2 in plants [31]. Arabidopsis plants (one-week-old) were treated with 200 mM mannitol for 0, 1, 3, and 6 h. The samples were incubated in 1 mg/ml DAB solution (pH 3.8) for 6 h, and then treated with 95% ethanol to remove chlorophyll. In the presence of H2O2, DAB is locally polymerised and produces visible brown stain.
Authors' contributions
JS carried out most of the experiments, and drafted the manuscript. LZ participated in southern blot analysis, RNA blot analysis and subcellular localization analysis of the study. HA, CW and XG conceived the experimental design and helped to draft the manuscript. All authors read and approved the final manuscript.
This work was financially supported by Genetically Modified Organisms Breeding Major Projects of China (2009ZX08009-092B; 2009ZX08009-113B) and Nation Natural Science Foundation of China (Grant no. 30970225)
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Louis Vuitton Runway Fall 2012
Louis Vuitton Fall 2012 — Plus, Accessories
>> Marc Jacobs's Fall 2012 collection for Louis Vuitton read like a love letter to the house's storied travel-luggage origins — right down to the real live steam engine that barreled down the runway at the start of the show. There were porters to carry the models' bags, and some of the clothes were even stitched in a checkerboard pattern reminiscent of the iconic "damier" that covers Vuitton trunks.
As for the collection, it was ornate and luxuriously detailed: giant jeweled brooches served as the buttons for coats, and sparkling appliques adorned lurex-threaded brocades that had been made by hand. The silhouettes had "a turn of the century feel, but with bits of the 1960s and 1970s," Jacobs explained. "We wanted to attenuate the height, so we started with the hats [by Stephen Jones] and then worked with a high waist, and a gentle flare just at the ankle."
Watch the video of the show below, courtesy of Telegraph UK.
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- Your favorite preppy style staple is making a comeback — Who What Wear
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- Long before Cara, Gigi, and Karlie, Christy ruled the runway — Style.com
- Is this the solution to your Mother's Day gift dilemma? — HuffPost Style
- Transform your outfit with these 5 accessories — InStyle
- It might be time to uncuff your jeans — Man Repeller

subset: commoncrawl


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B.C.'s auditor general asked to remain for two years
At first they didn't want him, but now they do.
Almost two weeks after a legislative committee voted not to give auditor general John Doyle a second term, it was decided Wednesday to ask him to remain in office for another two years.
"There's been an ongoing discussion over the last week and a half," said Eric Foster, Vernon-Monashee MLA and committee chairperson.
"We have to make sure we're doing everything by the book."
Doyle has not indicated yet as to whether he will accept the two-year extension.
The committee had been criticized for the decision not to renew Doyle's contract after six years on the job.
The committee consists of three Liberals and two New Democrats and it's been alleged one of the Liberals voted not to retain Doyle because he has been critical of the government. Reappointment must be unanimous among the committee.
Foster says a critical factor in the committee reconsidering its earlier decision was a recommendation from Premier Christy Clark that Doyle be kept on for another two years and that legislation be changed so future auditor generals are appointed for eight years with no chance for renewal.
"The idea of the legislation is what got me to ask the committee to reconsider," said Foster.
Foster has been criticized for chairing the committee determining Doyle's fate when Doyle had expressed concerns about financial documents Foster filed about renovations to his constituency office.
Foster insists that didn't play a role in the committee asking Doyle to stay on the job.
"I didn't know anything about it (Doyle's concerns about documents) when we made the first decision," he said.
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Under Joint Rules 61(b)(4) and 54(a), tomorrow is the last day for the introduction of bills in the California legislature. This means that in the next several months the legislature will be considering thousands of bills. In theory, the legislature should proceed in accordance with a schedule fixed by the state constitution, statutes and their own rules. However, the legislature historically has evaded the spirit, if not the letter, of some of these requirements.
Last week, Assembly member Bob Wieckowski introduced AB 1680 to amend Sections 1300 and 1301 of the Corporations Code, relating to dissenters' rights.
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CORESTAFF Services and AT&T Extend Relationship
Houston, Texas, February 27, 2007
AT&T Inc. (NYSE: T) today announced that CORESTAFF Services, a leading staffing services company, has selected AT&T to implement and manage an upgrade of its network services. The three-year $1.7 million contract extends and expands an existing relationship.
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Companies are increasingly turning to managed services to help address information technology (IT) needs. Managed services can reduce the total cost of ownership while allowing internal resources to be redirected to have the greatest impact on company performance.
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About CORESTAFF Services
CORESTAFF Services, a leading national staffing company headquartered in Houston, Texas, is part of The Corporate Services Group PLC, an international staffing firm headquartered in the United Kingdom. CORESTAFF provides a full range of supplemental staffing and outsourced solutions, including office support, light industrial, financial, engineering, library and records management, and information technology staffing. CORESTAFF has approximately 500 staff employees and an average of 10,000 temporary associates on assignment weekly throughout the U.S. The company currently operates over 100 branch offices and on-site client locations. CORESTAFF Services operates six Web sites: www.corestaff.com, www.it.corestaff.com, www.careertrust.com, www.techresources.corestaff.com, www.infocurrent.com, www.leafstonegroup.com and www.employmentzone.org.
About AT&T
AT&T Inc. is a premier communications holding company in the United States and around the world, with operating subsidiaries providing services under the AT&T brand. AT&T is the recognized world leader in providing IP-based communications services to businesses and the U.S. leader in providing wireless, high speed Internet access, local and long distance voice, and directory publishing and advertising through its Yellow Pages and YELLOWPAGES.COM organizations. As part of its three-screen integration strategy, AT&T is expanding video entertainment offerings to include such next-generation television services as AT&T U-verseSM TV. Additional information about AT&T Inc. and the products and services provided by AT&T subsidiaries and affiliates is available at www.att.com.
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Mon 16 Feb 2009Ashley_Mclennan is refusing to come down from the high of this weekend.... WINTERFEST 2009 was AWESOME!!! TURNED IT UP THAT DEG
Posted by Ashley_Mcl on Mon 16 Feb 2009 5:47:48 pm | no comments--------------------------------------------------------------------------------------------------------------- Wed 24 Oct 2007becasue GOD is the GREATEST POWER, we shall NOT be DEFEATED!notice the title of this blog.....it's actually in response to my first one Posted by Ashley_Mcl on Wed 24 Oct 2007 3:02:31 pm | no comments--------------------------------------------------------------------------------------------------------------- Mon 20 Aug 2007the big "C"ok.....you all know that when something bad happens to someone you barely know it doesn't really affect you. But when its one of the people closest to you it's as if your heart has been melted or ripped out and smashed into a million pieces. AS i returned home form summerfest on Sunday afternoon, i heard the horrifying news- my aunt had cancer... its was painful for me for a number of reasons. first, i had just come from one of the most amazing retreats of my life and i was so excited and never expected such bad news. secondly, of all my aunts, i owe this one the most... you see, if it wsn't for her i wouldn't know Christ as my personal savior. I would remember her bringing me to church when i was younger and i would go and do something rude and she would say to my mom (who wasn't saved at the time) "i'm not taking her back to church with me." But it would be a idle threat cuz the next Sunday i would have on my little dress and be off to church with her. From there, i grew to love the Lord and church to the point that if i was gonna be grounded the punishment would be that i couldn't go to church... and now the devil has decided to hit my aunt in the worst way...this is also horrible cuz her daughter who at this point is is between a rock and a hard place in her Christianity did not need to be hit with such hard news either. But i believe that my aunt is already healed... and that God only did this to "show off"...He just allows stuff to happen so that He can show us how great and marvelous He is... how "wonder-full" and "mercy-full" and "power-full" He is ( Bishop J. Bruno...thanx for that one...the fullness of God can HEAL) He wanted us to recognize that there is nothing impossible for Him... i think it is a major lesson for me cuz i have been praying for the baptism of the Holy spirit for years now and i think my main problem was my faith...God wants me to have faith...He wants me to believe that He can do anything...He wants a US to believe just as He said to Abraham, not asking him, but stating the fact "Is there anything to hard for the Lord????" So as a youth group...a community of faith...as the army of the lord....as Gideons army (as Bishop Steve stated on sunday morning of Summerfest) lets band together to pull down the strong holds of Satan cuz if God be for us, who DARE be against us????? There is a song that says "BECAUSE GOD IS THE GREATEST POWER WE SHALL NOT BE DEFEATED!" When you are in service or wherever you are if your worshiping or praising, please just send up a shout and a praise form my aunt cuz "at the shout of praise hell's foundation quivers" which means our praise can shake the very foundations of Satan and his demons....the demons of sickness and disease, the demons of doubt and fear, the demons of cancer, and whatever demons you or i might be facing. Please pray for my aunt cuz she really needs it... UNITED {IN CHRIST} WE STAND....DIVIDED {AGAINST ONE ANOTHER} WE FALL -God i kno u luv us & we luv & worship u--
Ashley Posted by Ashley_Mcl on Mon 20 Aug 2007 7:48:36 pm | 5 comments--------------------------------------------------------------------------------------------------------------- |


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Accoring to the US Census Bureau, Hoselton ranked #31,817 in frequency out of 151,671 surnames for which statistics were released from the 2000 Census. 686 people, or approximately 1 in every 393,239 individuals in the US had this surname in 2000.
Source: "Genealogy Data: Frequently Occurring Surnames from Census 2000", US Census Bureau.
Histopolis currently has 4 grave(s) with the surname Hoselton.
Search the Histopols Grave Index for the surname Hoselton.
The surname Hoselton has a Soundex code of H243. The following 89 surname(s) may sound similar to Hoselton since they share the same Soundex code.


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High-Throughput Screening Technique for Conversion in Hot Compressed Water: Quantification and Characterization of Liquid and Solid Products
Knezevic, Dragan and Schmiedl, Detlef and Meier, Dietrich and Kersten, Sascha and Swaaij, Wim van (2007) High-Throughput Screening Technique for Conversion in Hot Compressed Water: Quantification and Characterization of Liquid and Solid Products. Industrial and Engineering Chemistry Research, 46 (6). pp. 1810-1817. ISSN 0888-5885
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Abstract: | A high-throughput screening technique, developed earlier1 for reaction systems with primarily gaseous products, has been extended with quantitative and qualitative analysis of condensed reaction products. The technique is based on catalytically inert quartz capillaries (2 mm internal diameter) as microreactors, which allows for cheap and safe experimentation. To validate the technique, hydrothermal conversion (T 350 °C, P 200 bar) of beech wood and model compounds was used. Separation and sample preparation methods have been developed to recover the condensed reaction products. These methods, combined with the chemical analysis techniques, have shown to be accurate with respect to product recovery and detecting trends in the yield and composition of the products as a function of the operating conditions. |
Item Type: | Article |
Copyright: | © 2007 American Chemical Society |
Faculty: | Science and Technology (TNW) |
Research Group: | |
Link to this item: | http://purl.utwente.nl/publications/67413 |
Official URL: | http://dx.doi.org/10.1021/ie060900+ |
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Metis ID: 242752


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Earth CrisisEarth Crisis: Breed the Killers [reissue]Breed the Killers [reissue] (2008)I Scream Records Reviewer Rating: 3.5 User Rating: Contributed by: dktrdktr (others by this writer | submit your own) Anybody remember Earth Crisis? The straight-edge, vegan metalcore band from way back? Well, yes, I suppose a few of you do because they weren't actually bad, but I always found them most notable for vocalist Karl Beuchner being a person who wore a bandana and wasn't Axl Rose. Which you don't do. .
Anybody remember Earth Crisis? The straight-edge, vegan metalcore band from way back? Well, yes, I suppose a few of you do because they weren't actually bad, but I always found them most notable for vocalist Karl Beuchner being a person who wore a bandana and wasn't Axl Rose.
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theyve always been a step up from say, hatebreed, but thats not saying much.......theyve always been firmly rooted in the wifebeater/camo shorts/anti abortion sector of hardcore that i was never into..........day of suffering and slavearc are the only hardline vegan bands worth listening to anyways. your mom you cunt Does anyone even actually like this band? What a terrible review. Not even a serious fan of this band. Awful attempt. firestorm, my ass http://girthcrisis.blogspot.com Firestorm is the only good thing this band did. Everyone should shut the fuck up and go listen to "Gomorrah's Season Ends. Good Earth Crisis, no matter if you're sXe or not. "twat" sounds better with a british accent than an american one. the reviewer is british so he can pull it off. one of the worst reviews i've read in a while. Leaf by leaf, blunt by blunt... Toking it all up. I like the words beefy and tit face together. beefy tit face. Really? Cunt's pronunciation implies something more bold and assertive, twat just seems juvenile. I like the word "twat". i enjoy some of their early stuff but couldn't really get into this as much. Earth Crisis is a complete joke, what a horrible sound the make together. More like Ear Crisis. SXE crews are stupid. Someone choosing not to drink or do drugs however does not bother me. Also, DERP Crisis. If only there was a picture of one of their fans who looked totally ridiculous. Then you could be like,"This guy (in the picture) is stoked". inb4 earth crisis face tattoo Destroy the Machines> Sorry, but this is a horrible review. Like really, really bad. no thanks score is for straight edge. Score is for the word "twat". |
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BUZZFLASH: How could a company steal votes for one party and we would never know about it? HARRIS: Given inside access, which is available to software engineers and support techs, anything is possible. In California, according to internal memos we have obtained written by Diebold support techs and software engineers, in some elections no one looked at the software code AT ALL, except for a couple of programmers out of Canada. This is because the software that was certified and approved, and supposedly frozen and held in escrow, was replaced with different software for elections. All of the companies seem to do this: They allow their techs, and sometimes even elections officials, to replace or "update" programs, and you can't count on these "updates" being tested by anyone. In Georgia they did this repeatedly. Let me explain just how disturbing this is: The Diebold software that has been certified includes something called "GEMS" version 1.11.14 and also GEMS version 1.17.17. However, according to company memos, they rewrote "the guts of the program" when they made GEMS version 1.14.xx and then made even more changes, significant changes, with GEMS version 1.15.xx. (The last two numbers vary; none of these were certified.) These changes were made by programmers in Vancouver, Canada and stuck on an unprotected web site, where support techs went and retrieved them and put them on machines used in elections in California. |
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Bluestockings in Space
West End, London- Event date
- 04 May 2012
- Posted
- 1118 days ago
Science-fiction burlesque and cabaret from the Blue Stocking Society, in a show where geeks and showgirls mix, sometimes in the same body. Fantastic performances in every sense from Bluestocking co-founders Audacity Chutzpah and Tricity Vogue, with a heavenly array of special guests, including burlesque star and Double R Club co-founder Miss Rose Thorne, the fabulous Bettsie Bon Bon, and the beautifully geeky Helen Arney. Join us, as we Boldly Go Where No Bluestocking Has Gone Before.
Tickets from: http://cabaret.7onred.com/box-office/
Seven Dials Club
42 Earlham Street
Covent Garden
United Kingdom
Ad ID: 100216630


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Purpose:Workshop presentations are NOW available on the Computer Security Resource Center (CSRC) website. Also, links to the workshop agenda & minutes are also provided. Over the past decade, vendors have begun implementing ABAC-like features in their security management and network operating system products, without general agreement as to what constitutes an appropriate set of ABAC features. Due to a lack of consensus on ABAC features, users cannot accurately assess the benefits and challenges associated with ABAC. Posters and Demonstrations: Please check here for poster section registration and detail information. Poster and demonstration registration is due to Lisa Garlena email@example.com by COB July 12, 2013. Demonstration application and details here. Agenda:Agenda - Updated July 16, 2013 Related Project(s):Attribute Based Access Control (ABAC) Project webpages on the NIST Computer Security Resource Center (CSRC) website. Conference Web site:http://www.ibbr.umd.edu/EventDirections |
Details: Start Date: Wednesday, July 17, 2013
End Date: Wednesday, July 17, 2013
Location: IBBR, 9600 Gudelsky Drive, Rockville, MD
Audience: Industry, Government, Academia
Format: Workshop
Registration:Registration Contact:Teresa Vicente, 301-975-3883 Technical Contact:Vincent Hu, 301-975-4975 Vendor Contact: |


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Book online or call 800-328-1323 (001-970-496-4500) to speak with a local expert.
Guests can whiz down the tubing hill on the snow and then enjoy summer activities like hiking, mountain biking and mountaintop dining.
Type: Adventure, Outdoor, Kid Friendly, Groups, Grown Up
Location: Keystone Mountain
Summer Snow Tubing. Just like when you were a kid, only on a much bigger hill with its own tubing lift. Get ready to laugh until it hurts in this must-do experience for all ages.
Dates: Re-Opening June 13 for summer. Weather Dependent! Availability based on snow conditions.
Hours of Operation: 10:30 a.m. - 2:30 p.m.; Last tubing session @ 1:30 p.m. Weather Dependent! Availability based on snow conditions.
Cost: $31/person for 1 hour
Tube and Lunch $41 per person- 1 hour of tubing and $15 voucher for lunch at the Summit House- Lunch- Offering selection of sandwiches & BBQ including side dish and drink. One Run: $5 per run
Location: Adventure Point - top of the River Run Gondola
Reservations: Reservations are highly recommended. To reserve call 970-496-3000.
Hour long sessions start on the hour and half hour. You must be at least 42 inches tall to tube, which is around 4 years old and we recommend this activity for the more adventurous kid.
Check-In: Check-in is located at the Mountain Services Center in River Run's Gondola Plaza. We recommend that guests check in 30 minutes prior to your reservation.
Parking: Free parking will be available in the River Run Parking Lot. Free lots can be full on busy weekends and holidays so plan accordingly
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Welcome to SilverSingles.com
All free guest memberships include a free photo personal ad, free browsing, free matching, and the events calendar. Subscribing members access live chat, message boards, Mingle Mail, and more! To browse through thousands of photos and start meeting other singles, click here.
Last Login: Mar 22, 2015
Meet a discerningly interesting, open-minded, and creative; consultant, FEMA disaster assistance project office, and designer, who is an intimately involved, non-meddling, parent of two independent adult sons and "custodial" son for his dad who's in his "100th year".
I am affectionate, nurturing, patient, empathetic, gregarious, tolerant, caring, humanistic and politically active (against war, capital punishment and the diabolically insensitive and destructive Bush government), I can be humorous, some times ironic, irreverent and or silly, though emotionally mature.
Full of positive feelings, I am extremely ethical, trustworthy, sincere and honest with a great deal of integrity. People share their feelings and confide in me as a person whom they can and do rely on to, listen to and understand, them while keeping their "secrets". I am not only a "good weather" friend but am dependable in any kind of weather.
A great guy to have around the house: especially in the kitchen, the workshop and, in most emergencies!
I love live; to love and to make love; to dance (especially ballroom), swim, and to travel (I have lived, worked and or toured in: Africa, Europe, the Middle East, the Americas, the Caribbean and the Pacific islands); to cook; and especially, to be with my family.
Though I am very curious about life and relationships and believe that I have some of the answers, there always seem to be more questions and many enigmas.
I do not need to be" picked up after"; I return phone calls in a timely manner, and I, put down the toilet seat.
While I greatly value the truth I also make sacrifices to uphold it.
Appreciating the "bells and whistles" of the good life, I don't need them for my contentment.
Having said I a lot, I am a, we kind of guy.
If you thought, "YE SSS!" then I look forward to "hearing" from you.
If it was simply H'm, I still would like to hear from you.
Many single sites ask one to describe their dream date, which I consider somewhat absurd as I want a real date with a real woman (not a dream), that is one with whom I may in time; wake up with and not from. I want a person that is attracted by my profile and who wants to make sure that I am not a dream but a real person and a really terrific guy.
A woman who loves herself as depicted by caring for and about herself: physically, emotionally and spiritually, and is therefore capable of also loving someone else.
Someone who has the ability to express their feelings both physically and verbally, without anger and judgment. Someone who loves to cook and likes to share in the preparation of food.
An individual who thinks deeply and is deeply thoughtful, one who questions conventional wisdom, thinks creatively and iconoclastically. Someone that is sophisticated, passionate, fun, sexy, kind and self-aware, and who like myself, does well on their own as well, or perhaps even better, as together with their partner
About Me
Height | 5' 6" (167 cm) |
Build | Athletic |
Hair | Other |
Eyes | Brown |
Relationship Status | Divorced |
Children | 2 |
Children home | 0 |
Smoking | Never |
Drinking | Never |
Religion | I'm not affiliated with any religion |
Education | Master's degree |
Field of Work | consultant |
Ethnicity | Caucasian / White |
Languages Spoken | English |
Originally From | USA |
Fun Facts
Music | Soul/Rhythm and Blues |
Movie | Action/Adventure |
Indoor activity | Dancing |
Politics | Open Minded |
Timeliness | I am usually on time |
As for fashion | I dress to be comfortable |
Match Preferences
Age | Between 50 and 69 years old |
Distance | Within 50 miles of west chester, PA, USA |
Height | Between 5' 0" (152 cm) and 5' 6" (167 cm) |
Body Type | Athletic, Average, I should maybe lose a few |
Education | High School, Technical School, Associate degree, Professional degree, Bachelor's degree, Graduate Student, Master's degree, Doctoral degree |
Relationship Status | Single - never been married, Divorced, Widowed |
Religion | Agnostic, Anglican, Atheist, Buddhist, Confucian, Interdenominational, Jewish, Presbyterian, Protestant, Unitarian Universalist, I'm not affiliated with any religion, I'm not religious, Other |
Ethnicity | Any |
Smoking | Never |
Drinking | Never, Rarely, On occasion |


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Each week I meet with myself and plan out the week ahead. At least most weeks I do. Sometimes I miss this "meeting with me" and notice the chaos as the next few days get away from me. When I lay out the week before it hits, I get more done with less effort. Nice.
Each week I meet with myself and plan out the week ahead. At least most weeks I do. Sometimes I miss this "meeting with me" and notice the chaos as the next few days get away from me. When I lay out the week before it hits, I get more done with less effort. Nice.
Once a quarter, I comb through my business plan. I read through it, skimming over the pages and focusing on the items that have eluded me so far in the quarter, in the year…in my life. Now and then, I notice something that I have not gotten accomplished. I consider if I am making any progress at all in this direction. It's in the plan because at some point that goal made sense to me. Now, I question, "Do I care whether or not I accomplish this goal?" If it has ceased to inspire me, I scratch it from the plan.
So, my business plan is getting slimmer. Interestingly, the fewer goals on my list are getting bigger and bolder. That's pretty darn exciting, at least to me, and that's the idea. I get fired up reviewing my plan and focusing on the few things I am absolutely going to make happen.
You are smack dab into the middle of the first quarter 2009. Pull out your business plan and take a look at your mission, your vision and your goals for 2009. How's it going?
What? No plan? No problem. Just consider how last year went and that's what will probably happen this year. Only a little worse. Or a little better. If you want a quantum leap, or at least a major change of momentum, putting a plan together may help you think in dramatically different ways. Those thoughts of what you would rather have, what you really, really want, are what's required to revolutionize your life. Writing down what you want, from your business and your life, is a business plan. Easy enough.
Did you put a plan together once upon a time, and then stuffed it in a file somewhere? Maybe the plan was required to get a bank loan, or your wife's consent, to start your business. Before you started your business, the plan was perfect, a home run. Once the first shot was fired in biz battle, that plan might have gone into the file cabinet without another thought.
That's one of the tricky things about a plan. You have to launch your intentions in the uncharted waters of the future. Still, it is worth doing a plan, and updating that plan, because it is a powerfully positive tool for making what you want happen.
So grab your plan and a pencil…and start pruning.
Start with your Mission. Put your hand over the words. Do you have it memorized? I thought not. Count the words. Are there more than a dozen? Start pruning. Answer this question, "Why are you in business?" in under 12 words. Work it over a few times until you really care about your mission.
Now, who really cares about what you do? Who would be willing to pay you for it? Nail down your Target Market. Imagine your favorite customer. What does she drive? Where does she shop? What does she want from you? You might ask and find out. Put together a succinct paragraph describing your target market.
Next, review your Goals. Do you really want what you have listed? Do you believe in your goals? Cut the list down to the five most important goals. Include a financial goal and be specific in dollar and due date. Re-craft your goals using words that reflect your intention. Be specific. "More money" is not a goal. "Sales at $100K per month for Quarter 1, 2009" is a goal. Create goals that double as affirmations. Get rid of every goal that someone else wanted you to put on this list, if it doesn't mean much to you. Add a biggie, one that really gets your blood pumping. Why not? This is supposed to be fun.
On to the Organization Chart. Take your pencil and cross through the name of the person who needs to go…now. Shake off the denial and allow that person to go be a winner elsewhere. Next, look for someone who is not quite ready for a promotion but shows willingness and has "fizz." Promote him now, and see if he can fill in the gap between what he has and what he needs to do the job. Take a risk.
Review your Marketing Plan and Sales Processes. Upgrade the words that you use to communicate. For instance, I have added the word "explore" to my sales presentations. "Let's explore a few ideas for working together." It's friendly, non-threatening…and allows both parties to consider if working together is a good idea. Strike the word, "estimate" from your vocabulary…and all your sales and marketing systems. "Estimate" means "no one is buying today." Call it a "presentation" or a "visit." Pay attention to the words you use and the effect they have on the people who hear them. This small adjustment may have a big impact on you and your team.
Look at your Top Projects List. If you have a stalled out project, why not prune it? If you have delegated this project, ask, "What's the hold up?" Then, listen for the obstacles. Be willing to sit in silence until your team member comes up with something. Does the project need to be cut into smaller pieces? Consider what a next step could be and set the due date. You might move the project forward, or learn what another step could be. Consider also what would happen if you just aborted mission on it? If it is great opportunity and you really want it to happen, then make it happen. Otherwise, scratch it from the list.
Double check your Quality Department. Make sure your customers are well served. Ask them. Scrap your page-long customer survey and replace it with one question, "How did we do?"
May your pruned plan provide new focus and energy as you pave the way for next week, next quarter…the rest of your best year yet!
Here are my TOP FIVE Biz Plan Basics for building a successful business:
- Get a 3-ring binder. This is where you will store your business plan workbook. A binder is "old school" cool. It's a portable, practical way to get your ideas out of your head and onto the page. Sure, you can keep your plan on your computer as well. Still…nothing beats the binder.
- Adopt a simple business plan system. The Bare Bones Biz Plan is a fun, flexible, workable format for crafting your business vision and holding you accountable for getting things done. If you have a plan already, use that one and update it. There is nothing magic about the plan. The key is to use it.
- Hold yourself to one hour a day, five days a week, to work on your plan. In six weeks, you could have a living, breathing plan and your life and business could be totally transformed. This is easy…this is something you can do.
- Fill your head with powerful, positive thoughts. Are you ready to drop the baggage that is holding you back? It takes changing the words in your head. One way to do that is to listen to positive, business building, life-enhancing words. Play self-help cd's in your car. Listen to free podcasts and webinars on your computer. That which you think about comes about.
- Give it away to get it back. As you commit to your own success, commit to another's as well. Find a business owner buddy and vow to help each other put your business plans together. Teach your kids business planning as they consider a summer job or lawn mowing business. Teach your employees business plan basics as you learn them. What goes around comes around.


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The Criminal Defense Clinic provides a community service by representing defendants in criminal cases who would otherwise be unable to afford their own legal representation. Participants are limited to third year law students and a maximum of 8 students are selected for this clinic.
The Criminal Defense Clinic is offered as a full-year graded course during the Fall and Spring semesters. Participants earn six credit hours per semester (2 for classroom component and 4 for casework) and earn a total of twelve credit hours for the year.
The Criminal Defense Clinic offers effective counsel to indigent defendants charged with misdemeanors, felonies, and juvenile offenses in Lubbock County and the surrounding metropolitan area.
The Criminal Defense Clinic does not accept walk-ins or direct requests for representation


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Tweet Mizzou's Alden Retires Today UFC 186 Draws 10,154 In Montreal Pitt Names Barnes New AD Protests Erupt Outside Of Camden Yards Rockies To Celebrate 20 Years At Coors Field ESPN's McHenry Returns From Suspension Sterling Scandal's One-Year Anniversary Reached Oilers Hire Former Bruins GM Chiarelli Weekend Briefs
SBD/Issue 126/Events & Attractions
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Cowboys Stadium Draws 50,994 Fans For Pacquaio-Clottey Bout
An announced crowd of 50,994 watched Manny Pacquiao defeat Joshua Clottey at Cowboys Stadium on Saturday, a "historic night for boxing in Texas and the United States," according to Barry Horn of the DALLAS MORNING NEWS. The night featured Pacquiao, the "biggest star in the sport, fighting before an enthusiastic throng with Cowboys cheerleaders flitting in and out of view." And to make sure "no one could forget whose house it was, three Cowboys cheerleaders sang the national anthem." The crowd fell short of the "record for boxing paid attendance" in Texas, as the Pernell Whitaker-Julio Cesar Chavez bout in '93 drew 58,891 fans at the Alamodome (DALLAS MORNING NEWS, 3/14). ESPN.com's Dan Rafael noted it was the "third-largest attendance in U.S. boxing history," topped also by the 63,350 who attended the '79 Muhammad Ali-Leon Spinks fight at the Superdome. Saturday night's crowd "cheered Pacquaio's every move," and they "probably would have cheered had he been in the ring shadowboxing in the main event of the first card at Jerry Jones' sparkling" stadium (ESPN.com, 3/13). There was a "massive eruption of cheers" when Pacquiao first appeared entering the ring, and the "support continued whenever his mug flashed" on the HD videoboard (FT. WORTH STAR-TELEGRAM, 3/14). The AP's Jaime Aron noted only 45,000 tickets were made available initially, and "standing-room-only tickets were made available Friday." While not "every seat was great," every fan in attendance "felt close to the action" thanks to the videoboard (AP, 3/13).
A STAR PERFORMANCE: In Ft. Worth, Gil LeBreton wrote the "second-biggest story" behind Pacquaio's dominating victory "had to be the venue, a cozy little room with 72-foot TV screens and seemingly about 50,000 of Manny's closest friends." The videoboard was a "much-needed crutch Saturday night," and there was "atmosphere aplenty." For fans seated on the stadium floor, the ring itself "seemed like a thin and distant object," and the "giant video board was hard to see, if you were seated in the shadow of it." LeBreton: "But for sheer panorama and I-was-there value, Jerry Jones' house of punches delivered as promised" (FT. WORTH STAR-TELEGRAM, 3/14). The DALLAS MORNING NEWS' Horn today writes the "giant video board was mesmerizing and the presentation around the ring was electric" (DALLAS MORNING NEWS, 3/15). Boxing historian Bert Sugar: "When I saw this, I said to Jerry Jones, 'Just think what God could have done with this stadium if he'd had the money.'" In Ft. Worth, Jan Hubbard noted despite the "enormity of the stadium, it didn't seem too big." It was "hardly intimate, but a giant curtain was hung to hide seats in the upper deck" (FT. WORTH STAR-TELEGRAM, 3/14). SI.com's Bryan Armen Graham wrote, "We need more boxing in stadium-size venues." Cowboys Stadium was "electric from start to finish." With Miguel Cotto and Yuri Foreman set to fight at Yankee Stadium on June 5, "here's hoping we're seeing the beginning of a trend of boxing in larger venues" (SI.com, 3/14).
Texas To Set Aside $31.2M In Sales Tax Revenue For Super Bowl XLV
Texas state officials Friday said that they "would set aside $31.2[M] in sales tax revenue to help fund Super Bowl XLV" at Cowboys Stadium next year, meaning that local taxpayers "won't be on the hook for security-related costs for events in Dallas, Arlington, Fort Worth and Irving," according to Mosier & Nix of the DALLAS MORNING NEWS. The North Texas Super Bowl Host Committee "should have enough money to pay for its costs related to the game" on February 6, 2011. No decision "has been made about how the money will be split." The host committee and the cities are "all expected to tap into the fund." The fund taps "four sources -- sales, hotel, car rental and alcohol taxes -- paid by out-of-towners who come to the Super Bowl" (DALLAS MORNING NEWS, 3/13). Host Committee President & CEO Bill Lively said that the committee's budget is about $40M, and the group has "raised about $21[M] in cash and in-kind sponsorships." Host Committee Chair Roger Staubach: "With sponsorship and this, we should be able to fulfill every promise we've made." The $31.2M figure is "short of the $36.2[M] that the host committee had sought from the state fund," but Lively said that the "difference isn't meaningful and won't affect services." He said that the committee "will continue to sell sponsorships to cover expenses that aren't reimbursable." In Ft. Worth, Scott Nishimura noted the host committee "touted Texas' decision as an affirmation of an economic-impact study" the group had submitted with its request for funds. That study, conducted by California-based Marketing Information Masters, "estimated that Super Bowl XLV will generate a record $611.7[M] in economic impact" (FT. WORTH STAR-TELEGRAM, 3/13).


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How to keep it real and sustainable
Join the handmade movement and enter to win* sustainable artisan goods from around the world.
Across the world, the best things come from real people. Whether you're hiking with a guide along the Inca Trail to the lost city of Machu Picchu or learning to weave alpacca in a Quechua village, the best things in the world are personal.
Why we no longer ride elephants
Intrepid staff and travellers are a very passionate lot; unafraid to speak up about ethical and social justice issues they may be confronted with during their travels.
Human rights issues, poverty, environmental concerns…there are many, but one of the areas that Intrepid folk are most vocal about is animal welfare. From witnessing animals in distress, to captive animals in poor conditions or working animals, particularly if it's connected to tourism, Intrepid staff and travellers don't hold back on speaking up for the voiceless.
How to talk the walk on the Camino
Walking the ancient pilgrim path of the Camino de Santiago has attracted a myriad of nationalities for centuries.
Beyond the native Spanish, many languages are spoken on the way, but as discovered by Glenyce Johnson, Intrepid's General Manager – Business Development, the conversation generally started with much in common.
Finding a great future in Turkey
In its strategic east-meets-west location, Istanbul has attracted migrants and travellers for centuries.
Within the Fener and Balat districts, where the population is mostly rural immigrants, is a fabulous organisation receiving support through The Intrepid Foundation, and turning around the lives of young people from disadvantaged backgrounds.
More praise for Monty
We are thrilled that Intrepid's Dheeraj 'Monty' Bhatt is once again a finalist in the prestigious Wanderlust World Guide Awards.
To be recognised as one of the best in his business amongst such a talented team of guides is an achievement in itself, but to receive such high praise several times during his 8 years with Intrepid is testament to Monty's dedication and passion for adventure travel.
Walking the Camino – do you need a reason?
For over a thousand years, people have embarked on pilgrimages along El Camino de Santiago as an act of Christian spiritual devotion.
For Intrepid co-founder, Darrell Wade, the experience of walking on the Camino in Northern Spain was perhaps more on the self-gratifying or pleasurable side…
Intrepid Travel puts customers in focus
We all know what it's like to be a customer and we have all been on the receiving end of good and bad customer service.
If you pick up any trade or business magazine you'll read how many companies are these days claiming that "It's all about the customer", but are they really putting their clients first?
How travel makes you happy
March 20 is proclaimed as the UN International Day of Happiness. The day recognises the importance of happiness and well-being as universal goals and aspirations in the lives of people everywhere.
It was inspired by the country of Bhutan and their political philosophy of prioritising and measuring happiness amongst their people, over income.


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Created in this Craftsy class
Knit This: Mastering Lace Shawls
taught by Laura Nelkin
Learn to knit two beautiful lace shawls with Laura Nelkin as she sends you on a lace journey that is out of this world!
Other projects made as part of this class:
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Here are some details about my project: | |
Category | Knitting |
Type | Accessory |
Sizing | Women |
Style | Whimsical |
What an absolutely beautiful job you did!!! I have never made anything like this in my life but seeing this I believe I am going to give it a try. Thank you so much for sharing your finished product!
thank you so much!!! you should totally give it a try! it took me like a month but it was because I have a toddler who wouldn't let me knit unless she was napping hahaha. Anyways the class is really good in going step by step reading the chart and explaining the stitches!! lots of fun!!