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|Glutamine acts as a neuroprotectant against DNA damage, beta-amyloid and H2O2-induced stress.
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|PMID: 22413000 Owner: NLM Status: MEDLINE
|Glutamine is the most abundant free amino acid in the human blood stream and is 'conditionally essential' to cells. Its intracellular levels are regulated both by the uptake of extracellular glutamine via specific transport systems and by its intracellular synthesis by glutamine synthetase (GS). Adding to the regulatory complexity, when extracellular glutamine is reduced GS protein levels rise. Unfortunately, this excess GS can be maladaptive. GS overexpression is neurotoxic especially if the cells are in a low-glutamine medium. Similarly, in low glutamine, the levels of multiple stress response proteins are reduced rendering cells hypersensitive to H(2)O(2), zinc salts and DNA damage. These altered responses may have particular relevance to neurodegenerative diseases of aging. GS activity and glutamine levels are lower in the Alzheimer's disease (AD) brain, and a fraction of AD hippocampal neurons have dramatically increased GS levels compared with control subjects. We validated the importance of these observations by showing that raising glutamine levels in the medium protects cultured neuronal cells against the amyloid peptide, Aβ. Further, a 10-day course of dietary glutamine supplementation reduced inflammation-induced neuronal cell cycle activation, tau phosphorylation and ATM-activation in two different mouse models of familial AD while raising the levels of two synaptic proteins, VAMP2 and synaptophysin. Together, our observations suggest that healthy neuronal cells require both intracellular and extracellular glutamine, and that the neuroprotective effects of glutamine supplementation may prove beneficial in the treatment of AD.
|Jianmin Chen; Karl Herrup
Publication Detail:
|Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't Date: 2012-03-08
Journal Detail:
|Title: PloS one Volume: 7 ISSN: 1932-6203 ISO Abbreviation: PLoS ONE Publication Date: 2012
Date Detail:
|Created Date: 2012-03-13 Completed Date: 2012-08-02 Revised Date: 2013-06-26
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|Nlm Unique ID: 101285081 Medline TA: PLoS One Country: United States
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|Languages: eng Pagination: e33177 Citation Subset: IM
|Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey, United States of America. firstname.lastname@example.com
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|MeSH Terms
Alzheimer Disease
Amyloid beta-Peptides / metabolism*, toxicity
Cell Line
Cell Survival / drug effects
DNA Damage / drug effects*
Disease Models, Animal
Gene Expression
Glutamate-Ammonia Ligase / genetics, metabolism
Glutamine / metabolism, pharmacology*
Hippocampus / metabolism
Hydrogen Peroxide / metabolism*
Neurodegenerative Diseases / genetics, metabolism
Neurons / drug effects, metabolism
Neuroprotective Agents / pharmacology*
Stress, Physiological / drug effects*
|Grant Support
Reg. No./Substance:
|0/Amyloid beta-Peptides; 0/Neuroprotective Agents; 56-85-9/Glutamine; 7722-84-1/Hydrogen Peroxide; EC 220.127.116.11/Glutamate-Ammonia Ligase
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Journal ID (nlm-ta): PLoS One
Journal ID (publisher-id): plos
Journal ID (pmc): plosone
Publisher: Public Library of Science, San Francisco, USA
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Chen and Herrup. 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.
Received Day: 26 Month: 9 Year: 2011
Accepted Day: 5 Month: 2 Year: 2012
collection publication date: Year: 2012
Electronic publication date: Day: 8 Month: 3 Year: 2012
Volume: 7 Issue: 3
E-location ID: e33177
PubMed Id: 22413000
Publisher Id: PONE-D-11-18935
DOI: 10.1371/journal.pone.0033177
|Glutamine Acts as a Neuroprotectant against DNA Damage, Beta-Amyloid and H2O2-Induced Stress Alternate Title:Glutamine Protects Neurons against Stresses
|Jianmin Chenaff1*
|Karl Herrupaff1
|Hyoung-gon Leeedit1
Role: Editor
|Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey, United States of America
|Case Western Reserve University, United States of America
|Correspondence: * E-mail: email@example.com
Contributed by footnote: Conceived and designed the experiments: JC KH. Performed the experiments: JC. Analyzed the data: JC KH. Wrote the paper: JC KH.
Glutamine is the most abundant free amino acid in the human blood stream. It is typically classified as a 'non-essential' amino acid because it can be made from TCA metabolites by most cells. A more accurate classification of the body's need for glutamine, however, would be the term 'conditionally essential'. Many cell types are unable to survive in the complete absence of glutamine. Indeed, in certain B-cell lines supranormal concentrations are required. The value of glutamine is particularly apparent during stress. It becomes essential in organs or organ systems weakened by sickness, surgery or injury. Glutamine can regulate a variety of target genes involved in cell proliferation, differentiation and survival. It accomplishes this by altering the behavior of a range of transcription factors, including NFκB, through which the anti-inflammatory role of glutamine may be mediated [1]. A molecular explanation for the broad cellular dependence on glutamine remains elusive, but a key insight has emerged from recent studies showing that high intracellular glutamine is rate limiting for the uptake of several essential amino acids through the SLC7A5/SLC3A2 bidirectional transporter [2].
In brain, the majority of endogenous glutamine is produced by glutamine synthetase (GS), which catalyzes the formation of glutamine from glutamate and ammonia. Although all cells express GS to some extent, in the adult brain its levels are 40-fold higher in astrocytes than in neurons [3], [4], [5]. In adult brains, GS is neuroprotective [6], [7] and during embryogenesis functional GS is crucial for brain development. This can be seen from the finding that congenital GS deficiency causes brain malformation and neonatal death both in human and in mouse [8], [9]. GS responds to a variety of insults including oxidative stress, inflammation, and viral infection [10], [11], [12], [13], [14], suggesting a connection to neurodegenerative disease. Indeed, changes in GS level, activity and modifications have been documented in AD patients. Monomeric GS protein was found in 38 of 39 cerebrospinal fluid (CSF) samples obtained from AD patients [15], and the concentration of GS is significantly increased in AD CSF [16]. GS levels are also significantly higher in prefrontal cortex of AD patients than they are in non-demented controls [17]. Complicating the interpretation of these alterations in the amount of GS protein, the activity of GS is vulnerable to mixed-function oxidation which rises exponentially with age. Oxidized GS has reduced activity and is preferentially degraded [18]. This oxidation-induced loss of GS activity is brain region specific; it happens at double the rate in frontal lobe compared to the occipital lobe. Importantly, the decrease is more significant in frontal cortex from AD patients than from age-matched controls [19]. Proteomic analysis has identified GS as one of the cellular proteins most prone to oxidation after Aβ1–42 treatment in vitro[20]. In vivo, GS is one of the 3 hippocampal proteins (along with PIN1 and ENO1) that are found to be significantly oxidized in both MCI and AD compared to controls [13]. The suggestion is that although GS protein levels may rise, GS activity is compromised early in the Alzheimer's disease process, the deficit persists throughout the course of the disease, and perhaps like tau, the quantities in CSF increase as the GS protein in brain cells becomes increasingly dysfunctional.
In this study, we have explored the effect of glutamine and GS supplementation on the survival of both N2a cells and primary neurons. We compare the effects of altering endogenous glutamine (through manipulations of intracellular GS and its activity) with those found by altering the levels of exogenous glutamine. We document an unexpectedly complex relationship suggesting that both intracellular and extracellular glutamine are essential for neuronal health. To make the link to neurodegenerative disease, we extend these findings to the in vivo situation. We have used two mouse models of AD to test the effect of dietary supplementation of glutamine on the pathological features of the models. In the aggregate, our data suggest that glutamine may have significant neuroprotective effects that help restore homeostatic functions that are lost in AD.
Human frozen tissues and formalin-fixed sections were obtained from the Alzheimer's Disease Research Center, Washington University School of Medicine and the Alzheimer's Disease Research Center, Case Western Reserve University. Experimental procedures involving these samples were approved by the IRB of above two institutions and the Rutgers University IRB. All animal procedures carried out in this study were in accordance with Rutgers University IACUC standards, approval ID: 06-027.
Frozen brain tissues or formalin-fixed/paraffin-embedded 10 µm sections from AD and age-matched controls were obtained from the Alzheimer's Disease Research Centers at Case Western Reserve University and Washington University School of Medicine. Some of the cases used for this study had been clinically diagnosed with AD and were subsequently confirmed with standard pathological examination. Other cases died with no explicit diagnosis of their dementia, but were scored on neuropathological examinations as being either Braak stage V or VI. For the purposes of this study, we defined these cases as AD.
Two AD mouse models were examined [21] in this study. Both carry the entire human APP gene inserted into the mouse genome from a microinjected yeast artificial chromosome. Mice from the R1.40 line (B6•129-Tg(APPSw)40Btla/J) carry the human APP gene with the Swedish mutation for familial AD – K670N/M671L. Mice from the 8.9 line (B6•129S2-Tg(APP)8.9Btla/J) are similar to R1.40 animals but carry a wild type APP gene. Both transgenic lines express all mRNA and protein isoforms of the human gene in a correct spatiotemporal pattern.
Embryonic cortical neurons were isolated by standard procedures. E16.5 embryonic cerebral cortices were treated with 0.25% Trypsin-EDTA and dissociated into single cells by gentle trituration. Cells were suspended in Neurobasal medium supplemented with B27 and 2 mM glutamine, then plated on coverslips or dishes coated with poly-L-Lysine (0.05 mg/mL). All cultures were grown for a minimum of 5 days in vitro (DIV) before any treatment. Murine neuroblastoma N2a cells were purchased from ATCC (Manassas, VA, USA) and cultured in standard DMEM media supplemented with FBS (2% for differentiation, 10% for routine passage) or Neurobasal+B27 with or without glutamine. Synthetic amyloid-β peptide (Aβ1–42) was purchased from AnaSpec, Inc. (San Jose, CA). Peroxide oxidized Aβ1–42 dimer was prepared from synthetic human Aβ1–42 (5 µM) by incubation in PBS in the presence of hydrogen peroxide plus Cu2+ as previously described [22].
An expression construct encoding human GS was purchased from Origene (Cat# SC118847, pCMV6-XL5-GLUL, GenBank accession No.: NM_002065). The disease-related mutation (1021C→T) was introduced by means of site-directed mutagenesis using the system developed by Mutagenex, Inc. The success of the mutagenesis was confirmed by DNA sequencing using a GS internal forward primer (Flk-F): [gene: 5′-GACCCCTTCCGTAAGGACCC] and a vector reverse primer (pCMV6 Seq-R): [gene: 5′-TTAGGACAAGGCTGGTGGGCAC]. Sub-confluent N2a cells were transfected with either Lipofectamine™ 2000 or Lipofectamine™ LTX with PLUS™ Reagent (Invitrogen) following the manufacturer's suggested procedure.
For Western blots, protein extracts from tissues or cultures were made in RIPA buffer with protease inhibitors and phosphatase inhibitors, and then separated in 4–20% SDS–PAGE gel.
All antibodies used are commercially available. Antibodies against GS, 53BP1, ATM, phospho-S1981-ATM (PS1981-ATM), cleaved caspase 6, LC3, LC3B,, PCNA, cyclin A, PSD-95 and VAMP2 were all purchased from Abcam. Antibodies against cleaved caspase 3, ATR, phospho-S428-ATR (PS428-ATR), and phospho-S807/811-retinoblastoma (PS807/811-RB) were from Cell Signaling. The Map2 antibody was purchased from Sigma. Antibodies against γH2AX, Tau3R, Tau4R and phospho-tau (AT8) antibodies are all mouse monoclonals purchased from Millipore or Thermo. Synaptophysin antibody was from Invitrogen. β-actin antibodies were from Santa Cruz Biotechnology. Iba1 antibody was from Wako (Japan). The specificity of these antibodies has been established previously and confirmed by us through the use of Western blots.
The comet assay procedure was carried out using OxiSelect ™ Comet Assay Kit from Cell Biolabs following the procedure suggested by the manufacturer. Briefly, cultured cells were detached from the culturing vessels by trypsinization. Cells were then mounted in soft gels and electrophoresed for 15 minutes at 1 V/cm. After electrophoresis, slides were washed in water and dried at room temperature. Finally, slides were stained with Vista Green DNA dye (Cell Biolabs, Inc.) and visualized under a Leica DM5000B fluorescent microscope or a Zeiss LSM 510 confocal microscope using the FITC filter.
Each mouse was transcardially perfused with 4% paraformaldehyde under deep Avertin anesthesia, after which the brain was dissected from the skull and post-fixed overnight. For most material, the brain was embedded in paraffin and sectioned at 10 µm on a rotary microtome. The human materials were formalin-fixed, paraffin-embedded and sectioned at 10 µm. These were treated as described previously [23]. For immunohistochemistry, all paraffin sections underwent antigen retrieval with high temperature citrate buffer for 20 min, and then soaked in 0.3% hydrogen peroxide to remove endogenous peroxidase activity. Primary antibody was diluted in 10% goat serum with 0.5% Tween-20. Primary antibodies were detected using biotinylated goat anti-rabbit or anti-mouse secondary antibody (1∶400), avidin-biotin complex horseradish peroxidase and peroxidase substrates including DAB, DAB+Ni and VIP (Vector Laboratories). Some sections were counterstained with hematoxylin QS. For immunofluorescence, mouse brains were embedded in OCT (Tissue-Tek) and sectioned on a cryostat at 10 µm. Sections were pretreated with citrate buffer for 10 minutes, and then incubated with primary antibody. Alexa linked secondary antibodies were used to detect the presence of the antigens. Stained sections were photographed and viewed at a final magnification of 200 using Leica Application Suite/Leica DM5000B.
For both LDH assay and MTT assay, cells were cultured in media without phenol red. The LDH activity of the supernatant was measured by Promega CytoTox 96® Non-Radioactive Cytotoxicity Assay, according to the manufacturer's instructions, recording absorbance at 490 nm using absorbance at 650 nm as reference. Cell viability (MTT assay) was evaluated using a Promega CellTiter 96® Non-Radioactive Cell Proliferation Assay kit following the manufacturer's instructions, recording absorbance at 570 nm using absorbance at 650 nm as reference.
Neurons take up exogenous glutamine as part of the glutamate/glutamine cycle. Brain astrocytes clear glutamate from the synaptic cleft, convert it glutamine and secrete it to the extracellular space. Here it is picked up by neurons and converted back into glutamate for use as a transmitter or used as a precursor for other cellular components. Astrocytes typically maintain GS levels that are readily visualized by immunocytochemistry; in neurons under normal physiological conditions, GS is nearly undetectable. When exogenous glutamine levels drop, however, neurons can be induced to express GS [11]. Thus, although the levels of exogenous glutamine can be easily regulated, the levels of endogenous glutamine are a more complex function of uptake plus the activity of GS.
We found that in cultures of primary cortical neurons, reduction of exogenous glutamine had a small negative effect on neuronal viability (Figure 1A, C; quantified in Figure 1E). As the full effects of low media glutamine on the levels of intracellular glutamine might be offset by GS-induced glutamine synthesis, we tested the effects of the GS-inhibitor, methionine sulfoximine (MSO). MSO is the best known inhibitor of GS. It was originally isolated from nitrogenchloride-treated zein as the toxin responsible for the induction of convulsions, hysteria and epileptic fits in a number of animals (see reviews by Eisenberg et al. 2000) [24]. When added to neuronal culture media in the presence of 2 mM glutamine, MSO alone also caused the death of only a small number of neurons (Figure 1B, E). By contrast, when MSO was added to cells grown in glutamine-free medium (thus reducing both endogenous and exogenous glutamine), massive neuronal cell death was observed (Figure 1D, E). The reductions in exogenous and endogenous glutamine were synergistic. In normal medium there is a baseline level of caspase-3 labeling of about 7–8% of the total number of cells. MSO alone increased the rate of cell death by ∼3-fold; glutamine removal from the medium alone increased cell death by ∼2-fold. Applying MSO in the absence of glutamine, however, increased cell death by ∼8-fold. Thus, a healthy neuron requires glutamine both in the media and synthesized internally by the actions of GS.
In normal human hippocampus, astrocytes were the only cell type with detectable levels of GS by immunocytochemistry (Figure 2A). In AD patient samples, consistent with previous reports [25], we found numerous hippocampal neurons with elevated levels of GS (arrows, Figure 2A). Astrocyte staining was not noticeably altered in these AD samples. The percentage of GS-positive AD pyramidal neurons varied from subject to subject and from region to region, ranging from 1–50% of the total population. In the 4 control cases we examined, GS-positive neurons were rare (<1%). We validated these findings by Western blot using frozen brain samples from prefrontal cortex (Brodmann area 9 – BA9) of 20 additional subjects (12 AD; 8 control). As with the hippocampal immunohistochemistry, there was variability among the samples. These blots clearly indicate, however, that GS levels were elevated in frontal cortex of AD patients compared with controls (Figure 2B). When these results were averaged across all subjects, the increases in GS monomer and a higher molecular weight species that is likely to be a GS dimer were both highly significant (Figure 2C).
To verify that up-regulation of GS in neurons is triggered by glutamine deprivation, we cultured mouse primary cortical neurons (DIV 7–8) in Neurobasal media with or without glutamine, in the presence or absence of 5 mM MSO, for 3 days before collecting samples for Western blotting and immunocytochemistry. In normal Neurobasal medium with 2 mM glutamine, neurons express only trace amount of GS. As predicted by the in vivo work, GS expression increased robustly when primary neurons were deprived of glutamine either by removing exogenous glutamine from the media (Figure 2D, lane 1) or by blocking endogenous GS activity by MSO (Figure 2D, lane 4). The same relationships were found in N2a cells, a murine neuroblastoma cell line (Figure 2E).
Deprivation of exogenous glutamine can cause higher GS expression in neurons. The question we next addressed was whether this de novo expression of GS in neurons is protective or detrimental. Our results further underscore the complex nature of glutamine regulation in cells. We tested two GS constructs expressing the human GS transcript 1: wild type GS (GSWT) and an activity-deficient mutant, GSR341C. The GSR341C construct harbors a point mutation, R341C (a T→C change at nucleotide 1021 of human GS transcript 1), and encodes a peptide with reduced GS activity [8]. N2a cells were transfected with either construct or treated with only transfection reagents as a negative control. After 24 hours, culture media were replaced with differentiation media (Neurobasal without glutamine). After another 24 hours, cells were treated with three different stressors – 100 µM H2O2, 100 nM oxidized Aβ1–42 (oxyAβ), or 5 µM etoposide – for 24 hours in the same media. Then cells were processed for Western blotting or immunocytochemistry. Extensive degeneration of both processes and cell bodies was observed in GSWT but not GSR341C -transfected cells (Figure 3A). The levels of cleaved caspase-3 and caspase-6 were substantially higher in cells with high GS activity (GSWT transfection) than in cells expressing the GSR341C mutant (Figure 3B), even though comparable amount of the two proteins were present. We repeated the transfections using cells grown in medium containing 2 mM glutamine. Intriguingly, with ample exogenous glutamine, high GS expression reduced caspase-3 activity – as measured by the cleavage of key cellular proteins such as retinoblastoma (RB, Figure 3C, D). To directly evaluate cell death we treated N2a cells with H2O2 and measured the lactate dehydrogenase (LDH) activity in culture media, The LDH results confirmed the caspase 3 and caspase 6 data (Figure 3E). First, low glutamine increases cell death and second, the difference between wild type and mutant GS is only seen in low glutamine. Viewed together, our data point to the conclusion that high levels of exogenous glutamine are highly beneficial to neuronal cells whereas treatments that lead to high GS activity may be useful only in certain conditions.
The results in Figure 3 show that cells in low glutamine are more sensitive to the effects of a variety of different stressors. To explore this effect further, we treated N2a cells grown in normal or reduced glutamine with H2O2 (1 mM – a model of oxidative stress); ZnSO4 (0.2 mM – a model of heavy metal toxicity); and etoposide (20 µM – a model of DNA damage). Cells were stressed for 30 minutes then harvested for Western blot analysis. The results in Figure 4A illustrate that cells grown in any medium can respond to each of these conditions. They up-regulate their levels of several stress response proteins such as 53BP1 and appropriately modify other proteins either by phosphorylation (ATM) or cleavage (caspase-3). The magnitude of all of these responses, however, is considerably diminished in low glutamine. For example, in all conditions, the levels of 53BP1 protein increased in response to stress. In low glutamine, however, the final levels of protein were significantly less than in normal glutamine. Activation of ATM and ATR by phosphorylation revealed a similar picture. Under oxidative stress or etoposide-induced DNA damage, the activating phosphorylation of both proteins increased. Etoposide treatment drove robust ATM auto-phosphorylation, this change in phosphorylation led to an actual change in ATM kinase activity can be seen in the increased levels of γH2AX, while the phosphorylation of ATR was driven preferentially by ZnSO4. For each of these measures of stress response, however, the magnitude of the response in low glutamine was significantly less than in normal exogenous glutamine. One likely reason for this difference is that the levels of total ATM and total 53BP1 are reduced in low glutamine (Figure 4A–B). Viewed as a whole, the data in Figure 4A suggest that in many different domains, cells in low glutamine are not well prepared for stress.
We presumed that the findings in Figure 4A reflected the reduced levels of the various damage response proteins. This assumes, however, that the levels of damage induced by the various stressors are equivalent in low glutamine. To address this concern, we examined the response of the cells to etoposide since DNA damage can be easily monitored by Comet assay. In the absence of etoposide, DNA damage is minimal, even in low glutamine (Figure 4C). After 30 min in 5 µM etoposide, DNA fragmentation is easily revealed by the appearance of significant comet tails (top row, Figure 4C). We then removed the etoposide and followed the cells' ability to repair their DNA. Within 4 hours, the Comet tails found on cells in both culture conditions disappeared, indicating the cells were able to join the majority of their DNA breaks, with or without glutamine (Figure 4C). Yet while the DNA might have been "repaired" in both situations, there was significantly more cell death in the glutamine-deprived cultures as assessed by the number of pycnotic nuclei (Figure 4D). The majority of this death is likely necrosis not apoptosis because markers of apoptosis (activated caspase-3) and autophagy (LC3-II) were higher in cultures with glutamine than in those without glutamine (Figure 4A, B). To directly evaluate cell death and viability, we also performed LDH assay after a 6 hour recovery (Figure 4E) and MTT assay after a 24 hour recovery (Figure 4F) from a 30 minutes H2O2 challenge. Collectively, glutamine is essential for the stress response and post damage survival of cells.
N2a cells grown in low glutamine are sensitive to toxic effect of DNA damage and oxidative stress, likely due to low expression of stress response proteins such as ATM and 53BP1(Figure 4A–B). However, this impaired response capability is not unique to the N2a cells; similar reductions of ATM, ATR and 53BP1 are detected in primary neurons deprived of glutamine (Figure 5A). Like N2a cells, neurons grown in low glutamine were more sensitive to DNA damage as well (Figure 5B). The importance of glutamine to the survival of neuronal cells prompted us to examine the effect of glutamine supplement on Aβ-induced toxicity. Primary neurons were cultured in medium with 2 mM glutamine (replaced every 2–3 days) until DIV 10–15. Cells were then re-fed with fresh medium with or without glutamine. Twenty-four hours later, the cells were treated with 100 nM oxyAβ for additional 24 hours; oxyAβ has been shown to more readily form neurotoxic oligomers and is more potent than un-oxidized Aβ in its ability to induce cellular stress in neurons [22]. We monitored the neuronal response using the AT8 phospho-tau antibody. Although oxyAβ induced tau phosphorylation in cultures with or without glutamine, the levels of phospho-tau were significantly reduced in the presence glutamine (Figure 5C). The effects of glutamine were dose dependent as can be seen from the effects on tau phosphorylation (Figure 5D). Even neurons in long-term established cultures responded to 3 days of glutamine deprivation and Aβ treatment by showing more processes beading (Figure 5E) and less synaptophysin staining (Figure 5F) suggesting reduced synaptic density.
These in vitro findings provide multiple pieces of evidence that under conditions of neuronal stress such as is found in neurodegenerative disease, glutamine might have significant clinical value. The Aβ results further suggest that Alzheimer's disease might be a prime candidate for intervention. To test this concept in a preclinical disease model, we examined the effects of dietary glutamine on the phenotypes of the R1.40 and 8.9 transgenic mouse strains. Both lines carry an entire genomic copy of the region of human chromosome 21 with the APP gene [21]. The R1.40, mice develop amyloid plaques by 13 months (on a C57BL/6J background); 8.9 mice remain plaque-free throughout their lives on this background. Both lines develop neuronal cell cycle events in brain regions homologous to those affected in humans [26], [27]. These cell cycle events are robust outcome measures that are useful in tracking impending neurodegenerative events. They are induced by the AD-related brain inflammation as they can be prevented by NSAID treatment or advanced by an additional immune challenge such as LPS injection [28]. Given that LPS infusion in healthy human subjects causes a dramatic depletion of glutamine from their brains [29], we speculated glutamine may have a significant effect on LPS-treated R1.40 and 8.9 mice.
One-year-old R1.40 and APP8.9 mice were fed 4% glutamine in their drinking water for 10 days. The following day they were injected with a low dose of LPS to induce systemic inflammation [28], and sacrificed 2 days later for analysis. Control mice were treated the same way except that they were kept on regular drinking water. We had 3 mice in the glutamine supplement group and 4 mice in the control group. We did extensive comparison of all the markers we used and found no differences between APP8.9 and R1.40. The only exception was the transgene encoded APP; R1.40 mice had higher APP expression than APP8.9 mice, as expected. Instead, we found a significant difference between mice with or without glutamine in their drinking water. As expected, in mice fed with normal drinking water (Gln−), LPS treatment was accompanied by microglial activation, as can be seen from the thickening and shortening of their Iba-1-stained processes (Figure 6A). In mice supplemented with glutamine, however, LPS failed to induce activation. Microglia in these mice (Gln+) showed the normal resting phenotype with thin and long Iba-1-positive processes. Glutamine supplementation also led to a reduced level of tau phosphorylation, as well as ATM phosphorylation (Figure 6B–C). The expression of the cell cycle maker, PCNA (Figure 6B–C) was also reduced with glutamine supplementation. Immunostaining using cyclin A antibody corroborated the PCNA results (Figure 6D, arrows). VAMP2, one of the synaptic vesicle SNARE proteins that is reduced both in AD models and in AD patients [29], was restored by glutamine supplementation, as was synaptophysin (Figure 6B–C). The levels of PSD95, however, remained unchanged. Thus, a 10-day regimen of glutamine supplementation was sufficient to block the expression of several indices of neurodegenerative disease in two AD mouse models.
Our data provide strong support for a neuroprotective effect of glutamine supplementation in a variety of different situations. The molecular nature of this protection is complex and reflects the levels intra- and extra-cellular glutamine as well as the levels and state of oxidation of the synthetic enzyme, glutamine synthetase (GS).
Neurons in normal situations express little GS, but when exogenous levels of glutamine fall, we find that neuronal GS expression increases significantly. This is consistent with repeated observations that the activity of GS is regulated by cumulative feedback inhibition and is dictated by the overall cellular demand for glutamine [30]. It is also consistent with the finding that patients with congenital glutamine deficiency resulting from GS mutations (R324C and R341C) have much higher expression of GS message than either their parents or normal controls [8]. As the specific activity of R324C and R341C is reduced from wild type, the suggestion is that GS activity, rather than protein concentration, is the attribute that regulates the level of GS transcription.
These observations have direct relevance for neurodegenerative disease as reduced glutamine/glutamate levels have been reported in the AD brain [31], [32], [33]. As might be predicted, in regions affected by AD pathology, many neurons are found to express abnormally high levels of GS (e.g., Figure 2A and earlier reports [25], [34]). Manipulating GS levels, however, carries risk as a therapeutic target. While, increasing GS might help to raise intracellular glutamine, in an environment of low exogenous glutamine degeneration is induced (Figure 3). Further, the complex chemistry of the AD brain worsens the situation since increased levels of catalytically active GS renders a cell hypersensitive to the toxic effects of Aβ and other stressors, an outcome to be avoided in AD.
Manipulation of glutamine itself would seem to have fewer such risks. In addition to the direct neuroprotective effects, there is a significant anti-inflammatory action of glutamine supplementation (Figure 6A). Perhaps because the immune system must overcome this effect to be fully reactive, the pro-inflammatory cytokine, TNFα, has been shown to reduce GS expression in astrocytes [10]. This would be consistent with observations that systemic inflammation induced by LPS injections in human volunteers reduces the glutamine pool in brain [35]. Chronic inflammation has emerged as a candidate for the one of the central pathogenic features of AD [36]. These data suggest, therefore that glutamine supplementation may be beneficial to AD patients as it should be anti-inflammatory in the brain. In addition, it would appear that glutamine supplementation improves the DNA damage response (Figure 4). This may well explain the success of glutamine supplementation in mitigating the harmful side effects of radiotherapy for various cancers [37], [38]. This effect also has relevance for late-onset diseases such as AD as it has been argued that DNA damage is a direct cause of aging [39]. The cellular DNA damage response system is a complex network of proteins that protect cells by either fixing the damage or forcing cells into apoptosis or senescence. If the repair process is blocked, however, some DNA will stay unrepaired and the cells will live. This phenomenon is more prevalent in non-replicating or slowly replicating cells, such as neurons in the brain, which cannot rely on DNA repair mechanisms associated with DNA replication. This is not a small problem. In humans, approximately 800 DNA lesions occur per hour in each cell – about 19,200 per cell per day [40]; and all pathways of DNA repair become less efficient with age [41]. ATM expression goes down dramatically in aging tissues [42] and is further reduced in AD frontal cortex compared to controls (unpublished data). Our observations that expression of both ATM and its downstream target, 53BP1, are substantially lowered in cells deprived of glutamine suggest that low glutamine supply may contribute to this phenomenon. Viewed in a therapeutic light, however, the same data provide reason to believe that glutamine supplementation may restore the aging brain to a more youthful state when measured by the capability of its stress responses.
Our data also show that glutamine deprivation reduces autophagy under both basal and stressed conditions (Figure 4B). This observation is consistent with a recent study showing that glutamine increases autophagy under basal and stressed conditions in intestinal epithelial cells [43]. In fact, autophagy and apoptosis are positive stress responses of cells that ensure the health of cells and organisms by replacing spent or damaged cellular components [44]. Given the fact that autophagy in AD is reduced, we can interpret these observations to mean that in this domain as well, glutamine supplement will be beneficial to the AD patients.
While our findings underline the complexity of an organism's response to fluctuations in the levels of glutamine, they also demonstrate that glutamine supplementation in vitro enables nerve cells to resist stresses similar to those that affect the human brain during the course of Alzheimer's disease. More intriguing still is our finding that glutamine supplementation may have a protective effect on AD pathogenesis in vivo. Two different mouse models of familial AD responded in positive ways to a relatively short (10-day) nutritional supplement of glutamine in their drinking water. The levels supplied were comparable to those used clinically in human subjects and the strong interference with the neurodegenerative phenotype of induced neuronal cell cycle events and lost synaptic markers suggests that this strategy might be a useful and cost-effective adjunct to other therapeutic measures. In summary, an optimal level of glutamine is important for aging neurons to respond to stress. Our data provide strong support for the protective effect of glutamine supplementation to avoid the neurodegeneration of Alzheimer's and other degenerative diseases.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The work was supported by funding awarded to KH from the Alzheimer's Association – Zenith Award, NIA (AG024494), NINDS (NS20591), and Rutgers University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors wish to thank Yuxin Xie for her help with the Image J software, Timmy Cheung for his assistance with primary neuronal culture, and Noriko Goldsmith for her help with confocal microscope. The contributions of the AD cases and related controls from the Neuropathology Core of the Alzheimer's Disease Research Center at Washington University in St. Louis and the Alzheimer's Disease Research Centers at Case Western Reserve University are gratefully acknowledged. We also thank Drs. Changshun Shao, Jiali Li, Jie Zhang and Mark Plummer for valuable discussions during the preparation of this manuscript.
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|Zou J,Wang YX,Dou FF,Lu HZ,Ma ZW,et al. Year: 2010Glutamine synthetase down-regulation reduces astrocyte protection against glutamate excitotoxicity to neurons.Neurochem Int5657758420064572
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|Visalli V,Muscoli C,Sacco I,Sculco F,Palma E,et al. Year: 2007N-acetylcysteine prevents HIV gp 120-related damage of human cultured astrocytes: correlation with glutamine synthase dysfunction.BMC Neurosci810618062818
|Butterfield DA,Poon HF,St Clair D,Keller JN,Pierce WM,et al. Year: 2006Redox proteomics identification of oxidatively modified hippocampal proteins in mild cognitive impairment: insights into the development of Alzheimer's disease.Neurobiol Dis2222323216466929
|Lee A,Lingwood BE,Bjorkman ST,Miller SM,Poronnik P,et al. Year: 2010Rapid loss of glutamine synthetase from astrocytes in response to hypoxia: implications for excitotoxicity.J Chem Neuroanat3921122020034557
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|Tumani H,Shen G,Peter JB,Bruck W. Year: 1999Glutamine synthetase in cerebrospinal fluid, serum, and brain: a diagnostic marker for Alzheimer disease?Arch Neurol561241124610520940
|Burbaeva G,Boksha IS,Tereshkina EB,Savushkina OK,Starodubtseva LI,et al. Year: 2005Glutamate metabolizing enzymes in prefrontal cortex of Alzheimer's disease patients.Neurochem Res301443145116341942
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Question: The retail store in which I work is full of beautiful things. I have applied feng shui principles to the store and feel that it is in fairly good shape. The one problem is, that customers walk in the front door, look around in a dazed manner then walk right out of the back door without really stopping to take a good look. What feng shui treatment would you suggest to slow someone down a bit on their path through the store?
Answer: First, congratulations on applying feng shui in your retail store! If you take care of it consistently, meaning keep the quality of feng shui energy in your space fresh and flowing, you will definitely enjoy good results! You can also go deeper and apply feng shui for specific areas, such as attracting more wealth, or more recognition for your business; for this work you will need to know the bagua, or the feng shui energy map of your store.
Let me direct you to some quick tips on how to take care of the energy of any space, and then I will answer your question.
Now let me address your specific concern. From your question I assume that there are 3 feng shui retail points in need of attention:
1. You store has too many attractive items with no breathing room in between them or clarity as to their selection.
Feng shui tips: Create some breathing room and open space between the areas of interest, this will help avoid the "dazed look" you mentioned. People usually look this way when there is too much merchandise presented in a monotonous way. Spread out the beautiful items, or pair them in unexpected ways. In other words, jazz up the store!
2. Your store has a rushed, direct flow of feng shui energy from the main door to the back door, which leads to losing energy and potential sales.
Feng shui tips: You need to draw the customers in as they enter the store. Do that with strong focal points, clarity of the selections you offer, freshness of energy, etc. Do not have the back door strongly visible as you enter the store. If you have direct door alignment, meaning a straight line from the main door to the back door, arrange your displays in angled, or circular motion in order to remedy the direct line of energy.
3. There are no clear and fluid pathways for customers to explore the store.
Feng shui tips: Imagine your store as a park, or a little country of its own and develop/create more than one pathway to explore your store. Create unexpected moments, fresh combinations, colorful islands of interest, etc. In other words, satisfy your customers' sense of curiosity, but without creating more guessing work for them. Clarity, enough breathing room and beauty, some fun and surprise - these elements always work well in retail.
Last, but not least, do not hesitate to ask a customer, or a friend, to give you honest feedback as to why they would (or would not) spend more time in your store. Stay attuned to your customers' emotional needs and be willing to change your store as often as it takes until you no longer get that dazed look you mentioned.
Read this feng shui in retail article with basic feng shui tips for improving a retail space.
Good luck with transforming your retail store!
Continue Reading: 10 Feng Shui Tips To Improve Your Retail Store

subset: commoncrawl


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SAN FRANCISCO – The San Francisco Recreation and Park Department announced today the launch of a series of free holiday sports at Embarcadero Plaza. The Plaza already features the Holiday Ice Rink, located at heart of the Embarcadero Plaza. Now the Department is adding free, fun, drop-in, sports including Disc Golfing, Skateboarding, Rock Climbing, Slacklining, and Bocce Ball games on the south side of the Plaza. The goal is to offer free fun recreation for park constituents living, working and visiting the City's open space in the waterfront area.
"We are fortunate to live in a City where we can enjoy winter traditions such as ice skating at the same time as we bike, roller skate and rock climb," said Mayor Edwin M. Lee. "As the heart of both or our downtown and waterfront areas, the Embarcadero Plaza is a perfect spot to host such a wide array of activities and athletic events. Our residents and visitors will be able to enjoy a truly unique experience when they come to the Embarcadero this holiday season."
While there are permitted events celebrating special occasions and Artist Market at the Plaza year-round, Ferry Bocce League hosts fun and competitive games from Spring through Fall in the evening hours each year.
"We are so excited to be offering fun sports daily at the Embarcadero Plaza this holiday season," said Phil Ginsburg, SF Rec and Parks' General Manager. "We look forward to seeing everyone playing with their friends and families in the outdoors and enjoying the warmth of California winter sun."
The free sports activities will be available during the month of December from 11AM to 3PM, with the exceptions of rain, and the following dates: December 7th, 24th, 25th, 26th, 31st and January 1st. From 11:00AM to 1:00PM, Tuesdays are Disc Golfing, Wednesdays are Skateboarding, Thursdays, Rock Climbing, Fridays, Skateboarding, and Saturdays are Slacklining. And from Tuesdays through Saturdays 1:00PM to 3:00PM, Bocce Ball games will be available to everyone. All free activities are available at first-come-first-serve basis.
According to Christopher Pollock, SF Rec and Parks' Historian-in-Residence, in the late 1800s, the Plaza area was an early hub for Union Depot and Ferry House in San Francisco. The Embarcadero was the focus of the City's port for all cargos arriving and being shipped via sea. Later, in 1956, the board plaza was built as part of Redevelopment Agency's waterfront design efforts, and initially known as Ferry Building Park, the name was changed to Embarcadero Plaza during the project planning stage. Finally, in 1972, the Plaza construction was completed, a primarily brick and concrete hardscape, was designed by landscape architect Lawrence Halprin association with architects Mario Ciampi and John Bolles and Associates.
And just as the Plaza was about to open, the City's Recreation and Park Commission approved a resolution during their meeting in April of 1972, to rename the area formerly known as Embarcadero Plaza (Ferry Park), to "Justin Herman Park", and later formally changed to Justin Herman Plaza in October of the same year. At the time, Justin Herman was recognized for his work as the City's Redevelopment Agency executive director. Then in September of 2017, the San Francisco Board of Supervisors legislated and voted unanimously to urge the Recreation and Park Commission to rename the Plaza back to Embarcadero Plaza. Subsequently, in November, the Commission voted to support the Board of Supervisors' resolution, and officially renamed Justin Herman Plaza to Embarcadero Plaza.


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Brandt Works Vintage Copper Hill Wall Mirror BM031M3 31.5"x40.5"
A stunning choice for any bathroom or living space. This vanity or wall mirror features a warm copper and bronze wood grain look. The 3 inch frame stands out with a 1 inch raised profile creating a interesting yet classic look. Included is hardware for both vertical or horizontal hanging making it a functional and stylish decorating tool.
Proud to be American Made
Hanging hardware for vertical or horizontal installation included
Crafted by hand


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Become a Monthly Donor
Do you want to provide the crucial funding needed to advocate new missions of discovery, conduct valuable scientific experiments, and develop and launch our own mission to space?
|Donate by Phone
Please call (626)793-5100 or toll-free in the U.S. and Canada at 877-PLANETS to make your donation by phone.
You can do all that when you make your monthly gift as a member of The Planetary Society's Discovery Team.
Ensuring our future in space – exploring other worlds, understanding our own, and seeking life elsewhere – is a long-term and innovative process. To be successful, we rely on steady, predictable funding from passionate space explorers like you.
When you make a monthly gift to The Planetary Society, you ensure that, with you, we can move quickly – and work boldly – to shape space exploration.
Plus, as a Discovery Team member, you help The Planetary Society reduce fundraising expenses by eliminating renewal notices (your membership automatically renews), and you take us a step further in "going green" as we eliminate more paper from our day-to-day operations.
• You choose the amount of your monthly donation.
• Your first gift will be charged to your credit card today. Subsequent gifts will be charged on or around the same date each month, and you'll receive an email confirmation when charged..
• Each January, we will send you a statement of your total annual gifts (which can be used for tax purposes).
• Your membership will renew automatically each year.
• You can change the amount of your monthly gift at any time, or cancel your participation in the Discovery Team at any time.
Joining the Discovery Team is simple and secure, and is an effective way to support The Planetary Society.
You can use our safe and secure online form to join The Planetary Society's Discovery Team with your credit card today
Become a member of The Planetary Society's Discovery Team: Your monthly contributions will provide a cost-effective and stable flow of support for our crucial work to shape the future of space exploration.


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Ambergris - the technical term for hunks of sperm whale vomit - has long been a component in high end perfumes. The whales eat all kinds of things, including shells, bones and other hard or sharp objects that it can't digest. Instead of trying to pass the spiky bits, the whales coat them in this sticky substance and cough them back up like cats cough up hairballs. When that hunk-o-junk meets the seawater, a chemical reaction happens that turns the slimy mass into a little rock-like object that can be collected from the beaches.
It might seem like an unlikely candidate for perfumes, since the stuff apparently smells about as gross as it sounds. "To some people the odour is nauseous while to others it is attractive and even sensuous. There is certainly an animalic component, reminiscent of farm animals, or even a faecal note, perhaps like that of a well rotted manure heap," writes an online ambergris guide. But the perfume industry isn't using it for its smell. Instead, they use it because the little rocks have special chemistry: they bind fatty molecules together.
Fragrances are usually made of fats, also called lipids. These ambergris molecules are lipophilic - meaning they like lipids. So they can bind the lipids in the fragrance together and make sure they stick together and to skin.
Ambergris critics argue that it endangers the already vulnerable sperm whale from which it comes. Others dislike the use of animal products in their cosmetics in general. Not to mention its rarity, which drives up prices. One gram of ambergris goes for $20.
Enter: science. Researchers at the University of British Columbia have discovered a plant-based alternative to ambergris that is similarly lipophilic. "We've now discovered that a gene from balsam fir is much more efficient at producing such natural compounds, which could make production of this bio-product less expensive and more sustainable," Joerg Bohlmann said in the press release. So now, rather than crawling the beaches for little hunks of whale vomit, researchers could manufacture a similar compound in the lab.
Before you go check your perfume bottles, take solace that ambergris isn't generally used in American perfumes, according to Scientific American, but European companies still use it. For a long time it was in Chanel No. 5. The market is big enough that there are ambergris dealers, like Bernard Perrin. He sells stock to not only perfume companies but to wealthy families as well. "We also sell it to a royal family in the Middle East and they use it as an aphrodisiac. Apparently they take some milk, some honey, and grind up small quantities of the amber and put that in as well," he told Scientific American.
It's also worth noting that no one has ever actually seen a sperm whale vomit up ambergris. Some think it might come out the other end. Just another mystery science has yet to solve.
Via: The Atlantic
Images: Sperm Whale by Archibald Thorburn, Wikimedia Commons
Ambergris by Peter Kaminski, Wikimedia Commons
This post was originally published on Smartplanet.com


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U.S. Army
Post-Vietnam War Era
Addressing trauma leads to better quality of life
Renee was sexually assaulted in the Army. She tried to move past it but she felt angry and couldn't sleep. After she left the military she went to a PTSD program for Veterans who had experienced military sexual trauma. Later, her doctor recognized signs of depression. She began therapy and she feels she is in a much better place in her life.Find Local Support


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Let me introduce myself:
My name is Selina. I was born in 1994 and I live in a small village in Switzerland. I'm a passionate blogger and photographer (well, just a beginner to be honest - here's my portfolio). I study Publicity & Communication Science and Politics at the University of Zurich.
I'm sure you want to find out more about this blog as well, so here we go:
"Rose Kiara Peaches" was founded in March 2012. Since then, I regularly share my outfits with you lovely people out there. I add a song at the end of most of my outfit posts, because I feel like fashion and music are two excellent ways to express yourself. But not only that: I sometimes post my make up looks, hair care tips and reviews of beauty products, too. There's also a lifestyle section, where you can find travel pictures and reports, as well as posts about events that I attend.
And if you're wondering about the name of this blog:
No, Rose Kiara Peaches is not my real name. It's just a combination that I really like. "Rose" is my favourite flower, "Kiara" is one of my favourite songs and "Peaches" was the name of the cute baby mammoth in Ice Age and I really fell in love with this name.
Here are some questions & answers if you want to find out more about me:
How would you describe your style?
It's quite versatile. At the moment I like the whole minimalism trend, mixed with a boho touch. Things that I'll always love are flower patterns, black and white combos, pastel pink items and lace.
What are your hobbies?
I love watching Youtube videos, photography, blogging, raving and art.
What music do you listen to?
I enjoy a wide range of music genres. At the moment I enjoy listening to "Drum and Bass" and "Alternative" the most I'd say.
What are your favourite clothing brands?
My all time favourite is Spell Designs, an Australian boho brand. My favourite Swiss designers are Berenik and Steinrohner.
What is your favourite city?
London, followed by Barcelona and Berlin.
Information on collaborations
I know that readers trust bloggers like me, so I try to be as transparent and honest with you as possible. Here are my promises to you, concerning collaborations:
Information on collaborations
I know that readers trust bloggers like me, so I try to be as transparent and honest with you as possible. Here are my promises to you, concerning collaborations:
- I only accept collaborations with brands that I truly like and that have a good reputation. I will never recommend anything to you that I personally don't believe in.
- Currently, I don't use affiliate links. If I'm going to use them in the future, I'll clearly mark them.
- I always request products (for example pieces of clothing, beauty items and so on) for my posts, so I can try them and the service the brand offers.
- I don't accept collaborations where I get money for a post without trying the service or products of a brand.
- If I get products for free, I link them for you using nofollow links.
- Posts that were produced with a brand are marked using the label "Collaboration". Furthermore, I'll mention the cooperation in the text and at the bottom of the post.
Do you have any other questions? Feel free to leave them as a comment
Thank you so much for stopping by!
Thank you so much for stopping by!


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Dear winter, thank you for forcing me to slow down and enjoy the simple things in life.
With the increasingly cold weather keeping me inside, I've been taking every opportunity to cozy up with my kids in front of the fire place with some hot chocolate. I've never really been a big fan of winter, I prefer heat (beaches!!!), but I have to admit that since having kids, winter is a lot more fun; snow angels, snowmen, sliding, skiing... and most importantly, cuddling up on the couch! I also love the slower beat of things.
This year, for our Christmas tree decorating party, I wanted to give out Christmas themed party favors. That obviously meant gingerbread cookies and hot chocolate. I prefer things being personal and we all know I love making my own recipes. So I spent a bit of time trying to figure out the exact ingredients to my basic homemade hot chocolate mix. This is another one of those things where I usually just randomly dump ingredients together and just like magic, out comes deliciousness (that's a word right?).
It turned out quite delicious and smooth. If you want it sweeter, you can always add a bit more sugar, but I find it just right this way. I have every intention of making tons of variations of this basic recipe so stay tuned!
Hot Chocolate Mix
1 1/2 cup cocoa powder (pure, unsweetened)
1 1/2 cup sugar
2 tbsp corn starch
To make the mix is very simple. Just pour all the above ingredients into a container and shake. If you want to make sure it's homogeneous, you can mix the ingredients in a bowl first.
When you're ready to have some hot chocolate, all you have to do is add 2 tablespoons of the mix to 1 cup of cold milk and bring to a boil on med-high, stirring constantly (it shouldn't take more than 2-3 minutes). You could also microwave it, but I prefer the stove.
And if you want to give this as a gift, don't forget to make a pretty label with the instructions!
Single Serving Homemade Hot Chocolate
If you don't want to make a big batch and prefer just making a cup as you go, you can use the recipe below.
1 cup milk
1 tbsp cocoa powder (pure, unsweetened)
1 tbsp sugar
1/4 tsp corn starch


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Friday, October 31, 2008
Taking 1 for the Team
So Pogo (aka Aaron) is heading out to Olympia again this weekend to press the Syrah. I am too tired and a bit starved of family time and have elected to stay behind. I am sure he is just as pooped and sleep-deprived but he's taking one for the team. At times like this I realized I made the correct choice in who to join forces with to make wine.
Thanks man!
3 comments:
- mr aaron said...
Thanks for the props! I'll do my best not to F*CK it up.
- October 31, 2008 at 4:28 PM
- B-Muse said...
Let's see some pics, Pogo!
- November 5, 2008 at 10:32 AM
- mr aaron said...
off my back already - still recovering from the trip and the election
- November 5, 2008 at 3:09 PM
Subscribe to: Post Comments (Atom)


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You can purchase the packet by clicking HERE.
A second grade blog with many ideas, activities, and freebies for 2nd grade.
Morning Work for First Grade
Many people have asked me to make morning work packets for first grade. Here is a FREE sample of my newest creation. It can be used for morning work or homework. It is a spiral review of the first grade Common Core Standards. Click on the picture of the morning work for your FREE sample pages.
1 comment:
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I know many of you don't particularly wish to hear about my eye problems as that's about the only subject I have been going on about for the past three years. How do you think I feel about it having to live with it every single day? It has paused my career, caused me much pain, stopped me from going outside on many occasions, made me feel sad and unhappy about myself and last year drove me close to suicide from the extreme pain.
Now that work has dried up again and I think this is the time to focus on really promoting the show and getting out and doing more on location recordings, but I just cannot do much because of the flipping eye.
I do think a lot of people think that a small irritation in the eye is livable with and that it will eventually go away. But eventually is a ruddy long time and meanwhile life is passing me by and I feel wretched about myself and lost opportunities.
On more than one occasion I have asked the doctors to remove the eye and they have continually persuaded me to stick with it, but when I do their medicines and surgery just seems to fail. Having had three failing cornea transplants I am not terrible hopeful of having any more and beginning to wonder that I should simple demand a replacement plastic eye and be done with it.
Then perhaps I can give the rest of my life and the chosen field of podcasting a chance to succeed.


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Join Gordon Luckett for an in-depth discussion in this video Categories and rules, part of Learning Autodesk Vault.
- [Voiceover] Categorization of files and folders…enabled them to have rules applied.…Each category can be assigned to a folder or file…and a Rule will assign that category.…Categories drive the easier to find Properties,…Lifecycles and Revisions.…In the Vault Settings,…the Behaviors tab does not even exist…in the Vault Basic edition.…So you may have not have seen this…if you've seen any other videos in this course.…In the Categories area,…you'll see that there are Categories…and Rules to be applied.…Categories are the most important feature…inside the Behaviors.…
In fact, you should always do Categories first…and then do the Rules.…To assign Categories,…simply click the Categories Button.…Categories are the root of all the Rules,…the Revision, the Lifecycles,…and the Properties across the board…within Vault.…We configuring Folder Categories or File Categories,…you can change them in the drop down box…at the top of the screen.…Notice for the Folder Category,…you can see the name and description…of a lifecycle at the bottom underneath Behaviors.…
This course concentrates on the administrative side of Vault Basic. Gordon Luckett shows how to install a Vault server on a central Windows Server, and install the Vault Basic client on a computer with AutoCAD. The server and client are used to track how AutoCAD DWG and other non-CAD files are added, searched, and checked in and out of Vault. Gordon also covers autoloading files to Vault, and backing up and restoring the system. The final chapter covers the premium features in Vault Professional, such as categories, rules, and revisions.
- Installing the Vault server and client
- Organizing files and folders with Vault Basic
- Adding files
- Searching files
- Setting up client administration settings
- Connecting to Vault from AutoCAD
- Checking files in and out
- Autoloading files
- Backing up and restoring Vault
- Working with Vault Professional


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OPINION: Britain risks destroying human rights protection of millions
By Sam Grant, Campaigns manager, René Cassin
Did you catch the following line in the Queen's Speech on Wednesday: "My government will repeal the Human Rights Act and replace it with a British Bill of Rights"?
Given the bad press human rights have received, you'd be forgiven for thinking "so what?" or even "good riddance".
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But if your response is ambivalence or approval, spare a thought for vulnerable minorities in the UK and throughout Europe who may have cause to fear from this seemingly innocent, if anodyne, statement.
Much of the media seems to hate the Human Rights Act. Mud has been slung at it by the lorry-load, and a lot of it has stuck. Despite the work charities such as René Cassin have done to challenge them, sensational myths prevail: that the Act stopped the deportation of an illegal immigrant because he had a cat (not true); the Act allowed serial killer Dennis Nilsen to access hardcore porn in jail (not true); the Act means that the UK must give murderers the vote (again, not true).
You may have heard other human rights horror stories. I would urge you to delve beneath the headlines, because in the overwhelming majority of cases, the result will be the same, a mixture of gross exaggerations or outright misinformation. On the other side of the coin, good news stories about the Human Rights Act – and there are plenty – have failed to find traction.
The Act has helped elderly and disabled people receive the care they need to live with dignity; it has ensured that hospitals respect the rights of the mentally ill; it has obliged the state to protect us from crime; stopped the state from listening to our phone calls, recognised female genital mutilation as torture and much, much more. Fundamentally, it is about giving a voice to the voiceless.
However, it has also fallen foul of that other great media bête noire: Europe. The Act incorporates the European Convention on Human Rights into British law.
It is worth noting that the Convention is not a concoction of the EU, but was created by the Council of Europe, a body set up in the aftermath of the Second World War to promote democracy, human rights and cultural co-operation across a shattered continent.
The Convention is Europe's civilised nations' response to the horrors of the Holocaust. It demands that states will 'never again' be allowed to trample on the basic rights of their citizens.
Among its guarantees are the rights to life, freedom from torture, fair trial and freedom from slavery. The European Convention is not a foreign code foisted on Britain – it was co-drafted by British lawyers, is largely based on common law, and was supported by Winston Churchill.
So, where are we now?
In terms of the government's plans, the devil is not so much in the detail as in the fundamental architecture.
If a 'British Bill of Rights' retains the relationship with the European Convention and the Strasbourg Court, it will be a largely cosmetic change. However, the government has talked about 'breaking the formal link' with the European Court of Human Rights.
This would set the UK on a collision course with the Council of Europe and could lead to our expulsion, leaving us to join Belarus as the only other country between Lisbon and Vladivostock that is not signed up to internationally agreed human rights standards. By pursuing this course, Britain risks wrecking a system that protects the rights of millions throughout Europe.
The whole point of an international regime is that it exposes the actions of states to independent scrutiny. Not only do we worry about minorities in UK, but we worry about those in Hungary currently experiencing anti-Semitism and everybody who is subject to the whim of populist opinion.
René Cassin works to protect the rights of vulnerable minorities here in the UK.
We will be working to make the government aware of the worrying implications of their plans for millions of people here and abroad. Ultimately, our message is simple: 'We already have a British Bill of Rights – it's called the Human Rights Act'.
By Brigit Grant
By Jewish News
By Judy Elkus
By Judy Elkus


text:
Sunday, December 30, 2012
Monday, December 24, 2012
Out For Stats
Thanks for playing!
Thursday, December 20, 2012
Purse Thursday - Reghann
I am interviewing the purses of women again and again. These women do not know they are going to be interviewed, so catching them and their purse in their utmost natural presence is very important. I'm excited to learn more about the fashions and styles of women, see how much stuff they carry around, and just how clean or nasty they are. Shall we? Recently, I got to meet some very great people while working and touring with Crypts. One of them is not only the wife of the lead singer, but also my new personal hair stylist. This woman is filled with class and a whole lotta sassafras. She carries a purse bigger than most suitcases. A few weeks ago we drank with a group of friends all over The Hill. I give to you, Reghann and her purse. Reghann, 34. Black leather bag. 12:02am - Unicorn.Radjaw: What kind of purse is that?Reghann: Marc JacobsRadjaw: How long have you had it?Reghann: I don't know, like five years. It's my everything purse. I love it!Radjaw: What do you love about it?Reghann: I love the shape of it and after all this time it still smells the fucking same.Radjaw: Can I see what's in it?Reghann: Why?Radjaw: Because it's fucking huge!Reghann: OK, but Steve (her husband) needs a Jager shot first.Steve: Woooo!!!Reghann: Jesus, I have so much shit in here. Radjaw: Let's see!Downy spraySmall water bottle, emptyBig water bottle, half fullNail polishDog shit bags*Just then everything on the table falls onto the ground.Reghann: This is like the one time I went to a psychiatrist.Radjaw: ?...Crumpled up heart napkinWalletEmbassy Suites hotel cardReghann: Portland!Steve: Portlaaaaaannndddd!!!LipstickSmall water bottle, fullMedium water bottle, almost emptyRadjaw: You sure do have a lot of water bottles in there.Reghann: I like to stay hydrated, plus I get them at my job. Radjaw: It's like a personal recycling bin.Empty pack of gumChloe lotionBand-aids, emptyVictoria's Secret gift cardReghann: I don't wanna put that! I want to keep that.*Puts card into pocketNail fileHand lotionFace lotionLip glossReghann: (Yelling to bar) Someone left Yves Saint Lauren lip gloss on the table! It's mine now!Radjaw: Is that everything?Reghann: Yes.. time to photobooth!Radjaw: YIKES.
Posted by Radjaw at 6:04 PM No comments:
Labels: crypts, fashion, Interview, Purse Thursday, seven salon
Wednesday, December 19, 2012
Stop Biting
New compilation out now!! It is always great to see people working in their own elements. This a series of 8 small vignettes, capturing the studios and sentiment of the Stop Biting DJs featuring: OCnotes, WD4D, Suttikeeree, Specs Wizard, Ear Dr.Umz, Arthur Russell Simmons, Showbot, & Macros. This is super motivating and inspiring for me and my ship shop studio I'm putting together.
Posted by Radjaw at 11:58 AM No comments:
Labels: beats, lofi, ocnotes, specs, stop biting, truckasauras, video, wd4d
Wednesday, December 12, 2012
New Champagne Poster
Posted by Radjaw at 2:26 PM No comments:
Labels: Champagne Champagne, dtttc, dyme def, nectar lounge, Poster Art, shows, vox mod
Toro Y Moi "So Many Details"
Official video for Toro Y Moi's "So Many Details". The first single off his new album "Anything In Return" Out January 22.
Posted by Radjaw at 1:40 PM No comments:
Labels: cars, toro y moi, video
OCnotes - Food-$$-Booze-Xmas
"This record was made entirely out of christmas records. Listen to this with some bass for the proper experience. Your cheatin yourself otherwise. Funk. to the highest degree. Shout out to the architects before me, respect to the dons of the future. Im just playin my position. bump. respect. A christmas gift for 2012 for alllllll myyyyyy peeeeoooooppllleeee!!!1"
Costa Rica
I'm going in January.
Posted by Radjaw at 1:34 AM No comments:
Labels: costa rica, cumbia, dance, video
Tuesday, December 4, 2012
Gnartivity Scene
Now this is a Christmas compilation I can fuck with. The box sets contain two copies of the compilation, with a kit to gift the other copy to a friend or family member. They also include a high-quality zine with original holiday art by members of Gnar Tapes and the bands, as well as friends and family. There will be other goodies and surprises include and we are only making 35! White Fang plays The Crocodile this THURSDAY.
Posted by Radjaw at 4:26 PM No comments:
Labels: christmas, download, gnar tapes, holiday, White Fang
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Donec ullamcorper nulla non metus auctor fringilla. Vestibulum id ligula porta felis euismod semper. Vestibulum id ligula porta felis euismod semper. Sed posuere consectetur est at lobortis.
Frequently asked questions:
Can I order online?
Not currently. For information on catering, please email us at firstname.lastname@example.com. You can order merchandise online, though.
Do you make custom flavors?
Yes. If you can dream it, we will get in the kitchen and make it. Tell us the key flavors you want, and then we'll take it from there and craft you an amazing flavor - that before you, never existed!
Do you cater?
Yes. We cater all kinds of events; from happy to sad, there's never a wrong event for ice cream! For information on catering, please email us at firstname.lastname@example.com.
Do you have a store?
Yes! We're at 632 E Market St, Suite 101, Louisville, KY 40202.
Are all flavors available all the time?
Sadly, no. Since we work with making small batches we make flavors as needed. If you'd like to see if flavors are available for catering, please send us an email at firstname.lastname@example.com.
Where can I buy your ice cream?
You can buy our ice cream by the scoop at the following locations:
- Sister Bean's Coffee House 5225 New Cut Rd, Louisville, KY 40214
- Monnik 1036 E Burnett Ave, Louisville, KY 40217
- Bardstown Bourbon Company 1500 Parkway Drive, Bardstown, KY 40004
- Hell or High Water 112 W Washington St, Louisville, KY 40202
- The Pine Room 6325 River Rd, Harrods Creek, KY 40027
- Events where the Louisville Cream tent is at.
Five flavors of our pints can be purchased at the following locations:
- Rainbow Blossom 3738 Lexington Rd Louisville, KY 40207 (St. Matthews)
12232 Shelbyville Rd Louisville, KY 40243 (Middletown)
3046 Bardstown Rd Louisville, KY 40205 (Bardstown Rd)
3003 Charlestown Crossing Way, New Albany, IN 47150 (New Albany)
- Old Town Wine & Spirits 1529 Bardstown Rd, Louisville, KY 40205
- Four Points by Sheraton 2850 Crittenden Dr, Louisville, KY 40209
- The Marriott Courtyard Louisville Downtown 100 S Second St, Louisville, KY 40202
- The Omni Hotel Louisville 400 S 2nd St, Louisville, KY 40202
- Full Stop Station 1132 E St Catherine St, Louisville, KY 40204
- ValuMarket Highlands 1250 Bardstown Rd #1, Louisville, KY 40204
- Liquor Barn 4901 Outer Loop, Louisville, KY 40219
4808 Brownsboro Center, Louisville, KY 40207
If you would like to wholesale our pints contact us at firstname.lastname@example.com.


text:
Fans pouring into Buchanan ahead of motocross race at Red Bud
Posted: Jul 4, 2012 8:43 PM EST | Updated: Nov 6, 2014 5:10 AM EST
BUCHANAN, Mich. -- Motocross fans are pouring into Michiana for Saturday's main event.
The Lucas Oil Pro Motocross Championships are being held Saturday at Red Bud in Buchanan.
This will mark the half-way point in the motocross season.
Last season, Chad Reed raced to his fourth win in six races at Red Bud.
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