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This study provides a novel view on the interactions between the MS-KIF18A, a kinesin protein, and estrogen receptor alpha (ERα) which were studied in vivo and in vitro. Additionally, the regulation of MS-KIF18A expression by estrogen was investigated at the gene and protein levels. An association between recombinant proteins; ERα and MS-KIF18A was demonstrated in vitro in a pull down assay. Such interactions were proven also for endogenous proteins in MBA-15 cells were detected prominently in the cytoplasm and are up-regulated by estrogen. Additionally, an association between these proteins and the transcription factor NF-κB was identified. MS-KIF18A mRNA expression was measured in vivo in relation to age and estrogen level in mice and rats models. A decrease in MS-KIF18A mRNA level was measured in old and in OVX-estrogen depleted rats as compared to young animals. The low MS-KIF18A mRNA expression in OVX rats was restored by estrogen treatment. We studied the regulation of MS-KIF18A transcription by estrogen using the luciferase reporter gene and chromatin immuno-percipitation (ChIP) assays. The luciferase reporter gene assay demonstrated an increase in MS-KIF18A promoter activity in response to 10−8 M estrogen and 10−7M ICI-182,780. Complimentary, the ChIP assay quantified the binding of ERα and pcJun to the MS-KIF18A promoter that was enhanced in cells treated by estrogen and ICI-182,780. In addition, cells treated by estrogen expressed higher levels of MS-KIF18A mRNA and protein and the protein turnover in MBA-15 cells was accelerated. Presented data demonstrated that ERα is a defined cargo of MS-KIF18A and added novel insight on the role of estrogen in regulation of MS-KIF18A expression both in vivo and in vitro.
Citation: Zusev M, Benayahu D (2009) The Regulation of MS-KIF18A Expression and Cross Talk with Estrogen Receptor. PLoS ONE 4(7): e6407. doi:10.1371/journal.pone.0006407
Editor: Jose Vina, University of Valencia, Spain
Received: January 19, 2009; Accepted: June 17, 2009; Published: July 28, 2009
Copyright: © 2009 . This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by CellProm project, FP6th of the European Community (NMP4-CT-2004-500039) and internal grant from Tel Aviv University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Kinesins are microtubule-dependent motor proteins, with more than 45 members expressed in mammalian cells. Kinesins are classified based on three structural and functional regions: (i) a motor domain with microtubule binding site and a catalytic ATPase domain; (ii) central alpha-helical-coiled coil region which possesses protein-protein interactions and (iii) tail which interacts with cargo [1]. Different kinesin proteins share high homology of their motor domain but diverge considerably in the cargo-binding tail. Structural heterogeneity of kinesins is the basis for their diverse functions in various cellular processes including transport of cargoes such as membranous organelles, macromolecular complexes and mRNA [2]–[7]. It has been shown that kinesins play a role in trafficking directed towards the cell periphery, for example motility from the Golgi to the plasma membrane [8].
MS-KIF18A is a member of Kinesin-8 sub family [9] which was cloned from the marrow stromal cells and characterized by bioinformatic and biochemical means [10], [11]. Estrogen receptor alpha (ERα) was identified as a cargo for MS-KIF18A. We also suggested a role for this kinesin in estrogen signaling pathway [12]. Estrogen has pivotal functions in both female and male physiology and has been recognized as a regulator of bone remodeling in maintaining of bone mass and keeping the balance between bone formation and resorption [13]–[15]. Estrogen deficiency in vivo is recognized during post-menopause or following ovariectomy and associated with an increase of osteoclasto-genesis and decrease in osteogenesis that lead to bone destruction [16]–[19]. Estrogen hormone action affects cell proliferation and differentiation via the estrogen receptors (ERs). The ERs are expressed in various cells including osteoblasts [12], [20]–[24], osteocytes [25] osteoclasts [26] and mammary epithelial cells [27]. Specifically, ERα is identified in two isoforms: 66 kDa and 46 kDa, the shorter form lacking a ligand-independent activation function domain 1 (AF-1) [28], [29].
Steroid hormone binding to the receptors leads to a rapid (second – minutes) non-genomic signal transduction or to a prolonged genomic signaling [30]. The non-genomic pathway is mediated by activation of Mitogen Activated Protein Kinase (MAPK) proteins such as p38 and ERK1/2 [31] and increase in Ca2+ ion concentration [32], [33] or Inositol 1, 4, 5-trisphosphate (IP3) [34]. Such activation controls various cellular activities including cell proliferation, response to inflammation mediated via inhibition of NF-κB activation [35] and anti-apoptotic events [36]–[38]. The prolong estrogen action occurs within 30–60 minutes where the receptor is translocated to the nucleus and leads to genomic response. The ERα binds directly to estrogen response elements (EREs) [39] or indirectly via accessory proteins on AP-1 or Sp-1 binding sites [40] on promoters of target genes. The ERα translocation to the nucleus is a dynamic process regulated by ATP activity or by ligand-induced conformational changes and proteasome function. Depletion of ATP retards the intra-nuclear mobility of un-liganded ERα and causes the receptor redistribution to the cytoplasm [41]. When cells' treated with either 17βE2 or tamoxifen prior to ATP depletion the ERα was less mobile, more prominent in the nucleus and reduced the shuttling to the cytoplasm [42]. The ERα shuttling as ATP-dependent phenomena implies a role of motor protein in this process; however, thus far a candidate for such protein was not identified.
In this study, we presented two views on the MS-KIF18A - ERα cross talk: one aspect investigated the complex formation between MS-KIF18A and ERα and the second studied the regulation of MS-KIF18A expression under estrogen paradigm. The nature of interactions between ERα and MS-KIF18A was demonstrated using recombinant and endogenous proteins by immunoprecipitation (IP) and western blot (WB) assays. MS-KIF18A mRNA expression was analyzed in vivo in bone marrow cells or in vitro in a pre-osteogenic MBA-15 cells and breast carcinoma MCF-7 cells that are estrogen responsive cells. Estrogen effects on the binding of ERα and pcJun to MS-KIF18A promoter was studied by chromatin immunoprecipitation (ChIP) and the activation of the promoter was analyzed by luciferase reporter assay. The regulation of MS-KIF18A protein expression and turnover was explored by metabolic labeling and immunological analysis. The present research provides a novel view on regulation of MS-KIF18A and its' association with ERα and significantly contributes to the profound understanding of estrogen mediated activities.
The association between MS-KIF18A and a putative cargo; ERα was demonstrated in our laboratory in earlier study [12]. Currently, we elaborated on the interactions between these proteins using an in vitro pull down assay which applied recombinant proteins. We used three recombinant isoforms of MS-KIF18A: full length of MS-KIF18A (Figure 1A) and truncated forms: 1–635 AA that includes the motor domain and the coiled-coil region (Figure 1B) and 635–898 AA that contains the cargo binding domain (Figure 1C). The protein association between the three MS-KIF18A constructs and ERα was demonstrated by Co-IP and WB using three antibodies: a polyclonal anti-MS-KIF18A which identifies an epitope at the motor domain, a monoclonal anti-MS-KIF18A which identifies an epitope at the cargo-binding domain and anti-ERα (Figure 1D–1F). In addition, we previously demonstrated interactions of endogenous proteins in MBA-15 cells and revealed an association of MS-KIF18A with both 46 kDa and 66 kDa ERα isoforms [12]. Herein, we analyzed the 17βE2 effect on this complex formation in MBA-15 cells. Cells were pre-incubated in steroid-free serum for 48 h and then challenged with 10−8 M 17βE2 for 16 h followed with IP using either anti-MS-KIF18A or anti-ERα and analyzed by WB. In the treated cells, we detected a reduction in ERα appearance and an increase in MS-KIF18A-46 kDa ERα complex formation (Figure 2).
Schematic illustration of full length MS-KIF18A (A); truncated MS-KIF18A construct 1–635 aa (B); truncated MS-KIF18A construct 635–898 aa (C). (D–F) Co-IP experiments of MS-KIF18A constucts with recombinant ERα and WB with monoclonal anti-MS-KIF18A (1) and anti-ERα (2). Full length MS-KIF18A (D), MS-KIF18A constucts 1–635 aa (E), MS-KIF18A constucts 635–898 (F). Pull down with beads only (1); IP with anti-MS-KIF18A and WB with anti-ERα (2) (G). The results are representative from the set of at least three independent experiences.
MBA-15 cell lysates were IPed with anti-ERα or anti-MS-KIF18A, and analyzed by WB. Results are of representative experiment of a series repeated five times.
MS-KIF18A sub-cellular distribution and co-expression with ERα was analyzed at the cytoplasm (C) and nuclear/membrane (N/M) compartments. The ERα expression was identified at higher level in the nucleus, while MS-KIF18A was prominently localized in the cytoplasm (Figure 3A). IP with anti-MS-KIF18A and WB analysis with anti-ERα detected the MS-KIF18A-ERα complex mainly at the cytoplasm (Figure 3B). To elaborate on the role of MS-KIF18A in ERα signaling pathway we analyzed the interactions of ERα and MS-KIF18A with NF-κB (p65 and p50 subunits). Cell lysates were immunoprecipitied with antibodies towards p65 and p50 subunites of NF-κB and follwed with WB analysis using anti-MS-KIF18A. An association was found between MS-KIF18A and p50, whereas no interactions with p65 was observed (Figure 4A). However, protein complex was noted between ERα and both forms of NF-κB (Figure 4B).
Whole lysates (A) and IP (B) of fractionated cells were analyzed with anti-MS-KIF18A and anti-ERα. Results demonstrate a representative experiment of four independent repeats.
IP with anti-p50 (1) with anti-p65 (2) WB performed with anti-MS-KIF18A (A) with anti-ERα (B). Results revealed an association between MS-KIF18A and p50, but not with p65 while ERα interacts with both forms of NF-κB; p65 and p50. Results demonstrate a representative experiment of three independent repeats.
The role of estrogen on MS-KIF18A mRNA expression was analyzed in vivo and in cell culture. MS-KIF18A mRNA measured in vivo on RNA isolated from bone marrow cells harvested from rats and mice and correlated the expression levels in relation to animal age (Figure 5A, 5B). Studying mice, we measured 6-folds higher mRNA levels in young males then in old animals (p = 0.0019, Figure 5A). In rats, the expression of the mRNA was 4-folds higher in young male animals than in old ones (p = 0.0022, Figure 5B). In young female OVX-rats the level of mRNA was 3-folds higher compared to old ones (p = 0.0047, Figure 5B). All together, we noted a higher MS-KIF18A mRNA levels in bone marrow of young animals that was decreased with age. In addition, we analyzed sham rats that expressed MS-KIF18A mRNA levels 3-folds higher as compared to OVX-rats (p = 0.0084, Figure 5C). When OVX-rats were treated with 17βE2, a 12.5-folds increase in mRNA levels was detected (p = 0.0078, Figure 5C). These results provide the in vivo evidence of estrogen impact on the regulation of MS-KIF18A expression.
Total RNA from bone marrow cells were harvested from mice (A) and rats (B, C) and analyzed by qRT-PCR. (A) mRNA expression in bone marrow cells derived from young 4 month (white bars) and old 12 month (black bars) male mice; (B) Young 3 month (white bars) and 14 month old (black bars) male and OVX female rats; (C) Sham, OVX and OVX+E2 female rats. MS-KIF18A mRNA expression was normalized to G3PDH expression levels. Results are presented as mean values +/− SD obtained from triplicates for each data point.
The stimulatory effect of 17βE2 on MS-KIF18A mRNA expression led us to analyze the transcription regulation using a reporter gene assay. We analyzed in silico, the 1500 bp upstream to the TSS of the MS-KIF18A gene. This region was predicted as putative promoter and mapped for transcription factors (TFs) binding sites and regulatory elements. Bioinformatics analysis using MatInspector software enables to identify the promoter region and the TF binding sites and regulatory elements. Specifically, we mapped a non-palindrome half-site ERE that binds ER directly and AP-1 site that binds ER via accessory proteins such the pcJun. We amplified this genomic region by PCR and cloned into pGLuc vector upstream to luciferase reporter gene (pGluc-K) (Figure 6A). The cloned pGluc-K plasmid was co-transfected with β-galactosidase (β-GAL) plasmid in MCF-7 cells that were treated with 10−8 M 17βE2 or/and 10−7 M ICI 182,780 for 1 h or 24 h. The promoter activity was quantified by luciferase activity normalized to β-GAL (which indicates the transfection efficiency). In 17βE2 treated cells' we measured an increase in luciferase activity: 1.5-folds after 1 h (p = 0.0001) and 1.7-folds after 24 h (p = 0.01) as compared to control cells. Cells treated with ICI-182,780 demonstrated an increase of luciferase activity; 1.6-folds following 1 h (p = 0.02) and 1.5-folds after 24 h (p = 0.006) as compared to untreated cells. The combined treatment of 17βE2/ICI-182,780 resulted with a similar increase of luciferase activity after 1 h (p = 0.0012) and increase 2.5-folds after 24 h (p = 0.023) of treatment (Figure 6C). No activity in transfected cells with pGLuc-basic plasmid was detected (Figure 6B).
(A) Schematic illustration of MS-KIF18A promoter-luciferase reporter constructs. MCF-7 cells transfection with MS-KIF18A promoter cloned in luciferase reporter plasmid (pGLuc-K) or promoter less pGL3-basic along with β-galactosidase vector. (B) Cells treated (black bars) or not (white bars) with 17βE2 (10−8 M) for 24 h; (C) 17βE2 (10−8 M) or/and ICI-182,780 (10−7 M) were added to the cultures for 1 h (white bars) or 24 h (gray bars). Promoter activities are expressed as luciferase values normalized for β-galactosidase levels. A value of 100% was given to the basal promoter activity elicited by the pGLuc-K construct in the absence of any treatment. Results are mean±SD of 3 independent experiments, performed in duplicates.
ChIP assay was applied to correlate the endogenous regulation of promoter activity. We used antibodies to ERα and pcJun to measure their binding to MS-KIF18A promoter in MCF-7 and MBA-15 cells (Figure 7). In MCF-7 cells treated with 17βE2 for 60 min, we noted 4-folds increase in ERα binding (p = 0.0007, Figure 7A) while no change in the level of pcJun binding (Figure 7B) was observed. When the MCF-7 cells were treated with ICI-182,780, binding of ERα was 9-folds higher (p = 0.0001, Figure 7A) and the binding of pcJun was 2-folds higher (Figure 7B) as compared to untreated cells. Treatment of MBA-15 cells with 17βE2 for 2 h then ChIPed with anti-ERα revealed a 2-folds increase of ERα binding to the analyzed promoter (p = 0.0281, Figure 7C) as compared to untreated cells.
A-C Bar histogram of qPCR analysis of amplified MS-KIF18A promoter in MCF-7 ChIPed by anti-ERα (A) or by anti-pcJun (B), and in MBA-15 cells were ChIPed by anti-ERα (C). All the results presented as mean values +/− SD obtained from three different experiments each performed in triplicates for each data point.
The consequence of 17βE2 or ICI-182,780 regulation of ERα and pcJun binding to MS-KIF18A promoter and its' activation led us to study the MS-KIF18A mRNA expression level under this paradigm. The message expression level was quantified by comparative qRT-PCR (Figure 8). MCF-7 cells response to 17βE2 treatment for 1 h measured 2-folds increase (p = 0.0014) while treatment with ICI-182,780 induced 34-folds increase (p = 0.014) of MS-KIF18A mRNA expression (Figure 8A). MBA-15 cells treatment with 17βE2 resulted with 1.3-folds elevation mRNA levels after 2 h (p = 0.0001) and 1.8- folds after 24 h (p = 0.0053) as compared to untreated control (Figure 8B).
(C) Effect of Act D on the stability of MS-KIF18A mRNA in MBA-15 cells treated in presence of 17βE2 for 6 h. The MS-KIF18A mRNA is samples were normalized to G3PDH for each data point. Results are presented as mean values +/− SD obtained from three different experiments each performed in triplicates for each data point.
Actinomycin D (ActD) is an inhibitor of RNA synthesis. We measured the mRNA levels in cells treated by this drug in presence or absence of 17βE2 and noted a 25-folds increase in mRNA levels in ActD/E2 treated cells as compared to cells' treated by ActD only (p = 0.0001, Figure 8C) indicating mRNA stabilization in cells treated with estrogen.
Additionally, we followed the MS-KIF18A protein in MBA-15 cells treated by 17βE2 for defined periods from 1 h to 20 h. The cell lysates were separated on SDS-PAGE gel and analyzed by WB with anti-MS-KIF18A. We noted an increase in MS-KIF18A protein expression already after 1 h of treatment that was maintained until 20 h (Figure 9A). In addition, MS-KIF18A protein turnover was analyzed in presence or absence of estrogen applying metabolic labeling with Met/Cis-S35 on MBA-15 cells. The cells were pretreated for 6 h, 24 h or 48 h with 17βE2, radio-labeled for 1 h and then chased at 4 time points from 1 h to 36 h. At each time point cell lysates were IPed with anti-MS-KIF18A, separated on SDS-PAGE and exposed to developing film (Figure 9B). A 100 kDa band was identified and confirmed by IP and WB analysis as MS-KIF18A (Figure 9C). We have shown MS-KIF18A protein degradation in period of 24 h to 36 h after cells' labeling. The estrogen treatment induced an increase of MS-KIF18A synthesis (1 h chase), accelerated protein turnover (12 h chases) and shortened its half-life (36 h chase). In addition, other prominent proteins of 230 kDa and 45 kDa (earlier identified as actin [11]) were Co-IPed by anti-MS-KIF18A (Figure 9B).
(A) Lysates of MBA-15 cells challenged with 10−8 M 17βE2 for 1 h, 2 h, 4 h and 20 h were analyzed by WB with anti-MS-KIF18A and compared to the untreated control at 1 h (C 1 h) and 20 h (C 20 h). (B) MBA-15 cells were pretreated with 10−8 M 17βE2 for duration of 6 h, 24 h or 48 h, metabolic labeled with Met/Cis-S35, chased for various time periods from 1 h to 36 h, lysed, IPed with anti-MS-KIF18A and loaded on SDS-PAGE gel. (C) A 100 kDa protein confirmed as MS-KIF18A by IP and WB with anti-MS-KIF18A. Results are of representative experiment of a series repeated three times.
In summary, we demonstrated the association between MS-KIF18A and ERα recombinant and endogenous proteins analyzed in MBA-15 cells. These proteins' association was regulated by 17βE2 and the complex appeared more prominent in cytoplasm. At the molecular level, we demonstrated estrogen dependent activation of MS-KIF18A promoter measured by luciferase assay and an estrogenic increase in ERα and pcJun binding to the promoter. The regulation of MS-KIF18A mRNA and protein expression by estrogen was demonstrated in vivo in mice and rats as well as in cell systems.
MS-KIF18A is a member of kinesin family, which functions as motor protein that binds to microtubule and uses the energy derived from ATP hydrolysis to move along the cytoskeleton. Previous studies analyzed the MS-KIF18A using bioinformatics and biochemical tools suggested the estrogen receptor alpha (ERα) as a putative cargo [10]–[12]. The present study elaborated on two aspects; one highlighted on the interactions between MS-KIF18A and ERα and the second studied the expression of MS-KIF18A under the paradigm of estrogen which applied in vivo and in vitro models.
The complex formation between motor proteins and their cargo comes from the motifs that play a role in protein-protein interactions. Bioinformatics analysis of MS-KIF18A protein sequence revealed several motifs of interaction with nuclear receptors (NR-boxes and ΦXXΦΦ-like motifs) and a region homologous to ERα in the coiled coil and the cargo-binding domains of the kinesin. Co-transfection of MS-KIF18A and ERα in COS-7 null cells and also Co-IP of endogenous proteins in MBA-15 cells demonstrated such association [10], [12]. Here, we elaborated on the nature of these proteins interactions in in vitro assay using three constructs of recombinant MS-KIF18A and ERα. We have shown that full length MS-KIF18A or its truncated forms bind the ERα. Thus, we propose a putative function for the NR motifs localized at the coiled-coil region and cargo-binding domain of the MS-KIF18A, in mediating the proteins interactions.
Estrogen is recognized to activate numerous of transcriptional events in many cell types. Estrogen receptor localization was noted at various cell compartments that are down regulated in cells treated by 17βE2 [43], [44]. ERα shuttles between different cellular compartments including cell membrane, cytoplasm and nucleus [12], [30], [43]. A small portion of ERα is localized at the caveolar fractions of the plasma membrane [12], [45], [46], however the receptor is mostly localizes in the nucleus [21], [42], [47]. A connection between ERα mobility and a putative cytoskeleton protein was suggested, but no candidate protein has been identified thus far [43]. Herein, we have shown that fractionated cells analyzed by WB localizes the ERα is mainly at the nucleus/membrane compartments, while MS-KIF18A is prevalent in cytoplasm [11]. The association between MS-KIF18A and ERα is higher in the cytoplasm, confirming the kinesins' action in this compartment. We have also shown that the complex formed between these proteins is stimulated by 17βE2.
ERα signaling involves activation of MAPKs such as p38 and ERK1/2 [31], resulting with various cell response. pERK protein was earlier detected in association with ERα and MS-KIF18A, suggesting a role for the MS-KIF18A in non-genomic activation of ERα through the MAPK pathway [12]. Herein, we elaborated on the protein complex between MS-KIF18A and ERα revealing it regulation by estrogen. It is recognized that ERα binds NF-κB in various cells affecting cellular process such as inhibition of inflammatory or during cell apoptosis [35]. NF-κB transcription factor dimerize the p65 and p50 subunits to hetero-complex [48]. We detected an association between both NF-κB subunits and ERα, while MS-KIF18A binds only the p50 subunit.
Estrogen regulates numerous cellular functions including the remodeling of the cytoskeleton proteins and their composition. The cytoskeleton is a dynamic network of proteins that undergoes restructuring during cell division, formation of cell-cell or cell-ECM interactions and cell migration. Cytoskeleton plays a role in controlling of cells shape and influences gene expression [22], [49], [50]. We earlier reported that 17βE2 affects the composition of cytoskeleton proteins, such as thropomyosin and tubulin and reorganization of actin fibers in MBA-15 cells [22]. Estrogen induces the remodeling of both the F-actin and the intermediate filament [50]. It was shown also in vivo, in OVX-estrogen depleted rats an up-regulation of tropomyosin 2β and tropomyosin 1α expression by 17βE2 [51]. Earlier we have shown the association between MS-KIF18A and cytoskeleton proteins tubulin and actin [11]. The current study provides new insights on the impact of estrogen on the complex formed between ERα and MS-KIF18A.
The expression of MS-KIF18A mRNA and its' regulation by estrogen was analyzed in vivo in rats and mice. We have noticed an age difference in MS-KIF18A expression: mRNA was higher expressed in young animals then in old ones. Such differences may account for the decrease in estrogen levels with aging [17], [52], [53] suggesting a hormonal role in regulation of MS-KIF18A expression in vivo. This observation was strengthened using OVX-estrogen depleted rats which measured a decline in MS-KIF18A message as compared to sham rats. When the OVX-rats were treated with 17βE2 we have shown the restoration of MS-KIF18A mRNA levels.
To unravel the mechanism of estrogen effects on MS-KIF18A expression we analyzed the kinesin promoter activity by luciferase reporter gene and ChIP assays. It is known that ERα activates promoters when binds directly to palindrome ERE or half-site ERE [39], [54], [55] or indirectly via accessory proteins (such as Fos and Jun) at AP-1 binding sites or to GC-rich sequences via complex with Sp1 [40], [56]. ERα also stimulates gene expression via interaction with nuclear receptor NF-κB and this complex binding to promoters of target genes [48]. Herein, we used the cloned promoter of MS-KIF18A that contains non-palindromic half-site ERE, AP-1 and NF-κB binding sites. The MS-KIF18A promoter was cloned under luciferase reporter and transfected to MCF-7 cells. When these cells were treated with either 17βE2 or ICI-182,780 we have noted a similar inducible effect of the promoter activity while the combined 17βE2/ICI-182,780 treatment had an additive effect. MS-KIF18A mRNA levels were quantified by qPCR and have shown an increase in cells treated with either 17βE2 or ICI-182,780. The rational for the ICI-182,780 effect shown here lies in the fact that this drug is no more considered as an estrogen antagonist and reports present a role for its agonistic action. The agonist property of ICI-182,780 was observed in vivo on bone growth [57]. Cells derived from human breast tumor tissues were analyzed on HTS affymetrix gene chip resulted with an up-regulation of gene expression when treated by estrogen and even more by ICI-182,780 [58]. Modulation by ICI-182,780 resulted with up regulation of quinine reductase in MCF-7 cells [59], ERRα in SKBR3 cells [60] and spinophilin in hippocampus neurons [61]. The promoter activation by ICI-182,780 is proposed to act via ER binding on AP-1 sites, but not via ERE [62]. Moreover, the ERα-ICI-182,780 complexes that sequester transcriptional repressors away from AP-1 sites allow an unrestricted transcription [63], [64]. From the current study the indication is that binding of 17βE2 and ICI-182,780 to ERα increases the receptor binding to MS-KIF18A promoter and allows its activation, leading to an increase in message transcription. Using the ChIP analysis we have shown that ERα and pcJun bind to MS-KIF18A promoter in MCF-7 and MBA-15 cells that are regulated by 17βE2 and by ICI-182,780. In another study, we have shown that ERα and cJun are differentially regulated when bind to SVEP1 promoter. In cells treated by 17βE2 it was shown an increase of the TF binding but not when treated by ICI-182,780 [65].
Complementary with reporter gene and ChIP assays we analyzed the role of estrogen on MS-KIF18A mRNA and protein expression. It was noted that MS-KIF18A mRNA expression increased after cells were challenged with 17βE2 or ICI-182,780. Furthermore, inhibition of transcription with ActD and 17βE2 stabilized MS-KIF18A mRNA and decreased its' degradation. MS-KIF18A protein levels also increased following 17βE2 treatment. Metabolic labeling assay confirmed that 17βE2 accelerates MS-KIF18A turnover. Taken together, the results elaborated on the effect of estrogen on MS-KIF18A expression, lifetime and degradation.
In summary, we have shown the interaction between MS-KIF18A and ERα as its cargo in in vitro and in vivo assays using different biochemical and molecular approaches. Moreover, the involvement of kinesin in ERα signaling was demonstrated. Furthermore, we have shown for the first time regulation of MS-KIF18A mRNA expression and protein turnover by estrogen, implying the kinesins' function in estrogen-dependent manner. These results can lead to further investigation of metabolic regulation in the mesenchymal stem cells that play a role in estrogen regulated maintenance of bone in metabolic diseases or in cancer.
Materials and Methods
Animals and Experimental Design
ICR mice age 4-month (young) and 12-month (old) old (n = 10 in each group). Fischer 344 male and female rats age 90-day (young) and 11-month (old) old (n = 13–18 rats in each group from both sex). Female rats were subdivided into a control sham group (abdominal midline incision), ovariectomized (OVX) rats or OVX rats that were implanted with 90-day slow-release pellets of 17β-E2 [17]. Animals were maintained and treated according to the Institutional Animal Care and Use Committee at the Tel Aviv University.
Cell culture
We used two cell lines known for their response to estrogen: MBA-15, a pre-osteogenic stromal cell line [22] and MCF-7 breast carcinoma cell line. Both were cultured in growth medium; Dulbecco's Modified Essential Medium (DMEM) (Gibco, USA) with the addition of 10% heat-inactivated fetal calf serum (FCS) (Biological Industries, Israel), supplemented with 1% glutamine and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Before cells' were treatment with 10−8 M 17βE2 or 10−7 M ICI-182,780, they were incubated in 3% serum stripped medium for 48 h. Where specified, cells were treated with 5 µg/ml actinomycin D (Sigma, USA) a transcription inhibitor.
Bioinformatics analysis
60 kb of genomic sequence at 5′-flanking upstream the transcription start site (TSS) of MS-KIF18A gene was analyzed to identify the gene-putative promoter. The analysis applied Promoter 2.0 Prediction Server (http://www.cbs.dtu.dk/services/Promoter) for promoter definition and MatInspector software (http://www.genomatix.de) for transcription factor binding sites identification. All primers were constricted using Primer3 Software (http://frodo.wi.mit.edu/cgi-bin/primer3).
Putative MS-KIF18A promoter cloning and activity
1.5-kb 5′-flanking upstream the transcription start site (TSS) promoter sequence segment was amplified from genomic DNA using the 5′ TACCAAGACCAGCAGCACAC and 3′ TAAGGAGATCCCTGCCCTTC primers. The PCR fragment was verified by sequencing then restricted by Bgl II and Bcl I and a 1.3-kb segment was cloned upstream of a luciferase reporter gene into pGLuc-basic vector (New England BioLabs, USA). The MS-KIF18A promoter reporter plasmids named pGLuc-K.
MCF-7 cells seeded in 6-well plates, after 48 h cells were transfected with 1.3 µg of the pGLuc-K or promoterless pGL3-basic along with 0.3 µg of β-galactosidase expression plasmid (pCMVβ; Clontech, Palo Alto, CA), using the jetPEI™ transfection reagent (Polyplus Transfection, Illkirch, France). After 24 hrs media was changed to 3% serum stripped medium supplemented with 10−8 M 17β-E2 and 10−7 M ICI-182,780 for 1 h or 24 h then medium was collected and cells were harvested for luciferase activity (New England BioLabs, USA) and β-galactosidase activity [66]. Promoter activities are expressed as luciferase values normalized to β-galactosidase levels.
mRNA and gene expression analysis by real-time quantitative PCR
Total RNA was extracted from cells (EZ RNA kit, Biological Industries, Beit Haemek, Israel) and reverse transcribed to cDNA using Reverse-iT 1st Strand Synthesis Kit (ABgene House, AB-0789) and oligo-dT primer (Takara Shuzo Co. Ltd., Seta, Japan). The cDNA used as template for polymerase chain reaction (PCR), using primers for MS-KIF18A from human, rat and mouse (Table 1). Amplified PCR products were detected by SYBR Green (ABgene House, USA). Verification of a single product amplified was checked for each primer pair by analysis of product melt curves on (MxPro™ QPCR Software, Stratagene, USA). MS-KIF18A cDNA PCR products were subjected to dissociation curve analysis resulting with fluorescence peak corresponding to the MS-KIF18A product centered at 80°C in rats and at 82°C in mice and humans. Primer-dimmers were distinguishable at 76°C. Level of expression for PCR products was normalized to G3PDH gene expression. Experiments were performed with triplicates for each data point.
Chromatin immunoprecipitation (ChIP) analysis of regulatory factor binding to putative MS-KIF18A promoter in human and mouse cells [67]
For assay, DNA was extracted from input chromatin fractions and complex was immunopercipitated with anti-ERα (Stressgen, Canada) or with anti-pcJun (Santa Cruz Biotechnology, USA). DNA served a template to amplify promoter of MS-KIF18A gene, with specific primers (Table 2) using real-time quantitative PCR. Input DNA was used as a positive control and for standard curve. The fluorescence peak dissociation corresponding to PCR product centered at 85°C and was distinguishable from the peak of primer-dimmer centered around 79–80°C.
Immunological methods
Cells' were collected for immunoprecipitation (IP) or whole lysate (WL) analysis; SDS-PAGE gel and Western blot (WB). Cells were washed twice with ice-cold PBS and collected in presence of protease inhibitors (phenylmethylsulfonyl fluoride, PMSF, 1 mM; 1-chloro-3-tosylamido-4-phenyl-2-butanone, TPCK, 10 µg/ml; aprotinin, 10 µg/ml and phosphatase inhibitors cocktails I and II (Sigma, USA). Samples were spin down at 1500 rpm for 4 min, lysed in lyses buffer consisting of 50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40; protease and phosphatase inhibitors; incubated for 20 min at 4°C and centrifuged at 16,000×g for 5 min. For WL the samples were resuspended in Lamelli sample buffer and boiled for 3 min. For IP the 1 µl of the antibody of interest and 25 µl Protein-A sepharose beads (RepliGen, USA) were added to lysates and samples were incubated overnight at 4°C. Immunocomplexes were precipitated at 16,000×g for 1 min and washed four times with lyses buffer. The washed beads were resuspended in Lamelli sample buffer and boiled for 3 min. The proteins were separated on 8% SDS-PAGE for 2 h 30 min and transferred to nitrocellulose for 1 h 30 min. For Western blot, the membranes were blocked with 5% BSA in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20, Sigma, USA) for 1 h at RT or overnight at 4°C and then incubated with primary antibody. The membranes were washed with TBST and incubated with goat anti-rabbit or goat anti-mouse conjugated to biotin (Dako, Denmark) for 40 min at RT and with extravidin-peroxidase for 30 min at RT (Sigma, USA) for detection with chemiluminescent substrate (Pierce, USA).
Polyclonal anti-MS-KIF18A (1∶1300) [10], [12]; monoclonal anti-MS-KIF18A (1∶500) [11]; anti-ERα (1∶800) (SRA-1010, Stressgen, Canada), anti-p65 and anti-p50 (Santa Cruz, USA).
Metabolic Labeling and Immunoprecipitation
MBA-15 cells were grown to 70% confluence in 100 mm dishes, cells were pretreated with 10−8 M 17βE2 for 6 h, 24 h and 48 h. The medium was replaced with medium depleted of serum for 1 h and cells were metabolically labeled in the presence or absence of 17βE2 for 1 h at 37°C using Redivue promix S35 label (200 µCi/sample; Amersham) in methionine and cysteine-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 3% stripped FCS. At the end of each time point, cells were washed twice with PBS and with DMEM supplemented with 3% stripped FCS with or without 17βE2 for variable periods of time. Cells were lysed in 50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP40 and protease and phosphatase inhibitors and MS-KIF18A was immunoprecipitated overnight at 4°C using anti-MS-KIF18A antibody and protein A sepharose beads (RepliGen, USA). The immunocomplex was washed three time with lysis buffer, resolved in 8% SDS-PAGE, and detected using Kodak BioMax MS Film.
Cell fractionation
Cells (1×107) were washed twice with 3 ml of cold PBS, resuspended in 100 µl Buffer A (10 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.025% NP-40) with protease inhibitors (1 µg/ml aprotinin, 1 µg/ml TPCK, 1 µg/ml pepstatin A, 0.2 mM PMSF) and incubated on ice for 20 min followed with centrifugation at 7500 rpm for 10 min at 4°C and the cytoplasm extract was removed, frozen and stored at −80°C. The pellet was resuspended in 50 µl Buffer B (20 mM HEPES, pH 7.4, 420 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.2 mM EDTA, 25% Glycerol) with protease inhibitors. The nuclear suspension was stirred vigorously on ice for 30 min. The sample was centrifuged at 15 000 rpm for 12 min at 4°C, and the nuclear/membrane extract was frozen and stored at −80°C. The protein concentration of cytoplasm and nuclear/membrane extracts was determined by the NanoDrop (ND-1000 Spectrophotometer, NanoDrop Technologies, Inc., USA).
Binding assay for recombinant proteins
MS-KIF18A recombinant proteins were expressed at the Structural Proteomics Center, Weizmann Institute of Science, Israel. Recombinant ERα was purchased (Sigma, USA). For experiments we used 0.25 µg of protein and pull down was performed in presence of either poly or monoclonal anti-MS-KIF18A, anti-ERα with 25 µl Protein-A sepharose beads (RepliGen, USA) overnight at 4°C. Then beads were spanned down, re-suspended with loading buffer and protein complex was analyzed by western blot.
Statistical analysis
Statistical analyses were carried out by Student's t-test, where values of p<0.05 are statistically significant. * is p-value≤0.05, ** is p-value≤0.01, *** is p-value≤0.001.
This study is a partial fulfillment of Margalit Zusev towards her PhD thesis. We are grateful to Dr. I. Shur and Dr. H. Schayek for valuable advices.
Author Contributions
Conceived and designed the experiments: MZ DB. Performed the experiments: MZ. Analyzed the data: MZ DB. Contributed reagents/materials/analysis tools: DB. Wrote the paper: MZ DB.
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Roles and Responsibilities
Director, Centre for Urban and Regional Development Studies (CURDS)(2012-)
Co-Director, i-BUILD research centre (Infrastructure BUsiness models, valuation and Innovation for Local Delivery)(2013-)
Daxia (Great China) Guest Professor, East China Normal University, Shanghai, People's Republic of China (2014-)
Special Researcher, Institute of Spatial Planning and Regional Economy (ISPRE), National Development and Reform Commission (NDRC), Beijing, People's Republic of China (2014-)
Advisory Board Member, ESRC, BIS and CLG What Works Centre for Local Economic Growth (2013-)
Editor, Regional Studies (2005-2014)
Visiting Senior Research Fellow, History, International and Social Studies, Ålborg University, Denmark (2008-09)
Visiting Senior Research Fellow, Centre for Urban and Regional Studies, Helsinki University of Technology, Finland (2008-09)
External Examiner, MSc Urban and Regional Development, Cardiff University (2013-)
External Examiner, MSc Local Economic Development, London School of Economics (2008-13)
Visiting Senior Research Fellow, Centro de Estudos Geográficos, University of Lisbon, Portugal (2003-)
ESRC Virtual College Member
Founding Degree Programme Director MA Local and Regional Development (Research) and MA Regional Development and Spatial Planning (2002-2013)
PhD Geography, University of Liverpool
BA (Hons) Geography, University of Liverpool
Previous Positions
2003-2009 Senior Lecturer, CURDS, Department of Geography, Newcastle University, UK
1995-2003 Lecturer, CURDS, Department of Geography, Newcastle University, UK
1993-95 Research Associate, CURDS, Newcastle University, UK
Member and Fellow of the Regional Studies Association
IBG-RGS Economic Geography Research Group
Institute of Economic Development
Honours and Awards
Regional Studies Association Best Book Award 2012 (Handbook of Local and Regional Development edited with Andrés Rodríguez-Pose (LSE) and John Tomaney (UCL), Routledge: London)
Regional Studies Association Best Book Award 2010 (Whither Regional Studies?, Routledge: London)
Conferment as Academician of the Academy of Social Sciences (2009-)
Best Published Paper Prize Nomination, Association of European Schools of Planning and US Association of Collegiate Schools of Planning, 2007 ("What kind of local and regional development and for whom?" with Andrés Rodríguez-Pose and John Tomaney, Regional Studies)
Research Interests
My central research interest is the geographical political economy of local and regional development. This core provides the basis for two main strands of work. First, this research is concerned with the concepts and theory of the meaning and governance of development regionally and locally in an international context. In particular, this work seeks to question and broaden our understandings of 'development' beyond the economic to encompass the social and ecological in more sustainable and progressive ways and to begin more meaningfully to connect development locally and regionally in the global North and South.
Second, this research focuses upon the intersections between local and regional development and Economic Geography. This theme has been explored in ongoing work on the geographies of brands and branding, and collaborative work on evolutionary approaches and the geographies of financialisation.
My research builds upon close policy engagement and has informed local and regional development policy for international (e.g. European Commission, United Nations International-Labour Organization (UN-ILO), Organization for Economic Co-operation and Development (OECD), national (e.g. Government Departments – Business, Innovation and Skills; Communities and Local Government), regional (e.g. Local Enterprise Partnerships, Regional Development Agencies, trade unions, voluntary organisations) and local (e.g. Local Authorities, Development Agencies) organisations.
Google Scholar profile: http://scholar.google.co.uk/citations?user=fFRhgXEAAAAJ&hl=en
Current Work
My current work is focused on five areas of activity:
First, I have completed a book manuscript on 'Origination: The Geographies of Brands and Branding'. This has been published by Wiley-Blackwell – see http://eu.wiley.com/WileyCDA/WileyTitle/productCd-1118556402.html. Caribbean cuisine, Latin American coffee, American high-tech, Danish furniture, Thai silks, Catalan design, Hollywood films, Broadway theatre, Florentine leather goods, Hunter Valley wines, London insurance… Where branded goods and services commodities are from and are associated with is integral to their meaning and value. Internationalisation, even globalisation, has complicated the picture as brand owners, managers, marketers, consumers and regulators have grappled with questions of origin(s), provenance, authenticity and transparency. Yet, longstanding research has been fixated with the 'country of origin' effect on consumer behaviour and failed to develop ways of thinking about the geographies of brands and branding that encompass and extend beyond this national frame. Origination introduces and develops a new way of understanding and explaining the geographical associations constructed by actors related in spatial circuits – producers, circulators, consumers and regulators – in their attempts to cohere and stabilise meaning and value in goods and services brands and their branding in particular spatial and temporal market contexts. Connecting geographically political and cultural economy concerns, origination provides a means to address critical questions about how, why, where and by whom goods and services brands are associated with specific and particular geographical attributes and characteristics of spaces and places and why it matters for people and places.
Second, ongoing research focuses on the governance of uneven development. This work specifically examines the governance of economic development in England and is currently exploring the shift away from regions, regionalisation and regionalism toward rebalancing, decentralisation and localism under the Conservative-Liberal Democrat coalition and now Conservative government in the UK. This research includes studies of the demise of the RDAs and the emergence of the LEPs in England in collaboration with David Marlow (Third Life Economics and CURDS), Anja McCarthy (CURDS), Peter O'Brien (CURDS) and John Tomaney (UCL). This project was part of my involvement in the Spatial Economics Research Centre (SERC) funded by ESRC, Department for Business, Innovation and Skills (DBIS), and the Welsh Assembly Government (WAG). See www.spatialeconomics.ac.uk.
Third, the inter-disciplinary research centre i-BUILD (Infrastructure BUsiness models, valuation and Innovation for Local Delivery) involving the Universities of Birmingham and Leeds and funded by EPSRC and ESRC began in 2013. See: http://research.ncl.ac.uk/ibuild/. i-BUILD is developing new business models to improve the delivery of infrastructure systems and the services they provide. These new business models aim better to exploit the technical and market opportunities that emerge from the increased interdependence of modern infrastructure systems. i-BUILD focuses on infrastructure at the scale of neighbourhoods, towns and cities where infrastructure is most dense and interdependencies between infrastructures, economies and society are most profound. As cities, local authorities and local enterprise partnerships are given more powers for infrastructure delivery and to raise finances it is crucial to develop robust new business models to develop infrastructure related business and growth locally - to the benefit of the nation. I am Co-Director of the centre and lead on the workstream on the governance of infrastructure funding and financing. With Dr. Peter O'Brien, we have undertaken the first national comparative study of the City Deals in the UK and are now working on the governance of infrastructure funding and financing in London and the (re)valuation of local authority assets.
Fourth, we have undertaken a research study on tackling declining cities in the UK for the Joseph Rowntree Foundation with CURDS colleagues David Bradley, Tony Champion, Mike Coombes, Danny MacKinnon, Liz Robson and Colin Wymer and Andy Cumbers from Glasgow University. The geographical extent and multi-faceted nature of urban decline has seen it become an area of policy concern internationally. Policy responses have typically sought to counter decline through the promotion of urban growth and recovery, although a new strategy of seeking to manage and steer decline has emerged in recent years. Clear gaps in understanding, empirical evidence and policy evaluation remain. Potential learning from international experiences has yet to be systematically identified, despite the emergence of these new approaches. The evidence base is fragmented, relying upon either case studies of individual cities or quantitative analyses of the process of decline. In this context, the research is reviewing the UK evidence on long-term population and economic decline in cities and international policy responses.
Fifth, with the Regional Studies Association, CURDS has contributed to a policy intervention at the RSA Winter Conference 2014 in London setting out the case for a more balanced approach to spatial economic growth and development policy in the UK. The draft report has been produced by Ben Gardiner, Ron Martin and Peter Tyler from Cambridge University and Andy Pike from CURDS, Newcastle University. See http://www.ncl.ac.uk/curds/news/item/regional-studies-association-spatially-rebalancing-the-uk-economy
Future Research
I am continuing ongoing work in collaboration with Stuart Dawley (CURDS), Andy Cumbers (Glasgow), Danny MacKinnon (CURDS) and Robert McMaster (Glasgow) on elaborating evolutionary approaches to economic geography in a geographical political economy vein and undertaking empirical work on resilience and the adaptation, adaptability and adaptive capacity of local and regional economies in a comparative international context.
As part of a new EPSRC and ESRC-funded grant, I will be contributing to a research network on 'Building sustainable local nexuses of food, energy and water: from smart engineering to shared prosperity'. Local nexuses, involving localised food manufacturing and decentralised energy and water supply that interact with the food system, are a special chapter of re-distributed manufacturing (RDM) with a focus on the sustainable local alignment of resources, production and consumption. To-date, a comprehensive technological, economic, and social/political understanding of local production/manufacture of food, energy and water, or local nexus, is still yet to be developed. By collaboration between engineering and social sciences, the Local Nexus Network will carry out feasibility projects and events to (i) establish the technical and socio-economic state-of-the-art of local productions of food, energy and water, (ii) generate initial insights for guiding researchers, businesses, policy makers and communities who are enthusiastic about exploring the potential of local nexuses, (iii) develop an evidence-based research agenda, (iv) form an inclusive research and stakeholder community, and (v) inform other related research on RDM.
Research Roles
Editorial Advisory Board, Local Economy
Editorial Board, Finisterra - Portuguese Journal of Geography
Postgraduate Supervision
Laura Sariego (2015-, FT) 'Local institutions and local economic development in Latin America' (Costa Rican Government Studentship, Joint supervision with Danny MacKinnon)
Graham Thrower (2014-, FT) 'Private investment and local infrastructure funding, financing and governance' (iBUILD Studentship, Joint supervision with Jane Gibbons (NUBS) and Oliver Heidrich (Civil Engineering))
Diana Morales (2014-, FT) 'Regional co-operation and development in Colombia and Germany' (Colombian Government Studentship, joint supervision with Danny MacKinnon)
Liam Keenan (2013-) 'Financialisation and the decline of the local pub: An Anglo-German comparison' (ESRC Collaborative Studentship, Joint supervision with Paul Langley (Durham), Neill Marshall, Jane Pollard and CAMRA)
Fraser Bell (2012-) 'The reputation of place' (ESRC Collaborative Studentship, Joint Supervision with Stuart Dawley, Ray Hudson (Durham) and Tina Snowball, NewcastleGateshead Initiative)
Matt Jenkins (2012-) 'The social construction of economic statistics' (ESRC Collaborative Studentship, Joint supervision with Mike Coombes and Joe Painter (Durham))
Emil Evenhuis (2011-) 'The political economy of adaptation in old industrial regions' (ESRC Studentship, Joint supervision with Stuart Dawley and John Tomaney (UCL))
Anja McCarthy (2011-) 'The local state and economic development and regeneration' (ESRC Collaborative Studentship, Joint supervision with John Tomaney (UCL) and Newcastle City Council)
Tom Strickland (2011-) 'Financialising urban development? A UK-US comparison' (ESRC Collaborative Studentship, Joint supervision with Jane Pollard and Newcastle City Council)
Rebecca Richardson (2010-) 'Place branding for urban development?' (ESRC Collaborative Studentship, Joint Supervision with Anoop Nayak and Tina Snowball, NewcastleGateshead Initiative)
Sophie Yarker (2010-14) 'Urban regeneration and the transformation of local identities' (ESRC Studentship, Joint Supervision with Robin Humphrey and John Tomaney (UCL)) (Now Teaching Fellow, CURDS, Newcastle University)
Mai Stabell (2010-) 'Petroleum-related FDI and local economic development' (Joint supervision with Matt Davies (Politics) and Neill Marshall)
Ben Fisher (2009-) 'New industry path creation and evolution' (Joint Supervision with Stuart Dawley) (Now Business Development Manager, Newcastle University)
Cecilia Pasquinelli (2008-12) 'Place Branding' (Joint Supervision with Professor Nicola Bellini, Scuola Superiore Sant'Anna, Pisa, Italy)(PhD awarded 2012)(Now Post-doctoral Researcher, Department of Economic and Social Geography, Uppsala University, Sweden)
Xue Feng Wang (2008-09) 'Cities and the Knowledge Economy in China' (Chinese Government, Joint Supervision with John Tomaney) (PhD Awarded 2009) (Now Research Associate, CURDS)
Rosibel Víquez-Abarca (2008-11) 'Universities and Regional Development in the Knowledge Economy' (Costa Rican Government, Joint Supervision with Andy Gillespie and José Esteban Castro) (PhD awarded 2011) (Now Researcher, UNED, Costa Rica)
Young-Jin Shin (2008-) 'Regenerating the Coalfields in South Korea: lessons from Europe' (Joint Supervisor with John Tomaney)
Julie Lister (2007-) 'Economic Evolution in North East England' (ESRC Studentship, Joint Supervisor with Ranald Richardson)
John Edwards (2007-11) 'Social Capital and Territorial Development Institutions and Policy' (ESRC Studentship, Joint Supervisor with John Tomaney) (PhD awarded 2011) (Now Post-doctoral Researcher, European Commission Joint Research Centre, Seville, Spain)
Michail Biniakos (2006-11) 'Transition and New Regionalism in the Balkans' (Greece National Government Studentship, Joint Supervisor with Alison Stenning and John Tomaney) (PhD awarded 2011) (Now Local Government Official, Greece)
Pedro Marques (2006-10) 'Power and Territorial Innovation Systems' (Portuguese National Government Studentship, Joint Supervisor with Andy Gillespie)(PhD awarded 2010)(Now Research Associate, Cardiff University)
Chieh-Wen Liu (2004-) 'Culture and Regional Development in Taiwan' (Joint Supervisor with Frank Moulaert, School of Architecture, Planning and Landscape)
Alex Rowley (2002-05) 'Trade Unions and Lifelong Learning' (TUC-funded, Joint Supervisor with Frank Coffield, Institute of Education, University of London) (Now Councillor and Leader of the Labour Opposition, Fife Council)
Liz Dixon (2000-2004) 'The Role of the Private Sector in Regional Development and Governance' (ESRC CASE Studentship with EEF Northern Association; Jointly supervised with John Tomaney)(PhD awarded 2004)(Formerly Research Associate, CURDS)
Chris Potter (2000-2005) 'Host Economies and Transnational Corporations' (ESRC Competition Studentship)(PhD awarded 2005).
Peter O'Brien (1999-2004) 'The Role of Trade Unions in Regional Development and Governance' (ESRC CASE Studentship with Northern TUC; Jointly supervised with John Tomaney)(PhD awarded 2004)(Formerly Chief Executive, North East Local Enterprise Partnership and now Research Associate, CURDS)
Stuart Dawley (1998-2002) 'High-tech Investment Cycles and Peripheral Region Development' (ESRC CASE Studentship with North of England Microelectronics Institute; Jointly Supervised with David Charles)(PhD awarded 2002)(Now Senior Lecturer, CURDS)
2015-17 EPSRC/ESRC Re-distributed Manufacturing Network 'Building sustainable local nexuses of food, energy and water: from smart engineering to shared prosperity', Co-I (£500k, with Aidong Yang, Oxford University)
2015-17 Newcastle University-Xiamen University Joint Research Initiative, 'Adaptive Capacity, Resilience and Change: A Comparative Study of Local and Regional Development in Northern England and Fujian Province', Co-I (£150k, with David Bradley, Stuart Dawley, Danny MacKinnon, Peter O'Brien and Xuefeng Wang)
2014-15 Joseph Rowntree Foundation, 'Tacking Declining Cities in the UK', PI (£90k, with David Bradley, Tony Champion, Mike Coombes, Danny MacKinnon and Liz Robson)
2013-16 What Works in Local Economic Growth Centre, ESRC Co-I (£3m)
2013-2017 i-BUILD (Infrastructure BUsiness models, valuation and Innovation for Local Delivery), EPSRC and ESRC, Co-I (£3.5m)
2012-13 Local Economic Recovery and Development Planning in Iraq, United Nations – International Labour Organisation, PI ($20k)
2012 SME and Entrepreneurship Issues and Policies at the National and Local Levels in Russia, OECD, PI (€5k)
2012 The Contribution of Manufacturing to UK Resilience, BIS and Government Office for Science PI (£10k)
2011-13 Spatial Economics Research Centre II, ESRC/BIS/WAG, PI and Co-I (£20k)
2012-13 BIS/NIESR, Regional Economic Analysis, Co-I (£48k)
2010 OECD, SME and Entrepreneurship Issues and Policies at the National and Local Levels in Mexico, PI (€5k)
2011 European Spatial Observatories Network (ESPON), Knowledge Support System Expert Sounding Board Member (5.5k€)
2010 Henan Development and Reform Commission, China, 'Spatial and Industrial Development', (Co-I with Professor John Tomaney and Dr Xuefeng Wang) (£150k)
2010 Communities and Local Government 'Decentralisation and public service provision' (Co-I with Professors Tomaney and Rodríguez-Pose and Drs Torrisi and Tselios) (£70k)
2009 OECD 'Non-State Actors in Local Development Governance' (PI with Professor John Tomaney and Drs Gianpiero Torrisi and Vassilis Tselios) (1.5k€)
2009-11 ESRC 'Devolution and Economic Performance' (Spatial Economics Research Centre Project 5b) (PI with Professors Ian Gordon, Andrés Rodríguez-Pose and John Tomaney) (£80k)
2008-09 British Academy 'A 'British' Brand? The Case of Burberry' (Principal Investigator) (£6.7k)
2008-2011 ESRC, Department of Business, Enterprise and Regulatory Reform, Communities and Local Government and Welsh Assembly Government Spatial Economics Research Centre (Co-I with LSE, Glasgow, Oxford and Swansea) (£2.4m)
2008-2011 ONE North East 'Integrated Regional Strategy Peer Assist' (Co-I with Professor John Tomaney and Dr Nick Henry, GHK) (£190k)
2008-2010 The Northern Way 'Research Programme Peer Assist' (Co-I with Professor John Tomaney and Ranald Richardson) (£181k)
2008-09 ONE North East 'Evaluation of the Impact and Policy Response to Restructuring at Northern Rock' (Co-I with Professors Neill Marshall and John Tomaney, Drs Stuart Dawley and Jane Pollard and Regeneris Consulting) (£148k)
2008 South West Regional Development Agency 'Assessment of Regional and Local Development over the Last 20 Years' (Co-I with Professor John Tomaney and Dr Nick Henry, GHK) (£20k)
2008 ONE North East 'Economic Capacity Building' (PI with Professors Richard Harris (Glasgow), Ian Stone (Durham), Colin Wren (Newcastle) and Dr Henry Overman (LSE)) (£20k)
2007 OECD Local Economic and Employment Development Programme and Welsh Assembly Government 'Västra Götaland Regional Growth Agreement, Sweden' (PI) (£1k)
2007 OECD Local Economic and Employment Development Programme and USAID 'Strengthening Entrepreneurship Innovation and Economic Development at Local Level in Croatia: Institutional arrangements for the design and delivery of entrepreneurship policies' (PI) (£3k)
2007 The Northern Way 'Economic Linkages in the Northern City-Regions' (Co-I with Professors Alan Harding and Bryan Robson) (£80k)
2007 North Tyneside Council 'Economic and Social Change in North Tyneside' (PI with David Bradley) (£3.5k)
2007-2009 ESRC Seminar Series 'Changing Cultures of Competitiveness' (Co-I with Ngai-Ling Sum and Bob Jessop (Lancaster University) - see http://www.lancs.ac.uk/fass/events/changingcultures/ (£15k)
2006-2009 European Commission 'Social Innovation and Inclusion in the Knowledge Economy – KATARSIS' (Co-I with Professor Frank Moulaert (Leuven)) (£606k)
2006 ONE North East 'What Works in Regional Economic Development: Learning from International Best Practice' (Co-I with Professors John Tomaney and Mike Coombes and Drs Nick Henry and Stuart Dawley, GHK) (£47.5k)
2005 OECD Local Economic and Employment Development Programme and German Federal Ministry of Transport, Building and Urban Affairs 'Strengthening Entrepreneurship and Economic Development in Eastern Germany' (PI) (£3.5k)
2005-2007 ESRC Seminar Series 'The New Public Management and Regional Policy' (Co-I with Les Budd (Open University), Josie Kelly (Aston) and Joyce Liddle (Durham) (£14.1k)
2005 Office of the Deputy Prime Minister 'The Economic Viability and Self-Containment of Geographical Economies' (PI with Mike Coombes, Tony Champion, Lynne Humphrey and John Tomaney) (£32k)
2005-08 Regional Studies Association 'Regional Studies Editorship' (Editor-in-Chief with 9 other Editors) (£116k)
2005 ESRC Advanced Training and Development Award 'Researching Civic Engagement and Devolved Governance in Europe' (PI with the Regional Studies Association) (£10.2k)
2004 University of Newcastle Vacation Scholarship 'Microbreweries in North East England' (Co-I with Matthew Needham) (£1.5k)
2004 ESRC Advanced Training and Development Award 'Research Strategy and Methodology for Regional Development and Governance in an Enlarged Europe' (PI with the Regional Studies Association) (£9.4k)
2003-04 European Commission 'Regional Economic Sustainability and University Learning and Teaching Strategies - RESULTS' (Co-Investigator with Dr Hans-Peter Baumeister (Reutlingen University)) (£118k)
2003 University of Newcastle, Arts and Humanities Research Fund 'Recasting Steel Geographies' (Co-I with Alison Stenning, and Stuart Dawley) (£2.5k)
2003 North East Assembly 'Regional Governance Capacity Building Course' (PI with Professor John Tomaney) (£30k)
2003 University of Newcastle Vacation Scholarship 'Directly Elected Mayors and Local Economic Development' (Co-I with Graham Turner) (£1.5k)
2002 ESRC Advanced Training and Development Award 'Doing Regional Research in a Devolving State: Research Strategy and Methodology in Post-Devolution UK' (PI with the Regional Studies Association) (£6.8k)
2002 Government Office North East 'Regional Governance' (Co-I with Professor John Tomaney) (£7.5k)
2001 University of Newcastle Vacation Scholarship 'Pension Fund Capitalism and Regional Development' (Co-I with Felicity Wray) (£1.5k)
2000 UNISON 'The Economic and Social Implications of Best Value' (PI with Peter O'Brien) (£2.5k)
2000 University of Newcastle Small Grant 'Task Forces and the Governance of Economic Development' (PI with Stuart Dawley) (£1.5k)
Undergraduate Teaching
GEO2099 Economic Geographies
GEO3114 Local and Regional Development
GEO3099 Dissertation module
Postgraduate Teaching
GEO8002 Local and Regional Development Theory and Policy
GEO8007 Local and Regional Development: Methodology, Analysis and Statistics
GEO8009 Developing Local and Regional Strategies
GEO8018 Local and Regional Development and Governance
GEO9089 Research Dissertation


text:
Collected by Karl T. Gosnell For Mary C. Parler Transcribed by Frances Majors
Recited by Mrs. Horace Smith Springdale, Arkansas November 3, 1958
Reel 272, Item 7
Noah, Jonah and Captain John Smith
Noah, and Jonah and Captain John Smith,
Mariners, travelers, magazine of myth,
Sitting up in heaven, chewing and a-chawing,
Eating their tobacco, talking and a-jawing.
Sitting by a creek, spitting in the water,
Talking tall as Atlus, as saints hadn't oughter,
Lolling in the shade, baiting hooks and anglers, Occasionally friendly, occasionally wrangling.
Noah took his halo from his old bald head
And swatted off a hopper grass and knocked it dead;
And he baited off his hook and he spoke and said,
"When I was skipper of the tight little Ark,
I used to fish for porporpise, used to fish for shark.
I have catched in a single hour on Monday
Sharks enought to feed the family anyway till Sunday,
To feed all the serpents, tigers, and the donkeys,
To feed all the zebras, the insects and monkeys,
To feed all of the varmint, bears and orillas,
To feed all the camels, cats and armidillos,
To give all the pelicans stews for their gizzards,
To feed all the owls, catamounts, and lizzards,
To feed all the humans, their babies and their nurses,
All the hound dogs and hippopotamuses,
To feed all the oxen, feed all the asses,
Feed all the bison and the little hopper grasses.
Always I catched in half an hour on Monday
All that the family could gourmandize till Sunday."
Jonah took his harp, to strum and to string her,
And Captain John Smith touched his nose with his finger. Captain John Smith, he hemmed some and hawed some,
And he bit off a chaw and he chawed some and chawed some. "When I was to China, when I was to Guinea,
When I was to Java and also in Virginia,
I teached all the natives how to be ambitious.
I learned them my trick of catching devil fishes,
I fitten tigers, I fitten bears,
I fitten serpents and wolves in their lairs;
I fit with wild men and hippopotamuses,
But the perilous of varmints is the octupuses.Noah, Johan and Captain John Smith (Cont'd)
Reel 272, Item 7 (Cont'd)
I'd rub my forehead with phosphorescent light
And plunge into the ocean and seek them out at night.
I've catched them in grottos, I catched them in caves;
I used for to strangle them beneath the waves.
When they seen the bright light blazing on my forehead They used to rush at me screaming something horrid, Tentacles waving, teeth white and gnashing,
Hollering and howling, wallering and splashing.
I used to grab them as they rushed from their grots;
Crab all their legs and tie them into knots.
Noah looked at Jonah and said not a word;
But if winks made noises, a wink had been heard.
Jonah took the hook from a mudcat's middle
And strummed on the strings of his Hallelujah fiddle.
Johan gave his whiskers a backhand wipe
And cut some plug tobacco and crammed it in his pipe.
Noah and Jonal and Captain John Smith,
Fishermen and travelers, narrating myth,
Sitting up in heaven all eternity,
Fishing in the shade, contented as could be,
Spitting their tobacco in the little shaded creek, Stopping of their yarns for to hear the ripples speak.
I hope for heaven when I think of this.
You folks down hellward, a lot of fun you'll miss!
Jonah, he duplicates the mudcat's head
And gets his pipe for drawn, and this is what he said,
"Excuse me if your stories don't excite me much,
Excuse me if I seldom agitate for such;
You think you're fishermen, I won't argue none;
I won't even tell you the half of what I done.
You had careers dangerous and checkered,
All as I will say is go and read my record.
You think you're fishermen, you think you're great.
All I ask is this, has one of you been bait?
Captain Noah, Captain John, I heard ye holler When I asked this, has one of ye been swallowed?
It's mighty pretty fishing with little hooks and reels, It's mighty easy fishing with little rods and creels;
It's mighty pleasant catching mudcats for your dinners, But this here is my challenge for saints and for sinners: Which one of ye has voyaged in a varmint's innards?N oah, Johan and Captain John Smith (Cont'd)
Reel 272, Item 7 (Cont'd)
When I seen a big fish, tough as Methusulam,
I used for to dive into his ozzlie-goozilum.
When I seen the strong fish walloping like a lummox,
I used to follow them, dive into their stomach.
I could voyage and steer them, I could understand them, I used to navigate them, I used to land them.
Don't you pester me with any more narration,
Go get famous, get a reputation."
Captain John he grinned, his hat brim beneath,
Clicked his tongue of silver on his golden teeth;
Noah, and Jonah, and Captain John Smith,
Strumming golden harps, narrating myth;
Sitting by the shallows forever and forever,
Swapping yarns and fishing in a little river.
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text: Abstract — Signal to interference plus noise ratio, SINR, is one of the main factors that affects the quality of wireless communication. While the impact of white Gaussian noise on a wireless channel is well understood, impact of interference remains one of the less explored areas. With the deployment of dense mesh networks, the interference will be a dominant factor that affects the transmission errors.


text:
Has open source hit a tipping point?
Tipping Point
Open source in the news, at events around the globe, and now delivered in convenient flyer form.
Today I read an article in the Harvard Business Review called Open Source Software Hits a Strategic Tipping Point. Author Laurie Wurster writes, "Based on surveys my colleagues at Gartner and I have conducted over the past several years, mainstream adopters of IT solutions across a widening array of market segments are rapidly gaining confidence in the use of open source software, with many now stressing its valuable features more than its risks."
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LinuxCon North America Convenes in Toronto, Canada
Linux Foundation's big event celebrates the 25th anniversary of Linux
Linux Turns 25
Linux has evolved from a "won't be a professional" project to one of the most professional software projects in the history of computers.
Mirantis Joins Hands with SUSE To Offer RHEL Support; Red Hat Not Happy
Competitors get in the game with RHEL without Red Hat
Linux on Windows 10 Poses a Security Risk
Security researchers have already notified Microsoft; some fixes are available
Mirantis to Refactor OpenStack Fuel for Kubernetes
The company is collaborating with Google and Intel to use Kubernetes as an engine for Fuel
Canonical Joins Document Foundation Advisory Board
The upcoming release of LibreOffice will be available as a snap package
SUSE Joins the Microsoft Test Drive Program
Customers can take a free test drive of SLES for HPC on the Azure Cloud
SiFive Launches a Line of Open Source Chips
San Francisco-based chip company announces their first fully open source chip platform.
Fedora 24 Released
The whole distro gets rebuilt on glibc 2.3
Canonical Announces Cross-Distro Support for Snaps
Ubuntu Vendor tries to solve app packaging and distribution problem across distributions.


text:
As part of a "Healthy Communities" initiative the Town Manager requested CPW investigate the potential construction and installation of an outdoor skating rink located at Emerson Field. After discussions with the Recreation Director, Deputy Town Manager and Recreation Commission, as well as visiting other communities who have successfully installed similar rinks to learn more about installation techniques, work commenced on the "pilot" installation in 2010.
Since that time the rink at Emerson Field and at Rideout Field in West Concord have been prepared for the enjoyment of residents as weather permits.
Concord Public Works Staff Creating the Rink
The Rink being enjoyed by hockey and figure skaters on a brisk Saturday afternoon.
Rink being maintained by CPW Park and Tree Staff


text:
Cancer was just another bump in the road for Eagle resident and semi-professional athlete John Klish.
After being born with profound bilateral hearing loss, the 30-year-old engineer is accustomed to overcoming challenges. If anything, growing up deaf has given him greater insight toward accomplishing goals.
"Mountain biking and competitions have helped shape my personality," Klish said. "I've learned to keep myself calm at all times, even when people are passing me or when things are not going well. Otherwise I waste the energy and focus I need to complete my task. I apply this concept to my real life."
Klish currently works full time as an engineer for the Colorado Department of Transportation.
Part of his lifelong dream is to become a professional athlete. That's why he's been especially busy with competitions in 2011. He primarily competes in triathlons, biking and foot races, and many of those races are on consecutive days.
"This year I've participated in 23 races since January with 16 podiums," Klish said on Aug. 2. On Aug. 6, he took second overall in the XTERRA Indian Peaks Triathlon and seventh overall in the LA Sportiva 10K @ 10,000 feet on Aug. 7. "By the end of the year, I plan to have completed between 50 to 60 races - trail running, mountain biking, road biking, swimming, triathlons, cyclocross, snowshoeing, rando-racing."
Klish said he trains between 15 and 25 hours a week, depending on the phase of his season. He took a weeklong break for recovery at the end of July.
"He has all the makings of a great athlete," said Greg Mueller, Klish's personal coach. "He recovers really well. He finishes a tough workout and wants more. Motivating him is never an issue. He has the suffer gene - he's a beast."
The other part of Klish's lifelong dream is to be someone who contributes to the community. That's why he volunteers for Aspen Camp of the Deaf and Hard of Hearing, a program he enjoyed growing up.
"I hope I can be a positive role model for other deaf people, to maybe inspire them to take on something that scares them," he said. "That's how we get stronger."
When Klish was born in Durango, it didn't take long for his mother, Karen Fehringer, to notice something was different about her baby.
"Because of my line of work as an occupational therapist, I identified very early that there was a problem," Fehringer said. "He had hearing aids by the time he was 3 months old."
As a result, Klish started speech therapy as a baby. He learned how to speak and read lips years before he learned sign language, which he did at age 14.
"The biggest influence in my life has been my mother," Klish said. "She strongly believed in my potential when I was born and refused to let the doctors define my life."
Fehringer described an early doctor visit that was discouraging but also pivotal.
"A young audiologist did a hearing test on him and said he was so deaf that there wasn't much hope," Fehringer said. "She recommended we put him in a state school for the deaf and blind where he would be raised. She was basically saying to give up and forget about him. My reaction was to walk out the door. It was a very defining moment - I knew he could learn."
When Klish was 4, his parents and older sister Megan moved with him to Massachusetts so that he could attend a Clarke School for Hearing and Speech.
"We just did it - sold the house and found new jobs," said Fehringer, who now lives in Denver. "I knew that learning how to read, write and speak would set him up for everything else."
The family moved back to Grand Junction when Klish was 11. He attended a private Catholic middle school and then finished at a public high school.
After attending Clarke's, Klish helped his public school teachers learn how to educate others like him because he knew how to voice his needs.
"He came back from Clarke's with an entire manual," Fehringer said. "One of his sixth grade teachers said, 'There isn't one thing I have done in my classroom to teach John that hasn't made me a better teacher for every other student.'"
Klish remains close to all of his family. Megan lives in Louisville with her husband and children, and his dad, Michael Klish, now lives in Grand Junction.
Klish went on to college at Colorado School of Mines and Colorado State University.
While finishing his education at CSU, Klish continued to make an impact on people with his brains and athletic ability. That's when he met one of his best friends, Jeremy Rietmann.
"(Klish) was a sophomore when I was a freshman and he was dating a girl in my dorm," Rietmann said. "I was like, 'Who is this deaf guy I'm always seeing in the bathroom?' We got to talking and discovered we both had an interest in biking. We started hanging out and then joined the CSU cycling team together."
They've been good friends for nearly a decade.
"He's probably one of the most exuberant personalities I've ever met," Rietmann said. "The guy is ridiculously smart. You don't see many people taking a thermodynamics class with a sign-language interpreter."
In addition to their mutual interests, Rietmann said that his lips are easy to read, so that helped to foster their friendship early on. Rietmann also makes a practice of keeping Klish in the loop of a conversation with multiple people, which is something not everyone has the patience to do.
"You can miss meeting a really interesting person if you don't take the time," Rietmann said. "I think (Klish) is good about seeking people who are supportive."
By coincidence, Klish and Rietmann both live in Eagle County these days. Klish moved to Eagle about two and half years ago for his job with CDOT and Rietmann moved to Vail last January for his wife's job.
"We've been able to get out and ride together again," Rietmann said. "(Klish) is the best mountain biker I've ever biked with. We're both super competitive and we feed off each other's energy."
An active lifestyle is another thing Klish was born into.
"We were always bike riding or skiing. It was just who we were," Fehringer said. "He loved to be active and we'd go outside at least once if not twice a day. He took little-kid tennis lessons and had a high aptitude for it."
When he was 6, however, Klish decided that soccer was his main sport.
"I played soccer from age of 5 to 19," he said. "I stopped playing when the Colorado School of Mines soccer team cut me off the team because I had a recent knee injury (torn ACL and LCL)."
Due to the knee problems, Klish all but gave up running until recently.
"I always came home with sore knees or feet until last spring when I changed my running form," he said. "I just lean forward a bit more and keep my knees more bent rather than landing on my heels with my landing knee straight. Now I love running!"
Meanwhile, Klish's dad introduced him to mountain biking at age 14. That turned out to be one of his main passions in life. Now, if he has to choose between biking or running, it's usually biking.
"Most of the time I prefer biking because of the speed and adrenaline on the descents, and it covers a lot of ground," Klish said. "There are also days where I just want the simplicity of running, just putting on shoes and going."
Klish was diagnosed with testicular cancer about six years ago. He was finishing college and saving for a trip to Australia.
"Sometime in June 2005, I found a sore lump on my testicle so I went to see a doctor," Klish said. "The doctor thought at first it was just a 'sore bump' - 95 percent of the testicular cancer cases do not hurt. He prescribed some antibiotics, which helped temporarily, but the pain persisted. Three months down the road, we finally got an ultrasound and blood test ordered. We were able to confirm that I had cancer and I was hurriedly put into the operating room to remove the affected testicle in October 2005.
"We hoped we caught it in time, but the post-op blood work showed that the cancer cells had spread to the rest of my body. I was still in good shape since nothing had developed yet. I went through three rounds of chemotherapy, starting on the week of Thanksgiving 2005 and ending mid-January 2006.
"We waited until April and found a large tumor near my stomach, toward the back of my body, so I went through another surgery to remove it and the lymph nodes that were part of the testicular system. The tumor turned out to be benign, so I have been in remission since then," Klish said.
Fehringer said the family was in shock when the cancer was diagnosed.
"We didn't have any time for second breaths," she said.
Klish's family asked him how they could help and he said he wanted someone to be with him at every chemo treatment, which lasted four hours a day, five days a week.
Six weeks after his last surgery - the minimum healing time Klish's doctor prescribed before he could do anything rigorous - he did his first mountain bike race of the season and got fourth place in the expert category.
"I guess you could say I had fresh legs," he said.
He had done light training on a stationary bike in the months leading up to the race but there was an extra challenge.
"I felt a sore spot in my stomach where the tumor was removed," he said.
Klish now has a check-up every three months. After two years he'll have one every six months. The visits include CAT-scans and blood tests.
"It is scary sometimes," he said. "I wonder if I'm going to get cancer again. Then I remember that I have a great life and family. The reality is that you die at some point, so you might as well live every moment you have."
Klish met Mueller, his coach, in June 2010. Mueller quickly recognized Klish's potential.
"He is great to work with," Mueller said. "He's good about looking at the sport in terms of the big picture. He listens to directions and when he is tired is when he shines."
Mueller has been a personal coach since 2004 and never worked with a deaf athlete before.
"In some ways, I think being deaf gives him an advantage," Mueller said. "He is in tune and sensitive in other ways. He compensates for what he is missing. He says, 'This is an obstacle, how am I going to overcome it?' Then he overcomes it and asks, 'How can I do it better?'"
There are competitive disadvantages to being deaf, of course. Klish said he has to look over his shoulder in races more often to make sure he doesn't collide with anyone. For him, that's just how he has to run his race.
"I like to think that you can do anything you want to, that there's no limitation except yourself," he said.


text:
By Julian Pecquet and Jamie Klatell - 05/30/11 07:18 PM EDT
Electoral consequences
Then-RNC Chairman Michael Steele, Nov. 4, 2009
"In a state that overwhelmingly voted in favor of President Obama, this stunning defeat of Corzine sends a clear message to Democrats across the country. Americans have grown sick and tired of big government and reckless spending, and this vote is a sound rejection of the far-left policies that are hurting our nation."
Then-White House press secretary Robert Gibbs, Nov. 4, 2009
"It's hard to pick national trends out of local elections."
DNC Chairwoman Debbie Wasserman Schultz, May 24, 2011:
"Tonight's [special election] result has far-reaching consequences beyond New York. It demonstrates that Republicans and Independent voters, along with Democrats, will reject extreme policies like ending Medicare that even Newt Gingrich called radical. With this election in the rear-view mirror, it is my hope that Republicans will accept the message being sent by voters."
BoehnerJohn BoehnerRank-and-file Republicans fear lame-duck vote on pricey funding bill New Trump campaign boss took shots at Ryan on radio show Election reveals Paul Ryan to be worst speaker in U.S. history MORE, May 25, 2011
"Special elections are just that. They're special."
The effects of reform | On rationing | Electoral consequences
Life or death (part 1) | Life or death (part 2) | "Granny"
Scare tactics | Asking for civility | "Grown up" solutions


text:
Featured Ride in the
"A tour of a bunch of Buena Vista singletrack."
— Jason Lampe on Aug 2, 2013
|11.518.5 milesKilometers
|1,063'324 m Ascent
|8,452'2,576 m High
|4% Avg Grade (2°)
|50% Singletrack
|-1,070'-326 m Descent
|7,895'2,406 m Low
|22% Max Grade (12°)
Take a
Virtual Ride
Overview1:45 average time for 11.5 miles. Details
Starting and ending in the main parking lot, you'll enjoy a good taste of Buena Vista's singletrack. If you're looking for more miles, combine this loop with the Midland Bike Trail Out and Back.
DescriptionBegin at the Buena Vista riverside parking lot and cross the small bridge over the Arkansas river. Climb slightly along the Whipple Trail (lower portion), which is mainly smooth singletrack. Continue straight when you come to the fork heading uphill. After about a mile, you climb up to and cross Fire Road 304 leading you directly into 6032, which climbs up and then turns to head north. The terrain is rolling singletrack with a few bits of sandy and rocky areas. After about 1.5 miles, you return to Fire Road 304 and take it south and very slightly uphill for about 3 miles to a junction and another parking lot.
From here, you can extend this loop by taking the Midland Bike Trail Out and Back, or dive right into the Gentleman's Tail Trail. The Gentleman's Tail winds its way west and downward through relatively rocky and tight turns and bottoms out into a fire road. From here, you can cross directly over this fire road and begin the climb up 6034 or climb the fire road.
Both lead you back to Fire Road 304 where you can decide again about extending your loop with the Midland Bike Trail Out and Back. If not, head north on Fire Road 304 and drop into the Whipple Trail (upper portion) back to where you began.
ContactsLand Manager: BLM Colorado - Gunnison Field Office
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2.5 from 4 votesCairns marking the nondescript entrance of Gentleman's Tail (Buena Vista, CO)
2.0 from 4 votesCrossing the Arkansas looking south (Buena Vista, CO)
2.0 from 3 votesThe obvious start of 6032 (Buena Vista, CO)
1.8 from 5 votesThe start of 6034 (Buena Vista, CO)


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VIENNA, 12 March 2014 – Two new publications on biofuels, released by the United Nations Industrial development Organization (UNIDO), will help promote investment in biofuel production by explaining the social and environmental sustainability and economic feasibility of biofuels, and will offer a set of case studies in the field from different continents.
The publications were prepared in collaboration with the Food and Agriculture Organization of the United Nations (FAO), the United Nations Environment Programme (UNEP) and three research partners, IFEU Heidelberg, Utrecht University and Öko-Institut.
The publications were developed under the project "Global Assessments and Guidelines for Sustainable Liquid Biofuel Production in Developing Countries", funded by the Global Environmental Facility (GEF):
The Biofuels Screening Toolkit. Guidelines for decision makers was designed to assist in making informed decisions about biofuel projects. The guidelines will help encourage and promote investment in biofuel production when it is socially and environmentally sustainable, as well as economically viable.
"We seek to provide guidance to countries interested in sustainable biofuel production. The project team assessed 74 liquid biofuel pathways for both transport and stationary applications in developing countries," said Diego Masera, chief of UNIDO's Renewable and Rural Energy Unit.
The second publication, Impacts of Biofuel Production Case Studies: Mozambique, Argentina and Ukraine, features biofuel production case studies at different stages of industrial development. The environmental and socio-economic impact assessments of these biofuel production examples provide valuable lessons learnt for future project design.
The new publications capitalize on the experiences of UNIDO, GEF, UNEP and FAO in the field of sustainable biofuel development and build upon the recently published Global Assessments and Guidelines for Sustainable Liquid Biofuel Production in Developing Countries (Final Report and Executive Summary).
For more information please contact:
Diego Masera
Chief, UNIDO Renewable and Rural Energy Unit
The publications can be accessed here.