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Label-Free Proteomics Reveals Decreased Expression of
CD18 and AKNA in Peripheral CD4+ +T Cells from Patients
with Vogt-Koyanagi-Harada Syndrome
Liming Mao1, Peizeng Yang1*, Shengping Hou1, Fuzhen Li1, Aize Kijlstra2
1Laboratory of Ophthalmology, Chongqing Eye Institute, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People's Republic of China, 2The
Department of Ophthalmology, University of Maastricht, Maastricht, The Netherlands
Vogt-Koyanagi-Harada (VKH) syndrome is a systemic autoimmune disease. CD4+T cells have been shown to be involved in
autoimmune diseases including VKH syndrome. To screen aberrantly expressed membrane proteins in CD4+T cell from
patients with active VKH syndrome, blood samples were taken from five patients with active VKH syndrome and five healthy
individuals. A label-free quantitative proteomic strategy was used to identify the differently expressed proteins between the
two groups. The results revealed that the expression of 102 peptides was significantly altered (p,0.05) between two groups
and matched amino acid sequences of proteins deposited in the international protein index (ipi.HUMAN.v3.36.fasta). The
identified peptides corresponded to 64 proteins, in which 30 showed more than a 1.5-fold difference between the two
groups. The decreased expression of CD18 and AKNA transcription factor (AKNA), both being three-fold lower than controls
in expression identified by the label-free method, was further confirmed in an additional group of five active VKH patients
and six normal individuals using the Western blot technique. A significantly decreased expression of CD18 and AKNA
suggests a role for both proteins in the pathogenesis of this syndrome.
Citation: Mao L, Yang P, Hou S, Li F, Kijlstra A (2011) Label-Free Proteomics Reveals Decreased Expression of CD18 and AKNA in Peripheral CD4+T Cells from
Patients with Vogt-Koyanagi-Harada Syndrome. PLoS ONE 6(1): e14616. doi:10.1371/journal.pone.0014616
Editor: Wasif N. Khan, University of Miami, United States of America
Received July 23, 2010; Accepted January 3, 2011; Published January 28, 2011
Copyright: ? 2011 Mao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Natural Science Foundation Project (30973242), Program for the Training of a Hundred Outstanding S&T Leaders
of Chongqing Municipality, Key Project of Health Bureau of Chongqing (2008115), Project of Medical Science and Technology of Chongqing (2008-1-15), Key
Project of Natural Science Foundation of Chongqing (CSTC, 2009BA5037), Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003), and PAR-EU
SCHOLARS PROGRAM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.com
Vogt-Koyanagi-Harada (VKH) syndrome is an autoimmune
disorder mainly affecting systemic melanocytes including those in
the eyes, meninges, ears, skin, and hair [1]. It is one of the most
common uveitis entities in China as well as in the Far East of Asia.
The uveitis seen in this syndrome is mostly characterized by a
chronic granulomatous inflammation with recurrent episodes [2].
Although the mechanisms involved in VKH syndrome are not
fully elucidated, previous reports have showed that CD4+T cells
sensitive to melanocytes are responsible for the development of
VKH syndrome [3,4]. Recent studies have shown that molecules
related to CD4+T cell function, including Fas/FasL [5,6], T-bet
[7], IFN-c, RORct and IL-17 [7,8], are involved in the
pathogenesis of this syndrome. As CD4+ T cells exert their role
principally through a variety of receptors, adhesion molecules,-
transport proteins and costimulatory molecules,which have been
found mainly in the plasma membrane or endomembrane systems,
our study focused on the differentially expressed proteins in the
CD4+ T cell membrane of VKH patients.
Proteomics provides important tools for identifying molecules
involved in both normal and pathological processes [9,10,
11,12,13,14,15]. A recently established label-free strategy [16,17]
has been shown to offer a more robust protein identification and
quantitation, easy automation and large-scale analysis with greater
efficiency as compared to conventional two dimensional electro-
phoresis (2-DE) approaches [18,19,20,21]. In the present study, we
investigated the differentially expressed membrane proteins in
active VKH patients using this label-free proteomic method.
Western blot technique was used to validate the proteomic results.
Our results showed a significantly decreased expression of CD18
and AKNA in CD4+T cells from patients with active VKH
Materials and Methods
Clinical Samples
Ten active VKH patients and eleven healthy individuals were
included in the present study. CD4+T cells from five active VKH
patients and five healthy individuals were used for label-free
proteomics analysis. CD4+T cells from another group of five
active VKH patients and six controls were used for a validation
study.The diagnosis of VKH syndrome was made according to the
criteria revised for VKH syndrome by an international nomen-
clature committee [2]. The patients included in our study had not
been systemically treated with any immunosuppressive agent for at
least one week prior to blood sampling. The mainly clinical
features of VKH patients were shown in Table 1. The healthy
individuals are sex and age matched with VKH patients showing
not any past history of infectious or chronic diseases. An informed
PLoS ONE | www.plosone.org1 January 2011 | Volume 6 | Issue 1 | e14616
Page 2
consent (written) was obtained from all patients and the
procedures had been approved by the Ethics Committee of the
First Affiliated Hospital of Chongqing Medical University.
CD4+T Cell isolation and flow cytometry
The PBMCs were prepared from heparinized blood by Ficoll-
Hypaque density-gradient centrifugation and were rinsed for three
times in phosphate-buffered saline (PBS). Peripheral CD4+T cells
were purified using human CD4 microbeads according to the
manufacturer's instructions (Miltenyi Biotec, Palo Alto, Calif).
Briefly, PBMCs were suspended in 80 ml of PBS containing 0.5%
bovine serum albumin (BSA) and 2 mM ethylenediamine tetra-
acetic acid (EDTA) per 107total cells. A volume of 20 ml of CD4
microbeads was added to this suspension and incubation was
performed for 15 min at 4uC. The cells were then washed in 2 ml
of PBS containing 0.5% BSA and 2 mM EDTA and applied to a
magnetic column on a MiniMACS separation unit (Miltenyi
Biotec, Palo Alto, Calif). The CD4+T cell fraction was collected
and used in subsequent experiments. For flow cytometric analysis,
aliquots of 16106PBMCs and isolated CD4+T cells were stained
with PE-cy7-conjugated monoclonal antibody (mAb) against
human CD4 and appropriate isotype controls (eBioscience, San
Diego, CA, USA) for 30 min at 4uC in the dark. Flow cytometric
analysis was performed using FACS Calibur and CellQuest
software (BD Biosciences, SanJose, CA).
Membrane Protein Preparation
Membrane proteins were extracted using a membrane protein
extraction kit (Merck KGaA, Darmstadt, Germany) according to
the manufacturer's instructions. Briefly, 56106CD4+T cells were
pelleted by centrifugation at 300 g for five min. The supernatant
was carefully removed and discarded. Reagent A (150 ml) was
added to the cell pellet and a homogeneous cell suspension was
prepared by pipetting up and down and was then incubated for
10 min at room temperature with occasional vortexing. The lysed
cells were placed on ice. One part of reagent B with two parts of
reagent C were mixed. A total amount of 450 ml of the mixed
reagent B and C was added to each tube of lysed cells and then
vortexed. The tubes were incubated on ice for 30 min with
occasional vortexing. After centrifuging at 10,000 g for 3 min at
4uC, the supernatant was transferred to a new tube and was
incubated for 10 min at 37uC to separate the membrane protein
fraction. The tubes were then centrifuged at room temperature for
2 min at 10,000 g to isolate the hydrophobic fraction from the
hydrophilic fraction. The hydrophilic phase (top layer) was
removed carefully from the membrane-enriched phase (bottom
layer). The protein concentration of the hydrophobic protein was
determined using the protein assay kit (Bio-Rad, Hercules, CA).
The protein was aliquoted and stored at 280uC until use.
Protein Digestion
Protein digestion was performed as follows. The protein
concentration was adjusted to 5 ug/ul with lysis buffer. The
aforementioned membrane protein was chemically reduced for
2.5 h at room temperature by adding DTT to 10 mM, and then
carboxyamidomethylated in 50 mM iodoacetamide for 40 min at
room temperature in the dark. Endoprotease LysC (Roche,
Indianapolis, IN) was added to a final substrate: enzyme ratio of
100:1 (w/w), and the reaction was incubated at 37u for 3 h. The
urea concentration in protein samples was adjusted to 1.5 M with
25 mM NH4HCO3, and then 2 mg of trypsin was added to a final
substrate: enzyme ratio of 50:1 (w/w). The trypsin digest was
incubated at 37u for 20 h. The peptide mixture was acidified by
formic acid to 0.1% for further MS analysis.
Analysis by HPLC and MS
Separation of the trypsin-digested peptides was performed with
the Ettan MDLC system (GE Healthcare, Piscataway, NJ). Peptide
samples were first desalted through a Zorbax 300SB-C18peptide
traps (Agilent Technologies, Wilmington, DE) and then separated
by a 0.15 mm*150 mm (RP-C18) column (Column Technology
Inc, Fremont, CA) with micro spray mode. An aqueous 0.1%
formic acid solution was used as phase A and a solution of 0.1%
formic acid and 84% acetonitrile was used as phase B. The
Table 1. The clinical features of the investigated patients with VKH disease.
number SexAge Ocular Manifestation
AcuityExtraocular Findings
lesions odos
1Male 33
presentpresent 0.250.1
2 Male 34present present0.5 0.5
3 Male 39 presentpresent present0.080.06-
4Male24 present presentpresentpresent0.060.05
5 Female34 presentpresent present0.81.0-
6Male 18
7 Female27 present presentpresent0.60.01
8 Female50 presentpresentpresent0.3 0.05
9Male 46presentpresent 1.00.8-
10Male 41 present presentpresent present0.20.5
Proteomics in VKH Syndrome
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peptides were separated with a linear gradient of 4%,50% mobile
phase B over 2 hours at a flow rate of 1300 nl/min. The column
temperature was maintained at 170 uC.Both pooled samples were
analyzed in triplicate.
The separated peptides were analyzed on a Finnigan LTQ
linear ion trap mass spectrometer (Thermo Finnigan, San Jose,
CA) equipped with a nanoelectrospray ion source [16,17]. The
injection voltage was set to 3.0 kV and the normalized collision
energy was set to 35.0. The number of ions stored in the ion trap
was regulated by the automatic gain control. Each scan cycle
consisted of one full scan mass spectrum (m/z 0,1600) collected
in profile mode. Five MS/MS events were recorded in centroid
mode with the dynamic exclusion settings: repeat count 2, repeat
duration 30 s, exclusion duration 90 s. MS/MS spectra were
searched against the IPI human protein database (version 3.36;
69012 entries) and analyzed using SEQUEST of BioworksBrowser
rev. 3.1. The search was performed with one missed cleavage
permitted. The fixed and variable modifications were set to
Carbamidomethyl (C) and oxidation (M) respectively. The mass
tolerance for precursor ions was set to 3.0 Da. The mass tolerance
for fragment ions was set to 1.0 Da. The peptide matches were
filtered based on cross-correlation scores (Xcorr) of 1.9, 2.2 and
3.75 for charge states 1+, 2+, and 3+, respectively. All the peptides
with different charges were translated to single charge and
expressed as ''MH+'' (Supplemental Table 1). To determine the
false discovery rate (FDR), the data set was searched against a
sequence-reversed decoy IPI human version 3.36 database using
the same search parameters. FDR was calculated using the
formula reported by Xue et al [17]: FDR= Number of false
peptides/(Number of true peptides+Number of false peptides)
Label-free Detection and Quantitation of Peptides
The peaklist of peptides was generated using DeCyder MS
software version 1.0 (GE Healthcare, Piscataway, NJ) and the
quantitative analysis of peptides was performed. Peptide detection,
elution profile comparison, background subtraction and peptide
quantitation were carried out on the full scan precursor mass
spectra in fully automatic mode. Peptide quantitation is based on
MS signal intensities of individual LC-MS analyses. Different
signal intensity maps were matched using the pepmatch module
and then the peptide quantitative results were acquired. As there
was no internal standard, the intensity distributions for all peptides
detected in both samples were used for normalization. Throughout
these studies the mass tolerance in the software was set to 0.5
atomic mass unit and the retention time tolerance was set to
2 min.
Western blot analysis
Total protein of CD4+T cells from five active VKH patients and
six healthy individuals were prepared for Western blot analysis. Ten
micrograms of protein from each specimen were used for SDS-
PAGE. The gels were then transferred to a PVDF membrane.
Membranes were incubated with antibodies at dilutions of 1:1000
for both anti-human CD18 mAb (R&D systems, Minneapolis, MN)
and anti-human AKNA mAb (Genway Biotech, San Diego, CA).
Proteins were detected using the Phototope-HRP western blot
detection system (Cell Signaling, Danvers, MA).
Statistical analysis
For differential analysis of peptides from both VKH patients
and normal controls, student's t-test was performed using the
Decyder MS software. A p-value of less than 0.05 was considered
as a significant difference. In the validation study, the immunoblot
bands were quantified using Bio-rad quantity one 1-D analysis
software. Band density was normalized to that of an internal
control, b-actin. The differences of immunoblot band intensities
between both groups were analyzed by student's t-test using SPSS
software (version 13.0). A p-value of less than 0.05 was considered
as statistically significant.
CD4+T cell isolation
CD4+T cells were sorted from peripheral blood of ten active
VKH patients and eleven healthy individuals using CD4 microbe-
ads. The purity of CD4+T cells identified in both patients and
controls was 98% (Figure 1). The obtained CD4+T cells from both
groups were subjected to label-free quantitative proteomic analysis
followed by Western blot validation.
Figure 1. Representative flow cytometric map of CD4+ +T cells isolated from peripheral blood of active VKH patients. The PBMC and
freshly isolated CD4+T cells were stained with the indicated markers using fluorescence-labeled mAb and analyzed by flow cytometry. Before sorting,
the ratio of CD4+T cells in PBMC was 38.11% (A). After microbeads based sorting, the purity of CD4+T cells was as high as 98.06% (B).
Proteomics in VKH Syndrome
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Page 4
Data quality evaluation
Before conducting the relative protein profiling analysis between
the two pooled samples from VKH patients and normal controls,
several quality control measures were performed on the replicates
of both pools to determine analytical reproducibility. The mass
precision of the extracted peptide components was within 5 parts
per million (ppm) of mass error. The quality control analysis
revealed similar results between patients and controls concerning
the median and average mass errors (1.90 vs 2.80 ppm), intensity
errors (2.10% vs 1.95%), and retention time (0.70% vs 0.50%).
Reproducibility between duplicate runs was evaluated using a
binary comparison map. The horizontal (x) axis represents the
distribution of peak intensities of the first run, and the vertical (y)
axis represents that of the second run. The average intensity
correlation coefficient (CC) between the two runs was 0.92. A
representative binary comparison map is shown in Figure S1. The
expected distribution of the duplicate runs showed no obvious
change. These quality control measurements were performed on
all runs and both experimental pools.
Relative quantitation of peptides in total membrane
protein extracts
The digested membrane proteins of CD4+T cells prepared
from active VKH patients and normal controls were analyzed by
LC-MS/MS. Base peak ion chromatograms were acquired in
profile mode to get enough data points for evaluation by DeCyder
MS. A representative base peak ion chromatogram from the
analysis of the VKH pool is shown in Figure S2. The other runs
displayed similar profiles. The MS files were converted to signal
intensity maps, 2-D representations with m/z on the y axis and
retention time on the x axis. A representative intensity map
(CD18) is shown in Figure 2, A and B. The density of the grayscale
pattern was proportional to the signal intensity of a peptide peak at
a certain m/z, retention time, and charge state. The signal
intensity maps were analyzed using the PepDetect module of
DeCyder MS to detect and quantitate the peptide ions. Since each
sample was analyzed in triplicate, peptides from six signal intensity
maps were matched and analyzed. As the peptide content reflects
the protein level, relative quantitation for corresponding proteins
Figure 2. Decyder MS intensity graphs of CD18 derived from LC-MS/MS analysis of a total membrane protein extracted from both
normal group (A) and VKH group (B). The location of peptide ALNEITESGR in both samples was labeled with square frames in the total graphs
and the magnified graphs (C and D). Statistical analysis showed that the different expression of peptide ALNEITESGR was significant between VKH
group and normal group (E).
Proteomics in VKH Syndrome
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Page 5
was obtained after importing identification results. A p-value of
0.05 was used as a threshold to identify significantly up- or down-
regulated expression. The MS/MS data was collected in centroid
mode and was transferred to TurboSEQUEST software for
protein identification.
In total 354 peptides were identified to be significantly different
between both groups, in which 102 could be sequenced and which
corresponded to 64 proteins. A FDR of 3.77% at the peptide level
was obtained by searching against the sequence-reversed decoy IPI
human database, suggesting a high fidelity of this strategy. Thirty
proteins showed a difference of at least 1.5-fold, when comparing
active VKH patients with normal controls. Of these, eight proteins
were identified based on two or more unique peptides (Table 2)
and in 22 the identification was based on a single peptide (Table 3).
All the identified peptides and their sequences of each protein in
both samples are listed in the Table S1. For single-peptide-based
identifications, the MS/MS spectra appropriately labeled were
listed in Figure S3.
Validation of CD18 and AKNA by Western blot technique
Among the thirty proteins identified, two proteins, CD18 and
AKNA, were found to have a more than three-fold difference
between active VKH patients and normal controls. The different
expression of CD18 in both samples was shown in magnified
intensity graphs (Figure 2, C and D). Our study further validated
the expression of these two proteins using the Western blot
technique. The immunoreactive band intensities were quantitated
and were presented as intensity volumes (vol %). The results
showed that the expression of CD18 was about five-fold lower in
the active VKH patients as compared to normal controls. As for
the expression of AKNA, a similar result was observed (Figure 3).
The results for both proteins were generally consistent with the
quantitative data of the label-free method.
In this study, label-free relative quantitative proteomics was
employed to screen differently expressed membrane proteins in
CD4+T cells from active VKH patients and normal controls.
Thirty proteins were identified to be at least 1.5 fold differently
expressed between active VKH patients and normal individuals. A
significantly decreased expression of CD18 and AKNA was
further confirmed using the Western blot technique. The results
presented here may provide new clues for the study on molecules
involved in the pathogenesis of VKH syndrome.
CD4+T cells have been shown to be crucial in the pathogenesis
of this syndrome [5,6,22]. The present study aimed at searching
for the differently expressed proteins in these cells between active
VKH patients and normal controls. Proteomics has provided
useful strategies for the screening of differently expressed proteins.
The recently established label-free strategy has been proven to be
more efficient as compared to conventional 2-DE approaches
[18,19,20,21]. It has been used to identify urinary biomarkers
from antineutrophil cytoplasmic antibodies [23] and differently
expressed proteins from patients with ductal carcinoma [24] and
Parkinson's disease [25]. These studies have proven that the label-
free strategy is a powerful tool for identifying disease-associated
proteins. In this study, we employed the label-free approach to
identify differently expressed proteins in CD4+ T cells, an
important population involved in various autoimmune diseases,
between active VKH patients and normal controls.
To ensure the accuracy and stability of the label-free
quantitative analysis, several procedures were performed. For
the purpose of minimizing the effect of individual differences on
the experimental results, an equal amount of membrane proteins
of CD4+ T cells from five active VKH patients and five normal
controls were pooled respectively and subjected to the label-free
quantitative proteomics. For label-free proteomics, strategies using
three or four technical replicates have been reported [16,17,26].
As three technical replicates are a minimal requirement for
statistical analysis and have already been justified by a number of
studies, we chose three technical replicates in the present study.
Our results showed a great reproducibility in peptide quantitation
(Table S1). To acquire stable MS data needed for quantitative
analysis, several quality control measures were used [16]. The data
from each run of both VKH patients and normal controls
appeared to be reproducible in terms of mass accuracy, retention
time and intensity. Binary comparison maps used in the
experiment validated the reproducibility between duplicates.
Coefficient of variation (CV) analysis [17] was performed and
the result showed that the average CV of the fold changes was
23% for proteins with two or more peptides, which ensured the
accuracy of protein quantitation (Table S2).
Our label-free proteomic results showed that thirty proteins
were different in expression for more than 1.5 fold between active
VKH patients and normal controls,although these results need to
be confirmed by other techniques. By searching for the functions
of these proteins according to annotations in the Swiss-prot
database at http://expasy.org, we interestingly noted that these
identified proteins are involved in 54 biological processes (Table
S3) and 39 functional groups (Table S4). Importantly, we found
that a number of molecules, including vimentin, dystrophin,
Keratin 10, AKNA and CD18, are associated with an inflamma-
tory and/or immune response. An abnormal expression of certain
proteins has also been found to be involved in autoimmune
diseases or immune related diseases [27,28,29]. Enhanced
vimentin immunoreactivity was found in the spinal cord of rats
with experimental autoimmune encephalomyelitis [28]. Increased
expression of dystrophin was observed in experimental autoim-
mune myasthenia gravis [27].
By analyzing the data acquired using the label-free method, it
was interesting to note that a number of proteins identified in this
study, including vimentin and AKNA transcription factor, were
non-membrane components. A similar result has also been
observed in proteomic studies. These non-membrane molecules
could be explained as being a component associated with the
membrane or with membrane fractions [30,31]. It is worthy to
point out that the increased expression of keratins may be
involved in the pathogenesis of a number of diseases affecting the
skin [32,33,34,35]. It is not clear whether keratins are truly
induced and, if it is, how it functions in VKH syndrome, an
autoimmune disease frequently affecting the integumentary
False positive data always exist in proteomic studies. Therefore,
the proteomic results usually need to be confirmed using other
techniques. As protein with a big different expression ratio is
readily confirmed by another technique, we chose two proteins
with a three-fold different expression between VKH patients and
normal controls, CD18 and AKNA, as candidates in the
Westernblot identification. As expected, the Western blot result
revealed a five-fold lower expression of both proteins in active
VKH patients as compared with normal controls. It is worthwhile
to point out that both CD18 and AKNA were identified using the
one peptide based identification, as already reported previously
[36,37]. This could be due to the lower abundance of both
proteins in the tested samples.
CD18, also known as integrin beta 2 or ITGB2, is one of the
members of the integrin family, which is involved in cell adhesion,
Proteomics in VKH Syndrome
PLoS ONE | www.plosone.org5 January 2011 | Volume 6 | Issue 1 | e14616


text:
Hello, I'm Nick Fitzgerald. This is my weblog. You can also check out my shared items from Reader, moments on Twitter, and code on GitHub. Feel free to contact me about whatever.
June 26th, 2014
On Tuesday, June 3rd, 2014, I gave a presentation on debugging web performance with Firefox DevTools to the Velocity Conf 2014, Santa Clara. I'm not sure how useful the slides are without me talking, but here they are:
Proposal for Encoding Source-Level Environment Information Within Source Maps on July 22nd, 2015
Source Maps are an Insufficient Debugging Format for the Web on June 19th, 2015
A Compact Representation of Captured Stack Frames for SpiderMonkey on April 10th, 2015
Memory Management in Oxischeme on February 22nd, 2015
Naming `eval` Scripts with the `//# sourceURL` Directive on December 5th, 2014
wu.js 2.0 on August 7th, 2014
Come work with me on Firefox Developer Tools on July 8th, 2014
Debugging Web Performance with Firefox DevTools - Velocity 2014 on June 26th, 2014
Beyond Source Maps on March 12th, 2014
Memory Tooling in Firefox Developer Tools in 2014 on March 4th, 2014

subset: commoncrawl


text:
Gorgeous hair. She should book some commercials for conditioner.
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Video Description:
DaneJones Young girl gets so excited and wet for her experienced lover
All Comments (24)
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questa moretta è fantastica
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Who is she?
kinda amazing to watch
1oo Points


text:
Durham, NCUSA
October 3, 2010
Sounds like a good group.
South Durham geek. I launch helium balloons and high power rockets. Playing with xbees/arduinos to track both. I have yet to make it to any of the meetings (I play in the Durham Symphony and our rehearsals are Tue) but hopefully at some point.
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text: the place is still the same when i visited last 2006. it even got worse because it's old and dirty. it's crying out for renovation. Boring. It must have an impact saying It's fun in the Philippines after all. A guest would definitely have an unhappy impression, it's no longer worth it, the food is bad. Service is bad and the place is definitely not an unforgettable place where a guest would say i'd love to visit there again.


text:
Citing former Defense Secretary Robert M. Gates' memoir, the father of a National Guardsman whose son was killed in Afghanistan is blaming White House leaks about the Osama bin Laden raid for the Taliban's downing of a transport helicopter that killed his son, 17 members of SEAL Team 6 and 12 other U.S. troops.
Douglas Hamburger, whose son was killed in the Aug. 6, 2011, crash of Extortion 17, last week filed a statement with a House oversight subcommittee quoting Mr. Gates as telling White House aides he feared the Taliban would use military tactics contained in administration leaks to target Americans in Afghanistan.
He quoted a passage from Mr. Gates' memoir, "Duty," in which the former defense chief wrote that he had a pledge from White House aides that they would not release details about the SEAL Team 6 raid that killed bin Laden. Mr. Gates said the aides broke their promise and began "pouring out" tactics and methods just hours later.
PHOTOS: Elite U.S. Navy SEALs in action
Three months after the May 2011 bin Laden raid, the CH-47D Chinook helicopter was shot down by a Taliban fighter with a rocket-propelled grenade standing on a tower about 100 yards from what was supposed to be a secret landing zone.
"Releasing their identity put a target on their backs, along with any support troops that went into battle with them," Mr. Hamburger, father of Army Staff Sgt. Patrick D. Hamburger, 30, of Lincoln, Neb., said in the statement filed with the House Oversight and Government Reform subcommittee on national security.
Some families of those killed suspect that Afghans betrayed the SEALs in revenge for the bin Laden raid. Their suspicion is based not just on the Taliban's nearby positioning with a rocket-propelled grenade. They note the Taliban's ability to infiltrate Afghan security forces and then turn their guns on Western troops.
To Mr. Hamburger, Mr. Gates' warning proved prophetic: In his best-selling book, Mr. Gates recounted the debate on whether to unleash SEAL Team 6 on a compound in Pakistan that proved to be bin Laden's hideout. He wrote of the confirmation of the kill and of Mr. Obama leaving the White House War Room to tell the nation that al Qaeda's leader, the man who orchestrated the 9/11 attacks, was dead.
Within hours, a flood of leaks ensued. Republicans charged that the White House was putting troops in danger to reap political capital while the president's re-election campaign was underway.
In his congressional statement, Mr. Hamburger quoted from the Gates memoir: "Before we broke up and the president headed upstairs to tell the American people what had just happened, I reminded everyone that the techniques, tactics, and procedures the SEALs had used in the Bin Laden operation were used every night in Afghanistan and elsewhere in hunting down terrorists and other enemies. It was therefore essential that we agree not to release any operational details of the raid. That we killed him, I said, is all we needed to say. Everybody in that room agreed to keep mum on details. That commitment lasted about five hours. The initial leaks came from the White House and CIA. They just couldn't wait to brag and to claim credit. The facts were often wrong, including details in the first press briefing. Nonetheless the information just kept pouring out. I was outraged and, at one point, told [National Security Adviser Thomas] Donilon, 'Why doesn't everybody just shut up?' To no avail."
In the raid's aftermath, Mr. Gates publicly complained about bin Laden leaks in a clear message to the White House. But he did not elaborate on the fact that, at that time, he believed he had a firm agreement from Obama aides not to leak details.
The White House declined to comment.
"When it suits the White House political purposes, it discloses national security information in leaks," said Larry Klayman, a lawyer representing three families of those killed in the Chinook. "When they want to cover anything up, it becomes national security information. The entire procedure is dishonest and criminal."
The House national security subcommittee held Congress' first, and likely last, hearing last week on the Chinook's downing. Mr. Hamburger did not testify, but his statement was put into the investigative record.
"Never before had a president in office released the identity of any Special Forces Team involved in a covert operation," Mr. Hamburger wrote. "Until Bin Laden was taken out, no one had really heard of SEAL Team VI. Its name and missions were always kept quiet for protection for themselves and their families.
"With Bounties on their heads and a target on their back, [30 Americans] were shot down by the Taliban who were positioned in a tower of a building in the perfect place at the exact time to launch an attack on the CH-47D when it was most vulnerable — at landing. How can anyone justify putting our troops at such risk?" the bereaved father wrote.


text:
George Allgood
1 portrait of George Allgood
George Allgood
by Camille Silvy
albumen print, 24 May 1862
3 1/4 in. x 2 1/8 in. (83 mm x 55 mm) image size
Purchased, 1904
Photographs Collection
NPG Ax58133
Click on the links below to find out more:
Sitterback to top
- George Allgood (1827-1907), Army officer. Sitter in 1 portrait.
Artistback to top
- Camille Silvy (1834-1910), Photographer. Artist associated with 3819 portraits, Sitter in 23 portraits.
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Exhibitions and displays
- Curators' Choice: Photographs from the Terence Pepper Gift
Until 24 January 2016
Related pages
Events programme
- Portrait of the Day: Adelina Patti: The Unknown Diva
17 October, 12:30


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If you uses Ruby 1.8.7 or higher you can natively connect to Gmail and do not need to install a plugin as you used to with older Ruby versions .
Selenium uses webrat as default which causes problems under Snowleopard. Getting the latest version of Selenium and replacing the gem's JAR-file with it will solve this problem.
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The initializer block in environment.rb contains the line
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which is commented out by default. Here the lib folder can be added in order to have it reloaded.
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Your need to install he latest gem for Selenium RC from gem cutter. You then need to get the latest version of Selenium and replace the gem's JAR file with the latest one you just downloaded.
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Paperclip::Geometry offers methods to get the image's dimensions. However, if you want to access those for a new file, the earliest you can access these is with the after_save hook. Thus you might have to save the file twice.


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Advances in Microbial Physiology
Series Editor:
- Robert K. Poole, University of Sheffield, UK
The Editors of the Advances in Microbial Physiology series have always striven to provide a diverse range of top-quality papers on all aspects of microbial physiology. Coverage of 'holistic' topics or whole cell studies such as ion fluxes, stress responses and motility have gone hand-in-hand with detailed biochemical analyses of individual transport systems, electron transport pathways and many aspects of metabolism. Now edited by Professor Robert Poole, University of Sheffield, Advances in Microbial Physiology continues to publish topical and important reviews, interpreting physiology in its broadest context, to include all material that contributes to our understanding of how microorganisms and their component parts work.View full description
Microbiologists, biochemists, biotechnologists, and anyone interested in physiology, microbial biochemistry and its applications
Book information
- Published: September 2003
- Imprint: ACADEMIC PRESS
Praise for the Series: "This series has consistently presented a well-balanced account of progress in microbial physiology....Invaluable for teaching purposes." AMERICAN SCIENTIST


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Excellent service, excellent food and beautiful property. Quaint setting with a garden courtyard for eating. Rooms were great size and comfortable. Everything was within walking distance to the hotel.
Clean, comfortable and spacious room. Friendly, welcoming service. Good breakfast with the usual buffet, plus fresh eggs and pancakes to order. Easy to access internet included. I will stay here again when in the area,
Stayed at Hotel Goldinger and had breakfast each morning at the cafe. Was amazing food and staff was very helpful and kind and patient with our entire group of girls.
Would stay here again and recommend to anybody traveling with groups or just passing through for a comfortable place to stay and eat.
A very friendly, homey, and clean hotel located just 150 meters from the Landstuhl train station. Price includes a lovely breakfast and the adjacent cafe/bakery is wonderful. Also, they are just about the nicest people working there that I have ever run in to on any of my many travels.
I was welcomed immediately and warmly to Hotel Goldinger. Ut was cold weather so I was given extra blankets without even asking for them. The bed and the room were very comfortable. The bathroom was quite large. The cafe serves a wonderful European breakfast with a staff that go above and beyond to ensure you enjoy your meal. The cafe...
I was here on a work trip and this hotel was perfect for what I needed. Breakfast was delicious and coffee was great! Staff was always kind and very hard working. Cafe/bakery made the hotel always smell delicious. Clean room with free wifi.
This is a very good place to stay. The family treated me very well and allowed a very late check in and a late check out. Close to City Center so easy to get around by foot. If I return to Landstuhl I will check here first for my Hotel
This is a very cute family run hotel in Landstuhl. It's not far from the train station and is attached to a konditorei/restaurant. I booked the Goldinger because I loved the look of their dining area, which looks like a cross between a solarium and a garden. Also, it got very good ratings on Expedia and TripAdvisor.
We arrived at...
The rooms are comfortable, the staff is excellent, the location is good, and the included breakfast is outstanding. This was my third visit and I plan to return again. I highly recommend this hotel. Even the free parking is secure and convenient.
A splendid family hotel. Large, simple, but comfortable room with a good bathroom. Excellent breakfast in the delightful dining room with courteous, friendly service. good nearby parking. Well recommended.


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Antwain Britt On Rafael Feijao Strikeforce Fight watch video >>
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Publisher:Little Brown
Harry Bosch's life is on the edge. His earthquake-damaged home has been condemned. His girlfriend has left him. He's drinking too much. And after attacking his commanding officer, he's even had to turn in his L.A.P.D. detective's badge. Now, suspended indefinitely pending a psychiatric evaluation, he's spending his time investigating an unsolved crime from 1961: the brutal slaying of a prostitute who happened to be his own mother.
Even after three decades, Harry's questions generate heat among L.A.'s top politicos. And as the truth begins to emerge, it becomes more and more apparent that someone wants to keep it buried. Someone very powerful...very cunning...and very deadly.
Edgar Award-winning author Michael Connelly has created a dark, fast-paced suspense thriller that cuts to the core of Harry Bosch's character. Once you start it, there's no turning back.
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» Print Recipe
Meatballs with pasta are the Godfather of all Italian meals and a real family favourite. These follow the Italian method of combining two types of mince; venison gives these a delicious gamey flavour but you can use beef if you prefer.
In a large bowl, mix the minces together with the crushed chilli, parsley and garlic. Soak the bead in warm water the squeeze and drain. Add the bread and eggs to the meat. Season and mix well.
Shape into egg shaped balls and fry in 5 tbspns of olive oil for 4 to 5 minutes.
In a large pan, gently fry the onion, carrot and celery until golden. Add the meatballs and red wine. Once the red wine has evaporated add the passata and chopped tomatoes.
Season and cook on a low heat for an hour. Cook the pasta in boiling salted water. Drain and place the pasta into the sauce. Mix and finish with basil.
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We offer fresh cut bank instrument for lease, such as BG, SBLC, MTN, Bank Bonds, Bank Draft, T strips and others. Leased Instruments can be obtained at minimal expense to the borrower compared to other banking options. This offer is opened to both those and corporate bodies. We are RWA ready to close leasing with any interested client in few banking days, if interested do not hesitate to contact us via email: All relevant business information will be provided upon request. If Interested kindly contact me via Email:~ Contact : Mr. Petrovic Dorde Email: firstname.lastname@example.com Skype ID: petrovic.dorde
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Like Cosmopolitans? Try this flavorful and easy tequila version of the drink.
Add all the ingredients to a shaker and fill with ice.
Shake well and strain into a Martini glass.
Garnish with a lime wheel.
You must be logged in to post a comment.
" Patron, A Classy Tequila Creating New Classic Cocktails" That will never be your by line if you keep Co-Opting names of classic drinks to promote your product. Your marketing department needs a shake up and a wake up. Example:" Bartender, i'll have a Manhattan, but use tequila." Tequila!?!, howled the bartender, "that's not a Manhattan." " I know'," replied the customer, " but i love the word Manhattan." " Bartender, I'll have a Jack and Coke, but use baccardi Rum." "Baccardi?!, says the bartender in disbelief. "Look bartender I like the sound of JAck and Coke so make the damn drink. What do you think you are , a professional?" " Yes I am", repliied the bartender."and you won't be drinking here!"
i won't be drinking here either. i have been enjoying Patron(and other tequilas beofre its introduction) and cranberry. It is not a Patron cape Cod! It is called Cape Fear. I have created a drink that combines the elements of a Margarita and Mohito. It is not Patron Mohito. That is assinie.Name /create your own mixology history. Respect the craft and respect yourself.
One more time:" PATRON, A Classy Tequila Creating New Classic Cocktails."
love cosmos


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A project of People For the American Way dedicated to monitoring and exposing the activities of the right-wing movement. Learn More >
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10/6/15 @ 10:30am
With parts of North and South the Carolina experiencing devastating floods, former Rep. Michele Bachmann is weighing in by suggesting that the flooding is a sign of God's wrath for President Obama's foreign policy. "US turns its back on Israel, disasters following," she wrote... MORE
10/7/15 @ 11:23am
On yesterday's "Hagee Hotline," Matthew Hagee interviewed conservative San Antonio talk radio host Trey Ware about last week's mass shooting at Umpqua Community College in Oregon. Both men agreed that the problem with mass shootings in America has nothing to do with the preval... MORE
10/6/15 @ 5:34pm
Andrew Husband @ Mediaite: Ben Carson on Oregon Shooting: 'I Would Not Just Stand There and Let Him Shoot Me'. Michael Fitzgerald @ Towleroad: Confusing Southern Baptist Statement Rejects 'Ex-Gay' Therapy, Embraces 'Biblical Conversion Therapy'. St... MORE
10/6/15 @ 5:30pm
Liberty Counsel is very upset that the Associated Press dared to note that the organization has been designated an anti-gay hate group by the Southern Poverty Law Center. Also very upset is WND, which is mad at us for daring to note that "America's Prophet" Jonathan Cahn is not... MORE
Alabama Supreme Court Justice Tom Parker: State Courts Must Defy The Supreme Court's Gay Marriage Ruling
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On his radio program today, Bryan Fischer interviewed Alabama Supreme Court Justice Tom Parker, a former Religious Right activist and aide to Chief Justice Roy Moore who has become a radical justice in his own right, for two segments about the Supreme Court's gay marriage ruling earlier this yea... MORE
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Rick Wiles returned to "The Jim Bakker Show" today to once again warn about the purported threat of gay marriage, this time telling Bakker that people may die because conservative Christians haven't stood up and resisted the Supreme Court's landmark marriage equality ruling.... MORE
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10/6/15 @ 10:32am
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Marina Resort
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Big Bear Lake
... would pass signs advising against possibly snow drifts or winter sports shops or even a ski slope that looked very odd without all the snow! But I guess that's what it's like when the seasons you live in are so extreme!! We found the lake and decided after yesterday's hiking around the mountains to just enjoy sitting calmly by the lake... ...
Great Race of 2013 from St. Paul, MN to Mobile,AL
... speedometer and the instructions from the Race committee that we will receive daily, 20 minutes before the start, there are 250 to 280 different instructions; turns, stops, speed changes, etc. covering about 250 miles each day. The times are taken down to the .01 of a second. 3 minutes to early or to late result in disqualification for that leg of the race. And the reason for getting involved in this adventure... "It is ...
A Glorious Day
... reached into a clear sky before. On the eve of this Holy Day could this be a message from Our Lord?
The wind died down to a dull roar for a few hours then came back roaring like a lion. The wind finally died down after dark. We like wind but not when the wind blows so hard that our dog's ears don't resemble delta wings on a jet aircraft.
... can look down on the San Andreas fault. The park ranger offered to point it out to us on the map but I wondered out loud if this was really necessary. He said "don't worry, it's not going to be active today" How did he know? I'm not happy staying so close to one of the biggest fault lines on earth. Earthquakes are so common place in his area that the local paper reports the previous ...
More good times in SoCal!
... oncoming traffic in the form of a big, extra-wide motorhome!
Last night we enjoyed Sarah McLachlan and Friends in concert at nearby Fantasy Springs Casino - thanks to our friends Norma Jean and Gus. We felt badly that they had to make a sudden trip to Omaha, but appreciated their offering their tickets to us.
Lots of pictures to upload, but just time for the map pin right now.


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Heres a link to what im selling hope you find what you want
Heyo im selling a bunch of my paintball stuff. Most of it is brand new and i just didn't get to use it!
I have a brand new azodin kaos: 89.95$
I ripped the original Tom Kay troubleshooting video from the original VHS tape to DVD (At a much higher resolution) and uploaded it to YouTube.


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A sign for Prairie Dog Town, which is located near Oakley, Kansas.
Larry Haverfield on his ranch outside of Russell Springs, Kansas. Haverfield appreciates prairie dogs, recognizing that they help support other wildlife, such as the endangered black-footed ferret.
A black-tailed prairie dog peers up from its burrow at Prairie Dog Town, near Oakley, Kansas.
Kansas State University faculty and students, Fish and Wildlife Service employees, and volunteers prepare to locate and capture black-footed ferrets on the Haverfield ranch.
A ferret being vaccinated and tagged by a biologist at the Haverfield ranch in Kansas.
Ron Klataske of Audubon of Kansas helped get local ranchers involved in ferret recovery.
Prairie Dog Town, a tourist stop featuring the "world's largest prairie dog"—a 20-foot cement statue—ironically is located close to where officials are trying to eradicate the wild version.
Rancher Larry Haverfield and his wife, Bette, have been been fighting against the use of a chemical on their ranch that kills prairie dogs.
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Join the elite ranks of our bird-saving monthly donors.
Voice your support for U.S. Fish and Wildlife Service efforts to save the Greater Sage-Grouse.
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Stefania con suo marito sono stati degli ospiti molto gentili, è stato un vero piacere poterli ospitare, spero tanto che vorranno ritornare in valle d'Aosta e da noi.
Stefania et son ami ont séjourné pendant 10 jours à St Rémy de Pce, ils sont adorables et très sympa.
Je pense qu'il ont beaucoup aimé la Provence et leur séjour.
Je les recommande.
Stefania and her friend spent few days in my place. I didn't received them personally cause I was too busy. But everything was organized, and spent as expected thanks to her. She was very responsive to emails, came right on time, were friendly and left my place in great shape. Would welcome them anytime with pleasure.


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It was a pretty chill night and not much was going on, so we called up some friends and got the limo. The chilling time was over. Jmac and the crew took over the VIP spot, with all the ass and titties you can shake your dick at. There was some ass there that was so big it would dwarf your dick. Kristine and her awesome body almost broke Jmac. She was on a mission with her pantyhose all ready ripped in the crotch for the pussy to be spread wide open. And what a spread it was. She was a fucking machine and destroyed the dance floor. While Jmac was pounding her, she was reaching out for more. It didn't matter who it was from as log she was getting as much action as she could.
Fransheliz Vasquez
"Switch Hitters"
"Walk It Out"
Bianca Jacobs
"Champagne Room"
"Dancing For Dick"
Nikki Bell
"The Big Easy"
"Happy Halloween"
Sophia Ashton
"Pretty Pink"
Vanessa Cage
"Bouncing Booties"
Tori Black
"Forbidden Donuts"
Alanah Rae


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text: North West Makes Sounds! by Hilton Hater at May 12, 2014 11:20 am. Updated at May 26, 2015 12:41 pm. Comments Kim Kardashian shows off a gift from Kanye West in this video. We can hear little North West coo in it. Adorable! Share Tweet Email Edit Delete Follow North West? Facebook Disqus Star: North West Related Videos: North West Videos Related Post: North West Poses in Front of Flower Wall, Coos on Camera Uploaded by: Hilton Hater Uploaded: May 12, 2014 Modified: May 26, 2015 12:41


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|Project Name
|Price (AUD)
|Project Name
|Price (AUD)
|Record a set of recording audio for an English textbook -- 5
|Record a set of recording audio for an English textbook (British Accent) 1. We need a set of recording audio for an English textbook (British Accent). A female broadcaster and a male broadcaster are needed, so you'd better to be a professional studio to cooperate with. 2. There are totally about 1000 expressions, each with a sample sentence. The female broadcaster read the expression and th...
|Audio Services, Voice Talent, Audio Production
|Oct 4, 2015
|Record a set of recording audio for an English textbook -- 4
|Record a set of recording audio for an English textbook (British Accent) 1. We need a set of recording audio for an English textbook (British Accent). A female broadcaster and a male broadcaster are needed, so you'd better to be a professional studio to cooperate with. 2. There are totally about 1000 expressions, each with a sample sentence. The female broadcaster read the expression and th...
|Audio Services, Voice Talent, Audio Production
|Oct 4, 2015
|Voice recordings for automated phone system (German)
|I need short IVR recordings (for automated phone system) in German are required.
|Audio Services, Voice Talent, Audio Production
|Oct 4, 2015
|Background music/instrumental for animations
|Hi. I am looking for someone to work with for my animations. I am creating small animation videos every month. Some times we create 2 animation videos sometimes 3. So, we would like to work with someone on continuous basis. Each animation video will be like 3 to 4 minutes sometimes even less. My budget is very limited and I can't afford expensive bidders and I don't want to commit...
|Audio Services, Music, Sound Design, Audio Production
|Oct 4, 2015
|Voice Over Work. Male voice. 4 audios
|I need a Young native american/British Young Male voice over artist to create 4 sales video voice overs today. Total character length of all 4 scripts in total is 3000 words. That's all 4 script in total just to give you an idea. I'll provide the script, need 4 separate audio files. Simple task, nothing major. Can show examples of what I need. Need to get it done today. $100 to...
|Audio Services, Voice Talent
|Oct 4, 2015
|Audio Needed based on a tune
|Need an exception audio track based on a tune, preferably by intermixing with inspirational high-intensity drumbeats, to accompany a video trailer. It needs to be a very innovative fusion and creative remix. Music and creativity are the key factors needed for this job, besides the ability to edit music using software.
|Audio Services, Music
|Oct 3, 2015
|Voice Narration - (Youtube)
|If you have a microphone and a cool voice, and want to make some extra cash, this is for you! We are looking for someone to narrate videos to our Youtube channel with pop culture content as comics, anime, movies, etc. The channel will be an english version of www.youtube.com/einerdtv and it will have 2 weekly videos. $50.00 per script narration. The scripts has 1200 words on average which will ...
|Audio Services, Video Services, Voice Talent
|Oct 3, 2015
|Create instrumental version of a simple song
|Hello I'm looking for someone to make the "instrumental version" of a song. It is for a wedding but we can't find a instrumental version of the song. It should match the original as much as possible Please send me a demo (just a couple of seconds, I won't steal your work ;) I have included an MP3 of the song
|Audio Services, Music
|Oct 3, 2015
|Bulgarian Voiceover/Audio Files
|I need a few Bulgarian voice files to promote a free iphone website. Looking for a female voice. I will provide the text in Bulgarian you will have to make the audio file in MP3 format. The statements will be short like: Днес даваме на 100 български граждани възможността да спечелят iPhone. I will probably need 4-8 different phrases recorded. Each phrase will be a different audio file. T...
|Translation, Audio Services, Bulgarian
|Oct 3, 2015
|Phone recording hardware - needs PC interface
|Hi, I have a small box from China which is supposed to record phone calls (UK landline). The box sits between the BT wall socket and a landline phone, connecting with a PC via USB. The user interface / software that comes with it is useless. I am hoping to find someone who can work with the output from the box to store the recorded data on the PC. I can send you the box to work with.
|Audio Services, Manufacturing Design
|Oct 3, 2015
|voice over project american accent
|i need to make voice over in american accent on the attached script. happy bidding.
|Audio Services, Music, Voice Talent, English (US), Audio Production
|Oct 3, 2015
|Oct 3, 2015Ends - 6d 20h
|English native speaker for voice over1
|we are looking for English native speaker for voice over(short animation) small budget
|Audio Services, Voice Talent
|Oct 3, 2015
|Oct 3, 2015Ends - 6d 17h
|EDM Ghost Production needed
|We are looking for someone to help us produce a remix of Childish Gambino's hit song "Sober". We are looking for a 128bpm progressive or Groove House type Track (we can send you examples and sample packs that we use).
|Audio Services, Music, Audio Production
|Oct 2, 2015
|Oct 2, 2015Ends - 6d 6h
|Producing EDM songs
|I'm looking for a producer/DJ that, with me, would like to produce the best music 4 hands I got many songs already started, that need your touch I will ask you to be expert and some example of your production soundcloud, beatport, itunes are welcome
|Audio Services, Ghostwriting, Music, Audio Production
|Oct 2, 2015
|Oct 2, 2015Ends - 6d 4h
|Work with former Michael Jackson Producer and Engineer
|You will be writing a song to one of Eddie "Angelikson" Cascio's Beat's . Winner get's to use this song on all there social media outlets as promotion and sale. http://frescoentertainment.net/eddie-angelikson-cascio/
|Audio Services, Ghostwriting, Music, Audio Production
|Oct 2, 2015
|Oct 2, 2015Ends - 6d 3h
|Voice talent
|I am looking for voice talent to help me with simple 2d animations, and videos, the voice will be for sharks, and other types of fishes. the target audience will be children and the videos will be posted on youtube and similar sites, these videos will be monetized and if successfull the pay will be negotiated further.
|Audio Services, Animation, Music, Voice Talent
|Oct 2, 2015
|Oct 2, 2015Ends - 6d 1h
|Music for shark video
|I am a shark diver and shoot underwater video, i would like to find some original music to use that I can put with my footage, the biggest part of pay will be exposure. I am open to all music types and speeds. Your name and info will be credited at the end of the video. All music must be royalty free original made by you these videos will be posted on youtube and be monetized.
|Audio Services, Music
|Oct 2, 2015
|Oct 2, 2015Ends - 6d 1h
|Creating all software needed to create a free host server including but not limited to, instant account activation with user names and passwords, email verification, ip address recording, full administration area for eliminating fraudulent activity, account monitoring with full stats and webmaster activity, browser uploads, limit file size uploads for jpegs, gifs, mpegs, avi's and such, compl...
|Audio Services, Linux, Engineering, MySQL, Database Administration
|Oct 2, 2015
|Oct 2, 2015Ends - 5d 18h
|Create Seamless Mixes
|I have been DJing nearly every weekend for just over 11 years. I'm currently studying multiple courses, and trying to maintain my work administration, running events, study and playing gigs on the weekends is becoming increasingly difficult. I run a podcast, which has been neglected, and I would like someone to mix the tracks within the episodes together for me. This is something I'...
|Audio Production, Audio Services, Music, Sound Design
|Oct 2, 2015
|Oct 2, 2015Ends - 5d 13h
|Recording of Skype conversations
|You have to record Skype-Conversations between two people like in this example https://www.youtube.com/watch?v=AHCwgRcnpQA Then you have to do some postproduction like adjusting the audio volume and cutting out the parts that won't be used in the final video.
|Audio Services, Video Services, Video Production, Video Editing
|Oct 1, 2015
|Oct 1, 2015Ends - 5d 1h


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Forget Powdered Alcohol, Is Powdered Weed Next?
Now that powdered alcohol has been approved, is powdered marijuana THC next?
Nevada Will Vote on Legal Weed in 2016
Nevada voters will have the chance to determine whether the state will be the next to legalize recreational cannabis during the next Presidential election.
Mexico: Narco-Gang Decapitates Mayoral Candidate
Aide Nava, 42-year-old woman running for mayor in Mexico's conflicted southern state of Guerrero, was found decapitated March 11, a day after she was abducted in her hometown of Ahuacuotzingo.
Michigan Hopes to Legalize Recreational Weed in 2016
While Michigan lawmakers continue to lock horns in the legislature over proposed changes to repair the state's crippled medical marijuana program, an organization fighting for total reform has emerged from the sidelines with plans to put the question of a taxed and ...
Guacamole… or Ganja?
Over a ton of weed was found hidden in a shipment of avocado pulp in a cold-storage facility in Lyons, a suburb of Chicago, said Cook County sheriff Thomas Dart in a news release.


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Latest from The Spokesman-Review
Eye on Boise: Historical murals some think offensive will get new review
July 12, 2015 in Idaho on Page B1 Renewed debate is emerging over two historical murals inside the former Ada County Courthouse that depict the lynching of a Native American man. The building will reopen later this summer … 27
Eye on Boise: New Idaho law center will cover up lynching murals
June 28, 2015 in Idaho on Page B10 When the former Ada County Courthouse, which is also the former state Capitol Annex, opens as the new Idaho Law and Justice Learning Center later this summer, two controversial 1930s …
Eye on Boise: Schools could get $3 million a year more from endowment
June 21, 2015 in Idaho on Page B3 Idaho schools could collect nearly $3 million a year more from the state's endowment starting a year from now. Based on how investment returns are coming into Idaho's endowment, the …
Eye on Boise: Less sun will shine on campaign finances
June 14, 2015 in Idaho on Page B3 Idaho Secretary of State Lawerence Denney is less than enthusiastic about a resolution passed by the Idaho Republican Party central committee at its summer meeting last weekend calling for loosening …
Eye on Boise: Two judge replacements, two very different processes
June 7, 2015 in Idaho on Page B3 Here's an interesting contrast: While Idaho U.S. Sens. Mike Crapo and Jim Risch pursue a secret process to name a recommended replacement for longtime U.S. District Judge Edward Lodge, who … 4
Eye on Boise: Jerky quip earns an Oberto sampler
May 24, 2015 in Idaho on Page B3 Oberto Brands in Seattle, manufacturer of beef jerky, is planning to send Idaho Rep. Heather Scott a case of beef jerky. The company noticed a post on Eye on Boise … 5
Corrections Corporation of America cleared in Idaho prison scandal
May 21, 2015 in City, Idaho on Page A1 BOISE – Federal prosecutors on Wednesday declined to file criminal charges of fraud or public corruption in connection with Idaho's controversial private prison contract with Corrections Corporation of America. The … 13
Mike Crapo to seek fourth term in U.S. Senate
May 13, 2015 in City, Idaho on Page A6 BOISE – Idaho GOP Sen. Mike Crapo announced Tuesday that he'll run for re-election in 2016, seeking a fourth six-year term. Crapo named Gov. Butch Otter, Sen. Jim Risch, and … 2
Eye on Boise: Bipartisan panel picked Idaho's last federal district judge
May 3, 2015 in Idaho on Page B3 With all the questions being raised about the secret selection process Idaho Sens. Jim Risch and Mike Crapo are using to vet candidates for the state's next U.S. District judge, …
Women shut out of Idaho judge selection
April 28, 2015 in City, Idaho on Page A1 BOISE – It seems that women need not apply to the federal district court bench in Idaho. A secretive selection process that Idaho's two senators have launched to find a … 61
Eye on Boise: Gatherings give opposite sentiments on Islam
April 26, 2015 in Idaho on Page B2 Two different views of Islam were on display on a single day last week in Idaho, more than 400 miles apart. At one, more than 100 people gathered at the … 3
Accomplishments, or not, of Idaho legislative session
April 19, 2015 in City, Idaho on Page B1 BOISE – By the end of Idaho's legislative session, lawmakers had reversed years of bitter dissension over school reform and passed a $125 million, five-year plan to boost teacher pay; … 3
Eye on Boise: Lawmakers want no credit for representative's editorial
April 19, 2015 in Idaho on Page B2 A brouhaha over the child support enforcement bill killed on the final day of this year's Idaho legislative session is dividing the House GOP. First, Former Rep. Cindy Agidius, communications … 5
Eye on Boise: Session's closing brings poetic inspiration
April 12, 2015 in Idaho on Page B2 After two days of hot-and-heavy conference committee negotiations, lots of waiting around and plenty of uncertainty, Idaho's legislative session lurched to something of a climax on Friday. Lawmakers and legislative …
Idaho roads deal reached; child-support enforcement in jeopardy
April 11, 2015 in City, Idaho on Page A5 BOISE – In a tumultuous final day that ran long into the night, Idaho lawmakers on Friday brokered a transportation funding compromise that satisfied no one and killed critical child-support … 21
Eye on Boise: Otter prefers 'user-pay' funding for transportation needs
April 5, 2015 in Idaho on Page B3 As Idaho's legislative session pushes into its home stretch, Gov. Butch Otter has laid out "parameters" on transportation funding, one big issue on which the House and Senate are at … 1
Idaho bill raises gas tax, cuts grocery sales tax and credit
March 26, 2015 in City, Idaho on Page A5 BOISE – Idaho House Republican leaders introduced complicated legislation Wednesday to make big changes in Idaho's tax system – lowering the top income tax rate, removing the sales tax from … 19
Eye on Boise: Bill limiting kids' use of tanning beds heads to governor
March 15, 2015 in Idaho on Page B10 After years of debate, lawmakers have sent legislation limiting children's use of artificial tanning beds to Gov. Butch Otter's desk. HB 177 passed the Senate late last week on a …
Idaho seeks corporate sponsor for state park funding
Feb. 3, 2015 in City, Idaho on Page A6 BOISE – Idaho's state parks system is looking for corporate sponsors for signs, brochures, picnic shelters and the like as part of its effort to make the parks system largely …
North Idaho College asks for bigger funding increase
Jan. 28, 2015 in City, Idaho on Page A8 BOISE – North Idaho College is getting about the same level of funding from the state now that it got 12 years ago, NIC President Joe Dunlap told lawmakers Tuesday. …


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Tibus provides secure, managed hosting for important websites.
We offer technology and advice for UK & Irish companies to deliver on their digital strategies.
Colin Williams, Creative Director, Sixteen SouthRead full case study
Darren Monksfield, Project Director, MTV NetworksRead full case study
Jonny McMeekin, Marketing Manager, Irish FARead full case study
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Gareth Herron, Advisor to the Inquiry, Northern Ireland OfficeRead full case study
Matt Davis, Head of Digital, talkSPORTRead full case study
Tibus is fully certified under ISO 27001, the quality assurance marque for IT Security and Information Governance. The ISO 27001 framework allows Tibus to offer our clients an independently verified level of confidence in the way we look after their data and their applications.Find out more
Tibus offers a full range of cloud and hosting services to UK public sector IT buyers, under H.M. Government G-Cloud framework. Our focus is on IaaS, managed and un-managed.Visit now
There has been a lot of talk about ad blockers in recent days - so is it ethical to use one?Continue reading
Are you enjoying the Rugby World Cup at the moment? Did you know you can watch it live on a Tibus platform? We'll explain how in this blog post.Continue reading


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This image, according to CNN iReport contributor Matthew Dishler, shows three white students at New York state's Waverly High School wearing blackface during a skit at the school's homecoming pep rally:
Three white students were to perform a skit in black face, depicting Chris Brown and Rhianna [sic] most notibly [sic]. In this skit they would display acts of domestic violence as satire to an audience that included not only students but parents, faculty, and various members of the media and community leaders. None of them stopped the skit.
The story, published early Monday morning, has stirred up plenty of conversation in the Waverly community. Students and alumni have shown support and shame. But more often than not, the response seems to be defensive.
Apparently, this is not the first blackface sketch in Waverly — a village with a 97 percent white population, according to the 2010 census.
Check out more articles on BuzzFeed.com!
- California Republican Kevin McCarthy has dropped out of the race to be the next speaker of the U.S. House. ›
- The United Nations has proposed a national unity government for Libya after peace talks between the north African country's two rival factions. Libya has been in chaos since its dictator was overthrown in 2011. ›
- It could take more than a year for Volkswagen to fix all its cars with diesel engines rigged to evade emissions tests, the company's U.S. CEO said. ›


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Tribeca Film Picks Up 'The Broken Circle Breakdown'
Felix van Groeningen's romantic melodrama, which won two awards at this year's Tribeca film festival and another in Berlin, will be released later this year.
Tribeca Film has acquired all U.S. rights to director Felix van Groeningen's bluegrass-infused drama The Broken Circle Breakdown. A late-2013 theatrical release is planned for the film, written by Carl Joos and van Groeningen and produced by Dirk Impens.
A box office hit in Belgium, the film played the 2013 Tribeca Film Festival, where Joos and van Groeningen won best screenplay for a narrative feature film and lead actress Veerle Baetens won best actress in a narrative feature film. Earlier this year, it won the Panorama Audience Award at the 2013 Berlin International Film Festival.
REVIEW: 'The Broken Circle Breakdown'
Baetens and Johan Heldenbergh play a couple who fall in love at first sight. She has her own tattoo shop, and he plays the banjo in a bluegrass band. But when tragedy hits, their love is tested.
"Through flashbacks and flash-forwards, director van Groeningen seamlessly weaves a tapestry, detailing the story of a marriage and the pleasure and tragedy of being alive," Geoffrey Gilmore, chief creative officer of Tribeca Enterprises, said.
The Broken Circle Breakdown also will be released through on-demand platforms. The deal was negotiated for Tribeca Film by Nick Savva, director of acquisitions, and Alison Diviney, acquisitions manager, and The Match Factory's Brigitte Suarez on behalf of the filmmakers.
Never miss a story. Stay up-to-date with THR's Daily Headlines.
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A Widow's Threats, High-Powered Spats and the Sony Hack: The Strange Saga of 'Steve Jobs'
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'The Martian's' Matt Damon Talks 'Bourne' Return, Politics and PC Flubs: "Oh My God, I Look Like an Asshole"
The Insane Battle Over Sam Simon's Will — Should the Simpson's Co-Creator's Trust Pay $140,000 Per Year For One Dog's Care?
'Empire's' "Batshit Crazy" Behind-the-Scenes Drama: On the Set of TV's Hottest Show
Hollywood's Notable Deaths of 2015


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Last day in Ilocos. The sun shone! Wish we had that much sunlight at Blue Lagoon, but we'll take it!
Walking along Saud Beach.
Last day in Ilocos. The sun shone! Wish we had that much sunlight at Blue Lagoon, but we'll take it!
Walking along Saud Beach.
From the numerous conversations we had via email, I could only recall "waterfall" and "swimming" for Day 3. "Hiking" was an idea my brain completely blocked.
Anyway, our first stop was Kabigan Falls. I survived the trek wearing a pair of slippers, but do wear comfortable hiking shoes for safety.
Manang Taling was our guide for the day.
In our little group Day 2 was officially known as SAND DUNES DAY! But first, culture. Haha.
We packed up and left Casa Teofila, ready to be driven around by our tour guide Manong Arnel Aguila (contact number: 0926-5880666) and van driver Manong Joseph.
Ilocos! This is one of my favorite trips. Relaxed (since we partitioned the tour into four days instead of our usual frantic two days) and involved equal parts culture and craziness.
We met up on March 23, Friday, at the McDonald's near the Quezon Avenue MRT station. After dinner we took a cab to the Florida bus station in Sampaloc.
Back from Ilocos! And drowning in work emails!
But first, some quick links:
"Needle Rain" Part 2 can now be read on PGS Online
"When words are enough", an article about the poetry reading at the Ayala Triangle Gardens
And a preview of the Ilocos blog posts I will be drafting (once I get through all the work emails):


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Nice room with balcony overlooking the large swimming pool with swim up bar and jacuzzi, all amenities you needed and was kept spotlessly clean during our stay. Only downside wifi is chargeable, although most places outside the hotel offer wifi free of charge. Stunning beach front location with plenty of sunbeds. Shops and restaurants within walking distance and only a five minute Taxi ride into town.
- Official Description (provided by the hotel):
- This enchanting 4-star picturesque resort is sprawled over 13 acres of land, fronting on one of Langkawi island's finest beach, Pantai Tengah. Here, our 258 guest rooms and suites range from the simple elegance to the truly extravagant. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Expedia, Travelocity, Booking.com, Agoda, Orbitz, TripOnline SA, Fastbooking, Priceline, Odigeo, HotelTravel.com, Hotels.com, Asiatravel.com Holdings and Low Cost Holidays so you can book your Holiday Villa Beach Resort & Spa Langkawi reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Holiday Villa Beach Resort & Spa Langkawi Hotel Langkawi
- Holiday Villa Beach Resort And Spa Langkawi
- Holiday Hotel Beach Langkawi


text:
TrustPort Tools 2013
Quick Specs
Total Downloads:215
Date Added:May 15, 2013
Price:Free to try; $12.95 to buy
File Size:19.41MB
Downloads Last Week:1
From TrustPort: TrustPort Tools 2013 is a standalone solution for the protection of sensitive documents. Ideal for businesses and individuals who are not looking for a full security suite. It consists of modules for both offline and online data encryption and also from the Portunes module which allows you to safely store sensitive user data to the cloud. Your data is also synchronized between your computer and mobile devices, making them available to the user anytime, and anywhere. TrustPort Tools also offers effective data shredding.
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text:
Does it drive? Can it Fly? Chug? Chuff and Puff? Well if it does, this is where it will be. From classic pull back cars, to RC Planes and helicopters, to kits that let you build your own moveable creations, we have something for everyone. If it's related to a train, plane, or car, you'll find it here.
Designed with authentic details and made of die-cast metal with plastic parts, this NYPD officially licensed 3 Vehicle Set is perfect for those who simply love police cars! With realistic features, these vehicles will provide hours of fun, acting out imagined rescue scenes in the playroom or on the playground!
Includes 3 NYPD vehicles: car, jeep, and helicopter.
Each vehicle measures approximately 3" long.
Designed with authentic details and made of die-cast metal with plastic parts, this NYPD 5 Vehicle Set is perfect for those who simply love police cars! With realistic features, these vehicles will provide hours of fun, acting out imagined rescue scenes in the playroom or on the playground!
Includes 5 NYPD vehicles: car, tow truck, transport vehicle, jeep, and sport utility vehicle.
Help the officer race to the scene with his Playmobil Police Motorcycle. This police officer is ready to help prevent crime and when you press the button a blue light on the back of his motorcycle flashes, letting everyone know help is on the way!
Includes: police officer figure, police motorcycle with flashing light, helmet and police equipment accessory.
Requires button cell battery (INCLUDED).
Figure measures approximately 2.95" tall.
The Siku U.S. Patrol Car was created based on the Dodge Charger Patrol Car design in the U.S. A perfect addition to your Siku rcollection! This model is fitted with a red siren and front bumper bracket. The tires are made of high grip plastic with sport wheel rims. The body of the vehicle is made nearly entirely out of metal and the functioning doors are able to be opened and closed.
Siku models are built tough and made to last. Realistic enough to please a child while playing as well as the avid collector, all Siku models are made of quality die-cast metal with PVC-free plastic parts.
Measures approximately 3.5" long x 1.6" wide x 0.79" tall.
The U.S. Police Truck from Siku has a striking design with detailed lettering work. The truck is set high so it is ready for driving on rough terrain! The body is made nearly entirely out of metal with shiny wheel rims. The classic all red siren lights are depicted and the distinct paint job make this a wonderful addition to your Siku collection!
Ships in assorted colours according to availability (blue or black).
Measures approximately 3.1" long x 1.6" wide x 1.2" high.
Made almost entirely of durable die-cast metal and plastic, this Siku low loader with police helicopter is ready for any rescue! Built to remain strong during rough play and painted a lustrous grey with blue and black accents, it also includes a fold down ramp and rotating helicopter blades. Realistic enough to please a child during playtime as well as the avid collector!
Measures approximately 6.3" long x 1.2" wide x 1.6" high.
The busy policeman drives around town on his motorbike making sure the traffic moves along and everyone is safe. He also makes sure that people stop when the light is red.
LEGO and the LEGO logo are trademarks of the LEGO Group. ©2010 The LEGO Group.
Lamborghini Gallardo Police show car with extra wide tyres on sport wheel rims, die-cast metal body, plastic headlights at the front and rear, blue alarm light on roof and Polizia paintwork.
Measures approximately 2.8" long x 1.2" wide x 0.79" high.
Serve and protect the citizens of Playmobil city with your Police Car with Flashing Light! This police vehicle can seat up to 4 Playmobil figures and is ready to help keep drivers safe with the included road signs. Best of all this police vehicle has a working flashing light, so everyone will know you're on the scene to help!
Includes: 2 police officer figures, police car with flashing light, 2 road signs, camera, megaphone and police equipment accessories.
Requires 2 AAA batteries (NOT INCLUDED).
Each figures measures approximately 2.95" tall.
Gearing up for school and finding just the right backpack
and all the coolest stuff to fill your pencil case, makes this such a fun time
of year! Let's get ready for a whole school year's worth of possibilities.
Check out our favourite finds to help bring FUN back to school! …
read more...
We're now hiring staff for our brand new location in Winnipeg. Send
resumes to firstname.lastname@example.com
Come visit us at our new Edmonton location.
Details Here


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The abuse and neglect of children is a serious problem for everyone – not just children. We can prevent child abuse if everyone gets involved. That's why Pennsylvania Family Support Alliance is taking the lead to protect our children through education, information and action.
Upcoming Events, Programs & Training
How Can You Help?
Speak out about changes in laws and policies that impact children and families. Find out more about our advocacy efforts.
Stay Informed
Learn how to prevent child maltreatment, and how to recognize and report it when you suspect a child is being abused.
Donate Now Protect PA's Kids
Support our work with mandated reporters, parents and communities to protect children from abuse.
Join Our Mailing List
Sign up to receive newsletters and the most updated information on child welfare in PA.
Ways You Can Connect
09/10/2015 - Fall Newsletter Published
Current Newsletter - Fall 2015 Newsletter
Years ago, when I started my career as a parent educator, I was surprised to discover the number of parents who were referred to me by the child welfare system…
Pennsylvania Family Support Alliance
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Definitions for visualiser
This page provides all possible meanings and translations of the word visualiser
Princeton's WordNet
visualizer, visualiser(noun)
one whose prevailing mental imagery is visual
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Borderline Antiphospholipid antibodies and trying to conceive
lovenox is based on weight is my understanding. when I get pregnant again i am on 40 mgs daily..i weight about 122 lbs...5'4
ashlyn-my blessing daughter born 2/11, mc 12/4/13, mc 5/22/13,
mc. 5/11/4/13...... waiting for a rainbow baby this year!
My blood work showed the same results with borderline levels. I also have a history of generic auto-immune stuff and was recently found to also have two copies of the MTHFR gene mutation. I am currently on 40 mg of Lovenox but only 5 weeks along. I think that is the standard. I stumbled upon this Q and A online which I found interesting.
When did you start taking the Lovenox? Did you wait for a positive pregnancy test?
My antibody level dropped to low positive postpartum so my hematologist felt I did not need lifelong anticoagulation because I take immunosuppressants for uc and arthritis. I had a DVT in the past but that was before I was on immunosuppressants. Having uc no one was in a rush to put me on a therapeutic weight based dose of lovenox.
I was boderline for APS as well. No other explanation for my recurrent early miscarraige, so they put me on baby aspirin and 40 mg lovenox; but also progesterone until 12 weeks. Success the first time after 4 losses, and now I have a handsome healthy, almost month old, baby boy!
Me 41, DH 42, L- 2/22/10 born a little early due to mommy's HELLP syndrome, aPS, Lupus, AMH 0.16
cp-10/12, m/c9wks 1/13, 3 iuis, bfn, natural bfp10/13-M-6/22/14
I am not APS (that we know). I have high ANA 1:160 (specifically SSA-Ro came back as positive). According to my DR's, this shouldn't cause RPL and MAYBE my two in a row were flukes. They said to take Baby aspirin 81mg but I am worried that this isn't enough.
We are actively TTC and my appt. with MFM is later this month, so if we conceieve this cycle, I will know by then-- should I insist on Lovenox?
EP- Silver


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Scrubs: Season 4, Episode 22
My Big Move (12 Apr. 2005)
Ratings: 8.5/10 from 414 users
Reviews: write review
J.D. and Carla's secret is out and Turk isn't reacting as they thought he would. Jordon's brush with botox gives Cox ammunition and the Janitor is not a big fan of his new uniform!
Director:Victor Nelli Jr.
Writers:Bill Lawrence (created by), Bonnie Sikowitz
Stars:Zach Braff, Sarah Chalke, Donald Faison | See full cast and crew »
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