text:
Reports indicate that PDLIM5 is involved in mood disorders. The PDLIM5 (PDZ and LIM domain 5) gene has been genetically associated with mood disorders; it's expression is upregulated in the postmortem brains of patients with bipolar disorder and downregulated in the peripheral lymphocytes of patients with major depression. Acute and chronic methamphetamine (METH) administration may model mania and the evolution of mania into psychotic mania or schizophrenia-like behavioral changes, respectively.
To address whether the downregulation of PDLIM5 protects against manic symptoms and cause susceptibility to depressive symptoms, we evaluated the effects of reduced Pdlim5 levels on acute and chronic METH-induced locomotor hyperactivity, prepulse inhibition, and forced swimming by using Pdlim5 hetero knockout (KO) mice.
The homozygous KO of Pdlim5 is embryonic lethal. The effects of METH administration on locomotor hyperactivity and the impairment of prepulse inhibition were lower in Pdlim5 hetero KO mice than in wild-type mice. The transient inhibition of PDLIM5 (achieved by blocking the translocation of protein kinase C epsilon before the METH challenge) had a similar effect on behavior. Pdlim5 hetero KO mice showed increased immobility time in the forced swimming test, which was diminished after the chronic administration of imipramine. Chronic METH treatment increased, whereas chronic haloperidol treatment decreased, Pdlim5 mRNA levels in the prefrontal cortex. Imipramine increased Pdlim5 mRNA levels in the hippocampus.
Citation: Horiuchi Y, Ishikawa M, Kaito N, Iijima Y, Tanabe Y, Ishiguro H, et al. (2013) Experimental Evidence for the Involvement of PDLIM5 in Mood Disorders in Hetero Knockout Mice. PLoS ONE 8(4): e59320. https://doi.org/10.1371/journal.pone.0059320
Editor: Kenji Hashimoto, Chiba University Center for Forensic Mental Health, Japan
Received: November 23, 2012; Accepted: February 13, 2013; Published: April 8, 2013
Copyright: © 2013 Horiuchi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by JSPS KAKENHI (Grant Nos. 23390285 and 10212648) and Grant-in-Aid for Scientific Research on Innovative Areas (23129501) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The PDLIM5 gene encodes the enigma homolog (ENH). In total, 4 known isoforms of the human PDLIM5 protein have been identified: ENH1, ENH2, ENH3, and ENH4. The largest isoform, ENH1, contains 1 PDZ (postsynaptic density-95/discs large/zone occludens-1) domain and 3 LIM (Lin-11, Isl-1, and Mec-3) domains at the C-terminal end [1]. ENH1 is expressed in various tissues, such as the heart, brain, spleen, liver and kidney. In comparison, shorter isoforms that lack the LIM motifs are expressed in cardiac (ENH3) and skeletal muscle (ENH2, ENH3, and ENH4) [1], [2], [3]. ENH1 is expressed in various regions of the brain, including the hippocampus, cortex, thalamus, hypothalamus, amygdala, and cerebellum [4].
ENH1 was originally identified as a PKC-interacting protein [1] and which may activate conventional PKCs by directly binding them through its LIM domains [5]. The ENH1 LIM domain is one of the targets of protein kinase C (PKC), which has been shown to bind to the regulatory domain of PKCβ1 and ε· [1], [4]. ENH has been shown to be localized in the presynaptic nerve terminals [4], [6] and in the postsynaptic density [7]. ENH has been reported to interact with CaV2.2, which is a presynaptic calcium channel type that is known to be sensitive to enhancement by PKC [4] and may be an adaptor for the regulation of intracellular calcium levels by constructing a PKCε–ENH–N-type Ca2+ channel complex [8]. However, the mechanism by which ENH and Cav2.2 interact remains controversial [6]. In the postsynaptic density, PDLIM5 is reported to interact with spine-associated RapGAP (SPAR, SIPA1L1), and to promote the shrinkage of dendritic spines [7]. RNA interference against PDLIM5, or loss of PDLIM5 interaction with SPAR, causes an increase in spine head diameter. PKC activation promotes the delivery of PDLIM5 into the dendritic spines and increases its spine co-localization with SPAR [7].
Recent studies have shown that SNPs in the PDLIM5 gene are associated with schizophrenia [9], [10], [11], bipolar disorder [10], [12], [13], and major depression [14], [15]. In contrast, other studies have reported no significant genetic association between the PDLIM5 gene and schizophrenia [16], [17]. However, the associations have been replicated in independent populations with bipolar disorders [12], [13] and major depression [15].
The precise molecular roles of PDLIM5 in mood disorders remain unclear. An association between the SNP rs2433320 and bipolar disorders confirmed by a meta-analysis [12] was also observed in the genome-wide association study carried out by The Wellcome Trust Case Control Consortium (WTCCC) (p = 0.03) [18]. The SNP rs2433320, which is located approximately 2 kb upstream of PDLIM5, was reported to be associated with PDLIM5 mRNA levels in the prefrontal area [9]. Iwamoto and colleagues used DNA chip analysis to examine the prefrontal regions of postmortem brains of patients with mental disorders, and found that PDLIM5 was upregulated in patients with schizophrenia, bipolar disorder and major depression [19]. The upregulation of PDLIM5 in the prefrontal cortex and cerebellum has also been show by the Stanley Medical Research Institute Online Genomics Database for schizophrenia (p = 0.037) and for bipolar disorder (p = 0.38), though it did not show significant upregulation for bipolar disorder (https://www.stanleygenomics.org/stanley/geneDetail.jsp?gene_id=7370). The PDLIM5 mRNA expression levels in the peripheral leukocytes of medication-free patients with schizophrenia were significantly higher than in those of control subjects [16]. In contrast, decreased peripheral PDLIM5 mRNA expression in whole blood from manic patients with bipolar disorder type I was reported [20]. The authors also reported no significant difference in PDLIM5 mRNA expression following treatment with olanzapine treatment while reducing in the severity of manic symptoms [20]. In addition, significantly lower PDLIM5 mRNA expression levels have been reported in the peripheral leukocytes of drug-naïve patients with depression than in those of control subjects. Furthermore, after 4 weeks of paroxetine treatments, PDLIM5 mRNA expression significantly increased to almost the same level as the control subjects [17]. Thus, studies on PDLIM5 expression have shown variable results when using postmortem brains and peripheral leukocytes in patients with mood disorders. It should be noted that there is a difference in the expression level of the PDLIM5 gene in the peripheral blood cells of patients with major depressive disorder and schizophrenia who were undergoing treatment with antidepressants and antipsychotics [16], [17].
In terms of PKC activity, which is mutually regulated by ENH1, the prefrontal cortex of postmortem subjects with major depression exhibited significantly lower protein expression of PKCβ1 and PKCε compared to controls [21]. Postmortem studies have demonstrated that membrane-associated PKC and the stimulation-induced translocation of the cytosolic enzyme to the membrane increased in the frontal cortex of patients with bipolar disorder [22], [23]. A significant increase in membrane-associated platelet PKC activity has been demonstrated in manic patients, while enhanced PKC activity during mania is suppressed by mood-stabilizer treatments [24], [25], [26]. In comparison, decreased PKC activity and PKCβI and βII levels, but not PKCα or PKCζ, in the membrane and cytosol fractions of platelets from medication-free pediatric patients with bipolar disorder has been reported [27]. In general, many other studies support the involvement of PKC and its substrates in the pathophysiology of bipolar disorders and their use in the treatment of bipolar disorders [28], [29], [30], [31], [32].
Thus, the links between PDLIM5 expression and mood disorders, and between PKC and mood disorders, have been reported in humans, while the link between PDLIM5 and PKC has been reported for cell and in-vitro experiments. However, the experimental analysis of lowered Pdlim5 expression and the disruption of PKCε translocation on mood disorder in mouse models have not been reported. To obtain insights about the effects of PDLIM5 expression on the whole body of individuals with mood disorders, we examined the effects of constitutionally reduced levels of the ENH1 isoform of PDLIM5 generated by the ENH1 isoform of Pdlim5 knockout (KO) mice on behaviors associated with mania and depression. Because it has been acknowledged that single-dose methamphetamine (METH) treatment and chronic METH treatment may model mania and the evolution of mania into psychotic mania or schizophrenia-like behavioral changes, respectively [33], we evaluated behaviors in response to METH treatments in Pdlim5 hetero KO mice. The mood stabilizers lithium and valproate are known to reverse METH-induced hyperactivity and behavioral sensitization [34], [35], [36].
In addition, we evaluated the effects of administrating the PKCε-translocation inhibitor peptide (PKCε-TIP) on the response of mice to METH. The LIM domains of PDLIM5 have been identified as interaction sites for various PKCβI and ε isoforms, while the V1 region of PKC has been identified as critical for PDLIM5-PKC interaction [1]. PKCε-TIP, which is a small water-soluble peptide (8 amino acid residues) derived from the V1 region of PKCε [37], may specifically inhibit the effects of PKCε by blocking the translocation of PKCε from the cytosol to the membrane [4]. PKCε-TIP has been used to specifically inhibit the effects of PKCε in in vivo experiments [38].
As a parameter of depressive behavior, we examined the immobilization of Pdlim5 hetero KO mice in a forced swimming test, with and without the administration of imipramine.
Materials and Methods
Generation of Pdlim5-deficient Mice and the Mice Used in This Study
The mouse Pdlim5 gene-trapped ES cell line (XH199) was obtained from the Mutant Mouse Regional Resource Center (http://www.mmrrc.org/). The Pdlim5 gene, which contains 13 exons, specifies a PDZ domain and 3 LIM domains. Exon 2 of Pdlim5 specifies the end of the PDZ domain, while exon 9 specifies the beginning of the first LIM domain. The gene-trap vector, pGT1lxf containing the Engrailed 2 (En2) splice acceptor sequence, β-geo (β-galactosidase/neomycin phosphotransferase fusion gene) and a SV40 poly (A) signal sequence generated a mature Pdlim5 (exons 1–8)- β-geo mRNA, which was determined by 5′ RACE PCR at BayGenomics (http://baygenomics.ucsf.edu/). Thus, incorporation of the gene trap generates a fusion between the PDZ and the β-geo gene products (Fig. 1A–D), which contains 1–368 amino acids (aa) of the ENH1 isoform and 1–306 aa of the ENH2 isoform. The ES cell line was injected into C57BL/6J host blastocysts to generate chimeras. The chimeras were then crossed with C57BL/6J mice to determine germ line transmission. Total RNA and DNA were extracted from the tail tissues by using ISOGEN (WAKO, Tokyo, Japan). cDNA was synthesized using ReverTra Ace (Toyobo, Tokyo, Japan) and oligo dT primers. Genotype detection was carried out by PCR of cDNA by using 3 primer methods. To detect the 5′ end of the gene trap insertion, the primers used were as follows: Pdlim5_exon8Fc: 5′-CACCAGCGTCAAGTCACCTA-3′, bgeoRc: 5′-ATTCAGGCTGCGCAACTGTTGGG-3′, and Pdlim5_exon9Rc: 5′-TGCACAAGTGTGTCTTGGTCAC-3′. Routine genotyping was carried out by PCR of genomic DNA by using the following primers: bgeoR: 5′-ATTCAGGCTGCGCAACTGTT-3′, Pdlim5_ex8F: 5′-CACCAGCGTCAAGTCACCTA-3′, Pdlim5_exon9R: 5′-TGCACAAGTGTGTCTTGGTCAC-3′, and Pdlim5_exon9F: 5′-CCGCCTAGTGTTTGCATCATAA-3′.
(A) Insertion site of the gene-trap cassette in intron 8 of the Pdlim5 gene. The inserted sequence (gray box) includes a splice acceptor (SA), β-geo, which is a fusion of β-galactosidase and neomycin phosphotransferase II, and is followed by a stop codon and a polyadenylation signal (pA). (B) Schematic of the gene trap cassette. C) RT-PCR results and genomic genotyping of Pdlim5+/+, Pdlim5+/−. Samples were extracted from the brain and tail. (D) Expected domain structures of wild-type and PDLIM5 mutant proteins. (E) Real-time PCR analysis of Pdlim5 in the prefrontal brains of Pdlim5+/+ (n = 6) and Pdlim5+/− (n = 8) mice [F(1, 12) = 14.0, p = 0.003]. Values are shown as mean ± SD. (F) Representative western blots of Pdlim5 in the prefrontal brains of Pdlim5+/+ and Pdlim5+/−.
The animals were housed under a 12-h light/12-h dark cycle, with constant temperature (25°C) and humidity, and were allowed free access to food and water. All animal procedures were performed according to protocols approved by the Animal Care and Use Committee of the University of Tsukuba, Japan.
Unless otherwise specified, 6- to 8-week-old male mice were used in this study; the mice were derived from 6 to 8 backcrosses (N6∼N8) to C57BL/6J (Charles River Laboratories Japan, Yokohama, Japan) from the chimera (129 X C57BL/6). Each behavioral experiment was performed independently using a new group of animals, except for novelty-seeking, rota-rod, social interaction, and prepulse inhibition tests without drug administration. These tests were performed consecutively every week in this order, using mice of the N6 backcross generation; however, the number of mice tested differed among each of these tests.
Analysis of Pdlim5 Transcription in the Brain Tissue of Mice
The prefrontal cortex, midbrain, hippocampus, thalamus, and striatum were removed by dissection. Total RNA was then extracted from the brain tissues with ISOGEN Reagent (Nippon Gene Co, Tokyo, Japan). Pdlim5 cDNA was synthesized with ReverTra Ace (Toyobo) and the oligo dT primer from RNA. Expression was quantified by real-time quantitative PCR analysis with the TaqMan Gene Expression Assay and ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA), according to the manufacturer's instructions. Primers and probes were purchased from Applied Biosystems (Assay ID: Mm00517301_m1). Rodent glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal control, and the threshold cycle (Ct) was measured in triplicate. Data were collected and analyzed with Sequence Detector Software (SDS) version 2.2.2 (Applied Biosystems). The relative gene expression was calculated as the ratio of Pdlim5 to the internal control Gapdh. The Ct measurement was the average of 3 replicates. Six wild-type and 8 heterozygous, 8-week-old male mice of the N3 backcross generation were used in this Pdlim5 transcriptional analysis.
Western Blot Analysis
Protein was extracted from brain tissue with Laemmli buffer. The concentration of total protein was measured by using a Wallac 1420 ARVOsx multilabel counter (Perkin Elmer, Yokohama, Japan). Then, 2 µg of each sample was run on a Pro-Pure™ SPRINT NEXT GEL (Amresco, Solon, OH) and transferred to BioTrace™ PVDF (Nihon Pall Ltd., Tokyo, Japan). Human PDLIM5 Polyclonal Antibody (1∶100dilution) (MBL, Nagoya, Japan) was used as the primary antibody, or polyclonal antibody to beta-Actin (1∶500 dilution) (Imgenex, San Diego, CA) for normalization. Bound primary antibodies were detected with goat anti-rabbit IgG antibody HRP conjugate (MBL) in 1∶10000 dilution and Immobilon™ Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA) by LAS-4000UVmini (Fujifilm, Tokyo, Japan). LAS-4000UVmini directly measures the amount of both proteins on the membrane, and facilitates the calculation of relative Pdlim5 expression among subjects. Six wild-type and 8 heterozygous, 8-week-old mice of the N10 backcross generation were used in this Pdlim5 western blot analysis.
Drugs and Administration Schedule
METH (Dainippon Sumitomo Pharma Co., Ltd, Osaka, Japan) was dissolved in saline solution. The acute METH treatment group received a single dose of METH injection (3 mg/kg, intraperitoneal [i.p.]). The chronic METH treatment group was generated by the injection of 1 mg/kg METH after i.p. injection of 3 mg/kg METH once daily for 14 days and a subsequent 14-day withdrawal period. This chronic treatment developed behavioral sensitization to METH, which was confirmed by 1 mg/kg injection of METH. Haloperidol was dissolved in 0.9% saline solution with tiny drops of lactate, and was administered (1 mg/kg, i.p.) daily for 7 weeks. Imipramine was dissolved in saline solution and administered (20 mg/kg, i.p.) daily for 14 days. The control group for each experiment received saline injections.
Locomotor Activity Analysis
To evaluate the behavioral effects of METH treatment, spontaneous horizontal locomotor activity was measured using the Lime Light (Actimetrics, Wilmette, IL) CCD camera tracking system. The mice were placed inside soundproof and illuminated plastic cages (40 cm × 20 cm × 20 cm). Each mouse was placed separately in a box for a 60-min habituation period. Spontaneous activity was recorded for 60 min (habituation to the experimental procedure), and then the mice were injected with i.p. METH or saline solution, and locomotor activity was recorded for an additional 120 min immediately after injection. The total distance traveled was recorded. The effects of acute METH treatment on locomotor activity were measured for 13 wild-type and 13 heterozygous mice, and the effects of chronic METH treatment on locomotor activity was measured for 11 wild-type and 23 heterozygous mice. Locomotor activity without drug treatment was measured in 4 wild-type and 6 heterozygous mice.
Acoustic Startle Response (Prepulse Inhibition, PPI) Test
PPI refers to the reduction in the startle response produced by the antecedent presentation of a low-intensity non-startling stimulus, representing sensorimotor gating [39]. Sensorimotor gating deficits in patients with schizophrenia, as indexed by measures of PPI, have been well characterized and are suggested as meeting the criteria of a useful endophenotype in human genetic studies [40]. With respect to bipolar disorder, current research suggests that PPI deficiency is state dependent rather than a constant trait. Patients with bipolar disorder with acute mania symptoms and psychosis exhibit PPI deficits [41]. PPI is measured using an SR-Lab system (San Diego Instruments Inc., San Diego, USA). Wild-type and hetero KO male mice (age, 6–8 weeks) were pre-treated intraperitoneally with saline or METH (1 mg/kg) 5 min before testing. After treatment, they were placed in a Plexiglas cylinder test chamber. Experimental sessions consisted of a 5-min acclimatization period with 70-dB broadband background noise, which was continuous throughout the session, followed by three 120-dB acoustic pulses and PPI sessions. Sessions consisted of 10 blocks of 5 different trial types: startle pulse alone with a 40-ms duration at 120 dB (P120); and 4 prepulse+pulse trials with a 20 ms duration prepulse at either 78 dB (PP78), 82 dB (PP82), or 86 dB (PP86) followed by a 40-ms duration startle stimulus at 120 dB after a 100-ms delay. These were carried out in a pseudorandom order, with an average intertrial interval of 15 s. The startle response was recorded as maximum startle amplitude. PPI was calculated as a percentage score for each prepulse intensity, using the following equation: %PPI = 100× [(mean startle amplitude on pulse-alone trials – mean startle amplitude on prepulse trials)/mean startle amplitude on pulse-alone trials]. The startle magnitude was calculated as the average of all pulse-alone (p120) trials, excluding the first 3 p120 trials in each session. The effects of acute METH treatment on PPI were measured in 11 wild-type and 13 heterozygous mice, and the effects of chronic METH treatment on PPI was measured for 12 wild-type and 24 heterozygous mice. PPI without drug administration was measured for 10 wild-type and 10 heterozygous mice.
Forced Swimming Test
The forced swimming test was performed according to the method of Porsolt et al. (1977), with minor changes. The day before the test, mice were anesthetized with isoflurane, and wire rings (0.28 mm in diameter, 1.5 cm in length, and 10 mg in weight) were attached to both hind paws. A small magnet (1 mm in diameter, 3 mm in length) was attached to each of the wire rings. After a 12- to 18-h habituation, mice were subjected to forced swimming tests. Narrow plastic cylinders (18 cm in height, 11 cm in diameter) were filled with water (maintained at 25±1°C) to a depth of 10 cm. The cylinders were located in a MicroAct (Neuroscience Inc., Tokyo, Japan; see Inagaki et al. 2002) behavioral measuring system, which allowed the detection of motion by registering electric currents induced in a magnetic coil surrounding the cylinder by using magnets attached to the limbs of the mice. Mice were placed in the cylinders and motion was recorded for 6 min. The mice were judged immobile whenever they floated passively in the water; the judgment was based on threshold values. Electrical signals meeting the following criteria were classified as swimming: signals greater than 100 mV that continued for more than 0.1 s without gaps of more than 1.0 s. Immobility time was measured during the last 4 min of the 6-min session. Imipramine (20 mg/kg) or saline was injected for 14 days, and the test was performed 40 min after the last injection. The forced swimming test was performed on 24 wild-type and 9 heterozygous, 8-week-old male mice of the N8 backcross generation after saline injection, and on 12 wild-type and 12 heterozygous mice after imipramine injection.
Rota-rod, Novelty-seeking, and Social Interaction Tests
Rota-rod, novelty-seeking, and social interaction tests were carried out according to the methods described in the published literature [42], [43]. The rota-rod tests were performed in 10 wild-type and 20 heterozygous mice, the novelty-seeking tests were performed on 24 wild-type and 41 heterozygous mice, and the social interaction tests were performed on 8 wild-type and 21 heterozygous mice.
Surgery and Intracerebroventricular Infusion Procedure
Mice were anesthetized with sodium pentobarbital (40 mg/kg, i.p.), and mounted in a stereotaxic frame. A stainless steel guide cannula (23 gauge) was placed into the lateral ventricle (AP, 0.46 mm; ML, 1.01 mm to the bregma) for subsequent intracerebroventricular (icv) infusion. The guide cannula extended 4.0 mm below the surface of the skull. One Anchor Screw (EICOM, Kyoto, Japan) was placed into the skull and dental cement was poured over the exposed skull to hold the screws and cannula in place and close the incision site. A dummy cannula was placed into the guide. Mice were allowed to recover for 3 days prior to testing. Cannula placement was assessed in random animals via the icv infusion of xylene cyanol and the verification of dye in the ventricular system.
During infusions, the dummy cannulas were removed. An infusion cannula (30 gauge), which was attached to PE 20 tubing, was inserted into the guide cannula. A 10.0-µl Hamilton syringe was used to manually deliver Ringer's solution or PKCε-TIP (Merck KGaA, Darmstadt, Germany) over a 1-minute period. The infusion cannula was kept in place for an additional 30 s following infusion. Mice were then replaced into their original cages. The change in locomotor activity induced by METH injection was measured in 6 mice infused with Ringer's solution infusion and 10 mice infused with PKCε-TIP.
Statistical Analysis
Data were analyzed using JMP software version 7.0.1 (SAS Institute, Cary, NC, USA). The data of the expression analysis were analyzed by one-way analysis of variance (ANOVA) followed by post hoc analysis using a Student's t test to evaluate the effect of METH or imipramine administration in each brain area. Effects of genotype, METH treatment, and imipramine treatment on behavior were analyzed by ANOVA, followed by post hoc analysis using a Student's t test. The results were expressed as the mean ± SD. A p value <0.05 was considered significant.
Pdlim5 Deficient Mice
To generate Pdlim5 KO mice, Pdlim5+/− mice were crossed; however, Pdlim5 KO mice were not identified among 134 genotyped pups, indicating that null Pdlim5 results in embryonic lethality. No apparent phenotypic differences were observed between the wild-type and Pdlim5 hetero KO mice. No significant difference in body weight, brain size, or brain shape was observed between Pdlim5+/− and Pdlim5+/+ mice. Pdlim5 expression levels in the prefrontal cortex of hetero KO mice (Pdlim5+/−) were less than 50% of those in the wild littermate mice (Pdlim5+/+) (Fig. 1E). The expression of PDLIM5 protein decreased in Pdlim5+/− compared to the wild littermate mice (Pdlim5+/+) (Fig. 1F).
Behavioral Characteristics of Pdlim5 Deficient Mice
No significant difference in rota-rod (Fig. S1A), novelty-seeking (Fig. S1B), social interaction (Fig. S1C), locomotion (Fig. S1D), or PPI tests (Fig. S1E) was observed between Pdlim5+/+ and Pdlim5+/− mice.
Pdlim5+/− Mice in Mania Models
Single-dose METH treatment produced an enhancing effect on spontaneous locomotor activity in mice. The magnitude of enhanced locomotor activity by an single-dose injection of METH was significantly lower in Pdlim5+/− mice than in Pdlim5+/+ mice when comparing locomotor activities 30 minutes before and after METH injection [F(1, 24) = 9.4, p = 0.005] (Fig. 2A, 2B). An injection of METH (3 mg/kg) impaired PPI in Pdlim5+/+ mice, but did not appear to impair PPI in Pdlim5+/− mice, leading to significantly less impairment of PPI in the acoustic startle response of Pdlim5+/+ mice compared to Pdlim5+/− mice; ANOVA revealed a significant main effect of genotype [F(1, 70) = 5.1, p = 0.03] (Fig. 2C). No significant difference was observed between the Pdlim5+/− and Pdlim5+/+ mice for the startle response to 120-dB acoustic stimuli when treated with saline or acute or chronic METH treatment (data not shown).
Relative locomotor activity and prepulse inhibition after acute (A, B, C) and chronic (D, E, F) administration of METH in Pdlim5+/+ and Pdlim5+/− mice. Values are shown as mean ± SD. * p<0.05 and ** p<0.01 by ANOVA.
The effect of the METH injection (1 mg/kg), after 14-day treatment with METH (3 mg/kg) and 14 days of subsequent withdrawal, was also significantly lower in Pdlim5+/− mice than in Pdlim5+/+ mice when comparing locomotor activities 30 min before and after METH injection [F(1, 32) = 6.2, p = 0.02] (Fig. 2D, 2E). An injection of METH (1 mg/kg) also produced significantly less impairment of the PPI in the acoustic startle response of Pdlim5+/− mice compared to Pdlim5+/+ mice; ANOVA revealed a significant main effect of the genotype [F(1, 106) = 9.3, p = 0.003] (Fig. 2F).
Effects of PKCε-TIP on Locomotor Activity and PPI Response to METH
After being treated with 3 mg/kg METH (i.p.) for 14 consecutive days followed by 14 days of withdrawal, 2 µg of PKCε-TIP or Ringer's solution was injected via icv fusion for 7 consecutive days. After this treatment, a 1 mg/kg METH challenge was provided intraperitoneally on the 8th day. PKCε-TIP treatment produced a weakened locomotor response to METH injection compared to Ringer's solution treatment [F(1,14) = 4.1, p = 0.06] (Fig. 3A, 3B). PKCε-TIP treatment produced significantly less impairment of the PPI in the acoustic startle response compared to treatment with Ringer's solution [F(1,43) = 4.54, p<0.05] (Fig. 3C).
Relative locomotor activity (A, B) and prepulse inhibition (C) by the treatment of PKCε-TIP after chronic METH administration. Values are shown as mean ± SD. P values are based on ANOVA. * p<0.05 and ** p<0.01 by ANOVA.
Pdlim5+/− Mice in the Forced Swimming Test and the Effect of Imipramine Treatment
We conducted a forced swimming test, which is a common behavioral test for assessing depression in rodents and for testing the efficiency of antidepressant drugs, in Pdlim5 hetero KO mice. A significant increase in immobility time was observed in Pdlim5 hetero KO mice compared to the wild-type mice when they were treated with saline [F(1,31) = 7.3, p = 0.01]. Imipramine significantly attenuated immobility time in Pdlim5 hetero KO mice [F(1, 16) = 12.0, p = 0.003]. A decrease in immobility time was also observed in wild-type mice, but was not significant [F(1,33) = 1.5, p>0.05] (Fig. 4).
Immobility time of Pdlim5 hetero KO and wild-type mice in the forced swimming test with saline or chronic imipramine administration. Values are shown as mean ± SD. * p<0.05 and ** p<0.01 by ANOVA.
Changes in Pdlim5 Expression Levels in Mouse Brains Following METH, Haloperidol, and Imipramine Treatment
We investigated whether chronic METH administration induces changes in Pdlim5 expression in mouse brains. Pdlim5 expression levels in the brains were not significantly different in 7-week-old male mice (C57BL/6J) sacrificed 24 h after 1 dose of i.p. METH injection (3 mg/kg) or in saline-injected mice [F(5, 44) = 1.9, p = 0.11]. Pdlim5 expression levels in the brains were significantly different in mice 24 h after administering the last chronic injection (3 mg/kg for 14 consecutive days) and mice injected intraperitoneally with saline [F(5,44) = 4.8, p = 0.001]. Post hoc analysis revealed higher Pdlim5 expression levels in the prefrontal area of chronic METH-administered mice than in saline-administered mice (p = 0.03; Fig. 5A). The upregulated Pdlim5 expression in the prefrontal cortex of chronic METH-administered mice was confirmed by a separate experiment with different mice (p = 0.01) (Fig. 5B).
Seven-week-old C57BL/6J male mice were treated with an intraperitoneal injection (i.p.) of METH (3.0 mg/kg, once daily for 14 days) (A, B), haloperidol (1 mg/kg, once daily for 49 days) (C), imipramine (20 mg/kg, once daily for 14 days) (D), or vehicle-saline. The upregulated Pdlim5 expression in the prefrontal cortex of chronic METH-administered mice (A) was confirmed by a separate experiment using a different mice cohort (B). Values are shown as mean ± SD. * p<0.05 and ** p<0.01 by Student's t test.
Chronic administration of haloperidol did not produce a significant difference in Pdlim5 expression levels in the overall brain of mice sacrificed 24 h after administering the last chronic injection (1 mg/kg for 49 consecutive days) or in mice injected intraperitoneally with saline [F(5,93) = 1.3, p = 0.27]. However, post hoc analysis revealed lower Pdlim5 expression levels in the prefrontal area of mice, and higher Pdlim5 expression levels in the hippocampus and midbrain after chronic haloperidol administration compared to mice administered with saline (p = 0.05, 0.009, and 0.06, respectively; Fig. 5C).
Pdlim5 expression levels in the brains were significantly different for mice after chronic imipramine administration compared to mice after 14 days of i.p. saline injection [F(5,45) = 6.3, p = 0.0002]. Post hoc analysis revealed higher Pdlim5 expression levels in the hippocampal area of chronic imipramine-administered mice than in saline-administered mice (p = 0.03; Fig. 5D). Pdlim5 expression levels in the brains were not significantly different for mice administered 1 dose of imipramine compared to saline-administered mice [F(1, 44) = 1.9, p = 0.11].
To our knowledge, this is the first report about Pdlim5 KO mice. The removal of exons 9–13 eliminates binding determinates for the LIM domain, consequently disrupting the interaction of PKC. The targeted Pdlim5 allele generated mice that expressed a fusion protein lacking the LIM domain, thereby allowing the physiological role of the PDLIM5 LIM domain to be assessed. LIM domains are members of the zinc-binding domain family that is found in many key regulators of developmental pathways. The present study identified that embryonic lethality occurs when the PDLIM5 LIM domain is lost in mice. PDLIM5 is expressed in various tissues, including the heart, skeletal muscle, brain, lung, spleen, thymus, prostate, testis, ovary, small intestine, colon, leukocyte, liver, and kidney [3]. PDLIM5 preferentially interacts with PKCβ through the LIM domains in cardiomyocytes [1], and has an important role in heart development by scaffolding PKCβ to the Z-disk region [44]. It also binds to PKD1 and interacts with the cardiac L-type voltage-gated calcium channel subunit alpha1c in cardiomyocytes [45]. The silencing of Pdlim5 inhibits the alpha-adrenergic-induced increase of L-type calcium currents [45]. Although we did not examine Pdlim5−/− embryos, the lethal embryonic phenotypes of Pdlim5−/− mice may have arisen because of embryonic heart/circulation failure. In zebrafish, ENH knockdown appears to have a lethal effect on embryos shortly after the end of gastrulation, with some embryos showing elongation defects and disorganized somites [46]. This observation indicates that PDLIM5 has a crucial role in embryonic development.
This study aimed to evaluate the effects of decreased PDLIM5 levels on certain behaviors, specifically those related to mood disorders, because significant but not robust evidence for mood disorders in humans has been reported. We used Pdlim5 hetero KO mice, because an increase or reduction of PDLIM5 expression in human brains, peripheral lymphoblastoid cells, and lymphocytes has been reported to be linked with schizophrenia and mood disorders [16], [17], [19], [47]. Because differences in the size and weight of the brain and body, or other anatomical abnormalities, were not found in the present study in Pdlim5 hetero KO C57BL/6J mice, haploinsufficiency of Pdlim5 did not appear to cause the developmental abnormalities. In addition, the behavior tests (rota-rod, novelty-seeking, and social interaction tests) did not show any significant difference in locomotion between Pdlim5+/+ and Pdlim5+/− mice. Therefore, decreased Pdlim5 expression is unlikely to have a major effect on mouse behavior. However, when mice were treated with METH in the present study, significant differences in locomotion and PPI were observed. For instance, acute and chronic METH-induced hyperlocomotion and PPI were less severe in Pdlim5+/− than in Pdlim5+/+ mice. These findings suggest that lower levels of PDLIM5 prevent the development of a manic state or schizophrenia, which is consistent with the speculation from the observations in human postmortem brains, because higher levels of PDLIM5 have been reported in the prefrontal cortex of patients with schizophrenia and bipolar disorders [10], [19]. The mechanisms for these findings are subject to speculation. However, the transient inhibition of PKCε by pretreatment with PKCε-TIP produced similar effects on locomotion to those observed in Pdlim5 hetero KO mice in response to METH. These observations support the speculation that the involvement of PDLIM5 in the behavioral response to METH is partly via calcium or PKCε signaling.
A significant increase in the immobility time of forced swimming test was observed in Pdlim5 hetero KO mice compared to the wild-type mice. This observation could also be interpreted as being consistent with observations in human peripheral leukocytes. Iga and colleagues also reported that decreased PDLIM5 levels returned to normal after 8 weeks of medication in the peripheral lymphocytes of patients with depression [17].
The same group of authors reported an increased expression of PDLIM5 in peripheral leukocytes in medication-free patients with schizophrenia but not in patients administered antipsychotic medication [16]. These reports raise the possibility that antipsychotics reduce PDLIM5 expression levels, while antidepressants increase PDLIM5 expression levels. Such a possibility is partially supported by the findings obtained through the chronic administration of haloperidol and imipramine; haloperidol decreased Pdlim5 expression levels in the prefrontal area, while chronic administration of imipramine increased Pdlim5 expression levels in the hippocampus area. However, chronic administration of haloperidol also increased Pdlim5 expression levels in the hippocampus and midbrain, indicating the presence of a complex relationship between drugs and Pdlim5 expression. An alternative possibility is that these drugs do not directly influence PDLIM5 expression, but that PDLIM5 levels are related to the psychiatric state.
PDLIM5 may also be implicated in neural development. For instance, in the present study, Pdlim5 expression levels increased in the hippocampus of mice receiving chronic haloperidol or imipramine treatment compared to the control. Adult hippocampal neurogenesis has been implicated in antidepressant action [48]. Adult treatment with haloperidol is reported to increase dentate granule cell proliferation in the gerbil hippocampus [49], and to promote the survival of stem cells in the rat hippocampus [50]. Lasorella and colleagues reported that PDLIM5 is upregulated during neural development, and that it contributes to the differentiation of the nervous system through cytoplasmic sequestration of Id2, which binds to the PDLIM5 LIM domains [51]. The findings of the present study may support the possibility that some antipsychotics influence Pdlim5 expression levels, and that changes in Pdlim5 expression levels may contribute to the worsening or alleviation of some symptoms. Because we did not examine the brain of Pdlim5−/− embryos in this study, the effect of the absence of PDLIM5 LIM domains on brain development should be focused on in future studies.
This study was subject to several limitations, including a lack of analyses of Pdlim5−/− mice, a lack of specificity of PKCε-TIP to PDLIM5, a lack of measuring hypothalamic-pituitary-adrenocortical axis activity parallel to the behavioral phenotypes, and particularly limitations of a pharmacological model of bipolar disorder mania by METH administration. Despite these limitations, the present study indicates that the increased expression of PDLIM5 may cause behavioral changes to patients with mania or schizophrenia-like symptoms, whereas a decrease in its expression may cause depression. In conclusion, our findings support that PDLIM5 is involved in psychiatric disorders, including mood disorders.
Supporting Information
Figure S1.
Behavioral characteristics in Pdlim5 hetero knockout mice. The rota-rod, novelty-seeking, social interaction, locomotion, and prepulse inhibition tests were conducted in Pdlim5+/+ and Pdlim5+/− mice. These behaviors showed no significant differences between the 2 genotype groups. Values are shown as mean ± SD.
Author Contributions
Conceived and designed the experiments: YH HI TA. Performed the experiments: YH MI NK YT. Analyzed the data: YH MI YI. Contributed reagents/materials/analysis tools: HI. Wrote the paper: YH TA.
- 1. Kuroda S, Tokunaga C, Kiyohara Y, Higuchi O, Konishi H, et al. (1996) Protein-protein interaction of zinc finger LIM domains with protein kinase C. J Biol Chem. 271: 31029–31032.
- 2. Niederlander N, Fayein NA, Auffray C, Pomies P (2004) Characterization of a new human isoform of the enigma homolog family specifically expressed in skeletal muscle. Biochem Biophys Res Commun 325: 1304–1311.
- 3. Ueki N, Seki N, Yano K, Masuho Y, Saito T, et al. (1999) Isolation, tissue expression, and chromosomal assignment of a human LIM protein gene, showing homology to rat enigma homologue (ENH). J Hum Genet 44: 256–260.
- 4. Maeno-Hikichi Y, Chang S, Matsumura K, Lai M, Lin H, et al. (2003) A PKC epsilon-ENH-channel complex specifically modulates N-type Ca2+ channels. Nat Neurosci 6: 468–475.
- 5. Maturana AD, Nakagawa N, Yoshimoto N, Tatematsu K, Hoshijima M, et al. (2011) LIM domains regulate protein kinase C activity: a novel molecular function. Cell Signal 23: 928–934.
- 6.
Gardezi SR, Weber AM, Li Q, Wong FK, Stanley EF (2009) PDLIM5 is not a neuronal CaV2.2 adaptor protein. Nat Neurosci 12: 957–958; author reply 958.
- 7. Herrick S, Evers DM, Lee JY, Udagawa N, Pak DT (2009) Postsynaptic PDLIM5/Enigma Homolog binds SPAR and causes dendritic spine shrinkage. Mol Cell Neurosci 43: 188–200.
- 8. Chen Y, Lai M, Maeno-Hikichi Y, Zhang JF (2006) Essential role of the LIM domain in the formation of the PKCepsilon-ENH-N-type Ca2+ channel complex. Cell Signal 18: 215–224.
- 9. Horiuchi Y, Arai M, Niizato K, Iritani S, Noguchi E, et al. (2006) A polymorphism in the PDLIM5 gene associated with gene expression and schizophrenia. Biol Psychiatry 59: 434–439.
- 10. Kato T, Iwayama Y, Kakiuchi C, Iwamoto K, Yamada K, et al. (2005) Gene expression and association analyses of LIM (PDLIM5) in bipolar disorder and schizophrenia. Mol Psychiatry 10: 1045–1055.
- 11. Li C, Tao R, Qin W, Zheng Y, He G, et al. (2008) Positive association between PDLIM5 and schizophrenia in the Chinese Han population. Int J Neuropsychopharmacol 11: 27–34.
- 12. Shi J, Badner JA, Liu C (2008) PDLIM5 and susceptibility to bipolar disorder: a family-based association study and meta-analysis. Psychiatr Genet 18: 116–121.
- 13. Squassina A, Manchia M, Manconi F, Piccardi M, Ardau R, et al. (2008) A case-control association study of the PDLIM5 gene and bipolar disorder in a Sardinian sample. Psychiatr Genet 18: 128–132.
- 14. Liu Z, Liu W, Xiao Z, Wang G, Yin S, et al. (2008) A major single nucleotide polymorphism of the PDLIM5 gene associated with recurrent major depressive disorder. J Psychiatry Neurosci 33: 43–46.
- 15. Wong ML, Dong C, Andreev V, Arcos-Burgos M, Licinio J (2012) Prediction of susceptibility to major depression by a model of interactions of multiple functional genetic variants and environmental factors. Mol Psychiatry 17: 624–633.
- 16. Numata S, Ueno S, Iga J, Yamauchi K, Hongwei S, et al. (2007) Gene expression in the peripheral leukocytes and association analysis of PDLIM5 gene in schizophrenia. Neurosci Lett 415: 28–33.
- 17. Iga J, Ueno S, Yamauchi K, Numata S, Motoki I, et al. (2006) Gene expression and association analysis of LIM (PDLIM5) in major depression. Neurosci Lett 400: 203–207.
- 18. Consortium WTCC (2007) Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447: 661–678.
- 19. Iwamoto K, Kakiuchi C, Bundo M, Ikeda K, Kato T (2004 a) Molecular characterization of bipolar disorder by comparing gene expression profiles of postmortem brains of major mental disorders. Mol Psychiatry 9: 406–416.
- 20. Zain MA, Jahan SN, Reynolds GP, Zainal NZ, Kanagasundram S, et al. (2012) Peripheral PDLIM5 expression in bipolar disorder and the effect of olanzapine administration. BMC Med Genet 13: 91.
- 21. Shelton RC, Hal Manier D, Lewis DA (2009) Protein kinases A and C in post-mortem prefrontal cortex from persons with major depression and normal controls. Int J Neuropsychopharmacol 12: 1223–1232.
- 22. Wang HY, Friedman E (1996) Enhanced protein kinase C activity and translocation in bipolar affective disorder brains. Biol Psychiatry 40: 568–575.
- 23. Friedman E, Wang HY (1996) Receptor-mediated activation of G proteins is increased in postmortem brains of bipolar affective disorder subjects. J Neurochem 67: 1145–1152.
- 24. Hahn CG, Friedman E (1999) Abnormalities in protein kinase C signaling and the pathophysiology of bipolar disorder. Bipolar Disord 1: 81–86.
- 25. Wang HY, Markowitz P, Levinson D, Undie AS, Friedman E (1999) Increased membrane-associated protein kinase C activity and translocation in blood platelets from bipolar affective disorder patients. J Psychiatr Res 33: 171–179.
- 26. Hahn CG, Umapathy, Wang HY, Koneru R, Levinson DF, et al. (2005) Lithium and valproic acid treatments reduce PKC activation and receptor-G protein coupling in platelets of bipolar manic patients. J Psychiatr Res 39: 355–363.
- 27. Pandey GN, Ren X, Dwivedi Y, Pavuluri MN (2008) Decreased protein kinase C (PKC) in platelets of pediatric bipolar patients: effect of treatment with mood stabilizing drugs. J Psychiatr Res 42: 106–116.
- 28. Chen G, Manji HK, Hawver DB, Wright CB, Potter WZ (1994) Chronic sodium valproate selectively decreases protein kinase C alpha and epsilon in vitro. J Neurochem 63: 2361–2364.
- 29. Friedman E, Hoau Yan W, Levinson D, Connell TA, Singh H (1993) Altered platelet protein kinase C activity in bipolar affective disorder, manic episode. Biol Psychiatry 33: 520–525.
- 30. Manji HK, Etcheberrigaray R, Chen G, Olds JL (1993) Lithium decreases membrane-associated protein kinase C in hippocampus: selectivity for the alpha isozyme. J Neurochem 61: 2303–2310.
- 31. Manji HK, Lenox RH (1999) Ziskind-Somerfeld Research Award. Protein kinase C signaling in the brain: molecular transduction of mood stabilization in the treatment of manic-depressive illness. Biol Psychiatry 46: 1328–1351.
- 32. Young LT, Wang JF, Woods CM, Robb JC (1999) Platelet protein kinase C alpha levels in drug-free and lithium-treated subjects with bipolar disorder. Neuropsychobiology 40: 63–66.
- 33. Shilling PD, Kuczenski R, Segal DS, Barrett TB, Kelsoe JR (2006) Differential regulation of immediate-early gene expression in the prefrontal cortex of rats with a high vs low behavioral response to methamphetamine. Neuropsychopharmacology 31: 2359–2367.
- 34. Coccurello R, Caprioli A, Ghirardi O, Virmani A (2007) Valproate and acetyl-L-carnitine prevent methamphetamine-induced behavioral sensitization in mice. Ann N Y Acad Sci 1122: 260–275.
- 35. da-Rosa DD, Valvassori SS, Steckert AV, Ornell F, Ferreira CL, et al. (2012) Effects of lithium and valproate on oxidative stress and behavioral changes induced by administration of m-AMPH. Psychiatry Res 198: 521–526.
- 36. Feier G, Valvassori SS, Varela RB, Resende WR, Bavaresco DV, et al. (2012) Lithium and valproate modulate energy metabolism in an animal model of mania induced by methamphetamine. Pharmacol Biochem Behav 103: 589–596.
- 37. Johnson JA, Gray MO, Chen CH, Mochly-Rosen D (1996) A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function. J Biol Chem 271: 24962–24966.
- 38. Przyklenk K, Li G, Simkhovich BZ, Kloner RA (2003) Mechanisms of myocardial ischemic preconditioning are age related: PKC-epsilon does not play a requisite role in old rabbits. J Appl Physiol 95: 2563–2569.
- 39.
Geyer MA Dulawa SC (2003) Assessment of murine startle reactivity, prepulse inhibition, and habituation. Current Protocols in Neuroscience. Chapter 8.17.
- 40. Powell SB, Zhou X, Geyer MA (2009) Prepulse inhibition and genetic mouse models of schizophrenia. Behav Brain Res 204: 282–294.
- 41.
Kohl S, Heekeren K, Klosterkotter J, Kuhn J (2012) Prepulse inhibition in psychiatric disorders - Apart from schizophrenia. J Psychiatr Res.
- 42. Koga M, Ishiguro H, Yazaki S, Horiuchi Y, Arai M, et al. (2009) Involvement of SMARCA2/BRM in the SWI/SNF chromatin-remodeling complex in schizophrenia. Hum Mol Genet 18: 2483–2494.
- 43. Syu A, Ishiguro H, Inada T, Horiuchi Y, Tanaka S, et al. (2010) Association of the HSPG2 Gene with Neuroleptic-Induced Tardive Dyskinesia. Neuropsychopharmacology 35: 1155–1164.
- 44. Nakagawa N, Hoshijima M, Oyasu M, Saito N, Tanizawa K, et al. (2000) ENH, containing PDZ and LIM domains, heart/skeletal muscle-specific protein, associates with cytoskeletal proteins through the PDZ domain. Biochem Biophys Res Commun 272: 505–512.
- 45. Maturana AD, Walchli S, Iwata M, Ryser S, Van Lint J, et al. (2008) Enigma homolog 1 scaffolds protein kinase D1 to regulate the activity of the cardiac L-type voltage-gated calcium channel. Cardiovasc Res 78: 458–465.
- 46. Ott EB, Sakalis PA, Marques IJ, Bagowski CP (2007) Characterization of the Enigma family in zebrafish. Dev Dyn 236: 3144–3154.
- 47. Iwamoto K, Bundo M, Washizuka S, Kakiuchi C, Kato T (2004 b) Expression of HSPF1 and LIM in the lymphoblastoid cells derived from patients with bipolar disorder and schizophrenia. J Hum Genet 49: 227–231.
- 48. Malberg JE (2004) Implications of adult hippocampal neurogenesis in antidepressant action. J Psychiatry Neurosci 29: 196–205.
- 49. Dawirs RR, Hildebrandt K, Teuchert-Noodt G (1998) Adult treatment with haloperidol increases dentate granule cell proliferation in the gerbil hippocampus. J Neural Transm 105: 317–327.
- 50. Keilhoff G, Grecksch G, Bernstein HG, Roskoden T, Becker A (2010) Risperidone and haloperidol promote survival of stem cells in the rat hippocampus. Eur Arch Psychiatry Clin Neurosci 260: 151–162.
- 51. Lasorella A, Iavarone A (2006) The protein ENH is a cytoplasmic sequestration factor for Id2 in normal and tumor cells from the nervous system. Proc Natl Acad Sci U S A 103: 4976–4981.


text:
Pure Supps is one of the latest breakout brands to storm the Australian supplement market. With a streamlined range of protein powders and single amino acids, Pure Supps aim to cater to the essential supplement needs of your typical fitness enthusiast. 100% Shredded is their fat loss protein, which we review herein.
Pure Supps 100% Shredded Highlights
Outstanding value is at the core of the Pure Supps brand and as such, it must be said that above all, 100% Shredded is a great value fat loss protein for anyone conscious of saving their pennies. Other highlights include:
- Whey protein only fat loss protein
- Good meal replacement alternative with its full vitamin & mineral profile
- Caffeine free
Pure Supps 100% Shredded Ingredients
100% Shredded is one of the few fat loss proteins on the market to only use a whey protein blend for its protein source. Most fat loss proteins comes with a mix of whey and casein. Whether that's to reduce raw material costs or to provide the classic mix of fast and slow proteins it hard to say. But of all the proteins, whey protein certainly stands out as the protein with the most research behind it as an effective aid in weight loss/management. Recent studies have begun to uncover the mechanisms by which whey protein favourably modulates certain hormones to result in better blood glucose control, which is essential to effective weight management. 100% Shredded comes with a combination of some of the best fat burners in the form of carnitine, green tea, coffee bean extract and garcinia cambogia. Together these ingredients make an effective synergistic fat burning mixture.
Pure Supps 100% Shredded Effectiveness and Pros
Because it uses whey protein exclusively, 100% Shredded is one of the best anabolic fat loss proteins on the market. Whey is undisputed as the best protein for putting on muscle so for those individuals who don't want to compromise their gains while trying to drop fat, 100% Shredded is a great option. As stated above, 100% Shredded is also one of the best value fat loss proteins available.
Its relatively common for fat loss proteins to include added caffeine, however this is not the case for 100% Shredded. This will be seen as a bonus for those individuals sensitive to caffeine or looking to restrict their intake thereof.
Pure Supps 100% Shredded Cons & Negatives
The most notable drawback with 100% Shredded is the dose of fat burners. But in consideration of the fact that 100% Shredded is one of the best value fat loss proteins on the market, it hard to be overly critical about this, as higher levels of such actives obviously add to the raw material costs of the product.
Pure Supps 100% Shredded Taste & Mixability
All the flavours of 100% Shredded have a nice balance of full-bodied flavour and sweetness, which the average individual should find most enjoyable. There's no problems as far as mixing goes either, with a few quick shakes being all that is required.
Pure Supps 100% Shredded Verdict
All considered, 100% Shredded is a great value fat loss protein with a number of unique features that should appeal to a broad range of active and health-conscious individuals looking to lose weight while supporting muscle growth and recovery.

subset: commoncrawl


text:
This odd tattoo is interesting but slightly disturbing at the same time. This anonymous female takes the cake for what is possibly the weirdest tattoo.
The odd tattoo has been designed and placed in a way that it really looks as if you are looking at an x-ray image of a skeletal fetus inside of her womb. It is hard to say where the inspiration for a permanent tattoo like this could have possibly came from, as the tattoo is seriously morbid and unattractive.
There are many tattoos out there that represent a wide variety of anatomy-inspired themes -- hearts on the chest, brains on the top and side of the head and even exposed muscles. They are all fairly odd tattoos but this one is the strangest that I have seen yet.
X-Ray Fetus Tattoos
More Stats +/-
Anatomy-Inclusive Tattoo Art
Extreme Commitment Facial Tattoos
Father-Inspired Minimalist Tattoos
Elaborate Film-Inspired Tattoos
Evolutionary Tattoo Infographics
Free 2018 Report & eBook
Get the top 100 trends happening right NOW -- plus a FREE copy of our award-winning book.
Our Research Methodology
This article is one of 350,000 experiments. We use crowd filtering, big data and AI to identify insights.
This Odd Tattoo is Interesting But Slightly Disturbing at the Same Time
- By: Ryan FowlerJun 10, 2013
References: pinterest
References: pinterest


text:
from The American Heritage® Dictionary of the English Language, 4th Edition
- adj. Splendid: "The working genius of American design has been . . . a refining of utilitarian purity into a kind of splendiferous native simplicity" ( Jay Cocks).
from Wiktionary, Creative Commons Attribution/Share-Alike License
- adj. beautiful, splendid
from the GNU version of the Collaborative International Dictionary of English
- adj. Splendor-bearing; splendid.
from The Century Dictionary and Cyclopedia
- Splendor-bearing; splendid; brilliant; gorgeous.
from WordNet 3.0 Copyright 2006 by Princeton University. All rights reserved.
- adj. having great beauty and splendor
from The American Heritage® Dictionary of the English Language, 4th Edition
from Wiktionary, Creative Commons Attribution/Share-Alike License
Perlman, the "splendiferous" author of the current spotlighted review, wasted no time savaging my contribution.
We should have been very sorry if such a "splendiferous" phenomenon had been obscured by envious boa or pelisse, or lost to the proprieties of costume.
Blackwood's Edinburgh Magazine, Volume 56, Number 347, September, 1844
Scroggles looked round at his comrades, as if to appeal to their judgment as to the fitness of the word, but not receiving any encouragement, he thrust down the glowing tobacco in his pipe with the end of his little finger, and reiterated the word "splendiferous" with marked emphasis.
Little did I know that "splendiferous" means "tastes like the sweat off a dog's nuts."
You can download everything needed to hold your very own splendiferous Peach Party here.
For the reader I am -- and, if I remember accurately, the reader I was even then -- they're okay, but nothing magically splendiferous.
It was a splendiferous day outside – the glens with their meadows of sunshine and dew all around us.
In 1565, Eliseus Libaerts, a Flemish goldsmith working in the French Mannerist style, created a horse-and-rider ensemble for Erik XIV of Sweden, who never got to wear his splendiferous outfit.
Four Seasons The pool at the Four Seasons Resort Carmelo Colonia, in fact, would seem to be the most mysterious place in Uruguay—whose popular beach towns I've explored widely—had I not arrived from Carmelo, an hour's drive inland, where the Four Seasons has operated a splendiferous resort since 2001.
He sees it as a state with great strengths, which he is happy to rattle off in the vein of last October's Time magazine cover story on the state's splendiferous assets, and which anyone who pays attention to California can imagine.
The California as First "Failed State" Debate: Schwarzenegger, Davis, Whitman, and Jerry Brown
Doesn't belong here, but since I'm not a native speaker, you MIGHT forgive me that.
having great beauty and splender.
I love Phoenix Wright. But I have an OBJECTION! to that citation. The word's been around a lot longer than the games.
Creation of the character Redd White, taken from the Phoenix Wright videogame series.


text: I have shopped (something I hate to do!) and found a stainless steel stove that meets my criteria. Sadly, I decided I not only couldn't afford the have the gas company bring a line so I could have the gas stove I wanted but I figure husband and I are getting older and the electric is probably safer. Anyway, I found a stainless microwave to mount above the stove that also meets my needs. Hip Hip! So...what's the question??? Are you ready??? Where to heck does all the steam and smoke go when you are cooking if you replace your old hood with a microwave?? I thought that there was an exhaust fan/microwave combo! Maybe I better just replace the hood with a hood and keep my microwave on the countertop. That's why I hate to shop...you end up with problems that you didn't even know existed! LOL DDD


text:
After hitting the bookmakers with a 10/1 winner on Bank Holiday Monday, we're playing up our winnings on four fancied runners this afternoon / evening.
The Micky Hammond stable is on fire with a double at Cartmel on Bank Holiday Monday making it eight winners in the last fortnight, and his last 12 runners have achieved finishing positions of 133118119511.
Having tipped up the last of those winners here at 10/1 – hope you were on – I see no reason to change a winning formula and I'm napping the trainer's VERKO, who gets weight from all his rivals in the 3m handicap hurdle at Hexham this evening (7.40pm). Placed in a couple of Irish points, the six-year-old showed his best form for his current stable when upped to this trip at Newcastle in November, finishing second to a well-backed favourite. On that evidence he's up to winning a race of this nature and, while he's failed to reach the same heights on his last two starts, he has dropped in the weights as a result and useful claimer Craig Nichol takes a further 3lb off his back.
His stablemate ROXYFET can provide Hammond with a double when he runs in the 2m handicap chase run later on the card (8.10pm). A three-time winner in France, he has taken a while to acclimatise but he looked to be getting the hang of things at Sedgefield last time, when finding only subsequent winner My Friend George too good. Nichol's claim cancels out a 3lb rise for that effort and there ought to be plenty more improvement to come from the five-year-old.
The Verko-Roxyfet double pays just over 18/1 with Betfair, who were just one of two firms up at the time of writing – it could be worth shopping around in the morning.
No apologies for tipping up another Hammond inmate, but this time on the flat at Redcar. The trainer's sole runner at the seaside track is FORT BELVEDERE and the seven-year-old looks to have been given a right chance by the handicapper. Rated 90 when finishing third in an Ayr handicap back in June 2013, he has tumbled to a mark of just 70 as a result of some less than stellar effort since and a year-long absence due to injury. He would have needed his comeback run over course and distance last month – noted keeping on in the final furlong under today's pilot Graham Lee – and the stable is in much better form now. The 1m apprentice handicap (3.50pm) he contests won't take much winning.
Charlie Hills looks to have turned the corner after a quiet start to the season and his Little Voice will be a popular choice for the 5f maiden fillies' stakes at Leicester (3.00pm). A fair fourth at Windsor on her debut, she's not opposed lightly here, but I can't resist a bet on Mick Channon's JERSEY BREEZE at twice the price. She too ran a race full of promise on her racecourse debut, staying on to finish third at Salisbury behind a couple of well regarded and more experienced rivals. A 105,000 Euros sister to a 6f/7f juvenile winner, she looks sure to win one of these before too long and her trainer's comments in a recent Stable Tour make her an irresistible bet this afternoon.
He said: "Very nice, high-class filly, beautiful looking; she goes very well. We think a bit of her and she'll come on a ton for the race at Salisbury – she's a bit of a character and we thought she could get beat first time and there were a lot of fancied runners in the race. But she's got a great demeanour – she's smashing and she's been showing us plenty."
I think he likes her and so do I at the 6/1 on offer!
Leicester 3.00, Jersey Breeze @ 6/1 bet365
Redcar 3.50, Fort Belvedere @ 6/1 BetVictor
Hexham 7.40, Verko @ 9/2 Betfair
Hexham 8.10, Roxyfet 5/2 Betfair
All odds were correct at time of posting.


text:
Read this: Online music licensing revenues pass radio for first time - Technology - The Guardian
Summary: Ed Sheeran and other UK artists enjoyed a bumper 2012 as digital licensing revenues passed radio for the first time. New licensing agreements with Google Play, Microsoft Xbox and other online players helped UK digital revenues soar 32.2 in 2012, from 39.1m in 2011. The advent of downloads and streaming services, such as Apple iTunes and Spotify, has more than doubled UK online licensing revenues, from 23.3m in 2008. - guardian.co.ukRead full article: Online music licensing revenues pass rad…
Lots more recommendations to read at Trends - ukfree.tv.
Summaries are done by Clipped-Your articles and documents summarized.
CommentsYour comment please


text:
|South Africa's National Liberation Movement
The Limpopo African National Congress congratulates the newly elected leadership of the ANCWL which was elected over the weekend during their 6th provincial congress held at The Ranch Hotel, in Polokwane.
The ANC takes this opportunity to wish all the Grade 12 learners all the best with the Senior Certificate examinations. We are confident that your preparations for the examinations will carry you through.
The Provincial Executive Committee (PEC) of the ANC in Limpopo held its PEC meeting on Monday 13 October 2014 in Mopani region. The holding of the PEC meeting in the Mopani region is an implementation of the PEC decision to hold meetings in all regions of the Province. Such PEC meetings are preceded by interactions with ANC structures within the region, as part of strengthening the work of the RECs and the PEC deployees in the regions. Similarly, the PEC meeting in the Mopani region was preceded by meetings with ANC sub-regional task teams (sub-RTTs) and ANC branches.
I bring you warm greetings from the Provincial Task Team of the ANC with the knowledge and confidence that this gathering will achieve its intended objectives with commitment and vigor.


text:
Tag: iPhone
Gaming Apps: Ruzzle
Another word game against friends? Bring it on baby. Ruzzle is a game that you play against friends or opponents online unless you buy the ad-free version where you can do practice mode until it's your turn in a game.
Gaming Apps: Anagram Twist – Jumble and Unscramble Text
Anagram Twist. The game is what the game title says. It's a game about anagrams and you twist the tiles around to make words. Sounds simple enough, yes? Does it shine in its simplicity or does it falter? Released December
Gaming Apps: Temple Run: Brave Review
The makers of Temple Run in collaboration with Disney an old game comes alive with a new twist and a new name just in time for movie's debut on June 22, 2012. Is it enough of a difference to justify
Gaming App: DrawSomething Review
Pictionary gets with the times with the newest App called "DrawSomething." Whether you've played Pictionary as a child, played with your kid(s), or if you're new to the whole scene…there's fun to be had by all. It's time to let
Gaming App: Fashion Story Review
Fashion Story is a phone app game developed by Team Lava for Android and iOS. This is the 2nd game by TL that I've become addicted to. I started off with Bakery Story, and tried a few other long list
Gaming Apps: Temple Run Review
Just how quick can you swipe with your finger? Are your reaction times better than everyone else? Can you quickly act upon what you've just seen on the screen? In order to well in Temple Run, you'll need reflexes like
Gaming Apps: Oregon Trail: American Settler Review
Travelling the Oregon Trail was a hard battle to be won. You could break your leg, your arm, get sick, get caught in currents, or have a member of your party die. It wasn't easy but if you mastered the
Gaming Apps: Bakery Story Review
Mmm…cakes…pies…donuts… intrigued? Bakery story was released after the popular Restaurant Story Facebook game but I'll have to take people's word for it because I've never played and never had any interest in it. Released November 12, 2010 Published by TeamLava
Gaming Apps: Touch Cats
Have you ever wanted a cat but don't have the time, energy, or money needed to care for one? Touch Cats may be the answer to your kitty needs. This game allows you to care, dress, socialize, and play with
Gaming Apps: Doodle Jump
Doodle Jump. When you look for it in the App Store is will read, "Doodle Jump – Be Warned: Insanely Addictive!" When I looked at that name, I was a little leery. That's a whole lot of promise for an


text:
The Essential Way to Creativity, Einstein and Others:
Brought to You by Your Curated Information Blog, Create & Be Well
This is a wonderful share from Brain Pickings if you are interested in understanding how creativity happens. "Combinatory play" is Einstein's description of how we shape new ideas by connecting through intellectual play seemingly disparate things. Others offer similar explanations for the roots of creative development.
This is a process I realize I have used all my life in developing new ideas. programs, and organizations; it is how my book in progress "Shaping Lives, Transforming Communities: Creative Expression in the Dance of Life" is being built. This essay, with its references to other writings and authors is a gem for your weekend delight.
"Combinatory play seems to be the essential feature in productive thought."
Source: How Einstein Thought: Why "Combinatory Play" Is the Secret of Genius | Brain Pickings
The Kyle Group, Shaping New Worlds Through Creativity!


text:
- sweetdee89
- 4 years ago
So I have this cluster promise ring (almost looks like an engagement ring) and I wear it everyday on my right hand. It means a lot and I want to incorporate it with my wedding band. I was thinking oof melting it down and using the smaller diamonds to make an eternity band and the larger ones to enhance the side of my e-ring. Maybe use the middle stone in my F'S wedding band.
What are your thoughts? Anyone have experience with this? Price to do so? Also – post pics of your rings – I just love seeing everyone's rings 🙂


text:
Thomasville Home Furnishings Of Nanuet
location on Google Maps.
in new tab.
on social media.
The story of Thomasville began in Thomasville, North Carolina, in 1904. At the time, we offered just one product – a chair. The "Thomasville Chair" it was called. The chair was so beautifully crafted and well made that people responded by asking us to create other pieces as well. Now more than 100 years later, the Thomasville story is still being written. And now part of our story is helping you tell yours – with home furnishings, accessories and cabinetry that allow you to express yourself through your home.
Business Hours
Monday 10:00 AM - 6:00 PM
Tuesday 10:00 AM - 6:00 PM
Wednesday 10:00 AM - 6:00 PM
Thursday 10:00 AM - 6:00 PM
Friday 10:00 AM - 6:00 PM
Saturday 10:00 AM - 6:00 PM
Sunday 12:00 PM - 5:00 PM


text:
AIDS virus particles, computer artwork
M050/1180 Rights Managed
530 pixels on longest edge, unwatermarked
Request/Download high-res file
Uncompressed file size: 50.2MB
Downloadable file size: 5.1MB
Please login to use the price calculator
Credit: MEHAU KULYK/SCIENCE PHOTO LIBRARY
Release details: Model release not required. Property release not required.
Keywords: acquired immune deficiency, aids, artwork, biological, biology, black-and-white, coat, cutaway, enveloped, hiv, human immunodeficiency virus, icosahedral, illustration, knob, knobs, many, matrix layer, medical, medicine, membrane, membrane envelope, microbe, microbes, microbiological, microbiology, microorganism, microorganisms, monochrome, multiple, nucleocapsid, particles, pathogen, pathogenic, protein, protein spike, retrovirus, rna virus, spike, spiked, spikes, surface, syndrome, virion, virions, virology, virus, virus particle, viruses
Licence fees: A licence fee will be charged for any media (low or high resolution) used in your project.


text:
ANTHRAX IN KABUL: When the Monitor's Scott Peterson heard that there was an anthrax facility on the edge of Kabul, his first thought was "What a scoop." There is, in fact, a lab in the Afghan capital and it's supported by the International Red Cross. But its purpose is to make anthrax vaccine for domestic animals.
Anthrax is a common disease among sheep in Afghanistan and many developing countries. The original anthrax cultures arrived at the lab decades ago, from laboratories in India, Iran, and Turkey.
Still, Dr. Abdulkadir Raoufi, the lab director, was worried that after the anthrax attacks in the US, American bombers would target his lab. He heard a radio report that his vaccine-making facility was "still in the hands of the Taliban Mullahs."
His was not an unreasonable concern, given that every known vaccine and chemical laboratory in Iraq - where extensive chemical and biological weapons programs were underway for years - has been destroyed by American bombs and United Nations weapons inspectors.
But the strain of anthrax Dr. Raoufi works with is so harmless to humans that "I will wash my hands and face with it, if you want." And Raoufi says that during the five years of Taliban rule in Afghanistan, he never received a request from the Islamic militants of the regime or from bin Laden agents, to convert his laboratory to use the deadly variety of anthrax. When the Taliban were in charge, they appointed a non-scientist director who knew nothing about their work, who "drank tea, signed the register each day and went home," Raoufi says.
NO RULES REMODELING: Fred Weir's family owns a Moscow apartment, which they rent out. So, he has been following the Russian remodeling craze he writes about today (page 1) for years. "In our building, people have enclosed their balconies, and extended their living rooms and bedrooms. The result, from the outside, is a building with an amazing variety of protruding shapes and colors."
David Clark Scott
World editor
Let us hear from you.
Mail to: One Norway Street, Boston, MA 02115 via e-mail: firstname.lastname@example.com


text:
Find Hotels in Aberfeldy
Aberfeldy Hotel Deals from ZUJI
Looking for cheap accommodation in Aberfeldy, United Kingdom? ZUJI helps you compare hotels in destinations all over the world for any time of year, whether youre booking early to secure a great rate or trying to snap up a cheap last minute room. Compare TripAdvisor reviews, browse our comprehensive range of Aberfeldy hotels and make your travel booking with ZUJI today!


text:
On September 20, 2017 local time, the Ad-hoc Meeting of the Council of Foreign Ministers of the Member States of the Shanghai Cooperation Organization (SCO) was held in New York on the sideline of the 72nd UN General Assembly. External Affairs Minister Sushma Swaraj of the Republic of India, Foreign Minister Kairat Kudaybergenovich Abdrakhmanov of the Republic of Kazakhstan, Foreign Minister Wang Yi of the People's Republic of China, Foreign Minister Erlan Abdyldaev of the Kyrgyz Republic, Foreign Minister Khawaja Muhammad Asif of the Islamic Republic of Pakistan, Foreign Minister Sergey Lavrov of the Russian Federation, Foreign Minister Sirojiddin Aslov of the Republic of Tajikistan and Foreign Minister Abdulaziz Kamilov of the Republic of Uzbekistan attended the meeting.
The meeting was chaired by Foreign Minister Wang Yi of the People's Republic of China.
SCO Secretary General Rashid Alimov and Director Yevgeniy Sysoyev of the Executive Committee of the SCO Regional Anti-Terrorist Structure were present at the meeting.
Foreign Ministers pointed out that over the past 16 years since the foundation of the SCO, it has become a force of important influence on the international stage, with its great contribution to maintaining regional peace and safety, achieving sustainable development and consolidating good-neighborly friendship and partnership between countries. On June 9, 2017, Pakistan and India were officially accepted as members of the SCO in the SCO Astana Summit. Now the SCO is facing new opportunities and prospects of development, as well as the improvement of international prestige and role.
Foreign Ministers exchanged views on some issues including development prospects of the SCO, urgent international and regional issues, strengthening the SCO's cooperation with the UN and other multilateral organizations, deepening cooperation among SCO member states within the framework of the UN, as well as of other international and regional organizations.
Foreign Ministers emphasized that the SCO member states should continue to pursue "Shanghai Spirit" featuring mutual trust, mutual benefit, equality, consultation, respecting diverse civilizations and seeking common development, and pointed out that the SCO Charter, Treaty on Long-Term Good-Neighborliness, Friendship and Cooperation and other basic legal documents are of great importance.
Foreign Ministers supported the SCO member states to comprehensively deepen cooperation in politics, security, economy, people-to-people and cultural engagement, external exchanges and mechanism construction for the sake of further development of the SCO.
Foreign Ministers pointed out that related procedures are unstoppable against the background that a multi-polar world is forming, with new political and economic centers being increasingly consolidated. At the same time, the principle of international laws for non-interference in the internal affairs of other states is under attack because of the increase of global unstable factors and the revival of groupthink and Cold War mentality. Based on the adverse situation of economic globalization, as well as the unstable growth of global economy, the conflicts caused by imbalanced development and distribution injustice are continuously aggravated.
Foreign Ministers expressed that despite the uncertainty in the current international situation, the peaceful co-existence and sustainable development are still the themes of the times. We must resolve vital issues by upholding multilateralism and address various challenges and threats by jointly perfecting the global governance mechanism to build a community of common destiny for mankind and establish new type of international relations.
Foreign Ministers stressed that the UN is the most universal, representative and authoritative international organization. All parties indicated that they will firmly hold the purposes and principles of the UN Charter, and be ready to strengthen coordination within the UN framework and consolidate the UN's core role in international cooperation on global governance, safeguarding world peace, promoting common development, fighting terrorism, separatism and extremism. All parties supported the SCO's further cooperation with the UN and its special agencies to safeguard world peace, resolve hotspot issues and maintain peace, stability and sustainable development of the SCO regions and the whole world at large.
Foreign Ministers reiterated that they opposed to using "double standards" for terrorists as well as to any attempt to interfere in other countries' internal affairs and damage the leading roles of all countries' authorities by using anti-terrorism as an excuse.
In this respect, Foreign Ministers agreed that the SCO should approve the SCO Anti-extremism Convention as soon as possible, and determined to take further coordination actions and strengthen cooperation so as to address common security challenges and threats and protect national interests.
Foreign Ministers expressed concerns towards the situation of the Korean Peninsula, pointing out that the UN Security Council Resolution 2375 adopted on September 11, 2017 is a reasonable response to the nuclear test conducted by the Democratic People's Republic of Korea on September 3, 2017, calling on the SCO member states to obey the items of this resolution strictly to promote a political and diplomatic solution for the Korean Peninsula issue. Foreign Ministers stressed that it is necessary to launch the negotiation to ease the situation at an early date and to attach great importance to the opinion proposed by China and Russia.
Foreign Ministers pointed out that the government and people of the Islamic Republic of Afghanistan are endeavoring to achieve national peace, stability and economic prosperity while shaking off terrorism, extremism and drug crimes, and held that the UN should play a core coordination role in international cooperation on promoting stability and development of Afghanistan. Foreign Ministers welcomed that Afghanistan takes part in regional cooperation process and stressed that the SCO should carry out the consensus on the continuing work of the SCO-Afghanistan Contact Group reached by the heads of the SCO member states after the expansion.
Given the current economic climate, Foreign Ministers agreed to further develop and enlarge the economic and trade cooperation within the framework of the SCO and stressed that the intention of utilizing the unilateral restriction measures as a political and economic pressure tool to set barriers for economic, trade and investment cooperation among countries is of no constructive significance and in vein. Foreign Ministers supported the creation of advantageous conditions to gradually achieve the free transportation for commodities, capitals, services and technologies required by the SCO Charter.
In order to effectively implement prior cooperation projects of economic and investment within the SCO framework, Foreign Ministers were in support of active discussion on the topics about establishing SCO Development Bank and SCO Development Funds (special accounts).
Foreign Ministers believed that the meeting of the Council of Prime Ministers (the Heads of Government) of the member states of the SCO to be held in the Russian Federation in 2017 will propose prior tasks and specific measures to further strengthen economic, people-to-people and cultural cooperation.
Foreign Ministers emphasized that it is important to implement the achievements of the meeting of the Council of Heads of State of the member states of the SCO (June 9, 2017, Astana). They also welcomed the incoming presidency of the People's Republic of China during 2017-2018 and reiterated their support for it.
As the outcome of this meeting, all Foreign Ministers signed a series of documents related to international cooperation and administrative affairs of the SCO and its permanent bodies.
Foreign Ministers highly appreciated China's effective work to this ad hoc meeting of the Council of Foreign Ministers of the member states of the SCO and thanked the UN Secretariat for its assistance.
This meeting was held in the atmosphere of traditional friendship and mutual understanding among the SCO member states.


text:
Author has written 17 stories for Negima! Magister Negi Magi/魔法先生ネギま!, Yu-Gi-Oh GX, Tales of Symphonia, Tales of Vesperia, Megami Tensei, Persona Series, and Fire Emblem.
Some info about me:
I used to go by the penname Lunardragon2607. I am on YouTube, under the username lunardragon2607. The link: www.youtube.com/user/lunardragon2607
I'm just your average guy who loves games and stuff.
Some of my favorite authors:


text:
Available Condos & Townhomes for sale in Centennial, CO
Including Cluster & Patio style properties, for sale in Centennial, CO
Sorry no results were found. Go Back
Discover more homes for sale in Centennial, Colorado.
View community photos and find homes for sale in Centennial CO Neighborhoods. Interested in area restaurants and nearby schools? Explore Centennial.
Centennial, CO community expert real estate agents serve the greater Centennial CO area near Denver, Colorado.
Catch up on Centennial local happenings by visiting our Centennial Community blog. Connect with a trusted, local real estate expert who knows Centennial CO to request a showing.


text:
SALT LAKE CITY, April 7 (UPI) -- Carlos Ruiz scored the tying goal Saturday, getting his new FC Dallas Coach Steve Morrow a 2-2 draw with Real Salt Lake in the season opener for both teams.
FC Dallas's Kenny Cooper also found the back of the net.
Real Salt Lake's Jeff Cunningham, who was last year's leading goal scorer, picked up both goals for the team.
FC Dallas committed 17 fouls during the first of three meetings between the two teams this season.


text:
|$ in cart: 0.00 - Shipping Quote Edit/Complete Order
TF 1b/30 is a natural black tipped with golden auburn color. T1b/30 will have less streaks and just be a tipped color
Privacy StatementPage Design & HTML Code by Amphigory / Graphics by Amphigory / All copyrights and trademarks respected / (C) Copyright 1996-2018 Amphigory / WITH EXPLICIT RESERVATION OF ALL RIGHTS (U.C.C. 1-207)


text:
White chocolate sugarpaste made with luxury Belgian, white chocolate.
This high quality sugarpaste is very easy to work with and covers cakes and cupcakes without any cracking. Suitable for making delicate flowers and cake decorations too.
Chocolate sugarpaste is a pliable medium predominantly made from icing sugar which sets hard once in place and is used for covering cakes and cupcakes, including modelling finely detailed cake decorations.


text:
H.R.1622 - To provide for a program of research, development, and demonstration on natural gas vehicles.111th Congress (2009-2010)
BillHide Overview
|Rep. Sullivan, John [R-OK-1] (Introduced 03/19/2009)
|House - Science and Technology | Senate - Energy and Natural Resources
|Committee Reports:
|H. Rept. 111-206
|Latest Action:
|Senate - 07/22/2009 Received in the Senate and Read twice and referred to the Committee on Energy and Natural Resources. (All Actions)
|Roll Call Votes:
|There has been 1 roll call vote
This bill has the status Passed House
Here are the steps for Status of Legislation:
- Introduced
- Passed House
Subject — Policy Area:
- Energy
- View subjects
Summary: H.R.1622 — 111th Congress (2009-2010)All Information (Except Text)
Shown Here:
Passed House amended (07/21/2009)
(This measure has not been amended since it was reported to the House on July 14, 2009. The summary of that version is repeated here.)
Directs the Secretary of Energy (DOE) to: (1) conduct a five-year program of natural gas vehicle research, development, and demonstration; (2) coordinate with the Administrator of the Environmental Protection Agency (EPA) regarding such program; (3) coordinate with the natural gas vehicle industry to ensure cooperation between the public and the private sector; and (4) report to Congress on implementing such program.
Includes among program objectives: (1) the continued improvement and development of new, cleaner, more efficient light-, medium-, and heavy-duty natural gas vehicle engines for onroad and offroad applications; (2) the development and improvement of nationally recognized standards for the safe operation of natural gas vehicles and their components; (3) improvement in the reliability and efficiency of, and certification of, natural gas fueling station infrastructure; (4) the improvement of existing natural gas fuel storage systems and the development of new storage materials; and (5) the use of natural gas engines in hybrid vehicles.
Authorizes appropriations.


text:
Surround your Marquis hot tub with custom Environments™ modules to transform your outdoor living space into your personal hot tub utopia.
• Aromatherapy in your spa
• A powerful non-chlorine oxidizer
• Choose from four exquisite fragrances
• No more chlorine smell
Choose from 4 Experiences:
Island Pleasures, Divine Secrets,
Quiet Escape or Simple Rituals
The Cover Caddy gas spring assisted cover lift defines versatility and effortless spa cover removal. It's unique design allows for installation on an array of spa shapes and sizes including round, octagonal, and spas with large radius or cut corners. The Caddy's new under-mount base plate provides for a complete installation within minutes. The addition of a powerful gas spring makes lifting and lowering your spa cover easier than you ever imagined.
A Marquis hot tub is the ultimate hot tub experience! The moment you first set your eyes on a Marquis you'll be dazzled by the artistry with which it was sculpted - the sinuous, organic styling that threads throughout the body - the river rock footwell, hand-picked from an Oregon river - and the solid, exclusive DuraWood extruded from a hand-honed board. Marquis' eye for detail knows no bounds.
The Spirit™ relaxes your mind and rejuvenates your body. It accommodates three people in comfort. The full body lounge pays extra attention to your shoulders, back, thighs and feet. The deep therapy seat works on all the back muscles. Plus, there's a cool-down seat to get you out of hot water. But the highlight is the H.O.T. Zone Therapy Pillars of whirlpool jets that really bring you to life! The super convenient 120V plug-and-play system helps to enhance your carefree experience. Renew your Spirit™ every day.
The Nordic Retreat SE ™ offers unprecedented value. With seating for five, this spa can help you create quality family time or simply offer sanctuary from every day stress to couples or individuals.
Powerful jets provide invigorating hydrotherapy to tense areas of the body. Plus, Nordic's exclusive dual-therapy system (DTS™) offers gentler, whole-body therapy to complement these traditional, more targeted jets. The versatile bench seat can be used as full body massage from the Whirlpool Jet. The Retreat Sport Edition is delivered standard with our Northern Light Package including a waterfall.
Leizure World Pools, Hot Tubs & Billiards | 5213 Airline Rd | Muskegon, MI 49444 | P: 231.865.1208


text:
Home > Sport > Cricket
New bowling coach Walsh delighted with Bangladesh job
>> Reuters/bdnews24.com
Published: 2016-09-01 13:12:04.0 BdST Updated: 2016-09-01 13:12:41.0 BdST
Newly appointed bowling coach Courtney Walsh thinks the Bangladesh team are a 'talented bunch of players' and is looking forward to begin working with them.
Related Stories
Walsh new pace bowling coach
The West Indies fast bowling great agreed to a three-year contract with the Bangladesh Cricket Board which begins this week, the board said on Thursday.
The 53-year-old Walsh, the most successful bowler from the Caribbean Islands with 519 Test wickets, recently completed his role as a West Indies selector.
"Having watched Bangladesh cricket from afar over the years, they are a seriously talented bunch of players," Walsh said in a statement.
"Chandika Hathurusingha has done a wonderful job thus far as head coach so hopefully I can complement his skills and continue the positive progress.
"Obviously the West Indies is my home but the chance to go in a new direction on the coaching front and with such a talented group, was one I couldn't look past."
The Bangladesh job will be the first senior role for Walsh, who in the past has managed the West Indies Under-19 team and been a bowling consultant for the Jamaica Tallawahs in the Caribbean Premier League among others.


text:
Wastin away in Margaritaville...5:00 AM
14 comments
I super love these!! I want margarita nails!ReplyDelete
So cute, makes me want a margarita now in some beach-y location! xReplyDelete
That you actually thought to add the salt blows my mind!ReplyDelete
Mmmmmmargaritas! <3 these! :)ReplyDelete
totally love these - especially the salt on the rim!! xReplyDelete
pour me a glass, please!ReplyDelete
Margarita's are my absolute favorite!! I love these nails :)ReplyDelete
Love it! The little salt tips are perfect!ReplyDelete
Love the textured salt touch.ReplyDelete
Awesome job! The added salt makes it fabulous!ReplyDelete
oooooh Margarita Nails! You know I love these for sure!ReplyDelete
I love the subtle gradient! Making me thirsty!ReplyDelete
What a great idea - perfectly executed!ReplyDelete
Love love love these! The colors are perfect and the salt on the tips is perfect :)ReplyDelete
Thank you for visiting and commenting! I read all my comments and love to hear from you! Please DO NOT leave links in the comment box. ALL links which are not relevant to the post are subject to deletion. If you'd like me to check out your blog, please email me at email@example.com. Thank you again!


text:
via www.ellapublishing.com
January 14, 2013 in layouts, scrapbooking | Permalink
Comments (0)
January 09, 2013 in layouts, scrapbooking | Permalink
Comments (0)
Text Tricks for scrapbookers: Titles | Ella Publishing Co.
January 08, 2013 in family life, layouts, scrapbooking, Taylor | Permalink
Comments (0)
January 07, 2013 in layouts, scrapbooking | Permalink
Comments (0)
As a way of documenting and celebrating the countdown until Taylor returns I have been sharing some photos, layouts, and memories of Tay. As I have been looking at stuff I created I thought it would be fun to share some favorite boy pages. (not just about Tay)
Scrapbooking about boys
Continue reading "Countdown to Taylor: 3 days to go" »
July 30, 2012 in family life, layouts, Missionary, scrapbooking, Taylor | Permalink
Comments (0)
I am loving using circles in all my current porjects, can't seem to stop as a matter of fact. I like them whole, cut in half, layered, glittered, colored, even sticking out. Loving circles-some of my obsession came from this awesome scrapbooker, Vivian Masket and her top ten post here. Love these layouts!
July 28, 2011 in Caden, Jacob, layouts, scrapbooking | Permalink
Comments (0)
Thanks for stopping by via Francine's awesome blog. (if you want to start at the beginning of the blog hop go here) This month's theme is all about blooms and we got to use the same product, an adorable spring line by Making Memories called Dilly Dally.
To enter a giveaway for this awesome line head over here, but first finish the blog hop. The next stop is at Kimber's blog. Have fun and see you next month.
I am lucky to have a book published under the Memory Makers brand called Start Scrapbooking. Each month we are going to share some projects just for you.
April 11, 2011 in layouts, Memory Maker blog hop, scrapbooking, Start Scrapbooking | Permalink
Comments (2)


text:
Collection scam: SC hints at CBI probe
Read more below
- Published 24.04.14
|File picture of a vigilance raid on Sea Shore office at Patia in Bhubaneswar
New Delhi/Bhubaneswar, April 23: The Supreme Court today indicated that it would order a CBI probe into the swindling of over Rs 50,000 crore belonging to investors by 44 deposit collection companies, including Bengal-based Saradha group.
The scam that surfaced last year triggered a political storm in the state with the Opposition slamming the BJD, which ordered a crime branch inquiry into the goings-on of around 127 such money collection entities, apart from instituting a judicial probe. The Opposition, however, continued to insist on a CBI probe.
The bench of Justice T.S. Thakur and Justice C. Nagappan said a CBI inquiry was needed to track down the conspiracy in the embezzlement as the companies had operations not only in the state, but also in Assam and Tripura.
The court decided to order a CBI inquiry after former solicitor-general Gopal Subramanium appearing for the state government had initially opposed it but later relented when the bench assured him that it did not in any manner reflect on the state machinery's functioning.
The bench today also expressed its shock after counsel Sunil Mathews appearing for the petitioner Alok Jena, brought to the court's notice that the panchayati raj department had issued a circular to all self-help groups in the state to invest in Microfinance Corporation Ltd, which had heavily defrauded the investors.
While the petitioner had claimed that Rs 50,000 crore was involved in the scandal, the state insisted that it was only around Rs 3,000 crore.
The court minced no words in expressing displeasure at the inaction of regulatory agencies such as SEBI, RBI and Registrar of Companies in preventing the loot of public money and said their conduct should also be probed to unravel the conspiracy.
The apex court questioned various probe agencies for failing to prevent the scam and rejected the government's contention that the Justice R.K. Patra Commission and state police were making all efforts to refund the investors and book the culprits.
"You say properties worth Rs 500 crore had been attached. But where is the remaining Rs 2,500 crore, even if we are to take your figure of Rs 3,000 crore as being defrauded," Justice Thakur, heading the bench, asked Subramanium.
The counsel said the state had introduced the Odisha Protection of Interests of Depositors and Finance Act, 2013, which prescribed a maximum punishment of 10 years with fine, which he claimed, indicated the state's seriousness to deal with the issue.
But, the argument did not impress the court, which asked: "Tell us what you have done to unearth the money. There is no agency to detect where the money has gone. All your efforts so far have only led to collection of 20 per cent of the money."
The court also brushed aside Subramanium's claim that the Patra commission was seriously looking into the matter.
A deposit collection scam surfaced in the state last year when big-time operators such as Sea Shore group, Artha Tatwa, Saradha group, Astha and Flourish India were found to have duped investors of around Rs 2,000 crore. Artha Tatwa group chief Pradip Sethy and Sea Shore group chairman Prashant Dash are both in jail.
The state government, till recently, had been fighting shy of a CBI inquiry fearing repercussions in the elections. But today, BJD leader Kalpataru Das said the state government would extend all co-operation in the event of a CBI inquiry being ordered.


text:
Elegant solutions to common language problems
Interpreting | Translation | Safety
Alfonso Interpreting is a unique language service company offering interpretation, translation, and safety training.
Our mission is to improve lives by removing language barriers and replacing them with clear, accurate, and transparent communication.
We save lives by creating an inclusive safety culture that takes into consideration the linguistic and multicultural variety our clients enjoy.
Insistence on professionalism helps us avoid common pitfalls in the profession. Free-flowing communication promotes efficiency, commerce, and business success.
Do you know the difference between an interpreter and a translator?
Translators write. Interpreters speak.
For as long as mankind has spoken multiple languages, interpreters have existed. They have been present for peace treaties among nations and have always been essential for trade. Interpreters are necessary for due process in court and have aided medical professionals in saving many lives.
Shortly after humankind began to speak, they began to write. Diplomacy and trade documents require competent translation. Evidence in court must be translated in order to determine a just decision. Written medical instructions are useless if a patient can't read them, requiring the need of a skilled translator.
Both interpreting and translation are beautiful and noble sister professions!


text:
Audrey Focht and Robert G. Hetten Jr. exchanged wedding vows yesterday in SalemUnited Church of Christ, Catasauqua.
Connie Focht was maid of honor for her sister and Greg Buckman was best man.
The couple's parents are Mr. and Mrs. Peter J. Focht and Mr. and Mrs. Robert G. Hetten, all of Whitehall.
The couple are graduates of Whitehall High School. Mrs. Hetten also graduated from Lehigh County Vocational-Technical School and is employed at Laneco, Inc., Coplay. Her husband graduated from Pennsylvania State University and is employed by Boyle Agency, Inc., Whitehall.
They will reside in Catasauqua.


text:
Having consumers buy products online while watching one of their much-loved episodes is the ultimate dream for marketers, so Target decided to experiment with this idea. The brand turned the sitcom Cougar Town into an all-out Target ad, allowing audience members to click on featured items (if watching online). Once clicked, viewers would be led to a link where these products could be purchased.
Product placement is nothing new in advertising, but this strategy takes it to a whole new level. Integrating links and highlighting the products that can be bought within an episode makes it that much easier for consumers to buy, which is exactly what companies like Target wants. The only question is how much does it take away from the overall viewing experience? Only time will tell.
Product-Integrated Sitcoms
More Stats +/-
Target Couture
Affordable Luxury
Manhattan Pop-Up Shops
Mass-Market High Fashion
Modern Milk Container Storage


text:
There are so many ads being announced by the presidential campaigns and their surrogates that it's tough to tell what's really running these days. Most are pretty negative.
Not so Mitt Romney's latest spot, "Bringing People Together," which excerpts snippets his first debate. What makes the spot stand out was the pivot toward bipartisanship and the fact that it's a positive spot. It ran during 60 Minutes in the Miami media market -- Florida's most expensive.
Liberals will complain that this isn't the real Romney, the guy who trashed the so-called 47 percent. And it's not like Romney or his surrogates (or Obama or his surrogates) have run a positive campaign. Indeed, Karl Rove's American Crossroads latest Obama-basher ran a few moments after Romney's spot.
But Romney's looking all sunny these days. An unwritten rule of thumb in politics is that, if you're ahead, you stay positive. And by that standard (and the polls) Romney looks like he's ahead and that this is part of his closing argument.