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Glucose Metabolism via the Pentose Phosphate Pathway,
Glycolysis and Krebs Cycle in an Orthotopic Mouse Model of
Human Brain Tumors
Isaac Marin-Valencia1,2, Steve K. Cho3,4,5, Dinesh Rakheja6, Kimmo J. Hatanpaa4,6, Payal
Kapur6, Tomoyuki Mashimo4,5,7, Ashish Jindal8, Vamsidhara Vemireddy4,5,7, Levi B.
Good3, Jack Raisanen4,6, Xiankai Sun9, Bruce Mickey4,10, Changho Choi8, Masaya
Takahashi8,9, Osamu Togao8,9, Juan M. Pascual1,2,3,11, Ralph J. DeBerardinis1,12, Elizabeth
A. Maher3,4,5,7, Craig R. Malloy7,8,9,13,*, and Robert M. Bachoo3,4,5,7,*
1Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas
2Division of Pediatric Neurology, University of Texas Southwestern Medical Center, Dallas, Texas
3Department of Neurology, University of Texas Southwestern Medical Center, Dallas, Texas
4Annette G. Strauss Center for Neuro-Oncology, University of Texas Southwestern Medical
Center, Dallas, Texas 75390
5Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas
6Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas
7Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas,
Texas 75390
8Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas,
Texas 75390
9Department of Radiology, University of Texas Southwestern Medical Center, Dallas, Texas
10Department of Neurological Surgery, University of Texas Southwestern Medical Center, Dallas,
Texas 75390
11Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas
12McDermott Center for Human Growth and Development, University of Texas Southwestern
Medical Center, Dallas, Texas 75390
13Medical Service, Veterans Affairs North Texas Healthcare System, Lancaster, Texas 75216
*Corresponding authors: Robert M. Bachoo, MD, PhD, Department of Neurology, Annette G. Strauss Center for Neuro-Onoclogy, UT
Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75235, email@example.com; Craig Malloy, M.D.,
Mary Nell and Ralph B. Rogers Magnetic Resonance Center, 5323 Harry Hines Blvd., Dallas,TX 75390-8568.,
NIH Public Access
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Published in final edited form as:
NMR Biomed. 2012 October ; 25(10): 1177–1186. doi:10.1002/nbm.2787.
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It has been hypothesized that increased flux through the pentose phosphate pathway (PPP) is
required to support the metabolic demands of rapid malignant cell growth. Using an orthotopic
mouse model of primary human glioblastoma (GBM) and a brain metastatic renal tumor of clear
cell renal cell carcinoma (CCRCC) histology, we estimated the activity of the PPP relative to
glycolysis by infusing [1,2-13C2]glucose. The [3-13C]lactate/[2,3-13C2]lactate ratio was similar for
both the GBM and renal tumor and their respective surrounding brains (GBM: 0.197 ± 0.011 and
0.195 ± 0.033 (p=1); CCRCC: 0.126 and 0.119 ± 0.033, respectively). This suggests that the rate
of glycolysis is significantly greater than PPP flux in these tumors, and that PPP flux into the
lactate pool was similar in both tissues. Remarkably, 13C-13C coupling was observed in molecules
derived from Krebs cycle intermediates in both tumors, denoting glucose oxidation. In the renal
tumor, in contrast to GBM and surrounding brain, 13C multiplets of GABA differed from its
precursor glutamate, suggesting that GABA did not derive from a common glutamate precursor
pool. Additionally, the orthotopic renal tumor, the patient's primary renal mass and brain
metastasis were all strongly immunopositive for the 67-kDa isoform of glutamate decarboxylase,
as were 84% of tumors on a CCRCC tissue microarray suggesting that GABA synthesis is cell-
autonomous in at least a subset of renal tumors. Taken together, these data demonstrate that 13C-
labeled glucose can be used in orthotopic mouse models to study tumor metabolism in vivo and to
ascertain new metabolic targets for cancer diagnosis and therapy.
Intermediary metabolism; Mouse model; 13C NMR; brain; glioblastoma; renal cell carcinoma
The pentose phosphate pathway (PPP) provides ribose 5-phosphate for nucleotide synthesis
and NADPH as a cofactor for fatty acid synthesis. These metabolites are required for cell
proliferation and for this reason flux in the PPP is thought to be increased in cancer[1, 2].
This hypothesis is consistent with high uptake of 18fluoro-deoxyglucose in tumors detected
by positron emission tomography (18FDG-PET). However, flux in the PPP relative to
glycolysis is difficult to examine in cancer in vivo because of the complex connections
between the two pathways. Methods to monitor the PPP take advantage of the first few
biochemical steps. The oxidative portion of the pathway involves an initial reduction of
glucose-6-phosphate to form 6-phosphogluconolactone plus NADPH, followed by
conversion to 6-phosphogluconate which in turn undergoes decarboxylation at position 1 to
yield CO2, a second molecule of NADPH, and ribose-5-phosphate (Figure 1). All carbon
tracer methods probing the pathway rely on detecting the effects of this characteristic
decarboxylation reaction in downstream metabolites, particularly lactate. Traditionally 14C-
enriched glucose was used in rodent and human experiments [3-5] but more recent studies
have extensively evaluated the use of [2-13C]glucose or [1,2-13C2]glucose with detection of
lactate by either 13C NMR spectroscopy [6-9] or mass spectrometry [10]. Together, these
studies indicate that flux of glucose through the PPP is active in a variety of tissues and that
it is increased in cancer [11] or after brain injury[12].
Although established tumor cell lines have provided invaluable insights into the regulation
of metabolic pathways, recent molecular studies have shown that, in-vitro, especially under
serum enriched cell culture conditions, tumor cells may undergo marked genomic and
epigenetic changes [13, 14]. How cell culture conditions may modify tumor cell metabolism
or the extent to which these artificial conditions are able to replicate the in situ tumor
environment is unknown. To minimize non-physiological metabolic changes in the cell, we
developed an orthotopic mouse model of human brain tumors that provides a good
approximation of the cells' native microenvironment. Using this model, we analyzed the
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relative activity of the PPP compared to glycolysis in orthotopic glioblastoma and orthotopic
clear cell renal cell carcinoma (CCRCC) brain metastasis in situ by infusion of
[1,2-13C2]glucose followed by microdissection and analysis of the tissue extract by 13C
NMR spectroscopy. The distribution of 13C in lactate was used to assess flux through the
PPP relative to glycolysis essentially as described by others[10, 12, 15].
Flux through the PPP relative to glycolysis was not significantly different between the GBM
and CCRCC and their respective nontumor-bearing surrounding brains. As anticipated,
glucose was oxidized via pyruvate dehydrogenase and Krebs cycle in non tumor-bearing
surrounding brain tissue with label observed in glutamine as well as in the neurotransmitters,
glutamate and GABA. Surprisingly, oxidation of glucose in the Krebs cycle was observed in
both GBM and a CCRCC. Because GABA was detected in the renal tumor and CCRCC
brain metastasis and GABA synthesis is not characteristic of normal renal metabolism,
additional immunohistochemistry studies were performed to assess the distribution of
glutamic acid decarboxylase, the key enzyme in GABA synthesis, in the CCRCC brain
metastasis. Taken together, our data show that an orthotopic mouse brain tumor model can
be used to assess intermediary metabolism in the context of normal brain microenvironment.
This approach may enable detection of novel biomarkers of specific tumors.
Orthotopic Transplantation of Human Tumors into Mouse Brain
Brain tumor tissue was collected from three patients with a diagnosis of primary
glioblastoma (GBM) and one patient with a metastatic renal tumor of clear cell renal cell
carcinoma (CCRCC) histology, undergoing complete surgical resection at the University of
Texas Southwestern Medical Center. Written informed consent for use of tumor tissue was
obtained from all patients under a protocol approved by the Institutional Review Board.
Diagnoses were established by pathological examination. Tumors were collected in ice-cold
Hanks medium and were gently dissociated by enzymatic treatment with papain. A single
cell suspension (~1 × 105) was injected into the right caudate of 6-week-old female NOD/
SCID mice as previously described[16]. A cohort of 5 mice were injected for each of the 3
GBM tumors and one renal cell tumor (n=20 mice for this study). All tumor cell
transplantations were completed within 3 hours of surgical resection. Mice were examined
twice weekly for weight loss, loss of grooming, seizures, and focal motor deficits.
Magnetic Resonance Imaging and Positron Emission Tomography (PET)
Brain imaging was performed to document the presence of an expanding intracranial mass.
MR imaging was conducted with a 7-T small animal MR system (Varian, Inc, Palo Alto,
CA) with a 40 mm (I.D.) Horizontal Millipede™ Coil. All animals were anesthetized with
1-2% isoflurane (AERRANE, Baxter Healthcare Corporation, IL) mixed in 100% oxygen
and placed head first in the supine position with a respiratory sensor. The head was centered
with respect to the center of the RF coil. First, low-resolution multi-slice imaging was
performed on the head region to confirm the location and orientation of the brain.
Precontrast T1-weighted (repetition time/echo time, 500/10 msec; echo-train length, 2; field
of view, 25.6×25.6 mm; matrix, 256×256; in-plane resolution, 100×100 μm; slice thickness,
1 mm; gapless; number of excitations, 4; number of slices, 11) and T2-weighted (repetition
time/echo time, 2500/60 msec; echo-train length, 8; field of view, 25.6×25.6 mm; matrix,
256×256; in-plane resolution, 100×100 μm; slice thickness, 1 mm; gapless; number of
excitations, 4; number of slices, 11) multi-slice images were obtained with a fast spin-echo
sequence in the axial plane. Following an i.v. injection of 0.2 mmol/kg gadolinium contrast
agent (Prohance, Bracco Diagnostic Inc, Princeton, NJ), postcontrast T1-weighted images
were acquired with identical parameters as above. Total acquisition time for each animal
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was approximately 20 minutes. The mice were monitored for respiration during the imaging
session with an MR imaging-compatible small-animal respiratory gating device (SA
Instruments, Inc., Stony Brook, NY).
18FDG-PET imaging was performed in a small animal PET scanner with a full-width at half-
maximum resolution of 2 mm was used. GBM-bearing mice were anesthetized with 2%
isoflurane. Following positioning in the scanner, an attenuation data set was obtained; then
animals were injected with 35 mCi of 18FDG through the tail vein and maintained in the
scanner for 1 h for real-time acquisitions to ensure the generation of regions of interest
(ROI) (tumor vs normal contralateral brain) from which time-activity curves were
Intravenous Infusion of [1,2-13C2]glucose
Tracer studies were performed when mice developed focal neurological deficits. This
typically occurred at 4-6 weeks for NOD-SCID mice implanted with the CCRCC brain
metastasis and at 10-12 weeks for GBM orthotopic tumors. The right jugular vein was
aseptically cannulated under 2% isofluorane. The catheters were filled with glycerol to
maintain patency. After cannulation, animals were allowed to recover from anesthesia and
were loosely confined to a cylindrical Lucite cage. [1,2-13C2]glucose (99% enrichment;
Cambridge Isotope Laboratories, Cambridge, MA) was administered as a bolus of 0.4mg/g
(0.3 ml) infused in ~1 min, followed by a continuous infusion of 0.012 mg/g/min at 150 μl/
hr for a duration of 150 min. At the end of the infusion, blood was collected by retro-orbital
puncture (100-150 μl). The mice were then deeply anesthetized and decapitated, the brain
was rapidly removed from skull (<30sec), immersed into ice-cold Hanks media and cut into
1mm coronal sections on pre-chilled rodent brain block and examined by trans-illumination
under a dissecting microscope. Orthotopic CCRCC tumors were easily identified by a
distinct border between tumor mass and surrounding normal brain. GBM orthotopic tumors
were characterized by a large central mass surrounded by indistinct borders at the tumor
margins. The central tumor mass and the contralateral hemisphere were separately
microdissected and weighed (GBM: 217 ± 28 mg, renal cell: 44 ± 7 mg, surrounding brain
of GBM: 376 ± 53 g, surrounding brain of renal cell: 361 ± 50 g), frozen in liquid nitrogen,
and stored at −85°C. ~20 μL of blood was saved to measure blood glucose concentration
after the infusion with a Contour® glucometer (Bayer Healthcare, Tarrytown, NY)[17] and/
or an enzyme spectrophotometric assay (GAHK20-1KT kit, Sigma, St Louis, MO). The
same tumor extraction protocol was carried out for the sham-injected NOD-SCID mouse
Preparation of Tumor Extracts
The mass of CCRCCs ranged from 30-50 mg per tumor at dissection. In preliminary
experiments we had established that 150 mg (wet weight) of tissue was required to generate
a reasonable NMR spectrum (data not shown). Therefore, to have an adequate sample for
NMR analysis, we pooled 4 orthotopic CCRCCs. Pooled tumors and pooled non-tumor
bearing surrounding brain from each mouse were finely ground in a mortar under liquid
nitrogen. Perchloric acid (4%; 1:4 w/v) was added to each sample, followed by
centrifugation at 47800 g for 15 min. The supernatant was transferred to a new tube where
chloroform/tri-n-octylamine (78%/22%; v/v) was added in a 1:2 volumetric ratio to increase
the pH to ~6. The samples were centrifuged at 3300 g for 15 min, the aqueous phase
removed and transferred to a microfuge tube and then lyophilized. 100 μl of deuterium
oxide (99.96%, Cambridge Isotope Laboratories) was added to each sample and the pH
adjusted to 7.0 with 2-3 μL of 1 M of sodium deuteroxide (99.5%, Cambridge Isotope
Laboratories). The pH-neutral samples were then centrifuged at 18,400 g for 1 min and the
supernatant removed and placed into a 3-mm NMR tube for subsequent NMR analysis.
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Measurement of 13C Fractional Enrichment in Blood Glucose
Blood samples were processed to measure 13C-glucose enrichment by gas chromatography-
mass spectrometry (GC-MS). A 10 μL aliquot of whole blood was transferred to a 16 ×
10mm glass tube. Glucose was extracted by the sequential addition and vortexing of 2 mL
each of methanol, chloroform and water. Samples were then centrifuged at 2000g for 5 min.
Approximately 3.5 mL of the upper aqueous phase was transferred to a new glass tube and
extracted with 2ml chloroform. After centrifugation at 2000 g for 5 min, approximately 3 ml
of the upper aqueous phase was transferred to a screw-topped glass tube, and evaporated to
dryness under blown air at 42 °C. The samples were then derivatized by adding 100 μL Tri-
Sil reagent (Pierce, Thermo Fisher Scientific, Rockford, IL), vortexing, capping, and heating
for 30 min at 42° C. A glass Pasteur pipette was used to transfer each sample into an
autoinjector vial for GC-MS. A three-point standard curve was also prepared using mixtures
of un-enriched glucose with [1,2-13C2]-glucose such that 0, 50 or 100% of the glucose in the
final mixture was 13C-labeled. GC-MS was performed using an Agilent 6890N Gas
Chromatograph coupled to an Agilent 5973 Mass Selective Detector (Agilent Technologies,
Santa Clara, CA). One microliter of each standard or sample was injected and analyzed in
scan mode. Abundances of fragment ions 204 (unenriched) and 205 (enriched) were
determined for each standard and sample, and the ratio of 205/(204+205) ions was
calculated. A standard curve was generated and linear regression was used to calculate the
atom percent excess (APE) of glucose from the 205/(204+205) ratio for each sample.
13C NMR Spectroscopy
Proton decoupled 13C spectra were acquired on a 400 MHz Varian magnet and Varian
VNMRS Direct Drive console using a 5 mm auto-switchable broadband probe (Varian
Instruments, Palo Alto, CA). Proton decoupling was performed using a Waltz-16
sequence. 13C NMR parameters include a 45 degree flip angle per transient, a relaxation
delay of 1.5 sec, an acquisition time of 1.5 sec, and spectral width of 24.5 kHz. Samples
were spun at 20 Hz with the temperature regulated at an ambient 25 °C. A 2H field-
frequency lock was used.
Analysis of Spectra
NMR spectral analyses were performed with ACD/Spec Manager 11.0 software (Advanced
Chemistry Development, Inc., Toronto ON, Canada). Free induction decays were zero-filled
and multiplied by an exponential weighting function of 0.5 to 1.0 Hz prior to Fourier
transformation. Resonances were assigned based on chemical shift position referenced to the
glutamate C4 singlet at 34.2 ppm. Each signal was fitted with a Gauss-Lorentz function and
the area measurements for each fitted resonance peak and the multiplet areas estimated as
previously described[18, 19].
13C NMR isotopomer analysis of glutamate measures the labeling pattern of acetyl-CoA
feeding the Krebs cycle. The analysis of glutamine is equivalent to glutamate. Since GABA
is produced from glutamate, the 13C NMR spectrum of GABA also provides information
about acetyl-CoA labeling patterns. Specifically, the 13C labeling patterns in glutamate
carbons 2–5 are preserved in GABA except that the designation of labeled carbons is
reversed (carbon 1 in GABA corresponds to carbon 5 in glutamate, etc., see Supplementary
Figure 2). Because of the structure of the Krebs cycle and numbering conventions, this
means that the 13C enrichment in C1 of GABA is equal to the 13C enrichment in C1 of
acetyl-CoA and the 13C enrichment in C2 of GABA is equal to the 13C enrichment in C2 of
acetyl-CoA. The only oxaloacetate carbon positions that are detected in GABA are carbon 2
of oxaloacetate, equivalent to carbon 3 of GABA, and carbon 3 of oxaloacetate, equivalent
to carbon 4 of GABA. Since the 13C labeling patterns in glutamate carbons 4 and 5 encode
the labeling pattern in acetyl-CoA, it is a simple matter to examine substrate competition in
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GABAergic neurons, even under non-steady state conditions, using the principles described
for glutamate [18, 19].
Paraffin-embedded GBM and renal cell carcinoma specimens were obtained from samples
maintained in the Divisions of Neuropathology and Surgical Pathology at UT Southwestern
Medical Center. Tissue microarray constructs of 96 renal cell carcinoma specimens were
obtained from the Division of Surgical Pathology at UT Southwestern Medical Center. The
GBM tissue and human orthotopic tumor-bearing mouse brains were fixed in 10% formalin
and embedded in paraffin. Sections were cut at 4-μm thickness and deparaffinized. The
sections were incubated in 3% H2O2 for 10 minutes to block endogenous peroxidase
activity. The primary antibodies and their dilutions were as follows: MIB-1 (prediluted;
790-4286 Ventana Medical Systems, Tucson, AZ) and GAD67 (1:500; ab26116; Abcam,
Cambridge, MA), respectively. All immunostains were performed on a Benchmark XT
stainer using the CC1 pretreatment solution (95 °C for 30 min) and XT UltraView Universal
DAB detection system (Ventana). The sections were counterstained with hematoxylin.
Statistical analysis
The statistical analysis was performed with the non-parametric Wilcoxon two-sample test
using SPSS Graduate Pack version 18.0 (SPSS; Chicago, IL, USA).
All three orthotopic primary GBM tumor lines appeared hyperintense on T2 sequences,
hypointense on T1 with gadolinium contrast enhancement and showed avid 18FDG uptake
by 18FDG-PET imaging. The mouse MR and PET imaging data were similar to the imaging
data obtained pre-operatively in the GBM patients from whom the tumors lines were derived
(Figure 2A). These data suggest that the routine clinical imaging modalities used to asses
and follow GBM patients are reproduced in the orthotopic setting of NOD-SCID mouse
Histological analysis of orthotopic intracranial GBM tumors (n=3), were characterized by a
high mitotic index (Ki-67 50-70%), pleiomorphic nuclei, tortuous microvasculature and
diffuse single cell infiltration. All of these features are characteristic of high grade gliomas
(Figure 2B). The orthotopic CCRCs derived directly from a CCRCC brain metastasis
showed brisk proliferation (Ki-67>50%), monomorphic nuclei, scant cytoplasm and distinct
margins between the tumor mass and the brain parenchyma (Figure 2B); all features
consistent with the histological analysis of the resected brain metastasis from the patient.
Tumor cell-host interactions defined by diffuse single cell infiltration or a distinct tumor-
brain parenchyma interface are characteristic histological features of GBM and metastatic
brain tumors, respectively. These were observed in the orthotopic setting of the NOD-SCID
mouse brain.
Evaluation of Pentose Phosphate Pathway Activity In Vivo
Intravenous infusion of [1,2-13C2]glucose followed by 13C NMR analysis of microdissected
tumor mass and nontumor bearing surrounding brain, was used to assess flux of the PPP
relative to glycolysis. Mean blood glucose levels at the end of the infusions were 2.8 mM
(n=3, range: 1.2 to 5.7 mM) for GBM and 6.6 mM (n=4, range: 5.6 to 11.8 mM) for
CCRCCs. The enrichment of 13C-glucose in blood was 21.3 ± 11.6 % and 55.3 ± 17 % in
mice with GBM and CCRCC, respectively. The higher mean blood glucose levels and
greater fractional 13C-glucose enrichment in the CCRCC brain metastasis cohort suggests
less robust glycemic control. Whether this is the result of a rapidly growing, non-infiltrating
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tumor mass, which adversely affects hypothalamic-pituitary axis, is not clear. The 1H-
decoupled 13C NMR spectrum from the brain of a control NOD/SCID mouse infused with
[1,2-13C2]glucose is shown in Figure 3. Since glucose is the preferred substrate for energy
production in normal brain, the appearance of spin-coupled 13C multiplets in metabolites
exchanging with the Krebs cycle such as glutamate, glutamine, GABA and aspartate is
anticipated [20]. The presence of spin-spin coupling in the NMR spectrum of these
metabolites confirms their origin from infused [1,2-13C2]glucose. The natural
abundance 13C signal from taurine was also detected routinely in the normal brain. The
majority of the 13C-enriched lactate (inset) originated from [1,2-13C2]glucose via glycolysis,
indicated by the high doublet D23 relative to the singlet (singlet to doublet ratio 0.69).
Blood enrichment of 13C-lactate (from M+0 to M+3) was measured in GBM and CCRCC
animals (Supplementary Figure 3). Most of the lactate was unlabeled (M+0) or labeled in
two positions (M+2). However, the enrichment of lactate M+1, which, by definition,
includes lactate labeled in C1, C2 or C3, was <1% in all animals. This indicates that the
contribution of lactate M+1 to the singlets of lactate C3 and glutamate C4 was very low.
13C-NMR spectra from the brain of a non-tumor-bearing mouse and from the uninvolved
contralateral hemisphere from mice bearing GBM or renal cell tumor are shown in Figure 4.
The singlet-to-doublet ratio of lactate C3 was not significantly different between the GBM
and its surrounding brain, 0.197 ± 0.011 vs. 0.195 ± 0.033 (p=1), respectively, or between
the CCRCC brain metastasis and its surrounding brain, 0.126 and 0.119 +/− 0.033,
respectively. The relative increase in the Lactate C3 ratio in the surrounding brain of GBM
when compared to brain surrounding the CCRCC likely reflects the relative decrease in
brain perfusion with a large tumor mass (217 mg, GBM vs 44 mg for CCRCC). This data, in
addition to the presence of the same pattern in non-tumor bearing brain (large doublet
relative to the singlet) suggests that flux through the pentose phosphate pathway relative to
glycolysis is not increased in the tumors compared to the surrounding non-tumor bearing
Spectra from mice with an intracranial tumor showed multiplets in glutamate carbon 4 that
were dominated by the doublet resulting from coupling between carbon 4 and 5 (D45)
(Figure 4). This doublet was derived from [1,2-13C2]acetyl-CoA. The quartet (doublet of
doublets) resulting from glutamate labeled in positions 3 and 4 and 5 appears as a
consequence of turnover of the Krebs cycle. The singlet in glutamate C4 is due to natural
abundance 13C or to more complex pathways such as pyruvate recycling [21], which
produces [2-13C]acetyl-CoA. Importantly, there was no qualitative difference in the 13C
spectrum of GABA C2 (at 35.1 ppm) compared to glutamate C4 (at 34.2 ppm). If most or all
GABA is produced directly from brain glutamate, these multiplets should be identical.
Production of Glutamate and GABA in Orthotopic Tumors
In both GBM and CCRCC brain metastasis, 13C multiplets were also observed in Krebs
cycle -derived metabolites, such as glutamate and GABA. In GBM, GABA C2 had
essentially the same labeling pattern as glutamate C4 (Figure 5A). These highly similar
multiplets are expected for a direct precursor-product relationship, as predicted for GABA,
which is produced from glutamate by a single decarboxylation catalyzed by glutamate
decarboxylase (e.g. GAD67, glutamate decarboxylase). Extensive brain neurochemical
analysis has established that GAD67 is exclusively expressed in a population of cortical and
subcortical interneurons, so called GABAergic neurons that convert glutamate to
GABA[22]. This hypothesis is supported by immunohistochemical (IHC) evidence showing
inclusion of of GABAergic interneurons interspersed within the infiltrating GBM tumor
mass (Figure 5A, blue arrows).
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In contrast, the CCRCC brain metastasis, which is devoid of infiltrating normal brain
parenchyma (except, host derived endothelial cells), exhibited a higher doublet, D12,
relative to singlet, S, in GABA C2 compared to doublet, D45, to singlet, S, in glutamate C4,
resulting in a singlet-to-doublet ratio of 0.172 and 0.346, respectively (Figure 5B). Although
this observation was obtained from a single spectrum (produced by pooling CCRCC tissue
from the brains of 4 mice), these data indicate that GABA production in the CCRCC brain
metastasis originates from a subset of cells that do not produce the majority of the
GAD67 Expression in Human Clear Cell Renal Cell Carcinomas
To further explore the unexpected finding of GABA synthesis in the CCRCC brain
metastasis, we examined the orthotopic tumor tissue for expression of the 65 kDa and 67
kDa isoforms of GAD (GAD65 and GAD67), respectively, by immunohistochemistry. The
CCRCC brain metastasis tumors were strongly immunopositive for GAD67 (Figure 5B), but
not for GAD65 (data not shown). Furthermore, immunohistochemistry evaluation of the
human brain metastatic tumors from which the orthotopic tumors were derived revealed
extensive GAD67 staining (Figure 6). In contrast, GAD67 staining was confined to the
interneurons in the GBM (Figure 5A), consistent with the lack of expression of GAD67 in
GBM cells. As further strong evidence for GABA production by the CCRCC brain
metastasis, we obtained tissue sections from the patient's primary kidney mass, and
demonstrated that it also stained positively for GAD67 (Figure 6). Finally, to test whether
GAD67 expression is a common feature of renal tumors with 'clear cell renal cell
carcinoma' histology, we stained a tissue microarray of an additional set of 96 primary
human renal tumors with the same histological (CCRCC) diagnosis (supplementary Figure
1). Remarkably, 84% stained for GAD67. Seventeen (17.7%) demonstrated strong
immunoreactivity (3+/3), 34 (35.4%) demonstrated moderate (2+/3) immunoreactivity, 30
(31.3%) were mildly immunoreactive and only 15 (15.6%) showed no immunoreactivity to
GAD67 (0/3). The staining pattern showed no statistically significant correlation with tumor
grade, tumor stage, presence or absence of metastasis (data not shown). Normal human
kidney epithelium, the presumptive origin of clear cell carcinomas, showed no GAD67
immunoreactivity (data not shown).
13C-labeling in lactate during infusion of [1,2-13C2]glucose was used to assess the relative
activity of the PPP compared to glycolysis in orthotopic mouse models of GBM and
CCRCC that had metastasized to the brain. There was no significant difference in the
singlet/doublet ratio in carbon 3 of lactate between either the GBM or CCRCC and
surrounding non-tumor bearing brain tissue. Interestingly, all tumors showed excess uptake
of 18FDG on PET scan. Taken together and assuming that increased 18FDG signal indicates
increased glucose metabolism, these results indicate that the relative flux through the PPP
compared to glycolysis is not altered in these tumors but the combined rate of glucose
metabolism is increased. This estimation of the PPP relative to glycolysis was based on the
singlet-to-doublet ratio of lactate C3 derived from [1,2-13C2]glucose. This approach assumes
that the singlet arising from lactate C3 is the product of the oxidative branch of the PPP plus
the background natural abundance signal (1.1% of total carbon). In both the GBM and
CCRCC brain metastasis, the ratio of singlet to doublet in lactate C3 was similar between
the tumor and the surrounding brain. Although the current data could be interpreted as
excess flux through the PPP in both tumor and surrounding brain relative to normal brain, it
is not possible to draw that conclusion with confidence because of possible
compartmentation of lactate. Specifically, lactate in the tumor mass is present in at least
three potential compartments: in blood, in a slowly-exchanging pool in poorly perfused or
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metabolically inactive tumor cells, and in a rapidly-exchanging pool that reflects metabolism
of exogenous glucose. We have shown that the majority of lactate in blood is not enriched,
thus metabolism of lactate from blood does not contribute to the PPP. Alternatively, PPP
flux can be underestimated using this approach, as ribose 5-phosphate generated through the
oxidative branch of PPP may not reenter glycolysis. Instead, it can be used for nucleotide
synthesis, reducing the abundance of lactate C3 singlet.
Previous studies had suggested that while the bioenergetic demands of malignant cell
growth are met by enhanced glycolysis[1, 2], the PPP is preferentially activated to support
macromolecule biosynthesis that is necessary for cellular growth[23, 24]. It is of interest to
note, however, that estimates of maximum flux through PPP relative to glycolysis reported
here for GBM and CCRCC tumors (19% for and 12%, respectively) are comparable to those
reported in traumatic brain injury in a rodent model (9-12%) and humans (19.6%)[12, 15].
These estimates are significantly higher than the approximate 5% estimates of PPP activity
in brain under unstressed conditions [25, 26]. One possible explanation for why PPP activity
may be increased by TBI or by the presence of a large intracranial mass and TBI tissue is
that both circumstances are associated with vasogenic/cytotoxic edema. This could produce
a compressive force as the brain expands inside a rigid cranium compromising perfusion and
limiting oxygen diffusion as result of interstitial edema. Although speculative, it is possible
that the resulting oxidative stress (generation of ROS) may explain why both intracranial
tumor tissue and non-tumor bearing regions of the brain show increased PPP activity.
13C NMR analysis of both GBM and CCRCC brain metastasis, revealed 13C-13C coupling
in glutamate and GABA, suggesting that glucose was metabolized to acetyl-CoA and further
oxidized in the Krebs cycle. Although [1,2-13C2] labeled glucose is a suboptimal tracer for
assessing Krebs cycle intermediates since only 50% of acetyl-CoA becomes 13C labeled,
nevertheless the presence of multiplets in glutamate and GABA denotes presence of Krebs
cycle activity. This observation suggests that malignant tumors growing in the
microenvironment of the brain rely on oxidative metabolism in addition to glycolysis and
increased production of lactate, typical of the Warburg effect[2].
An unexpected observation in the CCRCC brain metastasis was that 13C multiplets in
GABA C2 displayed a lower singlet-to-doublet ratio (0.172) relative to its precursor
glutamate C4 (0.346), suggesting that GABA derived from a subset of the total glutamate
pool. This was not observed in spectra from normal brain or GBM. Conversion of glutamate
to GABA requires GAD67 (or GAD65) which, in the brain, is exclusively expressed by a
critically important subset of GABAergic inhibitory interneurons [22]. Although the
orthotopic metabolic data presented here was derived from a single CCRCC brain metastatic
tumor, further support for this novel finding comes from the demonstration that the patient's
brain metastasis, from which the orthotopic tumor line was derived, as well as the patient's
primary renal tumor mass, all showed strong GAD67 immunoreactivity. Moreover, the
importance of this discovery and its potential clinical relevance is demonstrated by the
finding that 86% of an independently generated, clinically annotated, tissue microarray of 96
individual renal tumors with CCRCC histopathology also showed GAD67
immunoreactivity. Since the expression level of GAD67 did not appear to correlate with
tumor grade or prognosis, it raises the possibility that upregulation of GAD67 expression is
an early event in the genesis of CCRCC tumors. The fact that elevated GAD67 expression
persists as CCRCCs accrue mutations and evolve into higher grade local and metastatic
disease, including brain, suggests that the ability to synthesize GABA may play an important
role in the pathophysiology of this disease. In support of the speculation that GABA
signaling may promote tumor growth, there is growing evidence that a wide variety of non-
CNS tumors express GAD67, synthesize GABA and express GABA-specific receptors
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(GABA-A and GABA-B) [27]. What specific role GABA plays in supporting renal tumor
growth, particularly brain metastasis remains to be elucidated.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
This work was supported by the NIH (RR02584, RC1 CA146641, DK072565, NS0760675), NINDS
(F32NS065640), CPRIT (HIRP100437), Fundación Caja Madrid and Billingsley Fund.
List of Abbreviations
carbon 13
18fluoro-deoxyglucose positron emission tomography
6-phosphogluconate dehydrogenase
atom percent excess
clear cell renal cell carcinoma
carbon dioxide
doublet 23
fructose 6-phosphate
glyceraldehyde 3-phosphate
glucose 6-phosphate dehydrogenase
gamma-Aminobutyric acid
glutamate decarboxylase
gas chromatography-mass spectrometry
glucose 6-phosphate
lactate dehydrogenase
monoclonal antibody to Ki67
magnetic resonance
nicotinamide adenine dinucleotide phosphate
nuclear magnetic resonance
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non-obese diabetic/severed combined immunodeficient
positron emission tomography
phosphoglucose isomerase
pentose phosphate pathway
ribose 5-phosphate
singlet 3
1. Gogvadze V, Zhivotovsky B, Orrenius S. The Warburg effect and mitochondrial stability in cancer
cells. Mol Aspects Med. 2010; 31(1):60–74. [PubMed: 19995572]
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the pentose pathway in liver. J Biol Chem. 1985; 260(29):15439–44. [PubMed: 3934159]
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7. Schrader MC, Simplaceanu V, Ho C. Measurement of fluxes through the pentose phosphate
pathway in erythrocytes from individuals with sickle cell anemia by carbon-13 nuclear magnetic
resonance spectroscopy. Biochim Biophys Acta. 1993; 1182(2):179–88. [PubMed: 8357849]
8. Laughlin MR, Thompson D. The regulatory role for magnesium in glycolytic flux of the human
erythrocyte. J Biol Chem. 1996; 271(46):28977–83. [PubMed: 8910548]
9. Delgado TC, Castro MM, Geraldes CF, Jones JG. Quantitation of erythrocyte pentose pathway flux
with [2-13C]glucose and 1H NMR analysis of the lactate methyl signal. Magn Reson Med. 2004;
51(6):1283–6. [PubMed: 15170851]
10. Lee WN, Boros LG, Puigjaner J, Bassilian S, Lim S, Cascante M. Mass isotopomer study of the
nonoxidative pathways of the pentose cycle with [1,2-13C2]glucose. Am J Physiol. 1998; 274(5 Pt
1):E843–51. [PubMed: 9612242]
11. Boros LG, Torday JS, Lim S, Bassilian S, Cascante M, Lee WN. Transforming growth factor beta2
promotes glucose carbon incorporation into nucleic acid ribose through the nonoxidative pentose
cycle in lung epithelial carcinoma cells. Cancer Res. 2000; 60(5):1183–5. [PubMed: 10728670]
12. Dusick JR, Glenn TC, Lee WN, Vespa PM, Kelly DF, Lee SM, Hovda DA, Martin NA. Increased
pentose phosphate pathway flux after clinical traumatic brain injury: a [1,2-13C2]glucose labeling
study in humans. J Cereb Blood Flow Metab. 2007; 27(9):1593–602. [PubMed: 17293841]
13. Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM, Pastorino S, Purow BW, Christopher N,
Zhang W, Park JK, Fine HA. Tumor stem cells derived from glioblastomas cultured in bFGF and
EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured
cell lines. Cancer Cell. 2006; 9(5):391–403. [PubMed: 16697959]
14. Li A, Walling J, Kotliarov Y, Center A, Steed ME, Ahn SJ, Rosenblum M, Mikkelsen T,
Zenklusen JC, Fine HA. Genomic changes and gene expression profiles reveal that established
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glioma cell lines are poorly representative of primary human gliomas. Mol Cancer Res. 2008; 6(1):
21–30. [PubMed: 18184972]
15. Bartnik BL, Sutton RL, Fukushima M, Harris NG, Hovda DA, Lee SM. Upregulation of pentose
phosphate pathway and preservation of tricarboxylic acid cycle flux after experimental brain
injury. J Neurotrauma. 2005; 22(10):1052–65. [PubMed: 16238483]
16. Marian CO, Cho SK, McEllin BM, Maher EA, Hatanpaa KJ, Madden CJ, Mickey BE, Wright WE,
Shay JW, Bachoo RM. The telomerase antagonist, imetelstat, efficiently targets glioblastoma
tumor-initiating cells leading to decreased proliferation and tumor growth. Clin Cancer Res. 2010;
16(1):154–63. [PubMed: 20048334]
17. Suh JM, Zeve D, McKay R, Seo J, Salo Z, Li R, Wang M, Graff JM. Adipose is a conserved
dosage-sensitive antiobesity gene. Cell Metab. 2007; 6(3):195–207. [PubMed: 17767906]
18. Malloy CR, Thompson JR, Jeffrey FM, Sherry AD. Contribution of exogenous substrates to acetyl
coenzyme A: measurement by 13C NMR under non-steady-state conditions. Biochemistry. 1990;
29(29):6756–61. [PubMed: 1975750]
19. Sherry AD, Malloy CR, Zhao P, Thompson JR. Alterations in substrate utilization in the reperfused
myocardium: a direct analysis by 13C NMR. Biochemistry. 1992; 31(20):4833–7. [PubMed:
20. Kunnecke B, Cerdan S, Seelig J. Cerebral metabolism of [1,2-13C2]glucose and [U-13C4]3-
hydroxybutyrate in rat brain as detected by 13C NMR spectroscopy. NMR Biomed. 1993; 6(4):
264–77. [PubMed: 8105858]
21. Cerdan S, Kunnecke B, Seelig J. Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo
and in vitro 13C NMR. J Biol Chem. 1990; 265(22):12916–26. [PubMed: 1973931]
22. Fogarty M, Grist M, Gelman D, Marin O, Pachnis V, Kessaris N. Spatial genetic patterning of the
embryonic neuroepithelium generates GABAergic interneuron diversity in the adult cortex. J
Neurosci. 2007; 27(41):10935–46. [PubMed: 17928435]
23. Cascante M, Centelles JJ, Veech RL, Lee WN, Boros LG. Role of thiamin (vitamin B-1) and
transketolase in tumor cell proliferation. Nutr Cancer. 2000; 36(2):150–4. [PubMed: 10890024]
24. Swinnen JV. Increased lipogenesis in steroid-responsive cancer cells: mechanisms of regulation,
role in cancer cell biology and perspectives on clinical applications. Verh K Acad Geneeskd Belg.
2001; 63(4):321–33. [PubMed: 11603058]
25. Ben-Yoseph O, Boxer PA, Ross BD. Noninvasive assessment of the relative roles of cerebral
antioxidant enzymes by quantitation of pentose phosphate pathway activity. Neurochem Res.
1996; 21(9):1005–12. [PubMed: 8897463]
26. Gaitonde MK, Evison E, Evans GM. The rate of utilization of glucose via hexosemonophosphate
shunt in brain. J Neurochem. 1983; 41(5):1253–60. [PubMed: 6619864]
27. Young SZ, Bordey A. GABA's control of stem and cancer cell proliferation in adult neural and
peripheral niches. Physiology (Bethesda). 2009; 24:171–85. [PubMed: 19509127]
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Diagram depicting [1,2-13C2]-glucose metabolism through glycolysis and the pentose
phosphate pathway (PPP) with the resulted labeling pattern of lactate C3. As a consequence
of the higher activity of glycolysis relative to PPP in normal cells, the contribution of the
doublet (D23) to the total NMR signal of lactate C3 is significantly more prominent than the
singlet (S3). HK: hexokinase, G-6PDH: glucose 6-phosphate dehydrogenase, 6PGDH: 6-
phosphogluconate dehydrogenase, PGI: phosphoglucose isomerase, LDH: lactate
dehydrogenase, Glc: glucose, Glc-6-P: glucose 6-phosphate, F6P: fructose 6-phosphate,
G3P: glyceraldehyde 3-phosphate, 6PG: 6-phosphogluconate, R5P: ribose 5-phosphate,
PYR: pyruvate, LAC: lactate. Open circles denote carbon 12, filled circles denote carbon 13.
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Orthotopic primary GBM mouse tumors share MR and PET imaging features seen in
original patient. In panel A, both human and orthotopic mouse GBM tumors appear as a
large hyperintense T2 mass, with significant mass effect (mouse MRI). Clinical and
orthotopic tumor FDG-PET images are consistent with enhanced glucose uptake relative to
the surrounding brain tissue. Panel B shows hematoxylin-eosin (H&E) and Ki67 staining
from the orthotopic GBM and orthotopic CCRCC brain metastasis. GBM tumors were
characterized by a high mitotic index, pleomorphic nuclei, tortuous microvasculature and
diffuse single cell infiltration. In contrast, the CCRCC showed brisk proliferation,
monomorphic nuclei and scant cytoplasm. Magnification of histological images is 20×.
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Section of the forebrain NMR spectrum from a non-tumor bearing NOD/SCID mouse
infused with [1,2-13C2]-glucose. Spin-coupled 13C multiplets in metabolites exchanging
with Krebs cycle intermediates (i.e., glutamate, glutamine, GABA and aspartate) is observed
in the 13C spectrum as a consequence of glucose oxidation in the brain. Of note, the labeling
pattern of glutamate C4 (GLU4) and GABA C2 (GABA2) were comparable as GABA
derives primarily from glutamate in normal brain. Insets display labeling patterns of lactate
C3 and from aspartate C3 to glutamine C4. GLU: glutamate, GLN: glutamine, ASP:
aspartate, LAC: lactate, TAU: taurine. 1: N-acetylaspartate C6, 2: GABA C3, 3: unassigned,
4: glutamine C3, 5: glutamate C3, 6: unassigned, 7: unassigned, 8: GABA C4, 9: N-
acetylaspartate C3, 10: taurine C1, 11: aspartate C2, 12: N-acetylaspartate C2, 13:
unassigned, 14: glutamine C2, 15: glutamate C2. S: singlet, Dxx: doublet, Q: quartet.
Marin-Valencia et al. Page 15
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text:
Miserable Monday Commute… Again!
Overnight snow has left Hudson Valley roads snow covered. As crews figure out where to put even more snow, you can expect slow going this Monday morning.
Overnight snow has left Hudson Valley roads snow covered. As crews figure out where to put even more snow, you can expect slow going this Monday morning.
A man was hospitalized after jumping from the second floor of a Hudson Valley shopping mall because of a dare.
A group of talented, young Hudson Valley students bring down the house during their performance of Don't Stop Believin'.
As we brace for another winter storm, check out this wild time-lapse footage of the recent snowfall in Poughkeepsie.
New York State Police stepped up traffic patrols and sobriety checkpoints for Superbowl Sunday. The increased enforcement lead to many New Yorkers receiving tickets.
Yonkers Motorcycle Club hosts their 30th Annual Chili Party with contests, free buffet and live music. It's a chilly Saturday turned Chili Saturday.
This batch of new releases is looking to be for the kids...and lovers of SpongeBob. Jeff Bridges could not save the Seventh Son, nor could Channing Tatum and Mila Kunis ascend Jupiter.
Hanna Davis was revealed as the cover model for this year's Sports Illustrated Swimsuit Issue. But some are saying she went a bit too far.
In the days before the Internet, the Swimsuit Issue was eagerly awaited by teenage boys everywhere
Boris won a free lunch for four at a Hudson Valley restaurant. Will his coworkers compete for a spot?
A local library has the honor of being named first finalist for the 2015 Best Small Library in America Award. The annual award is handed out by the Bill and Melinda Gates Foundation and the Library Journal.

subset: commoncrawl


text:
Granite seams at sink - it depends?
My fabricator nearly always seams at sinks, because a high percentage of the time when he doesn't the slab breaks during transport as there is so little material - it is easy for 4 guys to twist slightly and snap it.
But my slab is gneiss and has lots of long, lovely lines - so I'm worried getting a nice seam will be difficult. And my counters are extra deep at 29" with a sink cutout that will be less than 18" front to back. The run is 115", the sink will be about 35" from one end and only 28" wide. I do not think the gniess is brittle, the slab edges were perfect and my broken sample could be glued back without noticing the line, there was no crumbling. It is 128" long.
This is for the cleanup zone, on the left side. The island will be one slab, and the cooking wall run will likely have a bridge behind the KA rangetop.
Are there other factors to consider as I prepare to make my case for no seam?
kk, you saw my seams and i don't have,as much movement in my stone as you do in yours. You said you glued your broken piece back together. The problem is when they cut for the sink they cut out about 1/8th of an inch. So some of your pattern is missing when they put the seam together. Also some fabricators leave the surface chipped. The inside of the stone fits perfectly, but the top looks wider. Plus it is difficult to match the color. (thanks, old ryder!)
Look inside the hole cutout. the slab looks like it is close, but look how wide the seam is.
Can you get bookmatched slabs? We were able to and the fabricator was able to have the two pieces on either side of the sink flow together nicely. This joint is hardly noticeable, even with our heavy movement granite (Crema Bordeaux).
Are you doing an undermount or drop in sink? If drop in, they could just do the cutout for the sink on site. That way, you can have one continuous piece.
My slabs are already at the fabricators, and I doubt they are bookmatched. The sink is undermount and I want a slight positive reveal, so it will have to be done at the shop.
Peke, I'm so sorry you had fabrication problems, you deserve better after all the work and effort you put in!
I am a fabricator.
We do not seam at the sink except in special circumstances. It doesn't make sense to me to have a seam at the busiest location in the entire kitchen.
Fabricators "sell" the seam at the sink because it eliminates their risk of a cracked sink rail. However, the vast majority of the time a seam elsewhre is preferable.
sink rails can be "rodded", which involves epoxying a steel rod into the underside of the sink rail, to minimize the chance of a crack.
when I started my shop I made an arrangement with a local engineering college to do a test of rodded vs. unrodded. The materials science professor (one of my partners teachers) assigned the project to 3 senior engineering students. They built a hydraulic test fixture with computer data acquisition and confirmed that rodded rails are substantially less prone to cracking. (I mention this because there are a lot of "opinions" in the industry about the benefits of rodding and the people that assert that rodding doesn't help are simply wrong.)
With rodding and proper care during install most granites can have an undermount sink without a seam at the sink.
Thank you, oldryder (and all). The pressure to "sell" this plan is on me, since my fabricator is my GC is my cabinet maker/installer/electrician, etc. I'm trying not to be a difficult client, but I'm sure we TKO GWers are never the easiest clients.
Would you rod a run that was 115" by 29" if it had a cut out that was 28 x 18? It must help that front and back rails will be a combined 11", doesn't it? Would the type of stone affect your decision?


text:
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Nintendo 3DS
What Zelda game do you hate that everyone else likes?
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As you all know, everyone has their favorite Zelda game, and some Zelda games are praised as being the "best in the series." Each game tends to form its own following, and that following sometimes effects other peoples opinions as well. For example, there's the people who fell in love with OoT, and that in return caused other people to try it out and like it as well. Then again, this isn't always the case.
So, out of all of the zelda that have huge followings, which one do you personally not like at all/hate (despite what everyone else says)? As for me, I personally hate Link to the Past. Yep, I said it lol. Now I never thought it was a bad game or anything, it has solid gameplay, its the game that really set the standards for future Zelda releases, its story isn't bad, it has challenging dungeons, and a wide verity of tools. As a game in general, I cannot deny that it isn't a good one; however I still hate it.
I've tried many times to get into the game, and its just something I can't do. I've started the game at least 10 times now, and each time I start it I've gotten a little farther and farther. Every single time I make it close to the end, go a bit farther, and then stop. That's when normally I'd somehow either lose my save file, or I'd get ANOTHER version of the game (for example the GBA version or VC version). Despite playing about 75% of the game through over and over again, I just couldn't get myself to like it. The first time I played the game it felt like a chore, and I didn't have any fun, and every playthrough after I just tried to force myself to finish it; which I could never do. While the Dark World is a really big turn off for me, I think I just found the dungeons annoying. Although since I've played them so many times I can run through them blindfolded, I just hate them... I guess LttP just isn't my type of game.
So, now, what about you guys? What Zelda game do you hate that most people seem to love?
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I love them all.
Confession Time!
jRPGs are pretty much the best thing that ever happened to Video Games - Soanevalcke6
guedesbrawl posted...
I love them all.
Even the cd-i games?
Project Beat a Game a Day, along with my Daily NES Archive:
i really didn't like wind waker, i found it to be way too easy and the ship sailing to be way too boring. yet i hear a lot of people talk about it as one of the best
on the other end i really enjoyed twilight princess even though i always hear it get ripped on
When I was younger, I never really got into A Link To The Past.
Steam ID: jessegames1996 | 3DS Friend Code: 2750-1600-2747 | Nintendo Network ID: Transdude1996
x_stevey_x posted...
i really didn't like wind waker, i found it to be way too easy and the ship sailing to be way too boring. yet i hear a lot of people talk about it as one of the best
Yeah, it's actually my favorite XD. I understand why people would hate it though
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Hate is a pretty strong word. Out of the Zelda's I've played, the ones I don't like are TP and SS.
Don't really hate any, but OOT isn't my favorite by a long shot. Still enjoyed it though.
I feel like throwing the controller at Zelda II if that counts........I try to get back >.> but casually.
Own a Vita, you're a troll, own a 3ds, you're a fanboy, own an IOS device, you have no opinion...tis the curse on these boards.
Minish Cap.
I really didn't like Minish Cap and it gets a lot of praise. I don't know if I hated it, but I sure didn't like it.
Official Alice of the Shin Megami Tensei IV board


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Centennial Park Retirement Village in North Platte is hosting a Halloween walk from 3:30-4:30 p.m. on Oct. 31.
Centennial Park Activities Director Kathy Hagan urges children to bring a canned food item and receive a special treat of a homemade caramel apple.
Centennial Park Retirement Village will donate the canned foods to a food pantry to help the pantry get stocked before the holidays.
I recognized there was a need at the local food pantry and with the holidays coming up, it just made sense to try and help out, Hagan said. Our goal is to deliver a good variety of canned goods to the food pantry."
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Houston Gamer
Video games with Willie Jefferson
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Gospel of Gaming: Advanced Warfare. Destiny. Extra-Life. Skylanders.
So, with a few days before "Call of Duty: Advanced Warfare" drops into living rooms, dorm rooms, dens, Man Spaces, Man Caves and the like – bedrooms, too), here's something to ponder on – "Advanced Warfare" Season Pass. Here's the skinny on it: The Call of Duty®: Advanced Warfare Season Pass includes: · Four epic […] [Read More]
A simple solution to GamerGate: R.E.S.P.E.C.T.
Originally published on LinkedIn I started playing video games back in the 1980s. My mom picked up a small console that played just one game – PONG. Fast-forward to 2014 – I play video games. I review video games. I blog about video games for one of the biggest newspapers in the United States, the […] [Read More]
Review: Garden Warfare [PS4], Attack Boss Cheat Code
Plants vs. Zombies Garden Warfare Developed by: PopCap Games Published by: EA Available: NOW Available on: Xbox One, Xbox 360, PlayStation 4, PlayStation 3, PC Rating: E10+ Cost: $39.99 (PS4), $29.99 (PS3) Let's play a guessing game, shall we? What do you get when you combine a 3rd-person shooter, "Little Shop of Horrors," and multiplayer-only […] [Read More]
Houston Gamer M4d Ski11z graces "Wil Wheaton Project" clip
UPDATE: Unconfirmed sources are saying the screen capture was first shown on "Weekend Update" on "Saturday Night Live." Talk.about.random … So, a college classmate of mine posted this on my Facebook wall: Sure enough, I watch this clip And sure enough, right after chicken pox, lo and behold, a screenshot of the Houston Gamer, blowing […] [Read More]
Which game had a bigger impact? "Titanfall" or "Infamous?"
It's been a bit of a drought for gamers, but rejoice! Two video games, "Titanfall" and "Infamous Second Son" have made their way to the Xbox One and the PlayStation 4. Did either software sell and/or move systems? Hit the jump for details. Recently, Sucker Punch's "Infamous Second Son" hit store shelves. The video game, […] [Read More]
Is Xbox One's $50 price cut permanent?
Let's take a walk down memory lane, shall we? During Sony's E3 conference in Los Angeles in 2013, Wedbush analyst Michael Pachter and I chatted about the Xbox One and PlayStation 4 prices. Pachter and I agreed – $399 was the sweet spot for both consoles. I even made bold predictions that Microsoft would release […] [Read More]
Review: Does Titanfall bring A-game to table?
"Titanfall" Developed by: Respawn Entertainment Published by: EA Available: NOW (360 version will be released on March 25th) Available on: Xbox One, PC, Xbox 360 – March 25th Price: $59.99 Standard Edition; $249 for collector edition with Titan statue Genre: SERIOUSLY?! Rating: M for Mature In the past months, gamers who made the jump into […] [Read More]
Sony raises PlayStation 4 prices $50 – in Canada
It's about to get real … According to a report, video gamers in Canada can expect their wallets to be lighter soon. Microsoft and Sony are planning to increase the costs for video games ($70) to combat a *weak* Canadian dollar. Not only will video game price climb, but Sony is raising the prices for […] [Read More]
UNBOXING Xbox One headset, media remote, rare Skylander
This week has been a great week for video gaming. Respawn's epic mech shooter, "Titanfall," debuted on the Xbox One and PC Tuesday. The multiplayer-only shooter lets gamers pilot huge mechs or hit the ground and battle enemies in traditional first-person shooter style. I'm still playing through "Titanfall" and will post a review shortly. Stay […] [Read More]
Infamous Second Son UNBOXING and What I'm rocking
So, "Infamous Second Son" came in for review. The Sucker Punch game drops March 21st – next Friday – but Sony sent it early for review. W00t! I can not wait to break enemies off something proper! Next Friday, look for my full review. Here is the "Infamous Second Son" unboxing. Also, this weekend, what […] [Read More]
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by Humes, Edward
Biloxi, Mississippi, has a "strip" of nightclubs and casinos where prostitution, drugs, and crooked gambling flourish unchecked. An older couple who thought they were retiring to a quiet seaside town got too deeply involved with local politics and the Dixie Mafia and were murdered. The investigation would've sunk beneath the muddy swirl of graft and business as usual but for the tenacious efforts of the victims' daughter. Despite death threats and indifferent law enforcement officials, she hired a private detective and swore to do whatever it took to bring her parents' killers to trial. Horror/suspense writer Peter Straubfinds the story reminiscent of Elmore Leonard and Carl Hiaasen: "Like those writers, Edward Humes can make the wild, amoral, scheming sleazoids he parades before our eyes all but sing and dance on the page. Here is America, fat and happy, both hands crammed into the till." Mississippi Mud was a 1995 finalist for the Edgar Award in Fact Crime.
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Combat Stress
November 6, 2009 - 5:53 AMWe'll find out much more about Major Hasan over the next days and weeks. I wonder, though, where the Army psychiatrists and nurses, the Combat Stress heroes, go when they need counseling -- when one of their own kills.
Hasan is alive and in custody. Unlike so many other maniacs who shoot up innocent people, we'll learn why he did this.
Hasan is a Muslim from a Muslim family in Virginia. He joined the Army out of high school, spending eight years as an enlisted man. The Army put him through college and then through medical school. For that the Army requires that you spend a little time in the Service on its behalf.
Hasan is an Army psychiatrist. Somehow, over the course of his career, he has avoided service in either Iraq or Afghanistan over the past eight years. It may be that he had played all the angles but his number came up and his military butt was en route the war zone.
Psychiatrists in war zones - at least in the war zone I was in - work in a unit called "Combat Stress." Combat, by its nature, is stressful. Some people deal with it better than others. In my experience some people walked around the Green Zone in full "battle rattle" any time they left the palace, just because. Combat Stress helped them.
Some people see, hear, and smell things - like their buddy, sitting in the seat next to them in their Humvee, being blown up by an IED - which are more than any, any, normal human being can handle. That's when the Combat Stress folks get involved.
My first trip outside of Baghdad in 2003 was to the U.S. compound in Ramadi in the dreaded Anbar Province and was, at the time, the home of the 82nd Airborne. I flew up there in a helicopter with an Army psychiatrist who had, as Nidal Hasan had, gone through the military's medical school at Bethesda, Maryland.
Here's what I wrote at the time: "The psychiatrist is going out into the field for a year. A YEAR! This is a long, long way from being a psychiatrist on Park Avenue. In fact, this is about as far from Park Avenue as you can get and still be in the near Solar System.
"The First Sergeant of the Combat Stress unit, a reservist who is a former nurse and most recently was a member of the California Highway Patrol dealing with organized crime specializing in the Russian mafia in Los Angeles."
My point is, the Combat Stress folks - doctors and nurses - were real soldiers and they were plenty tough. It may be harder to deal with the kid whose buddy was blown apart than to be the surgeon trying to save the life of the buddy.
Hasan who, according to the NY Times, is 39-years old "started having second thoughts about his military career a few years ago after other soldiers harassed him for being a Muslim."
Hello? You mean to tell me that after 9/11 a guy named Nidal Hasan wasn't harassed for being a Muslim but sometime over the past "few years" his fellow officers turned on him because of his name?
If he is 39 years old and joined out of high school, he has been in the military for about 20 years. If he was going to be harassed for being a Muslim, it is more likely to have happened in his enlisted days than when he was an Army physician.
Hasan, I suspect, started having those "second thoughts about his military career" not because of late-arriving harassment, but because he didn't want to be deployed to Afghanistan or Iraq.
Who has wanted to be deployed? Ok, not including me, I mean.
If Hasan was a staff psychiatrist working in the medical center at Fort Hood, there have to be some questions about whether his colleagues - his medical colleagues - noticed anything about his behavior which might have led at least one of them to wonder whether he might be headed down this path.
If not, why not?
We'll find out much more about Major Hasan over the next days and weeks. I wonder, though, where the Army psychiatrists and nurses, the Combat Stress heroes, go when they need counseling - when one of their own kills.
On the Secret Decoder Ring page today: Links to the NY Times and AP backgrounders on Hasan. Also a Mullfoto of the only person I have declined to be my Facebook friend, and a Catchy Caption of the Day.


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Personalized I.D. Heart Charm Bracelet in Sterling Silver 8"
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Aqua Hotel Bertran
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Nosotros en España!
We are in Spain! Found a camping site in Barcelona for tonight and tomorrow to celebrate New Year's Eve. Can't wait to shower and do laundry! (After boondocking in parking lots and "Aires" - essentially roadside rest stops - in France for several days, anything with real showers and toilets feels like a palace!) This campsite comes with a bar and restaurant ...
... for young people, it's dead. The people are great though, and it is obviously better than Cooma. I've been meeting with people regularly, which is always good, makes things less lonely. I miss a few people back in aus, which I can't wait to see when I get back. Spain is honestly great though, sometimes I wish I could live ...
Last day here. We are going to the caves this morning and then having paella for lunch, so keen to finally have some traditional Spanish food. Last night dinner was quite good, we went to a restaurant and had muscles which were so yum! Also some fish soup which was strange but good. It's sunny here today and looks like a beautiful day. Let's see how the day pans out ...
... to walk down to the beach, which was about a 49 minute intense walk, so good. Reminded me of being in Australia. Once we got to the beach, we relaxed a bit and took photos like true tourists. The walk back was slow, I was racing ahead as I was super thirsty, but kids being kids slowed down this process. We had a few drinks once we got back to the lighthouse, this was good! I'm pretty into a few Spanish songs, after listening to them repeatedly on the car trips. ...
... a movie, finally not in Spanish! We had a nice lunch, finally with vegetables. That's what I miss most about cooking for myself, how healthy I could make it. The afternoon was relaxed, I had a bit of a headache, but I still played with Andreu and also studied some ******* computing. I'm hating slightly how I have an exam after my amazing winter, but I guess when I'm doing it I feel slightly productive. I've been talking to ...


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The agent of Sampdoria striker Andrea Caracciolo admits Lazio's interest is "real".
Claudio Pasqualin said: "The interest of Lazio is real. There has been contact, next week could be decisive for reaching a turning point.
"My client also is interesting Parma and Atalanta, while I've spoken with Roma."
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Case Conclusion Date:March 27, 2013
Practice Area:Criminal Defense
Outcome:Case Dismissed
Description:Client was charged with theft at Midway Airport. Allegedly he picked up an iPad at security and walked off with it to his gate. The State's Attorney had a video and two TSA Agents in court when we set the case for trial. The victim, who had always appeared in court ready to proceed, failed to appear. The State asked for a continuance which was summarily denied by the judge when we answered ready for trial. Case dismissed!


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Wohoo, was super lucky to get hold of Process 153DL 2015. Frame is just the same as 2014, but there is changes with specs. Biggest being that drivetrain is 1x11, also handlebar has 0.5 inch more rise. Other Hubs are better in my opinion and in general looks better with black spokes and cool colours on the saddle. Surprise is that the green color on the bike exactly the same as in 2014 model.
Did the last enduro race of the season with it last weekend. It's really good. I like the handlebar better, but the x01 drivetrain was the biggest surprise. It's clearly more quiet and of course was really nice when we had to pedal up to some of the stages.
Photo from the race
Process 153DL 2015, wheels are not the ones that come with the bike, I use this lighter set for racing and trail riding.
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There's an old saying that goes that the longer a couple stay together, the more they end up resembling one another. If that is indeed a truism, if donning a curly wig to look like her boyfriend, Rory McIlroy, is any indication, we should start feeling sorry for Caroline Wozniacki beginning…now. On Tuesday night, the couple named Wozzilroy, who were once the hottest sports power couple in all the land before Tiger Woods and Lindsey Vonn came along and ruined everything, were attending a player's party on the eve of the Sony Erickson Open in Miami, Fla. As you can see above, this is how the couple appeared as the two entered the event. And as you will see below, Wozniacki, who has never really regained her form after she started dating McIlroy (but nevertheless has looked great while cheering her boyfriend on at tournaments), threw on a curly, bushy wig inside the party in order to better resemble her golf star beau. GAH! Sweet Fancy Moses! That's awful, just awful. She looks like…like…um, Rory McIlroy's ugly younger sister. Seriously, I can't come up with anything better than that. Too early in the day, I guess. Here's the tweet that unleashed the photo upon an unsuspecting world: Here is me trying to do my best @mcilroyrory impression at the players party last night! twitter.com/CaroWozniacki/… — Caroline Wozniacki (@CaroWozniacki) March 20, 2013 Weird stuff, man. [H/T Beyond the Baseline] TweetThe post Gah! Caroline Wozniacki dons curly wig to mimic boyfriend Rory McIlroy (photo) appeared first on Sportress of Blogitude.
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Get free worksheets plus activities, articles and science projects.
Horse-lovers and nature-lovers alike will enjoy coloring in this serene outdoor scene. The horse on this coloring page is taking a break to get a drink of water. Can your child give some color to this pretty day?
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Today's executives know that success depends on a strong understanding of business principles. But more important is the ability to apply those principles and create change that resonates on many levels. The Elms Masters of Business Administration was devised with this in mind - providing students with the skills necessary to navigate the global economy as well as contribute to the surrounding community.
Three separate tracks of study - healthcare leadership, accounting, and management - are offered in a fast track, hybrid format that pairs online and on-campus courses.
Come for a visit. Elms MBA program will host information sessions on campus March 24, and April 7, 2015 in the Alumnae Library from 4-6 p.m. Register here.
We are a Catholic liberal arts college founded by the Sisters of St. Joseph of Springfield, Massachusetts.


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Caz and I will be presenting at design schools all over Australia in July and August. Check back here later this week to see if we are visiting your Uni/Tafe.
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Here's a Corvette that had its 15 minutes of fame in television history. For sale on eBay is the 1984 Corvette that was driven by Templeton 'Faceman' Peck of the A-Team series. It was the only Corvette used during the show's 1983-87 run and featured a custom red stripe that went around the entire car. The Corvette is documented with registrations from Stephen J. Cannell's production company as well as two empty blank shells found under the passenger seat.
Videos & Pictures
Wayne Bickley's 1996 Black Corvette and its supercharged 396 cubic inch V8 was the epitome of performance. Unfortunately, it was also a target of California's stringent emissions laws. So instead of fighting the man, Bickley went the opposite direction by dumping the gas-powered V8 engine for an electric motor and a batch of 16-volt batteries. How is the performance of that black Vette now? Check out these videos to see for yourself.
We're not sure what sparked the fire that caused this torch red C5 Z06 to burn to the ground, and the original poster didn't say, but judging by the way the flames are dancing along the ground, it definitely looks fuel related. A lot of money went into this car for the opportunity to play in the 1000 hp club and in just a few minutes it was all gone.
Chevy put out this brief preview of the Autobots that are featured in the next installment of the Transformers movie franchise. Sideswipe, the autobot that reverts to the Stingray Corvette concept is featured prominently in the preview.
Article and photos contributed by Steve Burns
We finally had a chance to check out the new 2012 color, Carlisle Blue, in person last weekend at the Bloomington Gold show. This was on the same car which we spotted on Lance Miller's Facebook page. A constant stream of people surrounded the new Grand Sport convertible and its 2012 stable mates in the GM booth most of the weekend. Click past the jump to see more of Corvette's newest shade of blue.
In case you were unable to make it to Carlisle, PA back in May for the Premiere of The Quest, the acclaimed documentary about the Briggs Cunningham Corvettes that raced for the first time at 24 Hours of Le Mans back in 1960, we've got good news. The movie has been booked for an encore showing during Corvettes at Carlisle. The movie will be shown Thursday, August 25th at 8:00 pm. Get your tickets now because this event is sure to sell out!
When GM returned to the Nurburgring to validate the 2012 Corvettes, the ZR1 wasn't the only car driven by test driver Jim Mero. Mero also drove a black 2012 Corvette Z06, outfitted with the Z07 chassis package, and posted a lap time of 7:22.68. The new lap time for the Z06 is just 3 seconds off the record-setting 7:19.63 time Mero recently recorded in the ZR1 while besting the previous Z06 record at the Nurburgring by nearly 20 seconds.
Tell me, is there anything more satisfying than the sound you hear when opening up your dual NPP exhaust valves under full acceleration? This custom Corvette ZR1 was surprising docile sounding under normal throttle, but once the traffic cleared in front of him, his foot hit the floor and we heard the monstrous LS9 open up in all it's glory. The Corvette was so loud, it shocked those around it including the guy who was filming.
How does a Le Mans driver prepare for the big race? Hear Corvette Racing drivers' stories of what it is like to be on and off the track at the biggest and longest race of the year.


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In Ghana and Côte d'Ivoire, regions that acquired great wealth from the mining and trading of gold, chiefs and other people of s...
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Hiroshige's 118 woodblock landscape and genre scenes of mid-nineteenth-century Tokyo, is one of the greatest achievements of Japanese art.
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Published by Xseed, Developed by FuRyu
Genres: RPG (1 players)
US release date: Jan 3rd, 2013 | EU release date: -
Prices: $29.99 (US)
Unchained Blades follows the story of the dragon emperor, Fang, who after being stripped of his powers by the goddess Clunea, embarks on a journey for revenge and to regain his true form.
Arrogant and vengeful, Fang is joined in his quest by a cowardly golem prince, a shy medusa priestess, a young phoenix clan maiden, and many other mythical beings seeking to meet with the goddess.
Due to the collaborative nature of the game's design, each of the 13 main characters was created by a different artist. The main character Fang was designed by Pako, most famous for his work on the Shining Force series, while other characters were fashioned by artists such as Toshiyuki Kubooka (Lunar), Shinichiro Otsuka (Summon Night), and Kazushi Hagiwara (Bastard!!), to name a few. As such, each character has its own distinct and signature style.
In Unchained Blades, players journey through the game with a party of up to four characters, moving through dungeons in a first-person perspective and engaging in turn-based combat along the way. Using the game's unique ?Unchain? system while in battle, players are able to try and convince monsters to follow them. If they are successful in using the Unchain system, the enemy monster will then join their party and help them in the future by blocking attacks or supplying players with special offensive abilities. Each character in the player's four person party can hold up to four ?unchained? creatures, ensuring plenty of combat variety. The game has also been completely redubbed with new English voice-overs and boasts over 60 hours of gameplay.
Unchained Blades has no review on Wii's World.
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Michael Jordan said:
No they're making it from the movie ... Speed Racer: The Video Game
daveoneal said:
Thanks Rob. I had no idea this one was published. I'm very happy about it! Yes, Jonathan, it is hard as all get-out, ... Street Fighter 2010: The Final Fight review
daveoneal said:
This was a very well-written review. Apparently Super Hexagon was developed by Terry Cavanagh (who made vvvvvv), so ... Cosmophony review
dirk said:
:p p ... TMNT: Smash Up
dirk said:
cool :p ... TMNT: Smash Up
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