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Long-chain fatty acids are an important source of energy in muscle and heart where the acyl-CoA dehydrogenases (ACADs) participate in consecutive cycles of β-oxidation to generate acetyl-CoA and reducing equivalents for generating energy. However, the role of long-chain fatty acid oxidation in the brain and other tissues that do not rely on fat for energy is poorly understood. Here we characterize two new ACADs, ACAD10 and ACAD11, both with significant expression in human brain. ACAD11 utilizes substrates with primary carbon chain lengths between 20 and 26, with optimal activity towards C22CoA. The combination of ACAD11 with the newly characterized ACAD9 accommodates the full spectrum of long chain fatty acid substrates presented to mitochondrial β-oxidation in human cerebellum. ACAD10 has significant activity towards the branched-chain substrates R and S, 2 methyl-C15-CoA and is highly expressed in fetal but not adult brain. This pattern of expression is similar to that of LCAD, another ACAD previously shown to be involved in long branched chain fatty acid metabolism. Interestingly, the ACADs in human cerebellum were found to have restricted cellular distribution. ACAD9 was most highly expressed in the granular layer, ACAD11 in the white matter, and MCAD in the molecular layer and axons of specific neurons. This compartmentalization of ACADs in the human central nerve system, suggests that β-oxidation in cerebellum participates in different functions other than generating energy, for example, the synthesis and/or degradation of unique cellular lipids and catabolism of aromatic amino acids, compounds that are vital to neuronal function.
The acyl-CoA dehydrogenases (ACADs; EC 22.214.171.124) are a group of mitochondrial flavoenzymes that catalyze the α, β-dehydrogenation of acyl-CoA esters using the electron transfer flavoprotein (ETF) as their physiologic electron acceptor. There are at least nine known members of the ACAD gene family active in branched chain amino acid catabolism or fatty acid oxidation. All of the ACADs are nuclear encoded proteins that must first be translated in the cytoplasm then imported into mitochondria and processed into a mature form. Mitochondrial β-oxidation of long chain fatty acyl-CoAs is well recognized as a primary metabolic pathway for maintenance of energy homeostasis and body temperature [1]. However, some of the symptoms in patients with mitochondrial β-oxidation defects (OMIM# 609105) are not readily explained simply by energy deficiency. For example, a high percentage of patients with mitochondrial trifunctional protein deficiency present with neurologic disease, including neuronal demyelination and retinal degeneration, that responds poorly to available therapies designed to restore energy homeostasis [2–5]. Recently we described a new disorder of ACAD9 deficiency (OMIM# 611126) [6]. The presence of chronic abnormalities in the central nervous system in these patients further underscores the importance of non-energy generating, alternative functions of mitochondrial β-oxidation [6]. Three ACADs have been described that are active with long chain acyl-CoA substrates, very long chain acyl-CoA dehydrogenase (VLCAD), acyl-CoA dehydrogenase 9 (ACAD9), and long chain acyl-CoA dehydrogenase (LCAD), but except for the role of VLCAD in energy generation through fatty acid oxidation, the spectra of the physiologic functions of these enzymes remains unknown. ACAD9 has also been implicated in the assembly or stability of complex 1 of the mitochondrial respiratory chain, raising the possibility of a duel roll as a molecular chaperonin for this metabolic enzyme [7]. In this study, we characterize two additional ACADs with long chain acyl-CoA activity, ACAD10 and ACAD11. These new enzymes appear to be compartmentalized mainly in human brain, in contrast to the localization of VLCAD and MCAD to energy generating tissues, suggesting that mitochondrial β-oxidation in human brain is tailored to different fatty acids in different regions of the brain. This in turn implies that β-oxidation of long chain fatty acids in the central nervous system is involved in functions alternative to energy metabolism, such as controlling the composition of fatty acids or catabolizing functional intermediates of aromatic amino acids. Thus the five different long chain ACADs are likely specific to the different functions of mitochondrial β-oxidation in various tissues in humans.
Total RNA was extracted from frozen post mortem liver using TRIzol reagent (Invitrogen, San Diego, CA) as per the manufacturer's instructions. Reverse transcription was performed using 1 μg of total RNA and Powerscript™ reverse transcriptase using random hexamer nucleotides as primers. ACAD11 (BC019607) sequences were amplified with the Yieldace PCR kit (Stratagene La Jolla, CA) with primers as follows: 5′CTCGAGACCT GTGGATGCCG CGTTGC3′ and 5′CGACTCGCGG CCGCAAGCTT ATATCTTGGC TGTC. The PCR products were purified using the PCR cleanup kit (Qiagen, Inc., Valencia, CA), subcloned into pcDNA2.1 vector, and sequenced by the Genomics Core Facility at the University of Pittsburgh.
ACAD 11 proteins were synthesized from 1 μg of plasmid DNA (pcDNA3.1 containing the various cDNAs by coupled in vitro transcription and translation (TNT) in the presence of 35S-labeled methionine (20 μCi, 1250 Ci/mmol) as described [8]. Liver mitochondria were freshly prepared from young male Sprague-Dawley rats by differential centrifugation [8]. The final pellet was resuspended in 500 μl of 5 mM HEPES (pH 7.4), 220 mM D-mannitol, and 70 mM sucrose (HMS buffer) and diluted to a final concentration of 20 mg/ml protein. Thirty-five μl of translation mixture containing 35S-labeled precursor proteins were mixed with 85 μl of mitochondrial suspension and 30 μl of HMS buffer to a total volume of 150 μl and incubated at 30 °C for 75 min. Mitochondria were collected by centrifugation at 9100 xg for 2 min at 4 °C and washed three times in cold HMS buffer. They were then resuspended in HMS buffer and treated with trypsin (0.5 μg/μl) for 0, 10, 20 min. Soybean trypsin inhibitor (3 μg/μl) was added to stop the reaction and mitochondria were re-pelleted as described [9]. The pellets, together with 1 μl of TNT reaction product were solubilized in 30 μl of SDS-PAGE loading buffer, boiled for 10 min, and resolved by SDS-PAGE. Gels were transferred to supported nitrocellulose and exposed to X-ray film overnight. For native PAGE, 4–15% Tris-HCl Criterion gels (Bio-Rad) were used following the manufacture's instructions.
The ACAD11 model was created with Insight II molecular modeling software, Accelrys Inc. (San Diego, CA), using the atomic coordinates of rat SCAD (1JQI) and human glutaryl-CoA dehydrogenase (GCD) crystal structures (1SIQ) as described previously [10]. An acetyl-CoA and FAD is modeled as the substrate and cofactor respectively.
Full length cDNAs corresponding to the predicted mature mitochondrial protein sequence of ACAD10 and 11 were cloned individually into the E. coli expression vector pET-21a(+) (Novagen, Madison, WI), with E.coli codon biased introduced in the first 100 base pairs and over-expressed as described previously for other ACADs [11, 12]. The lysate was treated with 1% n-Dodecyl β-D-maltoside detergent (Anatrace, Maumee, Ohio, USA) before enzyme activity assay.
ACAD activities in extracts from cultured skin fibroblasts and frozen tissues obtained 6–10 hours postmortem (Brain and Tissue bank for Developmental Disorders, Baltimore, MD) were measured as described using the sensitive and specific anaerobic ETF fluorescence reduction assay with the indicated substrates at 5 μM final concentration [13]. Mitochondrial matrix and membrane fractions were prepared as previously described and assayed in triplicate using palmitoyl-CoA as substrate [14]. Five normal liver samples were used as controls. Hexanoyl (C6:0)-CoA, octanoyl (C8:0)-CoA, decanoyl (C10:0)-CoA, dodecanoyl (C12:0)-CoA, tetradecanoyl (C14:0)-CoA, palmitoyl (C16:0)-CoA, stearoyl (C18:0)-CoA, and arachidoyl (C20:0)-CoA were purchased from Sigma (St. Louis, MO). Synthesis of racemic 2-methylhexadecanoyl-CoA [15] and 4,8,12-trimethyltridecanoyl-CoA (4,8,12-trimethylC13-CoA)[16] has been described before, S-methyl-hexadecanoyl-CoA (S- methyl C16-CoA), 2S-methylnonadecanoyl-CoA (2S-methyl-C19-CoA) was prepared in a similar way as described for 2S-methyl-pentadecanoyl-CoA (2S-methylC15-CoA) [ref B], using the 1-bromohexadecane as starting point.
Reverse transcription was performed with a 1 μg of RNA from a panel of total RNA pooled from 20 human tissues or total RNA from pooled human hippocampus purchased from BD Biosciences Clontech (Palo Alto, CA) and Powerscript™ reverse transcriptase (BD Biosciences Clontech) according to the manufacturer's instructions. cDNA corresponding to 20 ng of total RNA was used for real-time PCR on an ABI 7900HT Sequence Detection System (PE Applied Biosystems, Foster City, CA) using heat-activated Taq DNA polymerase (BD Biosciences Clontech). Specific primer pairs (sequences available upon request) for VLCAD, ACAD9, LCAD, MCAD, and SCAD were designed to target coding sequences near the catalytic domain of each enzyme. Primers for the housekeeping genes GUSB and 18S rRNA were used as controls. All primers (labeled with Fam fluorescence tag at the 5- end and MGB non-fluorescence quencher at the 3′-end) were synthesized by PE Applied Biosystems (Assay on Demand™). PCR assays were performed in 96 well optical plates following the manufacturer's recommendations. The amplification efficiency of each primer pair was tested using different dilutions of total RNA, and all were similar (close to 1). For quantitative analysis of the data, the CT (threshold cycle number) values were normalized to those of GUSB or 18S rRNA using the ΔΔCT method [17].
Mitochondrial membrane and matrix protein fractions from human tissues were separated as described previously and 100 μg of total protein from each tissue lysate were separated on a 12% SDS polyacrylamide gel, transferred to nitrocellulose, and immunostained and visualized with the indicated antibodies as described [12]. Rabbit anti human ACAD11 antibodies were raised by immunization with the synthetic peptide RKGQEVLIKVKHFMK, a peptide unique to human ACAD11 at the predicted surface of its ACAD homologous domain. The resultant antiserum was used at a 1:500 dilution.
Formalin-fixed, paraffin-embedded sections of normal cerebellum and muscle were provided by the Department of Pathology, Children's Hospital of Pittsburgh. 5 μm sections were cut at regular intervals and mounted on glass slides. Immunohistochemical staining was performed as described previously using ACAD9, ACAD11 and MCAD antibodies at dilutions of 1:200 to 1:300. Staining was performed using the avidin-biotinylated peroxidase complex (ABC) method (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin. . Human fibroblasts and neuroblastoma SK-N-SH were fixed with 4% formaldehyde on coverslips, permeabilized using either 1% Triton X-100 or digitonin (25 μg/ml), incubated with primary antibody, and visualized with fluorescently-labeled secondary antibodies following manufacture's recommendation. Mitochondrial ATPase antibody was purchased from Chemicon (Billerica, MA). Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA).
Two genes identified as ACAD10 and ACAD11 have been annotated in the human genome (Fig. 1A and B). They are widely conserved across evolution and share 46% identity. Both genes are located within apparently complicated gene loci containing multiple predicted exons that code for multiple predicted protein domains. Preceding the conserved ACAD domains, there is a predicted aminoglycoside phosphotransferase (APH) domain in both ACAD10 and ACAD11, which also could be a homoserine kinase type II domain. The N-terminus of ACAD10 is longer than ACAD11, and there is a predicted haloacid dehydrogenase-like hydrolase (HDL) domain ahead of the APH/homoserine kinase type II domain. The carboxy end of ACAD11 harbors a peroxisome targeting signal 1 (PTS1). A similar domain structure is found in the non-mammalian orthologues, such as C. elegans [NP_504508], that also contain a PTS1.
Database searches of transcribed sequences from these genes identified the presence of multiple transcripts that differed mostly at either the 5′ or 3′ end. To examine the presence of these transcripts in vivo, we used real-time PCR with primer pairs targeted to unique exon junctions in the different transcripts to measure expression level of alternative transcripts of ACAD10 and 11 in 21 different human tissues The probes targeting published unique mRNA product containing alternative exons 20′ and 14′ identified in database entries for ACAD11 (AK022654 and AL833721) did not detect expression in all of the human tissues tested, suggesting that those splicing variants are either rare or do not exist in vivo. Therefore we can not assess further the alternative transcripts in ACAD11. However, multiple transcripts from the ACAD10 gene were detected and the relative abundance of each transcript differed in the various human tissues (Fig. 1C). Primer set 7/8, specific for mRNA AL832043 targets the region corresponding to the predicted APH domain. Primers 13/14 target a region shared by most transcripts in the genetic databases as shown in Fig 1B and translated into the junction between the APH domain and conserved ACAD N-terminal domain. Primers 21/22 amplify a region translated to a conserved C-terminal ACAD domain containing the predicted catalytic base shared by AL832043 and hCT2309109. In contrast, primers 21/22', unique to AK092356, target an alternatively spliced species that eliminates exon 22, leading to loss of the putative ACAD catalytic domain. In all tissues, expression of the transcripts amplified by primers 7/8 was higher than those by primers 21/22. Thus, a large portion of the transcripts that contain sequences for an APH domain at the N-terminus do not have sequences for the ACAD domain at the C-terminus. Expression of the fragment defined by p21/22 was much higher than p21/22', showing that the majority of transcripts encoding ACAD domains contain sequences potentially supporting ACAD enzymatic function. However, the expression of AK092356 detected by probe 21/22' reflects the existence of ACAD10 isoforms without ACAD enzymatic function or mitochondrial targeting signal peptide. Interestingly, in some tissues (e.g., small intestine and thymus) the expression level of transcripts containing fragment, probe 7/8, was higher than those amplified by probe 13/14, suggesting that these tissues may express a previously unrecognized mRNA species that encodes a protein comprised of only an APH domain without ACAD sequences. Information currently available on both genes in the public databases is incomplete, hindering our ability to quantify each of the specific transcripts, particularly for ACAD11. Nevertheless, the expression profile of ACAD10 defined by our probe sets supports the existence of multiple transcripts and proteins as well as possible unique roles in different tissues and cellular compartments. Given that ACAD11 and ACAD10 are highly homologous, it is likely that multiple isoforms also exist for ACAD11.
To begin to understand the in vivo functions of ACAD10 and 11, we first examined the tissue expression pattern of the two genes in 21 different human tissues using real-time RT-PCR. We used probes p13/14 (ACAD10) and p11/12 (ACAD11) to target the sequences that are common to most of the identified transcripts, thus best presenting the total expression of each gene (Fig. 2A and B). ACAD11 had the highest level of expression in adult brain, 1.3 times the expression of the housekeeping gene GUSB (used as the internal control). Thereafter, we normalized expression levels in other tissues to the brain (Fig 2A). ACAD11 additionally had significant levels of expression in adult liver, heart, and kidney. ACAD10 had lower levels of expression than ACAD11 in liver and adult brain, with the highest level in fetal brain (1.5 times the expression of GUSB). Fig 2B shows the expression of ACAD10 in other tissues relative to fetal brain. ACAD10 was expressed more highly in fetal brain than adult, a pattern similar to that seen for LCAD [6] and also was highly expressed in heart and kidney.
Among the transcripts of ACAD10 and 11 identified in the public databases, ACAD11 entry BC019607 is similar to entry hCT2309109 originally identified in the proprietary Celera, Inc. cDNA database (Alameda, CA; now also represented in the NCBI public gene database). hCT2309109 is a whole length cDNA and is predicted to translate into an ACAD like protein containing only the conserved ACAD domains. We therefore amplified cDNA sequences corresponding to hCT2309109 sequence from human liver total RNA and cloned them into an in vitro transcription expression vector. In vitro transcription of this plasmid (referred to hereafter as ACAD11 variant 1) followed by translation with rabbit liver lysate produced two stable proteins (Fig. 3A, lane 2). The first one (57 kDa) was of appropriate molecular mass to correspond to a polypeptide starting with the first ATG in the expression insert, while the other protein of ~52 kDa could arise from internal initiation from the third ATG (A142 from the first ATG) of the expression insert. Computer predictions (ATGpr_sim at https://www.jbirc.aist.go.jp/FLJ/ATGpr/atgpr/index.html) indicated that the third ATG could be an equally strong translation start site as the first ATG.
Following in vitro transcription/translation of ACAD11 variant 1, the majority of the 52 kDa protein was imported into isolated mitochondria in an in vitro import assay and was processed to a smaller mature protein (Fig. 3B lane 3– 5), while the majority of the 57 kDa protein remained in the supernatant that was free of mitochondria (Fig 3A lane 4). We therefore subcloned the cDNA sequence predicted to correspond to the 52 kDa ACAD11 (designated as ACAD11 variant 2) and performed in vitro transcription and translation followed by mitochondrial import of the translation products. As expected, only the 52 kDa protein was efficiently imported into mitochondria and cleaved to a mature form as shown by its resistance to trypsin digestion after the mitochondrial import reaction (Fig 3B, Lane 3–5). There are a few other smaller trypsin sensitive protein bands identified in the reaction, which likely represent alternative transcript or translation starting sites. A mitochondrial peptide peptidase (MPP) cleavage site in ACAD11 is highly conserved across species (Fig. 3D). Non-denaturing electrophoresis confirmed that, in contrast to the precursor protein (Fig. 3D lane 6), the intra-mitochondrial form of ACAD11 (Figu.3D lane 7) was assembled into a stable multimeric protein as are other ACADs (Fig. 3C, lane 3 and lane 5). This further supports that mature ACAD11 assembled in mitochondrial are likely functional mitochondrial enzyme.
The precursor form of nuclear encoded enzymes that localized to mitochondria, including all of the ACADs, are not usually active when expressed in E. coli cells, but their mature forms can often be produced in this fashion. To facilitate study of the mature form of ACAD11, we cloned the mature coding sequence into a bacterial expression vector with substitution of E. coli codon bias for the first 40 amino acids. Following induction, crude cellular extract was tested for ACAD activity with the ETF (electron transfer flavoprotein) fluorescence assay using a broad range of acyl-CoA substrates. The highest activity was seen with saturated C22-CoA (docosanoyl-CoA) (Fig. 4E) and relative activity of 30%, 63%, 15% and 15% compared to C22-CoA with C20-CoA (eicosanoyl-CoA), C23-CoA (tricosanoyl-CoA), C24-CoA(tetracosanoyl-CoA), and C26-CoA (hexacosanoyl-CoA), respectively at 50 μM substrate concentration. The activity of ACAD11 in crude cell extracts was about 10 mU/mg of cellular protein, similar to the level of expression seen with other ACADs in our system. We also cloned and over expressed the predicted mature mitochondrial coding sequence of ACAD10 in the same fashion. Extracts from cells expressing a predicted mature mitochondrial ACAD10 insert were only active with R and S, 2 methyl-C15-CoA (R and S, 2-methyl-pentadecanoyl-CoA) among a broad series of other substrates including the optimum ones for all of the other ACADs. The measured activity (1.4 mU/mg at 150 μM substrate concentration) is considerably lower than that obtained with other expressed ACADs towards their optimum substrates, suggesting that the dehydrogenation reaction between ACAD 10 and R or S, 2 methyl-C15-CoA is unlikely the optimal function for ACAD 10 in vivo. Unfortunately, we could not purify either of these two proteins further to allow more formal characterization of the enzymes' kinetic properties.
ACAD10 and ACAD11 are 20–30% identical to other ACADs (Fig. 5A) and computer modeling predicts that they are structurally similar to previously characterized ACADs (Fig. 5A and B). Interestingly, the catalytic residue, a glutamate in all other ACADs, is not conserved in ACAD 10 and 11. Rather, computer modeling predicts that both ACAD 10 and 11 utilize an aspartate rather than glutamate for this function (Fig. 5C). Mutagenesis studies with other ACAD have confirmed that aspartate can indeed be substituted for the wild type glutamate but that the resultant mutant enzymes have altered substrate specificities [19]. It is likely that the shorter side chain of the aspartate in the catalytic pocket will allow ACAD10 and 11 to utilize substrates with more bulky and/or longer carbon backbones. Of note, the computer modeling revealed two hydrophilic amino acids (Arg 512 and His 509, NP115545 ) in the substrate binding pocket of ACAD11 (Fig. 5C), which are highly conserved in both ACAD 11 and 10 proteins from multiple species.
This is unusual since amino acid residues located in the substrate binding pocket of the ACADs are mostly hydrophobic in order to accommodate the hydrophobic fatty acid moiety of the substrates. The exception to this is a highly conserved hydrophilic amino acid, Arg 23, in glutaryl-CoA dehydrogenase (GCD), present in GCD's substrate binding pocket. This residue has been shown to facilitate catalysis of the CoA ester of the dicarboxylic acid, glutaryl-CoA. Of note in our computer created model, Arg512 in ACAD11 aligns with Arg23 of GCD (Fig. 5C) suggesting a common function. Whether ACAD11 can also act as a decarboxylase as GCD will require further investigation. The predicted structure of the bottom of ACAD11 substrate binding pocket aligns poorly with both GCD and SCAD, indicating it likely uses longer or bulker substrates (Fig. 5B).
The presence of multiple ACAD10 and 11 transcripts in various tissues raises the possibility that some might generate protein species that localize to different subcellular compartments. To examine this, a rabbit polyclonal antisera was raised against a synthetic ACAD11 peptide corresponding to a portion of the sequence of the N-terminal domain of the mature mitochondrial ACAD11. The peptide sequence is common to potential ACAD11 protein isoforms translated from all identified ACAD11 transcripts, but not homologous to other ACADs including ACAD10. Western blot analysis with this antiserum identified an ACAD11 protein species in mitochondrial fractions of both human cerebellum and kidney corresponding in size to the mature mitochondrial form seen in our in vitro import studies. This form primarily localized to the membrane fraction of mitochondria isolated from both human cerebellum and kidney (Fig. 2C), but not in the mitochondrial matrix fraction. Multiple ACAD11 positive bands were seen in other tissues, such as liver (date not shown). The specificity of our antibody toward the ACAD11 peptide was validated by peptide competition analysis, however, purified ACAD11 isoforms were not available to further characterize antibody interactions. Immunofluorescence staining of ACAD11 in human neuroblastoma and skin fibroblasts revealed a marked difference in subcellular location of ACAD11 proteins (Fig. 6A–J). ACAD11 protein in neuroblastoma cells co-localized with mitochondrial ATPase (Fig. 6A–C). In contrast, the majority of the ACAD11 signal in fibroblasts did not co-localize with mitochondrial ATPase (Fig. 6D–F). Interestingly, in both human skin fibroblasts and human neuroblastoma cells, additional ACAD11 specific staining was observed in punctate cytoplasmic vesicles. These were most evident with digitonin treatment (which preferentially permeabilizes the plasma membrane but not organellar membranes). Finally, ACAD11 antibodies strongly stained nuclei regardless of cell permeabilization conditions (Fig. 6H–J), a pattern also seen for ACAD9. The striking difference in subcellular location of ACAD11 among various cell and tissue types emphasizes that expression of the mitochondrial form of ACAD11 as the product of alternative splicing is tissue specific, in agreement with our RNA expression studies.
In light of the unusual tissue distribution of ACAD11 and the growing menu of ACADs active with long chain substrates, we next examined the full spectrum of ACAD activity in fractionated extracts from various tissues using the highly specific ETF fluorescent reduction assay (Fig. 4). Most of the activity measured with long chain substrates in cerebellum (Fig. 4A) and muscle (Fig. 4B) was detected in the mitochondrial membrane fraction, however, the chain length optima were different. The muscle mitochondrial membrane fraction was most active with substrates with carbon backbones of 12–18. In contrast, cerebellum had a sharp peak of activity with C16- and C18-CoA and significant C22CoA activity. Of note, mitochondrial membranes from human cerebellum showed no detectable activity with C10- or C12-CoA as substrate. The spectrum of membrane bound activity in human muscle corresponds to that of purified human VLCAD [6] (Fig. 4F), while the spectrum of activity in human cerebellum is consistent with a combination of ACAD9 and ACAD11 (Fig. 4E). In contrast to VLCAD and ACAD9, LCAD is a mitochondrial matrix protein and is the only long chain ACAD with significant expression in human lung [6]. ACAD activity in human lung (Fig 6C) was detected only in the mitochondrial supernatant fraction with a substrate pattern matching that of LCAD (Fig 6G). ACAD activity with short and medium chain substrates predominated in human liver mitochondrial matrix (Fig 6D), matching the activity profiles of MCAD and SCAD (Fig 6H). The C22/C20 activity ratio in a liver mitochondrial membrane fraction was much greater than that seen for purified VLCAD or ACAD9 enzymes (1.0 vs. 0.6, respectively), suggesting a likely contribution from ACAD11 (C22/C20 >3) in human liver as well. Our ACAD11 peptide derived antibody did not inhibit native ACAD11 activity, precluding additional immunoinactivation experiments.
To better characterize the distribution of ACADs in human cerebellum, immunohistochemical staining was performed using antisera against MCAD, ACAD9, and ACAD11 (Fig 7. A–L). We have previously shown that VLCAD immunostaining in human cerebellum reacts mostly surrounding the vascular regions (He et al, 2007). ACAD11 antisera uniquely stained the white matter of cerebellum (Fig. 7A & C) but only minimally stained Purkinje neurons or granular layer (Fig. 7B), and this was especially strong in oligodendric cells as determined by cell morphology (Fig. 7C). ACAD9 uniquely strongly stained the granular layer (Fig. 7E & F), particularly the dendrites of granular neurons (Fig. 7F), but not the white matter (Fig. 7G). Both ACAD11 and ACAD9 antisera showed a high level of staining in the dentate nucleus in the deep white matter of cerebellum (Fig. 7D and H), while MCAD specific staining was much weaker (Fig. 7L). MCAD uniquely stained the molecular layer (Fig. 7I), certain axons in the white matter (Fig. 7K), and some astrocytes scattered in different layers of cerebellum (Fig. 7J–L). Both ACAD9 and MCAD also strongly stained Purkinje neurons (Fig. 7F and J). Overall the specific and compartmentalized pattern of distribution of the three major ACADs in human cerebellum suggests that each cell type is utilizing mitochondrial β-oxidation for distinct functions.
In contrast to cerebellum, MCAD and VLCAD both stained muscle fibers in a similar punctate pattern indicative of mitochondrial localization (Fig. 8A and B). ACAD9 and ACAD 11 stained muscle fibers less strongly, however, both were present in the perivascular and vascular regions (Fig 8C and D) indicating a different function in muscle for these enzymes than for VLCAD.
A majority of the genes in humans are now recognized to generate multiple functional transcripts through alternative splicing and it is especially common for genes with primary expression in the central nervous system [20]. In this study, we characterize expression of two new genes with homology to the mitochondrial ACAD family (ACAD10 and 11). Both genes exhibit a complicated pattern of alternative splicing from complicated loci and generate at least one protein species that can be imported into mitochondria. As demonstrated by real-time PCR, the majority of the transcripts generated from the ACAD10 and 11 genes translate to proteins with a predicted APH domain, at the N-terminus and an N-terminal ACAD domain and a C-terminal ACAD catalytic domain at the C-terminus (Fig. 1). These predicted proteins do not have mitochondrial targeting sequences at their N-termini and therefore are likely to be localized to alternative cellular locations. Consistent with this observation, a global proteomic survey of subcellular organelles of six major organs in mouse localized ~70% of predicted ACAD10 protein to the membrane (microsomal) fraction and 30% to mitochondria [21]. A typical glycosylation site (NTO) is present between the APH and ACAD N-terminal domains in ACAD11 protein, suggesting that this large ACAD11 isoform may target to microsomes or the cytoplasmic membrane. But this predicted protein also contains a canonical peroxisomal targeting signal at its C-terminus that could override other localization signals. In support of this, one published proteomic study has identified ACAD11 variants with an APH domain in peroxisomes [22]. In the current study, although a small portion of ACAD11 in skin fibroblasts may be localized to peroxisomes (data not shown), the majority of the staining appeared in a punctate pattern, adjacent to the cytoplasmic membrane of the cell (Fig. 6). This finding suggests that some ACAD11 isomers may be localized to membrane associated vesicles, a location it shares in part with ACAD10. In contrast, we found that the majority of ACAD11 localized to mitochondria in human neuroblastoma cells (Fig. 6), highlighting the importance of considering tissue of origin when examining the physiological role of the products of these genes. Recent global analysis of alternately spliced genes has indicated that groups of tissue-specific alternative splicing events often function in a coordinated manner in a specific pathway or interaction network [20]. Of note, we have observed a similar tissue pattern of alternative splicing for the ACAD9, 10 and 11 genes in human cerebellum, fibroblasts, and liver [6]. Moreover, mitochondrial forms of ACAD9, and 11 all were found to be present in certain neuronal cell populations in human CNS (Figs. 7 and and8),8), while VLCAD was not. Therefore, we speculate that these three long chain ACADs and their isomers of unknown physiologic function act in a coordinated pathway in the CNS not related to energy metabolism, but related to their specificity towards certain groups of functional fatty acid CoA esters. Recently, Zolman et al. indirectly showed that the Arabadopsis homologue of ACAD10 is involved in the β-oxidation of indole-3-butyric acid to indole-3-acetic acid, a metabolite of tryptophan metabolism [23]. Both compounds function as plant hormones known as auxins. The metabolism of auxins is localized to peroxisomes in plants but is independent of the more general fatty acid β-oxidation pathway [23]. In humans, weak auxins such as phenylbutyrate and phenylacetate are currently in the spotlight for their roles as drugs, especially in treatment of neoplastic disorders and may provide a novel target for ACAD10 function [24–26]. It seems likely that in mammals, the function performed by Arabadopsis ACAD10 has been transferred to mitochondria and may be involved in metabolism of tryptophan derivatives such as serotonin, an important neurotransmitter. Little is known of the role of β-oxidation in aromatic amino acid catabolism, although it is well known that MCAD is active with substrates with aromatic rings such as phenylpropionyl-CoA, a tyrosine metabolite from gut bacteria.
The identification of multiple isoforms of ACAD10 and 11 led us to further investigate the enzymatic activity of the mitochondrial forms of these enzymes (Fig. 4). Using in vitro mitochondrial import experiments and homology searches, we constructed prokaryotic expression inserts for the predicted mature mitochondrial forms of both enzymes. Expressed mitochondrial ACAD11 utilized very long chain acyl-CoA substrates with optimal activity with C22CoA as substrate while ACAD10 had low activity with 2S-methyl C15-CoA and 2-methyl C16-CoA (both the R and S forms). Minimal, but detectable, activity was seen with ACAD10 using very long and branched chain substrates such as 2-methyl C19-CoA or long chain substrates with branch at other positions such as 3-methyl iso C15-CoA. Thus, we postulate ACAD10 will have maximal activity with substrates of shorter chain length than ACAD11 with specific branched structures. Unfortunately, such substrates were not available for further study of this enzyme. Very long chain fatty acids are not found in significant quantities in the human diet, however, they are abundant in neuronal tissues and are important components of myelin [27]. This is consistent with the tissue expression pattern of the mitochondrial form of ACAD11, which is highly expressed in adult brain, especially in the oligodendrocytes. Expression of ACAD10 in fetal brain may reflect a role in metabolism of the fatty acids unique to brain development [27].
Most interestingly, our results indicate that different ACADs predominate in mitochondrial β-oxidation of long chain fatty acids activity in human brain than are used in most other tissues. Immunostaining of human adult cerebellum identified the presence of MCAD, ACAD9 and ACAD11 as major ACADs in this tissue (Fig 7.A–L). Thus, these three ACADs are likely responsible for the three major activity peaks seen at C18, C22 and C8 in cerebellar extracts (Fig. 4A). Interestingly, each of these enzymes has a distinct cellular distribution pattern in cerebellum rather than all residing in the same cells types as is typical of VLCAD and MCAD in other tissues such as human muscle (Fig. 7A–L and Fig 8. A–D). We hypothesize that the unique cerebellar cellular distribution pattern reflects physiological function. For example, ACAD9 predominates in Purkinje neurons and granular neurons of the grey matter (Fig. 7E and F), while ACAD11 is mainly present in the oligodendrocyte-like cells of the white matter (Fig. 7A and C). Recent studies have revealed that the fatty acid composition of glycosphingolipid sulfatide, a component of myelin and some non-myelin forming cells in the brain, differs between white and grey matter. Neurons contain mostly long chain fatty acids with an emphasis on stearic acid (C18), corresponding to the optimal substrate of ACAD9. In contrast, sulfatides in myelin, normally synthesized by oligodendrocytes in white matter, have a majority of very long chain fatty acids (C22-26). We hypothesize that C22-CoA is metabolized in mitochondria by ACAD11, filling a recognized gap in long chain fatty acid catabolism by peroxisomal acyl-CoA oxidases, which have substrate optima for C24 and 26 substrates [28] Moreover, we suggest that multiple ACAD9 and ACAD11 protein isoforms are involved in controlling the fatty acid composition of cellular lipids through lipid recycling and synthesis and thus play a major role in determining the functions of these lipids. It is interesting that even in human muscle, both ACAD9 and 11 were found mostly in the perivascular regions, differentiating them from VLCAD and MCAD (Fig. 8A–D). Therefore we suspect that their roles even in tissues outside the CNS are other than energy production. Of note, the majority of long chain fatty acids in humans are actually recycled by β-oxidation rather than being consumed as fuel [29].
In the CNS, the cellular location of MCAD is strikingly different from ACAD9 and 11, being uniquely present in the axons of certain neurons (Fig. 7K) and the molecular layer of the cerebellum (which contain mostly dendrites of purkinje neurons) (Fig. 7I). Numerous proteins and processes critical for CNS plasticity require ATP including cytoskeletal components, ion-motive ATPase, and a myriad of protein phosphorylation reactions. In turn, it is mitochondria that produce the bulk of ATP in neurons, and concentration gradients of ATP are greatest immediately adjacent to the mitochondria [30]. Therefore, it is likely that the high expression of MCAD in neuronal axons and dendrites reflects a primary role for β-oxidation in ATP production in these cells. This of course is the major function of β-oxidation in tissues such as muscle and heart, but it is not well established for brain. Since medium chain fatty acids can readily pass the blood-brain barrier, they serve as an important potential fuel for ATP production in maximizing neuroplasticity, which could be important in neurodegenerative disorders.
ACAD9 has recently been postulated to have an alternative structural role as a respiratory chain complex 1 stability or assembly factor [7]. We have also demonstrated the direct, physical interaction of multifunctional fatty acid oxidation complex and the respiratory chain supercomplexes [31]. Thus the possible existence of non-enzymatic functions for both the mitochondrial and non-mitochondrial forms of ACAD10 and 11 must be considered in future studies.
In summary, we have identified two new mitochondrial ACADs, one of several protein isoforms produced by the ACAD10 and 11 genes. Our studies indicate that in combination with the recently characterized ACAD9, these enzymes are likely involved in serving novel physiologic functions in the central nervous system such as controlling fatty acid composition of cellular lipids and metabolizing aromatic amino acids intermediates. These findings provide an impetus for further studies on the role of β-oxidation in the metabolism of these novel groups of substrates with neuronal and immune functions.
We thank Professor J. J. Kim for providing purified human LCAD. We thank Dr. Ronald Jaffe for providing the control human tissues. This work was supported in part by National Institutes of Health (NIH) Public Health Service grant R01 DK54936 and DK78755 (to J.V.), and by a grant Geconcerteerde Onderzoeksacties (GOA2004/08) from the Flemish Government (to P.P.V.V)
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Democracy has delivered some surprising results on the doorsteps of the world's leading financial and political experts in the past year. What was marginally possible is now fact and liberals have learned that they must review their assumptions on how votes are cast. The extent of dissatisfaction with the establishment is dawning upon the world, and politicians are having a hard time coming to grips with their disconnection from popular reality – the veil of ignorance has an evil twin.
It looks like Italy's referendum this week will fall in line with Brexit and Trump's election. On December 4th, the country will be voting on constitutional reforms put forth by Matteo Renzi, the young Prime Minister.
The Problem
There are many things wrong with Italy at the moment. Youth unemployment has soared over 40% since 2013. GDP expansion has been mostly negative since 2008. Worse still, GDP per capita has stagnated since the 1990s. Government debt stands at 133% of GDP, the second biggest in the Eurozone area, and private debt at 117%. The banks are saturated with non-performing loans. If Italy follows Greece to the brink of default, it will be too big to be bailed out.
The IMF predicts Italy's GDP will lag behind its European counterparts again this year with a 1.1% increase. Reform inertia has perpetuated the longevity of these issues and the root of inertia is institutional, the government claims. They are not wrong.
The Referendum
Unlike most EU countries, the Italian Constitution prescribes "perfect bicameralism," meaning that the Senate and the Chamber have equal power. In the words of The Economist, this is a "recipe for gridlock." Unable to perform and always challenged, Italian governments have numbered 65 since 1945.
Mr Renzi's proposal, one he staked his job on, will shrink the Senate's numbers and powers and strengthen the Chamber, freeing their hands to carry necessary reforms. The upper house will no longer be able to overthrow the executive through a vote of no confidence.
Italy since 1945
It is the fine print that differentiates the plan from other European systems. Senators will be appointed, not elected, from the culprit of Italian corruption – regional and local assemblies. Renzi has also passed an electoral law that guarantees the majority party a 54% majority in the lower house.
A Yes vote does not enable the Chamber, but the government. If executive power were the solution to legislation sluggishness, France would be thriving. It is not.
The Belligerents
When Renzi's constitutional package was passed through the lower and upper houses in April, his achievement was celebrated as historical. The young Prime Minister, it was thought then, would breathe new life in the archaic method of governance. Things have changed.
The Five Star Movement is his primary opponent. It started as a web-based movement led by comedian Beppe Grillo in 2009. As its origins hint, it is a popular and populist party. Its combination of Euroscepticism and progressive environmental policy gave it the reigns of city halls in Rome and Turin.
Mr Grillo's party polls at 30% of the popular vote, threatening Renzi's hold on power and is expected to win the referendum by a small margin. "This is mad stuff," Grillo blogged, "it's an apocalypse for the big papers, the intellectuals, the journalists."
The Reaction
Rejecting the government's proposal will have everyone dancing to a familiar tune: a win for populism, a loss for the establishment, mass panic for the markets.
The rejection of Renzi's proposal and stepping down as he vowed, will bring political turmoil. It is not realistic for yet another election to be carried out. A new government will have to be formed. More than that, Renzi has convinced the EU and the US that the reform will allow Italy's economy to perform to EU standards. The expectations are so high that if the proposal fails Italy's credibility internationally might come crashing down.
Investor confidence will plummet, destabilising the Italian banking sector and reducing growth prospects. In truth, this is a certainty in the immediate aftermath of a No vote. The question is for how long and to what extent.
The Consequences
Italy's financial sector is treading on treacherous grounds as it is. Asset managers, insurers and banks set up a €5bn fund to bail out weak lenders. The political instability might limit their willingness to be involved in the Italian economy. The Financial Times asked officials and senior bankers what the backlash might be. Their answer was that up to eight big banks might be in trouble.
The possibility of an Italexit, on the other hand, is an exaggeration. Only 15.3% of Italians want to leave the Eurozone, a poll conducted by newspaper La Stampa concluded. But the Five Star Movement has blunted the edge of that sword lately.
The extent of the financial crisis, however, risks contagion to the rest of Europe.

subset: commoncrawl


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Courtesy of Dennis Keesee Collection
Boonslick's W.B. Cox
The pencil sketch, Our Mess, is of officers imprisoned with Cox at Johnson's Island at Ohio.
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W.B. Cox
W.B. Cox
The Boonslick's W.B. Cox Gets His Due
W.B. Cox endured the horrors of battlefields and prisons. In July 1861, he served as a captain with the Missouri State Guard (a pro-Confederate state militia) at the Battle of Carthage. At Wilson's Creek that August, Cox acted as adjutant and had two horses shot from under him. The following September, Cox participated in the successful siege of Lexington and became a lieutenant colonel before the battle at Pea Ridge, Arkansas, in March 1862.
After the dissolution of his Missouri State Guard unit, Cox joined the Confederate army as a captain, and in December 1862, he fought at Prairie Grove, Arkansas. In the summer of 1863, Cox traveled to Richmond, Virginia, to deliver muster rolls—incidentally, his first exposure to the city where he would blossom as an artist.
That September he was captured at Little Rock and began six months as a prisoner of war. He was first taken to Memphis, then to the Gratiot Street prison at St. Louis where he responded under oath to a four-page questionnaire respecting his suitability for a prisoner exchange.
His occupation was "painter." When asked, "Are you a southern sympathizer?" he responded, "I am." Asked whether he wanted the authority of the U.S. government restored over the secessionists, he said, "I do not." Citing Cox's honesty, the examining officer approved his request for exchange, according to National Archives documents.
However, Cox continued through the Union's prisoner-of-war system. After two weeks at Camp Chase, Ohio, he was taken to Johnson's Island—a Lake Erie prison for officers near Sandusky, Ohio—where he was held from November 14, 1863, into the following February.
Here, in January 1864, he produced a pencil sketch, called Our Mess, shown at left. One of the eleven depicted officers, Cox holds a sketchbook and is seated in the center of the group just behind two men playing chess. The others seem strangely stiff, withdrawn, and disinterested in the ongoing game of chess. Their stolidity may reflect wretched living conditions.
A Virginian, who was at Johnson's Island while Cox was there, wrote in Confederate Veteran that starving officers ate candles bought from the sutler and "rats that lived upon the sinks." Cox, however, relieves the somber mood by depicting a puckish, smiling officer waving a sketch of a mounted horseman. Reproductions of this sketch frequently illustrate accounts of the Johnson's Island prison and general articles on Civil War prisoner-of-war camps.
Cox was exchanged from Point Lookout, Maryland, on March 17, 1864, and went to Richmond. Before resigning his commission, June 3, 1864, Cox began painting Virginia battle flags. In May, he received $420 for painting two regimental flags and the centers, or insignia, for five others at $60 each. In January 1865, he was paid $600 for painting six flags.
During the war, Cox painted likenesses of Confederate leaders from photographs. The Richmond Times (August 2, 1865) called attention to a "full-length portrait of General Lee in the window of Mr. E.O. Townsend's bookstore. It has deservedly received much attention. … The artistic performance, the blending of light and shade, the almost tangible distinctness of the various articles of dress and uniform are the work of the most unusual talent. The artist is Mr. W.B. Cox, a young Missourian."
This may well be the portrait, signed "By W.B. Cox, Richmond, Virginia, 1865," depicting Lee outside his tent with his horse and orderly that hangs in the Jay P. Altmayer Collection in Mobile, Alabama. Altmayer purchased the 24-inch-by-20-inch oil painting in 1961 from a Charleston, South Carolina, antique shop.
Cox is thought to have painted a similar, unsigned, and undated portrait of Gen. Robert E. Lee with a hilly background that hangs in the Virginia Historical Society Museum at Richmond. Set against a mountain landscape with a few soldiers barely discernible in the background, Lee wears a military sash and dress sword in this 21.5-inch-by-17.5-inch likeness—said to be a favorite of Lee's daughter, Mildred. It was presented to the society in 1957 by the daughters of a Confederate major. For his Lee paintings, Cox depended on 1864 photos taken by Julian Vannerson at Richmond.
The West Point Museum is home to another of Cox's works, The Heroes of Manassas, which juxtaposes triumph and disaster. As Jefferson Davis and his victorious commanders (P.G.T. Beauregard, Stonewall Jackson, Joseph E. Johnston, and J.E.B. Stuart) confer on horseback after the first battle of Manassas, a shell with a burning fuse threatens to blow them to bits.
David M. Reel, the museum's Curator of Fine Art and Decorative Art, says late nineteenth-century photographs of the painting were reproduced for sale. The 18-inch-by 24-inch work, which has been restored recently, was presented to the museum in 1968; but it took till the 1990s to establish that Cox painted it. It too is undated and unsigned.
Cox returned to the Boonslick in time for the 1870 census and painted portraits of two Glasgow residents in 1871. He joined the Order of Good Templars there and delivered at least two temperance lectures elsewhere. He moved to Miami, Missouri; Brunswick; and Trenton, where he married and divorced. By the end of the decade, he had a studio at Sedalia where he gave violin lessons and published poetry.
He specialized in portraits, which might be in crayon for economy, with a few landscapes and depictions of historic persons. Five of his Missouri portraits have been located, two of which are owned by Friends of Arrow Rock. In 1874, he launched a project to paint a large picture of the Civil War Battle of Wilson's Creek and produce engravings of it. He completed a painting of Gen. Sterling Price for this work; however, there is no trace of a finished product.
Cox was popular in Miami, Missouri, where he spent long periods living in hotels and the homes of his patrons. He won medals for his paintings at several fairs. Despite his temperance efforts, he died of delirium tremens at an Iowa poorhouse in 1882.


text: Vine Monograms Version 2 - Applique Machine Embroidery Applique Designs & Satin Stitch This is a wonderful applique monogram set! We wanted to create a unique interlocking monogram, with or without applique fabric. Each letter 'interlocks' with the applique oval frame. The oval frame has a fabulous detail around it, to enhance each interlocking letter. We have also included with this SET 2.25" height side letters! We created this set to have versatility all its own - with applique fabric, without applique fabric, center letter only, and center letter with the side letters! The height for the applique oval frame with the letter inside is 3.00" The width of each applique letter will vary from 2.50" to 2.75" maximum width. The satin stitch letters are 2.25" height, the width will vary from 1.50" through 1.85" width With your purchase you will have the option to download the following formats, HUS, VIP, PES, JEF in ZIPPED format Questions or concerns, let me know, I would be more than happy to help Be sure to add us to your favorites, as I will be adding more machine embroidery designs! **APPLIQUE FABRIC NOT INCLUDED WITH PURCHASE** ** Note these are NOT patches, you must have an embroidery machine to utilize this item** WE ask that you NOT share, or sell or claim the embroidery design files as your own, however, you may sew as many as you like for sale or gifts. BASIC SKILLS NECESSARY Skills of basic applique, machine embroidery Ability to merge embroidery designs SIZING / FINISHED MEASUREMENTS See photos for sizes of designs :)


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Blogger Antosca makes a few points worth mentioning:
First, Meyer is being kind of alarming and weirdly noisy, but not inappropriate. It's really odd how the police try to stop him almost immediately. As one of the videos shows, Kerry pointed to him and indicated that he should ask his question... he's not speaking out of line. I wonder about the context here. Had something happened between Meyer and the police earlier? Is it just that he exceeded a one minute time limit -- that's why he was attacked?We'll set aside the questions regarding the alleged lack of alternatives to Kerry in the 2004 election - there were some after all, although they were all but ignored by the corporate media. The point that using a taser on a handcuffed human being is torture is salient - I'd add that using a taser period is a form of torture, and one that can easily prove lethal. Also, the moral obligation of the bystanders is worth highlighting - they too have an obligation to prevent torture, especially when it's right in their faces. Looking at the video footage, it's pretty obvious that at least one of the cops was really getting off on the pain being inflicted on the victim. There's something sick about a society that not only seems to condone torture, but in which folks like Kerry can just keep droning on and on about nothing while it's going on in front of him, or in which bystanders just watch while it happens.
Second, John Kerry did not handle this well at all. I liked him all right before -- I voted for him, but what were the alternatives? -- but now I really dislike the man. His behavior here is pathetic. Listen to him droning sonorously on in the background as a guy is dragged down the aisles and pinned the ground. He does say something like, "Officers, can we--" but then trails off ineffectually... and you can also hear him make what sounds like a joke about Meyer: "...unfortunately he's not available to come up here and swear me in as President..." At that point, fair enough, maybe it didn't seem as bad from the stage as it looks on the video. But then the guy is screaming in pain, and Kerry is still droning on, not agitated, nothing. He should have gotten off the stage and told the cops to get the hell off that guy. It's not what a politician would do, but it's what a fucking man would do.
And third, the police officers in this video are absolute scum. They should all be fired, and the one who used the taser should be put in jail. It was bizarre and irrational for them to attack Meyer, a display of utterly unprovoked violence. The sheer brazenness of it, in front of an auditorium and multiple cameras, chills me. You can't go to a microphone and question an elected official (and again, there was nothing abusive or even particularly combative about these questions) without fear of being dragged off and tased? It's heartening that some audience members followed the police downstairs, telling them to stop and assuring Meyer that they would testify in his behalf -- but frankly, as far as I'm concerned, people watching this happen a few feet away would be justified in defending the victim from the officers with the use of any weapon available. The use of tasers on handcuffed prisoners, particularly when, like Meyer and Mostafa Tabatabainejad, the prisoner has done absolutely nothing wrong and is unarmed, is straight torture. You can't stand around and watch some innocent guy get tortured by the police. You ought to do whatever you can to make it stop.


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214-254-1676 has 1112 ads in Dallas / Fort Worth since March 4, 2015 and 904 ads in 28 other cities. 0 fake pics were reported for this ad. A written review can be found here.
Livetsreviews.com is a Website that makes certain classified advertising that is published
available in one place by locating it, indexing and organizing, and providing


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The Dragon's Den judge managed to create a good healthy discussion in my mind when he discussed how parents should not help their children get jobs on the Daily Telegraph.
Realistically speaking, what parent would not use their connections and status to let their energetic and ambitious child get a foot in the door in a competitive industry? If my parents had a book of useful contacts, I'd be expecting them to fish it out and pitch my employability ASAP. Unfortunately, my reality does not reflect this scenario. This led me to whole heartedly agree with Caan. Those young students and graduates need to experience what hard graft is. It toughens you up and makes you accept the harsh realities of life, whilst feeling a sense of pride in knowing that you have gained some invaluable life skills. Those graduates who have it plain sailing because of a phone call to the director of a company their dad is best buddies with, will not know what assessment days, rejections and late night job applying is about. They miss out on a life chart depicting peaks and troughs of failure and success.
Here's the catch though. Whilst sharing these words of wisdom , the newly appointed social mobility tsar, forgot a very relevant fact – his daughter Hanah, spent a year working at his private equity firm Hamilton Bradshaw . Woops. What I find quite amusing is that Caan mentions that she underwent a "rigorous recruitment process." Now I for one, would love to know what constituted this recruitment process, because I really fail to envisage my own father grilling me about my achievements so far. I just don't see it. The whole thing has back fired hideously. Cann has been labelled hypocritical and this is bad timing as Nick Clegg has launched a scheme whereby top British companies are to be encouraged to take on board more people from less privileged backgrounds. Oh dear.
Personally speaking, Caan messed it up. If he had mentioned in the initial interview that he has actually helped his daughters, then I could have perhaps accepted his truth, albeit reluctantly. Hiding facts that contradict what you preach is something I can't quite accept. I've seen nepotism and was never quite aware that the big, wide world of work was rife with it. I've also realised the importance of the much chucked around phrase 'it's not what you know, it's who you know.' Two years ago, I wouldn't have been able to digest that these things affect how far you get in life. As I sit and write this with a wiser head on my shoulders, I accept that it's part and parcel of building your career. Caan might be looked at a little unfavourably today, but I understand he is only doing what any parent would do for their child, if they had that power. I still think that the taste of success achieved from your own hard work is sweeter. I'll happily take that.


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Wednesday, 14 November 2012
Long Blue Prom Dresses
Long blue prom dresses start to be a trend in many memorable occasions, especially prom night. Blue prom dresses in general always display the beauty of women. Blue is a color that symbolizes honesty, truth, wisdom, and inspiration; it has a calming effect and it is considered to be one of the most profound colors. If you like a sparkling appearance, you could pick up a long blue prom dress. The following are some of long blue prom dresses which shown here.
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Civilian Conservation Corps
All Posts categories: Camp-News, Collection, Event, History, Digital Library
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CCC Museum Open House
CCC Museum of South Dakota 2015 Open House. . . more
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Reunion Dedication
A.J. Williams, Iven Jacobson, Jay Hendrickson, Jayson Dykstra, Melvin Hermanson, Stanley Hawthorn and Walter Atwood talk about their time in the Civilian Conservation Corps.. . . more
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CCC Museum proud to be part of Hill City Extravaganza. . . more


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by Sherri Di Blasi
(Claremont, CA)
What is acceptable for compensation for someone who generates leads for the medical billing business? And is it a one time referral fee, or are they entitled to a portion of monthly revenue?
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SCOTTSDALE, Ariz. - Keystone Mortgage Partners LLC was hired to fund a $55 million bridge loan and a $46 million construction loan for the developers of a Scottsdale shopping center.
Both loans would have three-year terms. The construction loan would finance the second phase of the Scottsdale Promenade, and the bridge loan would replace the construction financing for the first phase, which has been built.
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Submission + - Satellite Uplinks for the Masses (ieee.org) 1
IMHO, these antennas may be as big a leap for mobile computing and remote communications as the invention of fractal antennas was for mobile phones.
The solution to this problem lies in where you place the constellation. Satellites in low earth orbit (a few hundred miles) don't take much power to talk to with a minimally directional antenna. Think 60 to 90 degree beam width; you just vaguely point it in the right direction. Takes care of the speed of light problem too: latency isn't so bad across a 1000 mile round trip.
Shooting at geostationary orbit 22,000 miles away that you have to share with 3/4ths of a continent just doesn't work well for 2-way com
Top Ten Things Overheard At The ANSI C Draft Committee Meetings: (8) I'm on the committee and I *still* don't know what the hell #pragma is for.


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Drives in the hills
Entry posted by Skimbo ·
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@Roflyoshi: Thanks a lot
@Reikhardt: You've got this ability, St.Andrews is a good example for that
@K50; Thank you *gives K50 some drooling paper for this update*
@Jamonbread: Thank you !
@Haydin1996: The website where I've downloaded this train does no longer exist, APTX closed it this november. But you can find a lot of JR trains over at Simcity China. The best trainskin quality I've ever seen!
@kench: thank you =) I've got theese buildings from different japaneese sites. Say which one and I try to give you a link. But most of them have to be made growable because they are ploppables only.
@ImVhOzzi: Thanks a lot Let's hope this evil "back-button" dissapears during you write your next long comment
@TekindusT: Thanks a lot =)
@Benedict: Thank you, I've managed that with SimcityConfig. A nice tool that can be found at SC4D.
U42
Heyho and welcome to the fourtysecond update of my CJDragon Islands, Today I will show you a Place of Dragon Islands I really like.
But first, we have to go there. By car, for once.
My car is that cobalt blue one marked with the arrow, Jump in! I'm heading out of Ji? xiàn (??) a small but international town.
But first we have to cross one of the local tracks. In earlier days this was the end of a main route, today it's only a local route taken by a railtaxi that connects the town with it's small industrial area.
Let's head along this road.
There is a rail depot next to the city, some of you might remember it. A couple of JR 300 and CRH1 are parking there.... It's also the place where a lot of HSR trains get repaired and serviced.
Bt we're heading on towards north, more or less, at least. First a long piece to the east, than some meters to the north, than about a kilometer west again and then, finally, to the north.... The disadvantage of mountains.
That's a nice place....
The founders called this golfclub "Dragon Golf Club". Typical name for this region
And it's this golf club I want to show you.
We arrive at the clubhouse.
Let's go and warm up at the Driving range. Some Drives, some Pitches, some Putts.
And now.... Enjoy your round on the hilly nine-holes-course
The club is located next to the village Wúyu
And an overview.
(My bad score )
I hope you've enjoyed this update, greetings, Skimbo.
*The DragonGC lots have been lotted for this update and will be avaiable on the STEX soon*


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Tag Archives: Executive
Intel Israel General Manager Maxine Fassberg is Today's Honoree for Monday April 4th, 2016
Maxine Fassberg is vice president of the Technology and Manufacturing Group, general manager of Intel Israel, and plant manager of Fab 28 at Intel Corporation. She is responsible for manufacturing operations, including planning, product output and cost effectiveness for Intel's … Continue reading
Posted in Award, honoree, Recognition, women Tagged 2016, application, April 4th, chemistry, computer, engineering, Executive, General Manager, GM, innovation, Intel, Intel Israel, math, mathematics, Maxine Fassberg, Monday, opportunity, Science, technology, Today's Honoree Leave a comment
21st Century Dads Foundation President David Hirsch is Today's Honoree
At a time when 24 million kids across America are growing up without their dads, David Hirsch and his 21st Century Dads Foundation have taken to the road for the inaugural Dads Honor Ride – a 21-day, 2,100-mile bike ride … Continue reading
Posted in Award, celebrity, honoree, Recognition Tagged 21st Century Dads Foundation, Accounting, ceo, Chicago, CPA, Dad's Honor Ride, David Hirsch, Executive, Give Back, IL, Illinois, investor, member, President, Today's Honoree Leave a comment
Publicist Jeff Green is Today's Honoree
Jeff was born in San Mateo, California following his father footsteps, joining the United States Navy for the next 7 years of his young adult life. After 1 year of his discharge, Jeff pursued his degree in Biology and a … Continue reading
Posted in Award, celebrity, honoree, Recognition Tagged Campaign Manager, Corporate communications, Director of Communications, Executive, fundraising, headhunter, J. Silas Green, Jeff Green, media strategist, PR, public relations, publicist, publicity, Silas Green Media, Today's Honoree Leave a comment
Leadership Mastery for Women CEO Felicia Davis is Today's Honoree
I am proud to be a wife of 20 years, mother of three, a leadership branding strategist and most importantly a child of God. I have been blessed with many successes including more than two decades of experience in Human … Continue reading
Posted in Award, celebrity, honoree, Recognition, women Tagged branding, branding strategist, brilliance, Called Upon, ceo, child of God, Executive, Felicia Davis, founder, HR, human resources, Known, leadership, magnetic, non profit, noticed, organization Leadership Mastery for Women, Today's Honoree Leave a comment
Leadership Coach Pamela Miller is Today's Honoree
Pamela Miller is an Executive Leadership Development coach, consultant, speaker, and facilitator specializing in Core Energy Leadership and Emotional Intelligence. Drawing from her experience in retail operations management and as a senior executive in corporate operations she has developed and … Continue reading
Posted in Award, celebrity, honoree, Recognition Tagged academics, achievement, community advocates, consultant, Core Energy, Corporate, Emotional Intelligence, Executive, facilitator specializing, leadership, Leadership Coach, Leadership Development coach, non-profit executives, Pamela Miller, scientists, speaker, success, Today's Honoree Leave a comment
Today's Honoree is Stanlee Stahl the Executive VP of The Jewish Foundation for the Righteous
Stanlee Stahl is currently the Executive Vice President of The Jewish Foundation for the Righteous. the Foundation, which seeks to fulfill the traditional Jewish commitment to hakarat hatov, the searching out and recognition of goodness, has two goals. The first … Continue reading
Posted in Uncategorized Tagged clothing, commitment, Department, Executive, extra help, Extra Helping, financial assistance, food, friendship, Gentiles, Health and Human Services, help, Holocaust, JFR, Jwesish, monthly, new jersey, NJ, non-Jews, programs, shelter, Stanlee Stahl, The Jewish Foundation for the Righteous, Today's Honoree, United States, US, VP, war Leave a comment


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- koreanjapanclothing.com 7-efashion missing items; would not refund shipping paid for the items; escalate in Paypal, Internet
- Government Liquidation MIssing Items from Auction / No Refund, Internet
- Bargainpack.com Prolonged shipping, no refund for out of stock and missing items Magna, Utah
- Performance Automotive Transmission Center .com Overcharge, no refund, paypal fees, slow shipping, excessive shipping costs, Internet
- email@example.com ripoff, slow, no contact, missing items, No refund or care. Internet
- Phatcampus.com Damaged goods, missing items, NO refund, NO customer service Ripoff Boca Raton Internet
- Free shipping. Com no service. Free shipping big scam. Paid freight plus $12.97. Sorry A--H--- Scammers!!'! Internet
- Overstock.com ripoff, won't refund on late x-mas gift, paid $40 for shipping, promised refund -- none Internet
- ATS computer systems fake promotion price, missing accessories, rude and unprofessional service, no receipt and tracking number, wrong shipping company, and delayed shipping. toronto Ontario
- FCKH8.com Items ordered not received. Contacted the company via phone messages and emails. No replies. Internet
- UPS420.com Kay is girls name that answered the phone They charged me for the items and didn't deliver. None Internet California
- Report: #1008666
MyOTCstore.comInternet United States of America
MyOTCstore.com Delayed Shipping, Missing Items, No Refund Internet
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When I finally got hold of MyOTC by phone I was told that my card would be refunded for the product that was not shipped. They gave no transaction number or confirmation email. Unfortunately I just trusted them and waited for the refund.
My credit card statement arrived, showing no refund. I called MyOTC and spoke to Sam, who said that my refund was now being refused. I'm now trying to get my credit card company to re-open the case, but it may be too late for action by them. I was duped!
Find a reputable company to order from, even if it costs you a few more dollars. Dealing with MyOTC is not worth the hassle, money, or wait.
This report was posted on Ripoff Report on 02/04/2013 01:23 PM and is a permanent record located here: http://www.ripoffreport.com/reports/myotcstorecom/internet/myotcstorecom-delayed-shipping-missing-items-no-refund-internet-1008666. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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SHINee is as popular a boy group as ever!
They dropped their groovy, retro MV for "1 of 1" just yesterday, but in less than 24 hours have gotten over 1 million views on YouTube!
The MV is fun with the boys in retro, colorful attire while dancing with a large group of stunning ladies. The dance moves seem fun, too. If you missed out, watch the MV above!
SEE ALSO: SHINee open up official Twitter account!
Tip: Sara


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Lyrics.com »
Search results for 'by x album the shadows'
Yee yee! We've found 841 lyrics, 46 artists, and 100 albums matching by x album the shadows.
Radiohead/album/album · The Shadows · Shadows of Knight · Brother Bones & His Shadows · Bobby Vee & The Shadows · Chicago Bob & The Shadows · Cliff Richard & the Shadows · Dreadful Shadows · The Shadows · Shadows Fall · MOUHAMADOU LAMINE CISSE NUMERIS POLICE NATIONALE HUMANIS ALBUM 3 – and 36 other artists »
The Shadows/Out of the Shadows (The Shadows) · Dance with the Shadows/Sound of the Shadows (The Shadows) · Moonlight and Shadows (Bonnie Guitar) · Out of the Shadows (Homewreckers) · Deep Shadows and Brilliant Highlights (H.I.M.) · Standing in the Shadows of Motown (Hutchinson, Hutch) · Out of the Shadows (Mike McAdoo) · Where the Shadows Lie (Battlelore) · Shadows Collide with People (John Frusciante) · The Shadows' Madame (Cadaveria) – and 90 other albums »
Poor White Folks [Instrumental] Uncle Pen Album By Bill Monroe & His Bluegrass Boys The entire album is 'Instrumental'. These include tracks: 1.
From the Album: * No promises no debts Tiger tiger bay â?? watch the shadow play Tiger tiger bay â?? what's on the menu today You got hungry again
And the moment that she left the room The album started skipping Goodbye to beauty, shared with the ones that you love A shadow that has fallen
awaken Since my baby's gone From the album "True Love" Written by: M. Parish & H. White
Ten coaches roll into the dust Chrome windows turned to rust 'Hand on inside they know they must Hanging on the green backed words "In God we
shadow dreams are equal Then why can't the next shadow dreamer be you That's the philosophy I live my life by When things get hard through the stress
of time Just get lost without a sign As long as you stand by my side In your shadow I can shine(x3) Shine You see my soul I'm a nightmare Out of control I'm
right, the symbol remains Into the darkness must must for the pains In the shadow of California Jam Revolution by night, ? in this future Kings
the sheaves [Chorus] Sewing in the sunshine Sewing in the shadows Nearing by the clouds Winter chilling all the green By and by the harvest
Loyal fans and newfound followers Whassup y'all? Hello how are you doin out there? You're chillin I'm winnin Oh by the way the album's out go get
This song appears on four albums, and was first released on the Earth Songs album. It has been released on The Flower That Shattered The Stone,
Daddy Rae Represent (represent) Magic Mike Yo, I represent, I represent I represent 10 albums at twenty seven years old Millions of copies sold
I won't be afraid anymore of the terrors by night Or the arrow that flies by day And though a thousand may fall at my side And though ten thousand
This song appears on two albums, and was first released on the One World Album. It has also been released on The Country Roads Collection album
In this world o' darkness, Evil rules by night, But somewhere in the shadows, someone seeking light No one knows his neighbor here, No one has
and Mr. Shadow A-K-A The Mayhem Click And we're talkin' about rumors Now Lil' Rob we understand you have an album out entitle the Crazy Life Can you tell
This song appears on three albums, and was first released on the flower that shattered the stone album, and has also been released on the stonehaven
Tragedy Thomas Wayne with the DeLons Words and Music by Gerald H. Nelson and Fred B. Burch Peaked at # 5 in 1959 Later charted at # 10 in 1961 by
*****Erasure Lyrics***** *****Version 1.0***** Albums and be-Sides -Painstakingly compiled and transcribed by John Setlik (email@example.com)
[Originally released by Mayhem in 1994 on the album "De Mysteriis Dom Sathanas".] [5:12] Every time this year This dark fog will appear Up from
and side by side They will take me beyond the light [Repeat x2] Death is a forecourt to hell, Where the lost of humanity dwell Through the shadows
What do you when love dies? Somebody help me, I'm losing my mind I run for the 1:10 uptown The show starts at two I'm surrounded by strangers But
when love dies? Somebody help me, I'm losing my mind I run for the 1:10 uptown The show starts at two I'm surrounded by strangers But I'm haunted by you
Don't feel bad Because the sun went down The moon is made of gold * (Oh you) See how the shadows Enhanced by the light above Dance in the night
more time You do your thing Homes but I'm gonna do mine [Chorus: x2] Look at all this rumors Started by jealous people And basically that's what


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Mark your calendar, get the weekend off, make sure the knobbies are in good shape and head on up to the pines for 2 days of National AMA Dual Sport greatness. July 15 & 16. A, B, & C routes (C is definitely big bike friendly). We promise great routes, good food, and fun times. Sign up for the weekend or just one day – we've got you covered.
July will be here before you know it so sign up now:
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Indian Springs attic blaze extinguished
November 19, 2013
A fire Saturday morning in a two-story home was extinguished before it could spread, thanks to a coordinated efforts by responding fire agencies.
Nevada County Consolidated firefighters responded to a report of a fire in the 14000 block of Oak Ridge Road off Indian Springs Road just after 6:45 a.m. Saturday.
Resources arrived to find smoke coming from the roof, according to a press release from Battalion Chief Jerry Funk. The fire was found to be well established in a wall between a masonry chimney and interior masonry fascia and was extending into the attic, Funk wrote.
Firefighters from Penn Valley, Higgins and Rough and Ready also responded to the fire. The responding agencies were able to contain the fire to the area of origin.
No injuries were reported; the cause of the fire remains under investigation.
Recommended Stories For You
Trending In: Local News
- Nevada County fire: The aftermath (VIDEO)
- Carlee D'Arata sentenced to 10 years in prison in Nevada County death of Antonette Thevenin
- For Grass Valley "super senior," helping children is a way of life
- Overnight closure planned over weekend for busy intersection in Roseville
- Nevada County Captures: Firefighters battle fires; scenes from local fires


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Search over a million flights, hotels, packages and more
No Expedia cancellation fee to change or cancel almost any hotel reservation
Search over a million flights, hotels, packages and more
No Expedia cancellation fee to change or cancel almost any hotel reservation
Since you're in Governor's Harbour, why not fly from Governor's Harbour? We've saved you some heartburn and listed some of the top flight deals for your home airport. Choose from trusted airlines, compare flight times and ticket prices, and be sure to note how close your hotel is to the top airports below.
No matter if you have an important meeting in town, are itching for new experiences, or simply want to get away, Governor''s Harbour is the optimal spot to visit.
You have arrived, now it's time to relax. No matter if you desire some place to stay for a stopover or are looking to remain close to the runway during your holiday, you can depend on tons of hotels in Governor''s Harbour, and Expedia can help you find the right one for your dream holiday.
Trust us; there are plenty accommodations for all of your needs.
With great locations and quick access to the hot spots in town, think of your Governor''s Harbour hotel as home base during your jaunt. The best part of visiting Governor''s Harbour is that you can't leave without a ton of stories to bring back home. You never know what you are going to hear, see, and experience--and that's a wonderful thing!
Come see what the fuss is about! Those chores can wait, that to-do list can wait, your life can't wait. It is time to reserve a trip to Governor''s Harbour! Find the best deals on airfare to GHB from Expedia, and get ready for a vacation you'll never forget.
When flying from Governor's Harbour (GHB), give yourself plenty of time to make your flight because nothing's worse than running to catch your plane. Getting to the airport early lets you check into your flight, drop off your luggage, pass through the TSA checkpoint, and leisurely arrive at your terminal gate with time to spare.
Before you head to the airport though, check your flight time. Check your flight status at GHB to determine if there are any changes in your itinerary. Once you've confirmed your GHB flight status, many airlines recommend you arrive at the airport at least two hours early for domestic flights and three hours prior to international flights.
When you have a flight from Governor's Harbour (GHB), take advantage of the various transportation options available. If you've bribed your friend to drive you the airport, use the convenient curbside drop-off or the short-term parking at GHB. Alternatively, if you prefer to drive yourself, the airport provides safe and secure long-term parking for your vehicle during your trip. Furthermore, you'll find rental car and taxi services at the airport offering easy and reliable transportation.
In the mood for spreading your wings? Allow us to assist you. Below you'll come across our easy-to-navigate list of flights from Governor's Harbour across the world to help inspire your next big trip. Sort through the listed regions and find the perfect destination to fly into from your designated city today.
Browse the most popular US domestic flight destinations from Governor's Harbour airport.
Browse popular international flights from Governor's Harbour, GHB airport.
Currently the following airline carriers are servicing the GHB, Governor's Harbour.
Governor's Harbour, also known by IATA (International Air Transport Association) code GHB and ICAO (International Civil Aviation Organization) code MYEM, offers flights from multiple airline carriers to many popular global destinations. Above you'll find some useful information about Governor's Harbour's airport that can help you in planning your next trip. Expedia offers flights from GHB airport on multiple airline carriers, and makes it easy and affordable for you to book low cost Governor's Harbour flights.
Expedia offers amazing deals on flights around the world. Find Cheap Flights from Portland OR and feel confident that your online booking experience will be fast and easy.
With tens of thousands of hotels to choose from including Hotels in Miami Beach you will always find the best place to stay on Expedia.
Find the best Vacation Packages in Myrtle Beach today. Get a flight, hotel and rental car at the best online rates.


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Venus Williams Sobs as She's Asked About Fatal Car Crash During Wimbledon Press Conference
Venus Williams broke down in tears Monday during her first public appearance since a fatal car crash earlier this month in which police say she was at fault.
After winning her first found match at Wimbledon, Williams spoke to reporters for the first time since the accident.
Her eyes filled with tears when asked about the crash and she couldn't go on during the post-match press conference.
"There's no words to describe," she said. "I'm speechless."
The tragic accident took place near Williams' gated home in Palm Beach, Fla., as she tried to cross a six-lane highway in her Toyota SUV.
Heavy traffic forced her to stop in the middle of the intersection and when she drove forward again it was right into the path of an oncoming car, killing 78-year-old Jerome Barson.
According to the police report, the tennis great was at fault for 'violating" the elder couple's "right of way."
She wasn't ticketed or charged but the accident is clearly taking its toll.


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What kinds of bath products are offered at InterContinental Hong Kong?
Answers from Our Experts (1)
The bathrooms in most guest rooms at InterContinental Hong Kong are stocked with products from Agraria, the San Francisco-based bath and fragrance brand. Suites and Club rooms feature Ferragamo toiletries. You'll find small bottles of shampoo, conditioner and body wash in the shower as well as another set of shampoo, conditioner, body wash, body lotion, hand soap and bath soap on the counter.
In addition, the Hong Kong hotel fills your bathroom with a variety of other amenities, including two dental kits with a toothbrush and toothpaste, two shoe mitts, two shaving kits with a razor and shaving cream, cotton swabs and pads, as well as two shower caps with hair ties. So, if you forgot anything at home, you'll be covered here. And if you don't find it here, just call housekeeping or your butler and either can help you get ahold of whatever it is that you need at the Forbes Travel Guide Five-Star hotel.


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March 14, 2010
Caribbean Fahion Week.
Well I hope everyone is having a great weekend. I have been resting a lot and doing chores in moderation, hoping I will start the week energized and refreshed. I leave you now with some of the highlights from our Caribbean Fashion Week, please enjoy.
3 comments:
Hey Lisa :)ReplyDelete
Now, this collection is definitely something I can work with! These fashion are very colourful, free-spirted and speak confidence. Have a lovely day :)
Wow that first dress is breath taking! It reminds me of a mermaid almost.ReplyDelete
Love these clothes and colors. What a lucky woman to live in this beautiful climate year round. Thank you so much for stopping by my blog and leaving a comment. I look forward to following your blog, as well.ReplyDelete
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Happy Thanksgiving and a last minute dessert.
Happy Thanksgiving everyone, and may their be happy memoirs made as we gather with family and friends . Take time to be grateful for the...
Swiveling Mounted Chalkboards at Bistro Vida, via here. A vintage carnival wheel and chalkboards are unique accessories to this countr...
Hello My Lovelies, I hope this post finds you well and looking forward to what the month of August will hold. Today I am in love with th...


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I am a Designer with four years of experience in 3D modelling and 2D drafting. Just give me your design and I will create 3D model for you.
Jobs by solidcreation
I will add your logo to your product and provide rendered images... Just give me images of the logo and product.. -images for online sales -images for promote your new product -images for your website -images for new innovations -images for presentations
I am a mechanical engineer. and I have three years of experience of 3D CAD modeling. I am good at 3D modelling using Auto CAD and Solid works. Just give me your design with sketch and I will create your 3D model I will provide you following services.... - 3D models of any objects - 3D models of machine oparts - 3D design files for 3D printing - models for CNC milworks - rendering images - add your logo on your product


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Bigjigs Goods Train
Make 4 interest-free payments of $7.25 fortnightly and receive your order now. Afterpay is Available for purchases up to $800.Sold Out
Bigjigs Goods Train
Brand New in Box
Variety is the spice of life and this wooden Goods Train brings essentials, special treats and crates marked Fragile to where they're needed most.
Bigjigs Rail wooden wagons feature detachable loads that can be lifted by a Bigjigs magnetic crane.
Supplied complete with two pieces of wooden Bigjigs Rail track.
Magnetic couplings ensure compatibility with all other engines and carriages in the Bigjigs Rail range.
Most other major wooden railway brands are compatible with Bigjigs Rail.
Product size: Length 34 cm
Suitable from 3 Years +
Be The First To Review This Product!
Help other Kids Mega Mart users shop smarter by writing reviews for products you have purchased.


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Pet Naturals of Vermont, Calming, for Cats, 3.8 fl oz (112 ml) (Discontinued Item)
- You earn loyalty credit for every order you place at iHerb!
- Loyalty credit is equal to 5% of the value of your order after discounts and credits applied, excluding shipping charges.
- Your loyalty credit will be applied towards your next order as soon as your current order has shipped.
- Your loyalty credits are valid for up to 90 days from the date of your last purchase.
Pet Naturals of Vermont, Calming, for Cats, 3.8 fl oz (112 ml) (Discontinued Item)
Shipping Weight:?0.35 lbsSwitch to Metric units
- Product Code: PEN-95064
- UPC Code: 026664950642
1.7 x 1.7 x 4.7 in, 0.3 lbsSwitch to Metric units
Featured Products
- Sealed For Your Protection
- NASC - The Benchmark of Quality
- Supports Stress Reduction
Calming Formula is for cats exhibiting nervousness, hyperactivity, anxiety or responding to environmentally induced stress. It is an advanced formula that contains a combination of factors, not found in other calming formulas, that supports balanced behavior.
Suggested Use
Shake well before each use.
Give 1/4 teaspoon twice daily.
During Times of Increased Stress:
It is safe to double or triple the above amount, as needed.
Safe use in pregnant animals or animals intended for breeding has not been proven.
If animal's condition worsens or does not improve, stop product administration and consult your veterinarian.
Store in a cool, dry place.
For use in cats only.
For animal use only.
Keep out of the reach of children and other animals. In case of accidental overdose, contact a health professional immediately.
|Active Ingredients Per 1/4 Teaspoon (1.25 ml):
|Thiamine (Vitamin B1)
|31.25 mg
|L-Theanine (Suntheanine brand)
|10.56 mg
|Colostrum Calming Complex
|5.28 mg
|Inactive Ingredients: cornstarch, glycerin, guar gum, maltodextrin, non-fat milk solids, silicon dioxide, sorbic acid, sucrose, vegetable oil, water.


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We've been to a lot of conferences in our day. Some have been unforgettable, but others…
You know how it goes. You go to sessions that are supposed to be helpful, but end up being thinly-veiled sales pitches. You eat the boxed lunches, trying to remember the last time you had Ruffles and a sandwich. And worst of all, you do all that awkward network, only to find that 9 out of 10 people aren't your ideal customer – and have zero interest in your company.
Thank goodness for Quartz Events. They host events without any of the B.S. Hyper-relevant sessions, awesome networking opportunities, and one-on-one sessions with decision-makers from ideal clients. Sounds pretty heavenly, right?
Attending a traditional trade show comes with a lot of work…. You have to book a hotel, research companies, find them among a sea of vendors and ultimately rely on pure luck to connect you with an appropriate contact. Try and do all that while attending important conference sessions and you are bound to have limited results.
But imagine this: All the planning taken care of. You simply show up and spend 100% of your time in useful ways.
Welcome to Quartz Events. Our invitation-only events combine peer led industry content with intelligently scheduled one-on-one meetings that address your company's immediate needs. Plus, we top it off with first class meals, and lively networking opportunities in some of the finest resort hotels in North America…
…and for qualified attendees? It's free.
We know what you're thinking. What's the catch?
To be honest, there isn't one.
You'll spend 75% of your time at our networking functions and in educational sessions created by your industry peers to maximize the relevancy of the content. 25% of your time is spent in targeted one-on-one meetings. Based on our extensive research and planning, we're confident these will be the most productive and useful part of your time with us.
So why is it free? Because it's a win-win… Our sponsors love the opportunity to educate you on their newest services and technology, so they're happy to make the premium investment that covers the cost of your attendance… you can even bring your spouse!
See? Win-win.
Animated video production by http://explainify.com


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Skip to main content
No.5 Thornlea Mews Holiday Cottage, Hope Cove, Devon
Inside Our Holiday Home
Availability Calendar
Around the area
Beaches in the South Hams
Days Out!
The South West Footpath
Cream Teas
Contact Us
Site Map
Devonshire Cream Teas
Devon is known for its Cream Teas and you will always find somewhere in the village offering a very enticing cream tea. Eat it the Devon way by splitting the warm, freshly baked scone in two, cover each half with clotted cream, and then add strawberry jam on top.
For a cream tea with a difference, try the
Picnic boat
in Dartmouth. This one hours cruise takes in castles and sails past the home of the late Agatha Christie. The Devon cream teas are all served on pretty china plates with proper napkins. Highly recommended!
A warm welcomes you at the
Castle Tea rooms at Dartmouth Castle
, With stunning views in this historic setting, the castle was built in 1856 to guide shipping into Dartmouth Harbour.
There is a large outdoor seating area which has panoramic views of the Dart Estuary.
The award winning Cream Tea at the nearby
Avon Mill Garden centre
at Lodiswell near Kingsbrige, offers a very cosy atmosphere where you can treat yourself. All the food is carefully prepared to a high standard and there is a delicious range of homemade cakes and scones. There are tables and chairs outside where you can relax on sunnier days.
For a luxurious treat, try the Cream Tea with Champagne at nearby
Soar Mill Cove Hotel
. The hotels spectacular setting is a flower-filled combe with views of the sandy bay and 2000 acres of dramatic National Trust coastline. Set in one of the unspoiled corners of South Devon.
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Port-Harcourt, Port Harcourt, Rivers Added via phone at 12:41, 19 June 2017, Ad ID: 1002696274
I have great creative and innovative money making ideas on ICT,HOSPITALITY,EDUCATION,BUSINESS.
Interested investors can collaborate with me on an idea or takeover the outright implementation personally.
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Mountains May Depart (2015)Shan He Gu Ren
- Actors:
- Tao Zhao, Yi Zhang, Jing Dong Liang et al.
- Directors:
- Zhangke Jia
- Availability:
- Not released
Join the Cinema Paradiso DVD rental and Blu-ray Rental service today and get a Free DVD rental trial. Sign up today!


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Scenic Byways - panel by Hoffman Fabrics$9.60
Call of the Wild - elk panel by Hoffman Fabrics$12.80
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