text:
Altered mitochondrial dynamics contributes to endothelial dysfunction in diabetes mellitus.
ABSTRACT Endothelial dysfunction contributes to the development of atherosclerosis in patients with diabetes mellitus, but the mechanisms of endothelial dysfunction in this setting are incompletely understood. Recent studies have shown altered mitochondrial dynamics in diabetes mellitus with increased mitochondrial fission and production of reactive oxygen species. We investigated the contribution of altered dynamics to endothelial dysfunction in diabetes mellitus.
We observed mitochondrial fragmentation (P=0.002) and increased expression of fission-1 protein (Fis1; P<0.0001) in venous endothelial cells freshly isolated from patients with diabetes mellitus (n=10) compared with healthy control subjects (n=9). In cultured human aortic endothelial cells exposed to 30 mmol/L glucose, we observed a similar loss of mitochondrial networks and increased expression of Fis1 and dynamin-related protein-1 (Drp1), proteins required for mitochondrial fission. Altered mitochondrial dynamics was associated with increased mitochondrial reactive oxygen species production and a marked impairment of agonist-stimulated activation of endothelial nitric oxide synthase and cGMP production. Silencing Fis1 or Drp1 expression with siRNA blunted high glucose-induced alterations in mitochondrial networks, reactive oxygen species production, endothelial nitric oxide synthase activation, and cGMP production. An intracellular reactive oxygen species scavenger provided no additional benefit, suggesting that increased mitochondrial fission may impair endothelial function via increased reactive oxygen species.
These findings implicate increased mitochondrial fission as a contributing mechanism for endothelial dysfunction in diabetic states.
- SourceAvailable from: Francesco Paneni
Article: Molecular mechanisms of vascular dysfunction and cardiovascular biomarkers in type 2 diabetes.[Show abstract] [Hide abstract]
ABSTRACT: Prevalence of obesity and type 2 diabetes (T2DM) is alarmingly increasing worldwide. Albeit advances in therapy have reduced morbidity and mortality in T2DM, cardiovascular risk is far to be eradicated and mechanism-based therapeutic approaches are in high demand. In this perspective, deciphering novel molecular networks of vascular disease will be instrumental to develop novel diagnostic and therapeutic strategies in people affected by diabetes. There is therefore a need to address current knowledge gaps in disease aetiology in order to support innovation in diagnosis and treatment. Unfortunately, we are still lacking cost-effective markers able to identify atherosclerotic vascular disease at an early stage. The issue of risk stratification deserves attention because not every T2DM patient carries the same degree of inflammation and oxidative stress. The diversity of metabolic phenotypes with different outcomes underscores the need for cardiovascular risk stratification within such heterogeneous population. Early predictors of vascular damage are mandatory to implement intensive treatment strategies and, hence, reduce cardiovascular disease burden in this setting. In this review we critically discuss novel molecular mechanisms of diabetic vascular disease and their possible translation to the clinical setting.Cardiovascular diagnosis and therapy. 08/2014; 4(4):324-332.
Article: Targeting prolyl-isomerase Pin1 prevents mitochondrial oxidative stress and vascular dysfunction: insights in patients with diabetes.[Show abstract] [Hide abstract]
ABSTRACT: Diabetes is a major driver of cardiovascular disease, but the underlying mechanisms remain elusive. Prolyl-isomerase Pin1 recognizes specific peptide bonds and modulates function of proteins altering cellular homoeostasis. The present study investigates Pin1 role in diabetes-induced vascular disease. In human aortic endothelial cells (HAECs) exposed to high glucose, up-regulation of Pin1-induced mitochondrial translocation of pro-oxidant adaptor p66(Shc) and subsequent organelle disruption. In this setting, Pin1 recognizes Ser-116 inhibitory phosphorylation of endothelial nitric oxide synthase (eNOS) leading to eNOS-caveolin-1 interaction and reduced NO availability. Pin1 also mediates hyperglycaemia-induced nuclear translocation of NF-κB p65, triggering VCAM-1, ICAM-1, and MCP-1 expression. Indeed, gene silencing of Pin1 in HAECs suppressed p66(Shc)-dependent ROS production, restored NO release and blunted NF-kB p65 nuclear translocation. Consistently, diabetic Pin1(-/-) mice were protected against mitochondrial oxidative stress, endothelial dysfunction, and vascular inflammation. Increased expression and activity of Pin1 were also found in peripheral blood monocytes isolated from diabetic patients when compared with age-matched healthy controls. Interestingly, enough, Pin1 up-regulation was associated with impaired flow-mediated dilation, increased urinary 8-iso-prostaglandin F2α and plasma levels of adhesion molecules. Pin1 drives diabetic vascular disease by causing mitochondrial oxidative stress, eNOS dysregulation as well as NF-kB-induced inflammation. These findings provide molecular insights for novel mechanism-based therapeutic strategies in patients with diabetes.European Heart Journal 05/2014; · 14.72 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Mitochondria are perhaps the most sophisticated and dynamic responsive sensing systems in eukaryotic cells. The role of mitochondria goes beyond their capacity to create molecular fuel and includes the generation of reactive oxygen species, the regulation of calcium, and the activation of cell death. In endothelial cells, mitochondria have a profound impact on cellular function under both healthy and diseased conditions. In this review, we summarize the basic functions of mitochondria in endothelial cells and discuss the roles of mitochondria in endothelial dysfunction and vascular diseases, including atherosclerosis, diabetic vascular dysfunction, pulmonary artery hypertension, and hypertension. Finally, the potential therapeutic strategies to improve mitochondrial function in endothelial cells and vascular diseases are also discussed, with a focus on mitochondrial-targeted antioxidants and calorie restriction.Frontiers in Physiology 01/2014; 5:175.
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Altered Mitochondrial Dynamics Contributes to Endothelial
Dysfunction in Diabetes Mellitus
Sherene M. Shenouda, PhD1, Michael E. Widlansky, MD2, Kai Chen, MD1, Guoquan Xu,
MD1, Monika Holbrook, MS1, Corey E. Tabit, MD1, Naomi M. Hamburg, MD1, Alissa A.
Frame, BA, Tara L. Caiano, BS1, Matthew A. Kluge, BS1, Mai-Ann Duess, BS1, Aaron Levit,
BS1, Brian Kim, BS1, Mor-Li Hartman, PhD1, Lija Joseph, MD1, Orian S. Shirihai, PhD1, and
Joseph A. Vita, MD1
1Evans Department of Medicine and the Whitaker Cardiovascular Institute, Boston University
School of Medicine, Boston, MA
2Cardiovascular Medicine Division, Department of Medicine, Medical College of Wisconsin,
Milwaukee, WI
Background—Endothelial dysfunction contributes to the development of atherosclerosis in
patients with diabetes mellitus, but the mechanisms of endothelial dysfunction in this setting are
incompletely understood. Recent studies have shown altered mitochondrial dynamics in diabetes
mellitus with increased mitochondrial fission and production of reactive oxygen species (ROS).
We investigated the contribution of altered dynamics to endothelial dysfunction in diabetes.
Methods and Results—We observed mitochondrial fragmentation (P=0.002) and increased
expression of fission-1 protein (Fis1, P<0.0001) in venous endothelial cells freshly isolated from
patients with diabetes mellitus (n=10) compared to healthy controls (n=9). In cultured human
aortic endothelial cells exposed to 30 mM glucose, we observed a similar loss of mitochondrial
networks and increased expression of Fis1 and dynamin-related protein-1 (Drp1), proteins
required for mitochondrial fission. Altered mitochondrial dynamics was associated with increased
mitochondrial ROS production and a marked impairment of agonist-stimulated activation of
endothelial nitric oxide synthase (eNOS) and cGMP production. Silencing Fis1 or DRP1
expression with siRNA blunted high glucose-induced alterations in mitochondrial networks, ROS
production, eNOS activation, and cGMP production. An intracellular ROS scavenger provided no
additional benefit, suggesting that increased mitochondrial fission may impair endothelial function
via increased ROS.
Conclusions—These findings implicate increased mitochondrial fission as a contributing
mechanism for endothelial dysfunction in diabetic states.
endothelium; mitochondria; mitochondrial dynamics; reactive oxygen species
Type 2 diabetes mellitus is an increasingly prevalent risk factor for atherosclerotic
cardiovascular disease.1 A key mechanism in atherogenesis is endothelial dysfunction,
which is characterized by decreased nitric oxide bioavailability and development of an
Address for Correspondence: Joseph A. Vita, MD, Professor of Medicine, Boston University School of Medicine, 88 East Newton
Street, C-818, Boston, MA 02118, 617-638-8742, 617-638-8712 (FAX), firstname.lastname@example.com.
NIH Public Access
Author Manuscript
Circulation. Author manuscript; available in PMC 2012 July 26.
Published in final edited form as:
Circulation. 2011 July 26; 124(4): 444–453. doi:10.1161/CIRCULATIONAHA.110.014506.
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inflammatory phenotype that promotes atherosclerosis.2 An improved understanding of the
mechanisms of endothelial dysfunction could stimulate new approaches for the prevention
and management of diabetic cardiovascular disease.
Prior studies implicate increased oxidative stress as a primary mechanism of endothelial
dysfunction in diabetes mellitus.3 Exposure of cultured endothelial cells or isolated arterial
tissue to high glucose concentrations increases production of reactive oxygen species (ROS)
that decrease nitric oxide bioavailability and increase expression of pro-inflammatory
genes.2,3 Several enzymatic sources of ROS have been implicated in diabetes mellitus,
including NADPH oxidase, aldose reductase, and components of the mitochondrial electron
transport chain.4–7
There is growing appreciation of the importance of altered mitochondrial dynamics in
diabetes mellitus.8 Mitochondria undergo cycles of fusion to form networks and fission to
form smaller individual mitochondria.8,9 Proteins controlling fusion include mitofusin-1 and
-2 (Mfn-1, 2) and optic atropy-1 (Opa1). Fission is regulated by dynamin-related protein-1
(Drp1) and fission-1 (Fis-1). Fusion may be beneficial by allowing for distribution of
metabolites, proteins, and DNA throughout the network. At the end of their life cycle,
dysfunctional mitochondria and damaged mitochondrial components are eliminated by
fission and subsequent autophagy.10 Under pathological conditions, including diabetes,
fission is increased and autophagy is impaired, which leads to a loss of mitochondrial
networks, accumulation of small dysfunctional mitochondria, and increased mitochondrial
Prior studies have demonstrated a loss of mitochondrial networks under hyperglycemic
conditions in a variety of cell types, including islet cells,12,13 hepatocytes,11 skeletal muscle
cells,14,15 circulating blood mononuclear cells,16 and endothelial cells.17–19 The functional
consequences of altered mitochondrial dynamics in diabetes mellitus, however, remains
incompletely understood in the human vasculature. The present study was designed to
investigate the contribution of altered mitochondrial dynamics to increased ROS production
and impaired nitric oxide bioavailability under diabetic conditions.
Study Subjects
Adult patients with Type 2 diabetes mellitus and healthy volunteers were recruited at Boston
Medical Center by advertisement. Diabetes mellitus was defined as fasting glucose ≥126
mg/dl or ongoing treatment for Type 2 diabetes mellitus. Healthy volunteers were taking no
medications and had blood pressure <140/90 mmHg, fasting LDL cholesterol <160 mg/dl,
fasting glucose <100 mg/dl, and had never smoked or had stopped smoking for more than a
year prior to enrollment. Fasting glucose and lipids were measured in the Boston Medical
Center Clinical Laboratory. The study protocol was approved by the Boston Medical Center
Institutional Review Board and all participants provided written informed consent.
Non-Invasive Vascular Function Testing
Brachial artery flow-mediated dilation was measured in each patient as previously
described.20,21 Briefly, Doppler flow signals and two-dimensional images were recorded
from the brachial artery before and one minute after induction of reactive hyperemia by 5-
minute cuff occlusion of the upper arm. We simultaneously measured endothelial
vasodilator function in fingertip vessels using digital pulse amplitude tonometry (Endo-PAT,
Itamar Medical Ltd., Caesarea, Israel).22
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Materials and Reagents
Please see supplementary materials for a list of the sources for materials and reagents for the
endothelial cell experiments.
Fresh Isolation of Endothelial Cells from Study Subjects
Endothelial collection was performed as previously described.23 Briefly, local anesthesia
was administered and an 18 or 20 gauge catheter was placed in an arm vein using sterile
technique. Endothelial cells were collected by gently abrading the luminal surface of the
vein with a 0.018" J-wire. For immunofluorescence experiments, endothelial cells were
recovered from the wire using a dissociation buffer, fixed with 4% paraformaldehyde, and
plated on poly-lysine coated slides (Sigma). Fixed slides were stored at −80°C until further
processing. For live cell studies, recovered endothelial cells were allowed to adhere to poly-
lysine coated glass bottom dishes (MatTek, Ashland, MA, USA) for six hours in EGM-2
medium (Lonza Inc., Walkersville, MD, USA) prior to imaging.
Endothelial Cell Culture
Human aortic endothelial cells (HAEC's) were purchased from Lonza, Inc. and maintained
using the EGM-2 Bullet Kit media (Lonza, Inc.) containing 5 mM glucose. Cells were
cultured according to the manufacturer's instructions at 37°C with 5% CO2. For
immunofluorescence experiments, cells were grown in BD Falcon 4-well chambered slides
(BD Biosciences). The effects of elevated glucose on endothelial cell function were
investigated by incubating cells in EGM-2 media with a final glucose concentration of
30mM (540 mg/dL). This concentration is relevant to diabetic patients with severe
hyperglycemia and has been used in many prior studies of the vascular effects of high
glucose concentrations. To control for the osmotic effects of high glucose, we also
completed studies using EGM-2 medium containing 5 mM glucose and 25 mM mannitol.
Cells were stored at −80°C until immunofluorescence, protein, or gene expression
measurements were performed.
Immunofluorescence Staining and Fluorescence Microscopy
Fixed samples were rehydrated with phosphate-buffered saline (PBS) containing 50mM
glycine and permeablized using 0.1% Triton X-100. After blocking non-specific binding
sites with 0.5% bovine serum albumin, slides were incubated with two of the following
primary antibodies: anti-von Willebrand's factor (vWF) 1:200, anti-cytochrome C 1:300,
anti-cytochrome c oxidase-IV (COX IV) 1:400, anti-Fis1 (1:100) or anti-phosphorylated
endothelial nitric oxide synthase (eNOS) 1:200. Slides were incubated with corresponding
Alexa Fluor-488 and Alexa Fluor-594 secondary antibodies and mounted using Vectasheild
containing the nuclear stain DAPI (Vector Laboratories). All staining was performed in one
batch to avoid any day-today variability in staining sessions.
Slides were examined using a fluorescence microscope (Nikon Eclipse TE2000-E) at 100x
magnification and cellular images were digitally captured by a Photometric CoolSnap HQ2
Camera (Photometrics, Tucson, AZ, USA). All images were captured at the same exposure
time and presented values are corrected for local background fluorescence. For the freshly
isolated patient samples, endothelial cells were distinguished from other cells by vWF
staining. Images were captured and intensity was measured using NIS Elements AR
Software (Nikon Instruments Inc, Melville, NY, USA).
Assessment of Mitochondrial Networks
We used a semi-quantitative scale to rate the extent of mitochondrial networks in patient
cells and in HAEC's under different experimental conditions. Cells were stained for
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cytochrome c and evaluated by two blinded observers. Mitochondrial network extent was
graded on a scale from 0 to 3, where 0 = wholly fragmented or punctuated mitochondria and
3 = clearly defined mitochondrial networks. The average score from 20 cells from each
patient or condition was determined by two people, read separately and the readings were
averaged. The coefficient of variation was 7.5% (n=20 cells, measured 3 times) for repeated
assessment of cells under the same experimental condition.
We also performed live cell imaging to further evaluate mitochondrial morphology. For
these experiments, mitochondria were labeled with 100nM MitoTracker Green FM
(Invitrogen) in HEPES-buffered physiologic salt solution (HB-PSS) in accordance with
manufacturer's recommendations. Images were captured at 100x magnification.
Mitochondrial ROS Production
Mitochondrial ROS production was measured using fluorescence microscopy in live cells.
HAECs were incubated with 5μM MitoSox and 100nM MitoTracker Green FM (Invitrogen)
for 30 minutes at 37°C and imaged according to the manufacturer's instructions (excitation/
emission 510/580 nm). Co-localization of MitoSox and MitoTracker Green signal verified
that ROS signal was localized to the mitochondria. Thirty cells were analyzed per
experiment to obtain an average intensity value. In some experiments the contribution of
intracellular ROS to vascular dysfunction was evaluated by treating cells with a superoxide
scavenger, tiron (5mM). The coefficient of variation for ROS measurement was 9.7%.
RNA Isolation and Quantitative Gene Expression
Total RNA was isolated from HAEC's using the miRNeasy Mini kit (Qiagen, Inc.)
according to the manufacturer's instructions. RNA was reverse transcribed to
complimentary DNA (cDNA) using TaqMan Reverse Transcription Reagents followed by
cDNA preamplification using TaqMan PreAmp Master Mix (Applied Biosystems)
according to the manufacturer's instructions. Quantitative real-time PCR was performed
using Taqman Gene Expression Assays (see supplementary materials) and the following
protocol: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for
1 min. Using GAPDH as a loading control, results were interpreted by the relative quantity
method (ΔΔCt).
Protein Isolation
Collected cell pellets were resuspended and briefly sonicated in a cell lysis buffer (Cell
Signaling) containing 1% protease inhibitor cocktail (Sigma). Cell lysates were spun at
10,000 rpm for 10 min at 4°C, and the resulting supernatants were stored at −80°C. Protein
concentration was determined by the Pierce BCA protein assay (Thermo Fisher Scientific).
Western Blot Analysis
Proteins were subjected to 8–12% SDS-PAGE and transferred to PVDF membranes (GE
Healthcare). Membranes were initially blocked (PBS, 0.1% Tween 20, 5% nonfat dry milk)
for 1 hour. Membranes were probed in blocking buffer containing one of the following
primary antibodies: anti-OPA1, anti-Drp1, anti-Mfn2 or anti-Fis1 antibody followed by the
appropriate HRP-conjugated secondary antibody. Immunoreactions were visualized with
Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare). Membranes
were stripped (62.5 mM Tris-HCl (pH 6.8), 100 mM β-mercaptoethanol, 2% SDS) for 30
min at 50°C and reprobed using anti-actin antibodies to verify equal protein loading.
Resulting bands were quantified by densitometry.
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Assessment of eNOS Activation and Endothelial cyclic GMP Production
eNOS activation was assessed by measuring phosphorylation of eNOS at serine 1177. After
exposure of HAEC's to 5 mM or 30 mM glucose for 24 hours, cells were incubated with the
calcium ionophore A23187 (1 μM), acetylcholine (1 μM), insulin (10 nM), or vehicle for
five minutes. Cells were then fixed and stained for phosphorylated-eNOS as described
Bioactivity of endothelium-derived nitric oxide was assayed as change in cyclic-guanosine
monophosphate (cGMP) concentration as previously described.24 Briefly, cells were
equilibrated for 30 min with 200 μM 3-isobutyl-1-methylxanthine (IBMX) to inhibit
phosphodiesterases. Cells were then exposed to A23187 or vehicle for 5 min. Cells were
lysed and cell supernatants and protein pellets were stored at −80°C prior to processing.
Supernatant cGMP was determined using a commercially available EIA kit (Cayman
Chemical) and corrected for total cell protein.
Suppression of Gene Expression using siRNA
HAEC's were transfected with double-stranded siRNA targeting Fis1 or Drp1 using siPORT
NeoFX Transfection Agent (Applied Biosystems) according to the manufacturer's
instructions. Forty-eight hours after transfection, cells were incubated with medium
containing 5mM or 30mM glucose for 24 hours. Cells were then processed as described
above for assessment of mitochondrial network extent, protein expression, gene expression,
or cGMP production. Non-specific scrambled siRNA and GAPDH siRNA were used as
negative and positive transfection controls, respectively.
Statistical Analyses
Statistical analyses were completed using Sigma Stat Version 3.1 (Systat Software, Inc.).
Clinical characteristics and vascular function in the diabetic and healthy control groups were
compared using the unpaired t-test or chi-square test for continuous and categorical
variables respectfully. For cell experiments, we used the Student's T test for two-group
comparisons. For experiments involving more than two groups, we used analysis of variance
(ANOVA) with Student-Newman-Keuls (SNK) multiple pairwise comparisons. P<0.05 was
considered to be statistically significant. Data are expressed as mean ± SEM.
Study Subjects and Vascular Function
We enrolled ten patients with diabetes mellitus and nine controls. The clinical characteristics
and measures of vascular function are shown in Table 1. As expected, the patients with
diabetes mellitus had higher fasting glucose levels, higher body mass index, and lower HDL
levels. Endothelium-dependent flow-mediated dilation of the brachial artery, and flow-
induced changes in pulse amplitude in the fingertip were lower in the diabetics compared to
the controls. Baseline diameter, baseline flow, and extent of reactive hyperemic flow volume
were similar in the two groups, suggesting impaired flow-mediated dilation was not
attributable to differences in arterial geometry or the stimulus for dilation.
Mitochondrial Network Extent in Freshly Isolated Endothelial Cells
Figure 1A shows mitochondrial morphology in freshly isolated endothelial cells. Cells from
the healthy controls displayed elongated, thread-like mitochondria in complex networks
while cells from diabetics displayed smaller punctate mitochondria. Group data show that
patients with diabetes mellitus had lower mitochondrial network extent compared to controls
(0.77±0.09 vs. 1.2±0.06, P=0.002, respectively) as judged by blinded assessment of
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cytochrome c-stained cells (arbitrary scale 0 to 3). Figure 1B shows Fis1 protein levels in
the freshly isolated endothelial cells. Diabetic individuals have significantly higher Fis1
expression compared to healthy controls (1154±60 vs. 568±68, P<0.0001, respectively).
These results suggest altered mitochondrial dynamics in endothelial cells in patients with
diabetes mellitus.
Effect of High Glucose on Mitochondrial Networks and Dynamic Proteins in HAEC's
To investigate potential mechanisms and functional consequences of altered mitochondrial
dynamics in the endothelium, we exposed cultured HAEC's to 30 mM glucose as a model of
hyperglycemic conditions. As shown in Figure 2A, exposure to high glucose induced a
marked and sustained loss of mitochondrial networks over 24 hours. In separate
experiments, exposure to 5mM glucose plus 25 mM mannitol for 24 hours had no effect on
network extent, suggesting that the effect of glucose exposure is not attributable to an
osmotic effect.
The observed change in mitochondrial morphology was accompanied by a shift in
mitochondrial dynamics gene and protein expression favoring fission. Exposure to 30 mM
glucose increased message (Figure 2B) and protein levels (Figure 2C) of the fission protein
Fis-1. Drp1 protein expression was increased (P<0.05) and there was a trend for increased
Drp1 message (P=0.07). There was a modest increase in message level (P<0.05), but no
significant change in protein level of the fusion protein Mfn2 (P=0.11). There were no
significant changes in message or protein expression for the fusion proteins OPA1 and
Effect of High Glucose on Mitochondrial ROS Production, eNOS Activation and NO
We examined the effects of high glucose concentration on mitochondrial ROS production in
HAEC's. As shown in Figure 3, high glucose concentrations induced a marked increase in
mitosox fluorescence that co-localized with Mitotracker Green fluorescence, consistent with
a mitochondrial source of ROS.
We next examined the functional consequences of high glucose concentrations on eNOS
activation and production of bioactive NO (Figure 4A-4C). In cells exposed to 5 mM
glucose, acetylcholine, insulin, and calcium ionophore (A23187) individually activated
eNOS, as reflected by an increase in eNOS phosphorylation at serine 1177. Exposure to 30
mM glucose markedly impaired eNOS phosphorylation in response to all three agonists
(P<0.001 by ANOVA), consistent with impaired eNOS activation vasodilator function.
Calcium ionophore increased cGMP levels in endothelial cells exposed to 5 mM glucose
(Figure 4D), consistent with production of bioactive nitric oxide that is capable of activating
guanylyl cyclase. High glucose exposure blunted the cGMP response to A23187, consistent
with impaired bioactivity of endothelium-derived nitric oxide (P<0.001 by ANOVA).
Silencing Fis1 or Drp1 Protects Mitochondrial Networks and Blunts ROS Production
We next sought to determine whether increased mitochondrial fission contributes to altered
mitochondrial morphology and ROS production in the presence of high glucose. To test this
hypothesis, we used siRNA to silence the expression of the fission proteins Fis1 or Drp1 in
cultured endothelial cells. As shown in Figure 5, message levels were markedly reduced 48
hours after transfection with siRNA targeting Fis1 or Drp1, and there were corresponding
decreases in protein expression.
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As shown in Figure 6A, silencing Fis1 or Drp1 prevented the loss of mitochondrial networks
following exposure of endothelial cells to 30 mM glucose. Fis1 or Drp1 siRNA treatment
did not alter mitochondrial network extent in cells exposed to 5 mM glucose. As shown in
Figure 6A, silencing either of these fission proteins markedly reduced ROS production in
endothelial cells exposed to 30 mM glucose. There was no effect of Fis1 or Drp1 siRNA
treatment on ROS production in cells exposed to 5 mM glucose.
Silencing Fis1 or Drp1 Prevents Glucose-Induced Impairment of eNOS Activation
The effects of Fis1 or Drp1 siRNA on eNOS activation are displayed in Figure 7. As shown,
silencing these fission proteins did not alter the basal expression of phosphorylated eNOS.
However, silencing Fis1 or Drp1 expression prevented glucose-induced impairment of
eNOS phosphorylation in response to calcium ionophore, acetylcholine, and insulin. As
shown in Figure 8, silencing Fis1 expression also prevented glucose-induced impairment of
agonist-induced cGMP production in endothelial cells. Collectively, these results suggest
that glucose-induced alterations in mitochondrial dynamics have broad effects on
endothelial function.
Decreased ROS May Account for the Protective Effects of Fis1 and Drp1 siRNA
We tested the hypothesis that reversal of endothelial dysfunction by inhibition of
mitochondrial fission under high glucose conditions might be explained by the observed
decreased in ROS. eNOS phosphorylation (Figure 7) and cGMP production (Figure 8) in
response to calcium ionophore was preserved in the presence of 30 mM glucose when cells
are incubated with the cell permeable ROS scavenger, tiron. A similar effect was produced
by Fis1 or Drp1 siRNA. When combined with siRNA treatment, tiron had no additional
benefit beyond the effect of Fis1 or Drp1 siRNA. These results are consistent with the
possibility that glucose-induced alterations in mitochondrial dynamics affect eNOS
activation and nitric oxide bioavailability via excess ROS.
In this study, we observed altered mitochondrial morphology, reduced network extent, and
increased Fis1 protein expression in endothelial cells from patients with diabetes mellitus
with endothelial dysfunction compared to healthy volunteers. In cultured cells exposed to
high glucose, we observed a similar loss of mitochondrial networks accompanied by
increased expression of fission proteins, and the effect were not attributable to an osmotic
effect of glucose. The observed alteration in mitochondrial dynamics was associated with
increased mitochondrial ROS production and a generalized impairment in agonist-stimulated
eNOS activation and nitric oxide bioavailability. Silencing Fis1 or Drp1 expression blunted
glucose-induced alterations in mitochondrial networks, ROS production, eNOS activation,
and nitric oxide bioavailability. An intracellular ROS scavenger provided no additional
benefit, suggesting that increased mitochondrial fission may impair endothelial function via
increased ROS. These findings implicate altered mitochondrial dynamics as a contributing
mechanism for endothelial dysfunction in diabetic states.
Several prior studies have demonstrated altered mitochondrial dynamics in non-vascular
tissue from experimental models of diabetes and patients with diabetes mellitus or insulin
resistance. For example, exposure of rat hepatocytes to high glucose concentrations induces
mitochondrial fragmentation and increases ROS production, and the effect can be inhibited
with dominant-negative Drp1.11 Similar findings are observed in pancreatic islet cells
exposed to high glucose or free fatty acids that can be prevented by Fis1 siRNA.12
Mitochondrial fragmentation and decreased expression of Mfn2 is also observed in the
skeletal muscle of obese, insulin resistant rats and diabetic humans.14,25 Recently, we
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observed fragmented mitochondria and increased ROS production in peripheral blood
mononuclear cells from patients with type 2 diabetes mellitus.16
Recent studies have shown that diabetic conditions alter mitochondrial dynamics and
morphology in endothelial cells. Mitochondrial fragmentation occurs when cells from an
immortalized endothelial cell line (EAhy926) are exposed to high glucose.17 Incubation of
rat retinal endothelial cells with high glucose concentrations induces a loss of mitochondrial
networks and increases apoptosis, which could contribute to diabetic retinopathy.18 In a
recent study by Makino and colleagues, endothelial cells isolated from the coronary arteries
of diabetic mice displayed fragmented mitochondria and increased ROS production.19 Those
changes were associated with increased expression of Drp1 and decreased expression of
OPA1, consistent with a shift toward mitochondrial fission. Drp1 siRNA prevented
mitochondrial fragmentation under high glucose conditions, implicating increased fission as
a cause of mitochondrial fragmentation.
The present study extends our understanding of the functional importance and clinical
relevance of altered mitochondrial dynamics in the diabetic endothelium. We observed
increased expression of Fis1 and a protective effect of Fis1 siRNA on mitochondrial
networks in cultured human cells exposed to high glucose. We gained information about the
clinical relevance of these mechanisms by showing a loss of mitochondrial networks in
endothelial cells freshly isolated from diabetic patients that displayed impaired endothelium-
dependent dilation. Most importantly, our study provides information about the functional
consequences of altered mitochondrial dynamics in the diabetic endothelium by showing
that increased mitochondrial ROS, impaired eNOS activation, and loss of nitric oxide
bioavailability can be prevented by inhibiting mitochondrial fission.
Our study provides insight into the importance of mitochondrial network formation as a
regulator of mitochondrial ROS generation in the endothelium. High glucose concentrations
drive the electron transport chain to hyperpolarize the mitochondrial membrane and increase
ROS production at complexes I and III via uncoupled respiration.7 In hepatocytes, mild
membrane depolarization prevents ROS generation, but does not prevent glucose-induced
mitochondrial fragmentation, suggesting that increased mitochondrial ROS is a consequence
of mitochondrial fragmentation and not the cause.11 Consistent with this prior work, we
observed that inhibiting Fis1 or Drp1 expression was sufficient to completely inhibit
glucose-induced ROS production and network fragmentation in endothelial cells.
Collectively, these findings support the idea that network formation limits mitochondrial
ROS production under conditions of increased fuel, possibly by allowing for appropriate
distribution of mitochondrial components, including uncoupling proteins and antioxidant
enzymes.8 It also has been argued the fragmentation alters the spatial orientation of the
electron transport chain enzymes in a manner that promotes uncoupled respiration and ROS
production.11 On the other hand, Makino and colleagues showed that scavenging ROS
prevents glucose-induced mitochondrial fragmentation in mouse endothelial cells,
suggesting that ROS is a trigger for fission under these conditions.19 Further studies are
needed to elucidate how high glucose concentrations stimulate mitochondrial fission and
whether ROS is a cause, consequence, or exacerbating mechanism.
The present study suggests that mitochondrial fission is a major cause of endothelial
dysfunction in the setting of hyperglycemia, likely via increased mitochondrial ROS. In
addition to directly reacting with nitric oxide, ROS may lead to the uncoupling of eNOS,
oxidation of cofactors, and oxidative modification of target enzymes.2,3 Another
consequence of increased oxidative stress in the setting of high glucose may be O-linked N-
acetylglycosylation of serine 1177 on eNOS, which blocks phosphorylation at this site and
attenuates nitric oxide production.26 In our study, inhibiting the expression of fission
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proteins was sufficient to normalize agonist-induced eNOS phosphorylation and production
of bioactive nitric oxide. The beneficial effects were observed when eNOS was activated by
insulin, acetylcholine or calcium ionophore, suggesting that the effect did not depend on the
signaling mechanism for eNOS activation. Further studies will be needed to determine
whether there is a relation between mitochondrial dynamics and other enzymatic sources of
ROS such as NADPH oxidase, which also has been implicated in endothelial dysfunction in
diabetes5–7 and has been shown to be localized to mitochondria.27
Our study has a number of limitations. First, the sample size for the clinical study was
relatively modest, which precluded multivariable analysis to adjust for confounding factors.
The analyses were completed in venous, rather than arterial cells, reducing the relevance to
atherosclerosis, although diabetes mellitus is a systemic disease that affects the endothelium
in both arteries and veins. In addition, a prior study demonstrated significant correlations
between findings in venous and arterial endothelial cells.23 Secondly, we may have
overestimated the effects of high glucose on absolute mitochondrial ROS production by
using an excitation wavelength of 510 nm as recommended by the manufacturer rather than
395 nm for the MitoSox experiments as has been suggested by Robinson and colleagues,
although our studies focused on the relative changes in ROS according to glucose
concentration.28 Further studies are merited to define interactions between mitochondrial
and non-mitochondrial sources of ROS under diabetic conditions. Finally, 24-hour glucose
exposure does not fully mimic the endothelial pathology observed in patients who have had
diabetes mellitus for many years, although our model is well accepted for study of
mechanisms of diabetic endothelial dysfunction. These limitations are balanced by the novel
information about mitochondrial dynamics in endothelial cells from human subjects and
evidence for a mechanistic link between mitochondrial fission and impaired eNOS
Our study may have important clinical implications. A large body of work indicates that
endothelial dysfunction contributes to the pathogenesis of atherosclerotic disease in diabetes,
but the mechanisms of impaired nitric oxide bioavailabity in this setting remain
incompletely understood. Pharmacological inhibitors of mitochondrial fission are under
development.29 Identification of increased mitochondrial fission as a mechanism of
endothelial dysfunction in diabetes mellitus suggests that such drugs might have utility as
therapy to prevent and manage diabetic vascular disease.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
The authors gratefully acknowledge the intellectual support of the Mitochondrial Affinity Research Collaborative at
the Boston University Medical Campus Evans Center for Interdisciplinary Biomedical Research.
Funding Sources
Drs. Tabit, Hamburg, and Vita receive support from the NIH-sponsored Boston University Medical Center
Leadership Program in Vascular Medicine (K12 HL083781). Dr. Widlansky is supported by an NIH training grant
(K23HL089326). Dr. Shirihai is supported by NIH grants DK035914 and DK074778. Dr. Vita is supported by NIH
grants HL083801, HL081587, HL083269, and HL75795 and HL102299. Itamar Medical, Ltd provided the
laboratory with an EndoPAT device and probes for measuring endothelial function by digital pulse amplitude
Shenouda et al.Page 9
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1. Center for Disease Control and Prevention. 2007 National Diabetes Fact Sheet. 2007.
2. Tabit CE, Chung WB, Hamburg NM, Vita JA. Endothelial dysfunction in diabetes mellitus:
molecular mechanisms and clinical implications. Rev Endocr Metab Disord. 2010; 11:61–74.
[PubMed: 20186491]
3. Cohen RA, Tong X. Vascular oxidative stress: the common link in hypertensive and diabetic
vascular disease. J Cardiovasc Pharmacol. 2010; 55:308–316. [PubMed: 20422735]
4. Tesfamariam B, Cohen RA. Free radicals mediate endothelial cell dysfunction caused by elevated
glucose. Am J Physiol. 1992; 263:H321–H326. [PubMed: 1510128]
5. Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T,
Naruse M, Sano H, Utsumi H, Nawata H. High glucose level and free fatty acid stimulate reactive
oxygen species production through protein kinase C--dependent activation of NAD(P)H oxidase in
cultured vascular cells. Diabetes. 2000; 49:1939–1945. [PubMed: 11078463]
6. Hink U, Li H, Mollnau H, Oelze M, Matheis E, Hartmann M, Skatchkov M, Thaiss F, Stahl RA,
Warnholtz A, Meinertz T, Griendling K, Harrison DG, Forstermann U, Munzel T. Mechanisms
underlying endothelial dysfunction in diabetes mellitus. Circ Res. 2001; 88:E14–E22. [PubMed:
7. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001;
414:813–820. [PubMed: 11742414]
8. Twig G, Hyde B, Shirihai OS. Mitochondrial fusion, fission and autophagy as a quality control axis:
the bioenergetic view. Biochim Biophys Acta. 2008; 1777:1092–1097. [PubMed: 18519024]
9. Liesa M, Palacin M, Zorzano A. Mitochondrial dynamics in mammalian health and disease. Physiol
Rev. 2009; 89:799–845. [PubMed: 19584314]
10. Twig G, Elorza A, Molina AJ, Mohamed H, Wikstrom JD, Walzer G, Stiles L, Haigh SE, Katz S,
Las G, Alroy J, Wu M, Py BF, Yuan J, Deeney JT, Corkey BE, Shirihai OS. Fission and selective
fusion govern mitochondrial segregation and elimination by autophagy. EMBO J. 2008; 27:433–
446. [PubMed: 18200046]
11. Yu T, Robotham JL, Yoon Y. Increased production of reactive oxygen species in hyperglycemic
conditions requires dynamic change of mitochondrial morphology. Proc Natl Acad Sci U S A.
2006; 103:2653–2658. [PubMed: 16477035]
12. Molina AJ, Wikstrom JD, Stiles L, Las G, Mohamed H, Elorza A, Walzer G, Twig G, Katz S,
Corkey BE, Shirihai OS. Mitochondrial networking protects beta cells from nutrient induced
apoptosis. Diabetes. 2009; 58:2303–2315. [PubMed: 19581419]
13. Lowell BB, Shulman GI. Mitochondrial dysfunction and type 2 diabetes. Science. 2005; 307:384–
387. [PubMed: 15662004]
14. Bach D, Pich S, Soriano FX, Vega N, Baumgartner B, Oriola J, Daugaard JR, Lloberas J, Camps
M, Zierath JR, Rabasa-Lhoret R, Wallberg-Henriksson H, Laville M, Palacin M, Vidal H, Rivera
F, Brand M, Zorzano A. Mitofusin-2 determines mitochondrial network architecture and
mitochondrial metabolism. A novel regulatory mechanism altered in obesity. J Biol Chem. 2003;
278:17190–17197. [PubMed: 12598526]
15. Zorzano A, Liesa M, Palacin M. Role of mitochondrial dynamics proteins in the pathophysiology
of obesity and type 2 diabetes. Int J Biochem Cell Biol. 2009; 41:1846–1854. [PubMed:
16. Widlansky ME, Wang J, Shenouda SM, Hagen TM, Smith AR, Kizhakekuttu TJ, Kluge MA,
Gutterman DD, Vita JA. Altered mitochondrial membrane potential, mass, and morphology in the
mononuclear cells of humans with type 2 diabetes. Transl Res. 2010; 156:15–25. [PubMed:
17. Paltauf-Doburzynska J, Malli R, Graier WF. Hyperglycemic conditions affect shape and Ca2+
homeostasis of mitochondria in endothelial cells. J Cardiovasc Pharmacol. 2004; 44:423–436.
[PubMed: 15454850]
18. Trudeau K, Molina AJ, Guo W, Roy S. High glucose disrupts mitochondrial morphology in retinal
endothelial cells: implications for diabetic retinopathy. Am J Pathol. 2010; 177:447–455.
[PubMed: 20522647]
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19. Makino A, Scott BT, Dillmann WH. Mitochondrial fragmentation and superoxide anion production
in coronary endothelial cells from a mouse model of type 1 diabetes. Diabetologia. 2010
20. Vita JA. Nitric oxide-dependent vasodilation in human subjects. Methods Enzymol. 2002;
359:186–200. [PubMed: 12481571]
21. McMackin CJ, Vita JA. Update on nitric oxide-dependent vasodilation in human subjects. Methods
Enzymol. 2005; 396:541–553. [PubMed: 16291261]
22. Hamburg NM, Benjamin EJ. Assessment of endothelial function using digital pulse amplitude
tonometry. Trends Cardiovasc Med. 2009; 19:6–11. [PubMed: 19467447]
23. Colombo PC, Ashton AW, Celaj S, Talreja A, Banchs JE, Dubois NB, Marinaccio M, Malla S,
Lachmann J, Ware JA, Le Jemtel TH. Biopsy coupled to quantitative immunofluorescence: a new
method to study the human vascular endothelium. J Appl Physiol. 2002; 92:1331–1338. [PubMed:
24. Huang A, Vita JA, Venema RC, Keaney JF Jr. Ascorbic acid enhances endothelial nitric oxide
synthase activity by increasing intracellular tetrahydrobiopterin. J Biol Chem. 2000; 275:17399–
17406. [PubMed: 10749876]
25. Bach D, Naon D, Pich S, Soriano FX, Vega N, Rieusset J, Laville M, Guillet C, Boirie Y,
Wallberg-Henriksson H, Manco M, Calvani M, Castagneto M, Palacin M, Mingrone G, Zierath
JR, Vidal H, Zorzano A. Expression of Mfn2, the Charcot-Marie-Tooth neuropathy type 2A gene,
in human skeletal muscle: effects of type 2 diabetes, obesity, weight loss, and the regulatory role
of tumor necrosis factor alpha and interleukin-6. Diabetes. 2005; 54:2685–2693. [PubMed:
26. Du XL, Edelstein D, Dimmeler S, Ju Q, Sui C, Brownlee M. Hyperglycemia inhibits endothelial
nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest. 2001;
108:1341–1348. [PubMed: 11696579]
27. Block K, Gorin Y, Abboud HE. Subcellular localization of Nox4 and regulation in diabetes. Proc
Natl Acad Sci U S A. 2009; 106:14385–14390. [PubMed: 19706525]
28. Robinson KM, Janes MS, Pehar M, Monette JS, Ross MF, Hagen TM, Murphy MP, Beckman JS.
Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc Natl Acad
Sci U S A. 2006; 103:15038–15043. [PubMed: 17015830]
29. Lackner LL, Nunnari J. Small molecule inhibitors of mitochondrial division: tools that translate
basic biological research into medicine. Chem Biol. 2010; 17:578–583. [PubMed: 20609407]
Shenouda et al.Page 11
Circulation. Author manuscript; available in PMC 2012 July 26.
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Clinical Summary
Type 2 diabetes mellitus is an increasingly prevalent risk factor for atherosclerotic
cardiovascular disease, and dysfunction of the vascular endothelium contributes to the
development of diabetic vascular disease. We studied a previously unrecognized
mechanism of endothelial dysfunction in human diabetes mellitus, and show that an
alteration in mitochondrial homeostasis is important. In addition to serving as the primary
source of ATP in the cell, mitochondria also participate in many other cellular functions.
In the past, mitochondrial were viewed as discrete oval shaped organelles, but recent
studies have shown that mitochondrial fuse to form complex networks within the cell and
that these networks are important for normal mitochondrial function. We observed that
endothelial cells collected from patients with diabetes mellitus show a loss of normal
networks and marked fragmentation of mitochondria. These changes were accompanied
by increased levels of Fis1, a protein that controls mitochondrial fission. When
commercially available endothelial cells were exposed to high glucose concentrations in
tissue culture, we observed a similar loss of mitochondrial networks, increased Fis1, and
impaired endothelial function. When we prevent mitochondrial fragmentation by
blocking expression of Fis1, we maintain normal mitochondrial networks and prevent
endothelial dysfunction. This study suggests a new target for therapy in diabetes mellitus
and raises the possibility that a drug that prevents mitochondrial fission might protect
against the development of vascular disease in diabetic patients. Such drugs are currently
under development.
Shenouda et al. Page 12
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Page 13
Figure 1.
Diabetes mellitus is associated with increased mitochondrial fission in endothelial cells.
Venous endothelial cells were collected from healthy volunteers (n=9) and patients with
diabetes mellitus (n=10) as described in Methods. A: Mitochondria were labeled in live
endothelial cells with Mitotracker Green FM (Invitrogen) and images were captured at
100X. A representative endothelial cell from a healthy control (left) shows a complex
network of threadlike mitochondria, while a representative cell from a diabetic patient
(right) shows smaller, punctate mitochondria. Fixed cells were stained for cytochrome c and
mitochondrial network extent was rated in a blinded manner using a semi-quantitative scale.
Network extent was lower in the diabetics (*P=0.002). B: Freshly isolated human venous
endothelial cells were fixed and stained for Fis1 protein expression. A representative cell
from a diabetic patient (right) shows markedly higher Fis1 expression compared to a cell
from a healthy control (left). Pooled data show that Fis1 levels were significantly higher in
the diabetics (n=6 per group, *P<0.0001).
Shenouda et al.Page 13
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Figure 2.
High glucose concentration induces mitochondrial fission in endothelial cells. As described
in Methods, human aortic endothelial cells were incubated with 5 mM, 30 mM glucose, or 5
mM glucose + 25 mM mannitol (as an osmotic control) for 24 hours. A. To assess network
extent, cells were fixed at the indicated time points, stained for cytochrome c, and rated on a
semi-quantitative scale. Left panel: High glucose was associated with a rapid and sustained
loss of mitochondrial networks (interaction P<0.001 by repeated measures ANOVA). Right
panel: Mannitol had no effect on network extent, arguing against an osmotic effect. B. Gene
expression was assessed by quantitative real time PCR after 24 hours of glucose incubation,
and there were significant increases in Fis1 and Mfn2. C. Protein expression was measured
by Western blot analysis after 24 hours of glucose incubation. Expression of Fis1 and Drp1
increased significantly. There was no significant change in Mfn2 or Opa1. AU = arbitrary
units. Data are shown as mean ± SEM for 3 experiments (*P<0.05 versus 5 mM glucose).
Shenouda et al.Page 14
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Figure 3.
High glucose concentration induces mitochondrial ROS production. As described in
Methods, HAEC's were incubated with 5 mM or 30 mM glucose for 24 hours.
Mitochondrial ROS production was assessed using 5 μM Mitosox (red fluorescence) and
mitochondria were localized using 100 nM Mitotracker Green (green fluorescence). A.
Representative fluorescence images showing increased ROS production that co-localizes
with mitochondria in endothelial cells incubated with high glucose. High glucose is also
associated with mitochondrial fragmentation. B. Pooled data showing that ROS production
is higher in cells exposed to 30 mM glucose. Data are shown as mean ± SEM for 3
experiments (*P=0.01 versus 5 mM glucose).
Shenouda et al.Page 15
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NIH-PA Author Manuscript


text: ESPN Feed: jrue holiday http://assets.espn.go.com/i/tvlistings/tv_espn_original.gif ESPN en-us 10 ESPN Inc. http://espn.go.com/ Copyright 2010 ESPN Inc. ESPN's NBA Front Office takes over the New Orleans Pelicans and discusses ways to help their young superstar Anthony Davis.]]> December 10, 2014 03:10:13 PST Jrue Holiday's 2013-14 season was cut short in January after sustaining a stress fracture in his right tibia. Holiday has spent the last eight months ...]]> September 12, 2014 05:49:20 PDT The NBA Summer Forecast is uncannily accurate, but there are teams that make a surprise postseason run every year. Bradford Doolittle offers up three for the 2014-15 season.]]> September 11, 2014 05:20:03 PDT Injuries shortened the seasons of the New Orleans Pelicans' nucleus last season, from Anthony Davis (ankle, back) to Jrue Holiday (leg) to Eric Gordon...]]> August 22, 2014 11:30:36 PDT Ryan Anderson had cervical spine surgery In April to repair the herniated disc. At that time the Pelicans gave a 4-6 month timeline for his complete r...]]> June 20, 2014 04:33:24 PDT David Thorpe and Amin Elhassan redo the 2009 draft. Blake Griffin still goes No. 1, but after that it's wide open and their opinions reflect that volatility.]]> May 13, 2014 04:57:57 PDT The New Orleans Pelicans splashed the cash last summer when they assumed the four years and $43 million remaining on Jrue Holiday's contract and paid ...]]> March 20, 2014 07:15:59 PDT The New Orleans Pelicans have been without starting point guard Jrue Holiday for about the last six weeks and Friday came news he won't be available u...]]> March 01, 2014 05:21:50 PST Pelicans starting point guard Jrue Holiday has been out since Jan. 10 due to a stress fracture to his right tibia. Head coach Monty Williams spoke to ...]]> February 20, 2014 04:59:03 PST According to a report Tuesday by Darrell Williams of The New Orleans Advocate, Jrue Holiday "is expected to return in a week, if not sooner." Obvio...]]> February 18, 2014 10:29:16 PST Brian Roberts has been the starting point guard for the New Orleans Pelicans since Jrue Holiday was ruled out indefinitely due to a right tibia stres...]]> January 29, 2014 04:11:47 PST The New Orleans Pelicans are lacking depth at point guard due to Jrue Holiday's injury (out indefinitely - right tibia stress fracture) and they do ha...]]> January 16, 2014 03:57:53 PST Amin Elhassan breaks down the impact of Jrue Holiday's injury on the New Orleans Pelicans. Minus their star guard and likely out of the playoff race, should the Pelicans tank?]]> January 13, 2014 04:28:51 PST With Jrue Holiday now sidelined indefinitely due to a stress fracture in his right tibia, the door could be open for 2013 second-rounder and D-League ...]]> January 10, 2014 10:33:22 PST Should you trade Marshall or keep him? Last week after Jordan Farmar went down with another hamstring injury, I suggested picking up Kendall Marsha...]]> January 07, 2014 11:46:23 PST

subset: commoncrawl


text:
Welcome to Jodrey School of Computer Science
The opportunities for computer science graduates over the next 10 years are going to be exceptional as the generation of baby-boomers moves into retirement. Acadia University, rated consistently as one of the top three undergraduate universities in Canada, provides excellent degree programs in computer science. We teach a solid foundation in computer science and develop our students' interpersonal and team skills through course projects and cooperative education options.
We invite you to explore our website, investigate the various degrees and certificate programs, our areas of research, funding opportunities, and also our cooperative education program. Most importantly, get to know our faculty and students through the many videos and photos on the website. From this we think you'll discover why our motto is "come as a student, leave as a colleague".
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text:
Goodreads helps you keep track of books you want to read.
Start by marking "America on Film: Representing Race, Class, Gender, and Sexuality at the Movies" as Want to Read:
America on Film: Representing Race, Class, Gender, and Sexuality at the Movies
A lively introduction to issues of diversity as represented within the American cinema. A synthetic and historical text, America on Film provides a comprehensive overview of the industrial, socio-cultural and aesthetic factors that contribute to cinematic representations of race, class, gender and sexuality.
Paperback, 392 pages
Published October 20th 2003 by Wiley-Blackwell
(first published October 17th 2003)
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(showing 1-30 of 127)
May 02, 2012 MM rated it 4 of 5 stars
It's hard to rank this book because it's a textbook -- introductory, vocabulary-defining, that kind of thing. But as a textbook, yes! 4 or 5 stars! I liked it and will definitely consider adopting it for an intro class in film/gender studies. The format makes sense, they quite a lot of ground without stretching it too thin (it's not an all-inclusive text, even for an intro class), and they define concepts in a way that doesn't oversimplify or insult readers' intelligence. I'm curious to try it o ...more
Dec 21, 2012 Sarah rated it 2 of 5 stars
I am reading this book for my graduate class on Women and Film. So far, I am not too impressed. Although I am learning new concepts, the way it is presented is definitely for undergraduates and overly repetitive. I guess that is fitting since the class is cross-listed with an undegraduate class... The photographs are great and I particularly enjoy the case studies.
Dec 06, 2012 Casey rated it 5 of 5 stars
Couldn't put this down.
Read every inch. Especially appreciated the reiteration of terms and concepts throughout later chapters.
Wish all academic texts were written this effectively.
Read every inch. Especially appreciated the reiteration of terms and concepts throughout later chapters.
Wish all academic texts were written this effectively.
Jun 26, 2008 Chole Allyson rated it 2 of 5 stars
This is a great read for someone who is interested in the history of film when representing race, class, gender, and sexuality.
Nov 04, 2012 Salome Wilde rated it 4 of 5 stars
Wonderful introduction to cultural theory and the interpretation of Hollywood cinema.
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text:
The ever-shrinking Lindsay Lohan keeps things basic with a ultra-low cut, navy mini-dress. But something about the black tights and the pointy black ankle boots just doesn't work.
And can someone please condition that over-processed blonde hair?
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text:
Average Rating: 5/5 stars (1 review)
100% would recommend it to a friend.
View Product Page Write a Review
I am absolutely head-over-heels for this stamp. It's large and elegant and isn't so Christmasy that I can't use it on other things. Ink comes off of it pretty well although you have to be sure to get into the tiny crevices - there's a lot of intricate detail that is hard to see in the pictures.
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text:
- Cooking Time:
- Servings:
- Preparation Time:
- 1 teaspoon olive oil
- 1 cup diced zucchini
- 1/2 cup minced onion
- 1 clove garlic, peeled and minced
- 2 cups diced fresh tomatoes
- 2 tablespoons chopped fresh basil
- 1/4 teaspoon salt
- 1/4 teaspoon ground black pepper
- 4 (6 ounce) halibut steaks
- 1/3 cup crumbled feta cheese
- Preheat oven to 450 degrees F (230 degrees C). Lightly grease a shallow baking dish.
- Heat olive oil in a medium saucepan over medium heat and stir in zucchini, onion, and garlic. Cook and stir 5 minutes or until tender. Remove saucepan from heat and mix in tomatoes, basil, salt, and pepper.
- Arrange halibut steaks in a single layer in the prepared baking dish.
- Spoon equal amounts of the zucchini mixture over each steak.
- Top with feta cheese.
- Bake 15 minutes in the preheated oven, or until fish is easily flaked with a fork.
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text:
Anime Fans of modDBFans & Clans group with 2,126 members, open to all members
This group has been formed to gather the people who enjoy Japanese anime and all of its facets, to salute the people from the land of the Rising Sun.
2 comments by Sticky on May 4th, 2009
Sup kiddies?
Yep, as it turns out, a little over a year at Pwned, there was some major drama, and I got sick of it real fucking quick, so I'm back here where the drama is usually hilarious and petty instead of major-league like over at Pwned, and I came to the realization I sunk FAAAAR too much time into that site. So, what's new around here?
This group has been formed to gather the people who enjoy Japanese anime and all of its facets, to salute the people from the land of the Rising Sun.
This is where all the Awesome or Cool people of moddb linger, awesome people are asked to join, the cool ones are those that request to join. Are you...
Have your homepage set as the Forums? Join the legions of those addicted to sweet, sweet, forums.
If it's information about ModDB and ramblings about mods in general that you are after - then you have come to the right place. Welcome to the official...
For all the lovers of literature who lurk about the dark caverns of modDB, find haven here. Share the latest books your reading, discuss styles and trends...
For all the old skool modders that have stuck by Mod DB from v1 to v4. A lot has changed as over the years as the site moved from a dark v1 to a bright...
Senior Members are members of the ModDB community held in high esteem for their contributions to the site or their work on the staff.
The Escapist Magazine is home to some of the best Game reviewers on the net, and I'm here to post it all here onto this Group.
Well they wouldn't suck so much if all of you cool guys didn't have to leave.
Do you need to abandon the forums all together?
Alex, remember The Suffering? They made it free on a bajillion sites.
For instance Gamershell.com
But they are ad ridden and plagued with nasty hiccups. PM me for the ad removal and you can add this baby to your archive if you don't have it.
how is my old arch enemy from a few years ago....
I have no idea what pwned is but this place has surely changed god damn it
wheres aaron and tomato killer? Xd
Good review and relation of Pwned and ModdB! I also feel that the site needs a different approach, in order to appeal to it's mixed user base. People are so different here, yet neither the groups nor the forums create enough incentive to communicate. There are hardly any new topics, which stay in the discussion for long enough to become interesting and informative.
What helps me most of all on Moddb is browsing through active users and finding out about their interest and skills. I believe there could be more done in this direction and modding could become a little more personal as a consequence. Right now you will find many people don't submit much info on themselves and aren't eager to share information, either.
I like ModdB a lot, but even in the prior version it seemed more lifely and the forums were still thriving. I don't even think we have less forum posts now, it's just the quality is often poor and I see less contributions by the more interesting mods and indie games (what about Dreamscape, Talon 1-5 and Infinity - where have their updates gone?). So the truly inspiring stuff is missing sometimes - and too often.
That's what you think. >.>
*Pulls dime out of ear*
/me casts a spell on sticky
You will never leave the forums again. Muahahahahaha!!!
What's your Xbox Live name?
Nevermind, I forgot that I'd already added you.


text:
Officium Business Server 1.1
Officium Business Server is a revolutionary web-based office collaboration server that boasts 20 powerful business tools including a full blown email server and 100% compatible MS Office suite of tools. Includes Email, Contacts, Diary, Tasks, Timesheet, Bookings, Messaging and more all in a powerful 'web application' interface.
See also: office suite, thin client solution, office solution, free download evaluation, office collaboration server, email server, intranet software, intranet, web based office suite, intranet, SMS Gateway, SMS Server
TERAVoice Server 2004
TERAVoice is a high-performance telephony platform for Windows that can implement any type of telephony application like voicemail, automated attendants, voice menus, waiting queues for call centers and custom IVR applications.
TERAVoice can also act as a VoIP (Voice over IP) gateway for bridging calls from PSTN to H.323 terminals and computers for inbound and outbound calls.
See also: teravoice, voice, mail, voicemail, ivr, h.323, h323;voip, bridge, gateway, gatekeeper, isdn, tapi, tapi, dialogic, modem, sms, mwi, api, vbscript, javascript, server, telephony, pstn, pbx
acesms activex 3.6
Ace sms ActiveX SDK is a great tool to send receive Unicode SMS via GSM phone or Modem from windows system,AceSMS ActiveX is designed to work with a wide range of phones (Nokia:
Siemens:MotoFalcom: GSM Modem Wavcom Modem TC35 Modem
AceSMS ActiveX is based on a widely accepted Technical Specification known as :
GSM0705 All phone and modem support GSM0705 GSM0340 can use this controls to receive/send Unicode SMS,write/read Unicode PhoneBook
See also: SMS activex Send receive SMS server sms gateway SMS sdk
Mobizware-Lite MMS Server, SMS Server 1.22
Freeware MMS Server, SMS Server. Send MMS SMS from PC using GSM modem or mobile phone. Receive MMS file content and save to PC. Send applet, video and MP3. Send Wap Push. Interface with your own programming tools using VB,VS,Delphi, PHP,ASP.
See also: SMS server, MMS server, SMS Gateway, MMS Gateway, Send MMS, Send SMS, GSM, GPRS, Modem, Send Video, Send MP3, Send Message
SMSgee SMS Bulk Sender 2.0.2
SMS Marketing tools. Send SMS from PC using UMTS/GSM phone/modem. With user friendly GUI easily manage SMS traffic up to 10.000 SMS per day.Support import/export phonebook and SMS to CSV/XML file,create personalized SMS for each recipient very useful for SMS marketing, promotion campaign,customer helpdesk system or any other purpose using SMS.
See also: SMS, SMS GATEWAY, SMS GATEWAY SERVER, SMS TOOLS, SMS SERVER, SMS TRAFFIC, SMS BROADCASTING, SMS RECEIVING, SMS BULK, GSM MODEM, GSM PHONE, HIGH TRAFFIC, SMS ALERT, SMS CRM, SMS ERM, SMS FEATURE, SMS APPLICATION, SMS MARKETING, SMS SENDER
SMSgee PC SMS Gateway Server 4.2.6
Send/receive large number of SMS from PC using multiple GSM phone/modem connected to PC. Support 5 device and more, SMS traffic up to 50 thousand SMS/24h. Create personalized SMS for each recipient very useful for SMS marketing, promotion campaign,customer helpdesk system ,etc. Support import/export phonebook and SMS to CSV/XML file. Could be integrated with other application to add SMS functionality. Support running as windows service.
Ozeki SMS Server Software 184.108.40.206
PC-to-phone SMS Gateway software to send and receive SMS messages. This two-way text messaging solution helps the developers, who want to add SMS functionality to their application SQL, Files, XML, PHP, ASP, Delphi, Java, etc are supported. It is very easy to create solutions like autoreply services, automated SMS services with this tool. Features: Addressbook, Logfiles, Message tracking, special SMS types. Excellent support by the developers.
See also: SMS Gateway software, SMS Server, SMS Software, PHP SMS, SQL SMS, SMS sending receiving, download SMS software, buy sms software, Delphi SMS Component
IRCAS Alarm 3.4.5
IRCAS Alarm is a PC based camera alarm system and surveillance system for Windows. It detects motion from cameras, uses loops for magnetic switches for doors and windows and on/off key switches. Alert you by email and SMS. It runs as a service on the background with build in client, http and MJPEG video server.
See also: email, SMS, PDA, phone, logon, webpage, server, homepage, http, ftp, emergency, services, secure, doors, loops, detector, movement, detect, capture, cam, cameras, Electronics, Rombouts, try, free, system, alarm, home, based, IRCAS
MERCUR Messaging 2005
MERCUR Messaging 2005 is a versatile communication platform for MS Windows. Well known standard protocols, like SMTP, POP3, IMAP4 or HTTP provide the basis for a world wide communication. These protocols are easy to handle and compatible with the frequently used messaging products. A special interface to Norman Virus Control offers a proactive solution against new and unknown viruses. The Webmail interface enables access from all over the world.
See also: IDN, FIREWALL, SPAM, SYMANTEC, KASPERSKY, NORMAN, SERVER, MAIL, FAX, SMS, IMAP4, POP3, SMTP
Airquace SMS-Scheduler Demo 2.0
Airquace's SMS Software allows you to: - Send SMS and email messages from your PC - Send messages to multiple contacts/groups - Send personalized messages - Pre-fix schedules to send out your messages - Schedule messages on daily, weekly, monthly, yearly and events basis - Automate sending of scheduled messages - Manage incoming and outgoing messages. - Often used for: appointment reminders, birthdays wishes, product promotions, etc
See also: sms, email, marketing, gateway, broadcast, group, bulk, mass, singapore, asia, canada, europe, airquace, estate agent, send, text messag, free, restaurant, crm, retail, accounts, customis, customiz
AXIGEN Gateway Mail Server 1.2.3
AXIGEN Gateway is a secure mail relay server, available for many Linux distributions, FreeBSD, OpenBSD and NetBSD. AXIGEN uses firewall-like connection rules and is capable of integrating with most virus / anti-spam applications. By separating security functions from real mail server usage, you improve both server effectiveness and communication security.
During the evaluation period and first year of use, users have free tech support.
See also: FreeBSD, SMTP server, email server, secure server, server architecture, mail protocols, mail route, Listener, spam filter, virus filter, mail filter, gateway server, mail relay, IMAP, linux, POP3, SMTP, webmail, mailserver, mail server, ISP mail, OpenBSD, NetBSD
BLANCHESOFT SMSJoy 1.0.2
Online SMS processing, means you will pay a lot less for your messages than using a normal cellular/mobile phone. Simply create an account with CLICKATELL or KAPOW, set the username, password and Api ID (CLICKATELL) inside the application and you are ready to send SMS to anywhere in the world.
See also: SMS, sms gateway, cellular, mobile, instant, message, messages, messaging, online
SMS Gateway - Enterprise 3.12.1
SMS gateway for high capacity SMSC connections over SMPP, UCP, CIMD2 or GSM Modems. Ideal platform for SMS notification, SMS paging, two way SMS systems, or for becoming an SMS service provider. It supports SMS API for SQL, .NET, PHP, ASP and for OEM vendors. It can be configured as an HTTP SMS Gateway for WEB SMS applications, as an E-mail SMS Gateway for E-mail SMS alerts. For SMS trasnfer IP SMS protocols and GSM Modem AT commands are used.
See also: SMS Gateway software, SMPP SMS, UCP SMS, CIMD SMS, SMS Software, PHP SMS, SQL SMS, HTTP SMS, SMS sending, SMS receiving, download SMS software, buy sms software, Delphi SMS Component
Text Message Software 18.104.22.168
SMS sender device sends multiple SMS quickly while provides facility to connect multiple GSM mobiles with your computer device. www.sendfreesmstomobiles.com company website presents Text Message Software which delivers bulk SMS with facility to maintain list of contact numbers during message sending procedure.
See also: Broadcasting, SMS, sends, group, bulk, multiple, SMS, business, alerts, product, service, brand, promotion, information, ads, deliver, text, national, international, worldwide, global, network, mobile, gateway, cell, phone
Mobile Text Message Software 22.214.171.124
Mobile Text Message Software is available at www.freetextmessageonline.org website and enables school and colleges to announce unplanned holidays among students and teachers by sending them multiple text messages.
See also: Compose, forward, Mobile, Text, computer, desktop, skip, duplicate, message, sms, internet, gateway, cell, phone, sends, broadcast, number, entry, Unicode, multiple, bulk, individual, laptop
Text SMS Software 22.214.171.124
Want to send bulk sms simultaneously? Install user friendly PC to phone messages transmitting tool from URL www.mobiletextsms.com that empowers non technical users to broadcast large numbers of text messages without need of sms modem gateway. Technically advance Text SMS Software provides option to save all delivered messages at user defined location on PC.
See also: Messages, send, internet, connection, sms, gateway, broadcast, multiple, campaign, product, information, service, transmit, application, convey, wide, notifications, contact, numbers, mobile, phones, device, desktop, laptop
Free Mobile Messaging Software 126.96.36.199
Free Mobile Messaging Software from www.freemobilemessaging.org provides facility to broadcast business SMS to international smart phones, PDAs etc. from your desktop after it is connected with GSM mobile devices.
See also: Message, broadcasting, delivers, bulk, SMS, worldwide, mobile, subscribers, network, gateway, sends, news, job, event, alerts, greetings, business, advertisements, desktop, GSM, CDMA, PDA, cell, phones
PC to Mobile SMS 18.104.22.168
Do you have any idea how to send unlimited text messages from PC to the list of contact numbers? Then download affordable PC to Mobile SMS program from website www.pctomobilesmsgateways.com which is highly effective to deliver notification as well as standard messages in minimum time.
See also: mobile, unlimited, bulk, SMS, GSM, technology, cell, phone, device, deliver, news, event, notification, alert, compose, send, messages, promote, business, products, campaign, worldwide, globally, internet, connection, gateway
SMS Software Download 184.108.40.206
SMS Software from www.smssoftware.org delivers SMS advertisements, news alerts, job notifications or personal messages to international mobile gateways from windows OS based laptop to GSM cell phones quickly.
See also: Instant, messaging, delivers, bulk, SMS, greetings, broadcasts, business, advertisements, event, invitation, news, job, alerts, notifications, laptop, GSM, mobile, gateway, network, PDA, global, international, national, cell, phones
Send Bulk SMS 184.108.40.206
Now successfully promote your business products and increase sales or revenues by delivering mass number of text messages at national or international mobile users just in single mouse click using Send Bulk SMS Software from www.sendbulksms.org trusted website. Sms Software provides option to skip duplicate entries while delivering mass amount of text messages.
See also: Download, mobile, text, messaging, broadcast, group, sms, deliver, seasonal, greetings, mass, event, notifications, national, international, cell, phone, personal, computer, internet, gateway, promote, business, products, clients


text:
Jordan's Fate
On Nov. 23, Jordan was shot and killed as he sat in an SUV with friends at a Jacksonville, Florida, gas station. Michael David Dunn, 45, allegedly fired eight shots into the vehicle because the group of teens refused to turn down their music, killing Jordan, who was sitting in the back seat and unarmed. (Photos: CNN)
Sports Buzz: Miami Cheers LeBron James, Heat Beat Cavs 12/26/2014
Best NBA Instagram Photos of the Week 12/19/2014
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Stop The Presses - Biggest Sports Stories of 2014 12/15/2014
Still Got It: Star Athletes 35 and Over 12/14/2014
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Best NBA Instagram Photos of the Week 12/12/2014
Best NBA Instagram Photos of the Week 12/05/2014
Sports Buzz: Kobe Bryant, Rondo Breakfast Stirs Up Rumors 12/05/2014
NFL Coaches Who Will Likely Be Fired 12/04/2014
Potential Landing Spots for Ray Rice 12/02/2014
Sports Buzz: Richard Sherman Leads Seahawks Over 49ers 11/28/2014
Athletes React to Michael Brown Grand Jury Decision 11/25/2014
Big Baseball Contracts Gone Wrong 11/24/2014
NFL's Biggest Disappointments This Season 11/23/2014
Best NBA Instagram Photos of the Week 11/21/2014
Sports Buzz: Oakland Raiders Upset Kansas City Chiefs 11/21/2014
Sports Stars Who Have Love/Hate Relationships With Media 11/19/2014
College Basketball Players to Watch in the 2014-15 Season 11/18/2014


text:
Today's Post
Star Trek Online
Federation Shipyards
VA Ship Shopping
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Lt. Commander
Join Date: Dec 2007
Posts: 120
# 1
VA Ship Shopping
Have 2 chars up to VA now (plus a Lt Gen, but no sharing there) and after grinding far too much dilithium was looking at picking something special out of the C-Store for account-wide unlock. Not sure what though, and thought I'd ask people who've already taken test drives.
One character is an engineer in a recon science ship, and I love the durability and all the fun tricks I can pull out and just really play around with. But when it comes to the actual damage-dealing, a sci ship just doesn't do that especially well. My newer character is a tac captain in an advanced escort, with more than twice the damage of the science ship. However without all the gadgets to play with she's more effective but not as enjoyable. I bring up the characters and my appraisals of the styles as reference, both of what I have and how I like to play.
So first I looked at the MVAE, but then I realized it was identical to the regular advanced escort but for a BOFF difference and a fancy console. Same hull same speed same weapons, even one less console slot because of the gimmick. Is the seperation really that effective/powerful, or is it just 'neat?' Half a million dilithium for a console seems a lot unless its really cool.
Then I looked at the Excelsior, which could turn into a big single-cannon cruiser with lots of engineering tricks. But its a hundred years old (style is important) and I'd lose all the points I've sunk into my science abilities and have to massively rearrange my Boffs and their skills.
Now the Odyssey is out, and I adore the Boff options but its so slow and there are 3 options out there to choose and the only comments about the things I've heard are broad complaints about the price and useless pet AI.
So I guess I have 3 questions really:
1 When one of my characters is already flying an Advanced Escort, just how much better is the multi-vector ability? How effective is it, really?
2 Anyone who's flown both the Excelsior and the Odyssey, how do they compare?
3 Which Odyssey? How effective is the Aquarius vs the Worker Bees vs Saucer Sep?
And of course any other related insightful commentary if ya got it. Thanks! (I hope)
Archived Post
Lt. Commander
Join Date: Dec 2007
Posts: 120
# 2
The MVAM is not a gimmick. when you seperate you have 2 combat pets following you and your even more maneuverable. Since you like the science vessel the mvam is the only escort with a Lt commander boff slot.
if you like your engineer in a science vessel you can check out the nebula. it has a Lt commander engineer slot so you have more options again including eject warp plasma 1.
If you want to get one ship that both can be effective in the you have the galaxy-x. This is a tricky ship to set up. this will give your engineer more dps. It is also the only fed cruiser that can equip dual heavy cannons. It also turns like the oddy so you may not want to do a cannon build if you are in PvP. In the stf's I run a cannon build on it and it works well. The borg are the only enemy that moves slower. For a tactical officer the phaser lance is brutal. Just make sure you run phaser weapons on that ship with 3 phaser relays to get the biggest bang.
The excelsior is similar to the oddy but it has transwarp to all sectors. the big advantage is the Lt Commander Tactical boff.
I suggest you take a look at the spidermitch chart before you decide. Its Big.
Archived Post
Lt. Commander
Join Date: Dec 2007
Posts: 120
# 3
Between the options you have presented, the excelsior I think would be the best fit for you. The reason is that you seem to like the survivability of the sci ship, but want more damage output. And the reverse is true in the escort. The excelsior is really the middle ground between the dmg and survivability that you're looking for, without lossing tons of manuvering power.
The multi is definately not that much better than the adv escort, although you can use grav well with the multi. Otherwise the difference is pretty marginal, and the seperation does NOT offer a significant dps increase.
The ody has the BO options, and is very durable, but it is VERY slow, and the dmg output isn't very good. And to get the most out of the ody you'd have to spend 4k cstore points so you can get all 3 consoles, which I don't think is worth it.
Last thing to note is that if you do pick up the excelsior (1600 points) consider picking up the thunderchild class as well for 800 more points. Grab the point defense console from it and you can dismiss the thunderchild (you can always reclaim it for free). Run all phasers (cannon + turrets) on the excel, along with console. Activate all your damage buffs and activate the point defense (5k max range) and it will amaze you.
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An easy to follow fondue recipe, ideal for the weather
With a variety of good cheese now available in many places across Delhi, what can stop you from trying your hand at making a fondue?
Following is a recipe picked from the recently published "The Sood Family Cookbook" (Collins) put together by Aparna Jain.
Aparna documented 101 recipes from her family for the book. She says, "I sat with the older generation and tried to structure and document their instinctive approach to cooking. I worked with cousins who got their misen en place done by the cook but over time developed their own incredible fusion recipes. I began to collect recipes from Himachal Pradesh (since we are from Kangra and eat pahaadi khana at home), Sindh, Mangalore, Punjab, Tamil Nadu and Thailand."
She says the book was first ollated into a private family cookbook which they distributed to the family and close friends. Later, when, she thought of updating it, Harper Collins suggested she publish it for larger audience. This recipe of fondue, the national dish of Switzerland, "was strangely enough, popular at home" when they were kids.Winter Swiss Fondue
400 g Vacherin Fribourgeois cheese
400 g mature Gruyere cheese, grated
1/14 cups dry white wine
1 tbsp cornflour
1 garlic clove, minced
2 tbsp Kirsch (cherry liqueur)
1/8 tsp nutmeg
1/8 tsp black pepper
1 large baguette or other crusty white loaf, cut into cubes
In a fondue pot, combine both cheeses and heat until the cheese starts melting. Separately, whisk together the wine and cornflour. Once the cheese has melted, stir the wine and cornflour mixture into the cheese in the fondue pot.
Bring to a boil, stirring continuously.
Add the garlic, Kirsch, nutmeg and pepper.
Set the fondoue pot's heat source to low to maintain
a gentle simmer and place on the dining table.
Dip the bread cubes in the melted cheese and enjoy!
Keywords: chef's corner, Aparna Jain, Cheese recipes, The Sood Family Cookbook


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text: This download was added on 11/2/2010Need an extravagantly elegant, vintage-inspired accent for this year's Christmas projects? Our Decorative Label Punch has "the look," and creates a perfect backdrop for the Dec. 25 image from the Tags til Christmas stamp brush set. Like all My Digital Studio punches, the Decorative Label Punch is a vector file, which allows it to be scaled up or down without losing image resolution. This set coordinates with the traditional Decorative Label Punch from the Holiday Mini Catalog.


text:
Black KitchenAid® Artisan® Stand Mixer
Keen cooks with an eye for design will no doubt already have KitchenAid® appliances on their radar … Instantly recognisable, they give durable, uncompromising performance to a commercial level but their looks are a far cry from anything seen in a catering kitchen. Clear a space on your worktop - these are desirable pieces that anyone will be proud to have on display.
Retro good looks and the smooth, rounded design put this mixer on top of many a kitchen 'wish list'. It looks stunning, doesn't take up a huge space, is reliable, fast and thorough, and now it's available in a choice of colours, so you can match it to your personality and kitchen palette too!.
- Patented planetary mixing action.
- Single attachment hub for ease of use.
- Simple, intuitive controls.
- 4.8 litre stainless steel bowl with ergonomic handle.
- Wire whisk, dough hook and Artisan® flat beater.
Cat Ref: SKU 16269
22 x 36 x 35cm H. (8¾" x 14" x 13¾"). 300W.
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Best mixer ever" by Christine
Black KitchenAid® Artisan® Stand Mixer
This is an expensive piece of kit, but you get what you pay for and the quality here is amazing. Beautifully designed, can handle small and big jobs. I bought it principally for making bread dough, but now use it regularly for cakes and pastry (the results are excellent). It's a joy to use, easy to clean and looks good too.
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text:
Question from a reader --
"I have a project that needs to be done in 5 days. I've made solid progress on it.. but definitely will need to deathmarch / burn the midnight oil as you call it to get it done.
I still need to finish content creation, create videos, and upload them to the site. As with any project.. it's hard to say "I'm Done".. though I know shipping is necessary.
Any recommendations?
On my wall I have these reminders
1. Eat properly.
2. Drink enough water.
3. Good sleep schedule.
4. Decide on the most important thing to do the night before.
5. Reviewing and working on things first thing in the morning.
6. Limit alcohol, sugar, wheat.
7. Exercise: workout, walk, jog, and sketch.
8. Recharge!"
Okay. On a more positive note, if you're going to be undersleeping (well, try not to, but if you must...) -- take large doses of Vitamin C. If you start to get a little ill, you might consider taking a small amount of Tylenol and decently flavored non-narotic cough drops towards the end. Maybe it's an old wives' tale, and maybe not, but my grandmother was a nurse and she was always recommended heavy on Vitamin C, Tylenol, and Halls coughdrops when you feel an illness coming on. It seems to work for me. The Vitamin C, at least, is darn useful. Dose up highly on it -- there's no real downside to heavy doses of Vitamin C for a few days.
You'll of course do better if you have an accountability buddy.
You might consider scheduling in a short break or two for around an hour, maybe something that's even highly useful at a scheduled time like talking to a friend who has good perspective, or a mentor.
Be able to switch between different modes of work at different times. As sleep deprivation and exhaustion comes in, natural circadian rhythyms are amplified. You'll feel (relatively) much sharper during your high-awareness and times, and (relatively) much much worse during the low times. Balance what you're doing accordingly.
Very important -- if you have low-level stuff to do (going to the printshop, etc), then make damn sure you don't do it during one of those highly creative times, since you're going to need all you've got.
Don't exercise too heavily unless you're already a highly trained athlete and know your body very well. Heavy exercise usually requires more rest/recovery, and isn't done well on limited sleep/exhaustion.
Loud enjoyable music without lyrics helps stay awake.
So does showering. If you need to think clearer, grab a shower.
Most importantly -- try not to do this, and don't do it very often. Most of life is a marathon, not a sprint. But I know it happens occasionally -- good luck and godspeed. By the way, nobody mistake any of this for medical advice, this is explicitly bad for your health, I'm not advocating any of this (just recognizing that people will and have done it in the past, and describing what I did), and definitely go talk to a physician if you're doing stupid high-exhaustion overwork stuff with any regularity.
Final two pieces of advice:
*Make a list of what bad habits you're picking up, and quit them immediately when this is over.
*Schedule a 3-5 day vacation as soon as the dust clears, literally ASAP, and enjoy it.
I know it's not good to overwork yourself, but I gotta say, that "mental overdrive" you experience when working with a tight deadline can be quite invigorating, at least when used sparingly.
Updated my time tracking recently. Before we get started, allow me to quote "How to Live 24 Hours Per Day" -
"To "clear" even seven hours and a half from the jungle is passably difficult. For some sacrifice has to be made. One may have spent one's time badly, but one did spend it; one did do something with it, however ill-advised that something may have been. To do something else means a change of habits."
"And habits are the very dickens to change! Further, any change, even a change for the better, is always accompanied by drawbacks and discomforts. If you imagine that you will be able to devote seven hours and a half a week to serious, continuous effort, and still live your old life, you are mistaken. I repeat that some sacrifice, and an immense deal of volition, will be necessary."
As always, I recommend you start with a low amount and build up, as I described in "The Evolution of My Time/Habit/Life Tracking."
Okay, here's my new daily tracking -
In a reply to a reader, I found myself elaborating on things about Vitamin D3 that I didn't go into in my post. My answer is a bit tinged by my own worldview, a bit off-topic (surprise!), and opinions are solely my own, but don't let that dissuade you from doing your research into this powerful vitamin.
Glad to meet a fellow D-bomber who doesn't parrot the party line of kidney damage (which happens with non D3 types). I've only taken 4,000-8,000 IU for about 3 years (depending on if I forget to take that 2nd one per day) with no supplemental sunlight and last I tested my levels were only at 55 I think. I use Carlson's Vitamin D 4.000 IU Cod liver oil gelcaps. After seeing your dose I definitely have to up a notch :D. It's funny but anything over 20 or 30 I think in the 'conventional' medical paradigm worries the docs who aren't in on the benefits of D3. They always tell me of kidney damage and that one new england milk study (which used d2 instead of d3). I'm not immortal but ever since I've started C-D bombing as I call it (1-2k grams Vit C per day and 4-8k IU Vit D per day and maybe triple that when sick) I have only gotten sick once a year or so if even that. Now doing some no/low carb action to supplement that been feeling great so far - recovered from near metabolic syndrome myself.
W-in-H: D-bombing is a most fitting descriptive :) It's heartening to meet another soldier that has seen beyond the entrenched view on recommended Vitamin D3 doses.
Don't get me started on the establishment. They're dogged by super-slow regulatory processes, research bias, and a legal minefield around consumer protection (that last one we need, without the mission creep). To that, add the preventative nature of D3, the fact that it's not Rx, and all those faulty studies, and I'm not surprised they'd shirk at recommending higher doses. They're also liable if their recs go wrong, so why mess it?


text:
Quoizel DRE8405 Drew 1 Light Small Down Lighting Outdoor Wall Sconce
This down lighting outdoor wall sconce from the Drew collection features etched glass which allows for beautiful diffused light. This contemporary fixture is perfect for the entrance to your home.
Quoizel DRE8405 Features:
- Design: Contemporary / Modern
- Shade: Etched Glass
- Collection: Drew
- Shade Dimensions: 4" x 10"
Quoizel DRE8405 Specifications:
- Requires (1) x 100 Watt A19 Medium Base Bulb (Not Included)
- Voltage: 120
- Height: 12.5"
- Width: 5"
- Extension: 5.5"
- Item Weight: 4 lbs.
Quoizel creates more than lighting and home accessories, they create timeless pieces designed with you in mind. This kind of dedication, integrity and quality not only goes into the design of their products, but it's in the way they do business as well. It's why Quoizel has grown from a small company to become one of the nation's leading manufacturers of fine decorative lighting and home accessories.
Additional Quoizel Links
Our SKU: Quoizel DRE8405
*Denotes a finish or option that has been discontinued
Installation Resource Center
Whether you are installing the product yourself or hiring a professional, our installation Resource Center has everything you need to get your project done. Visit the Installation Resource Center to get started.