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Genome-wide analysis of androgen receptor binding and gene regulation in two CWR22-derived prostate cancer cell lines
Department of Medical Microbiology and Immunology, University of California Davis, Davis, California 95616, USA.Endocrine Related Cancer (Impact Factor: 4.81). 12/2010; 17(4):857-73. DOI: 10.1677/ERC-10-0081
Source: PubMed
Prostate carcinoma (CaP) is a heterogeneous multifocal disease where gene expression and regulation are altered not only with disease progression but also between metastatic lesions. The androgen receptor (AR) regulates the growth of metastatic CaPs; however, sensitivity to androgen ablation is short lived, yielding to emergence of castrate-resistant CaP (CRCaP). CRCaP prostate cancers continue to express the AR, a pivotal prostate regulator, but it is not known whether the AR targets similar or different genes in different castrate-resistant cells. In this study, we investigated AR binding and AR-dependent transcription in two related castrate-resistant cell lines derived from androgen-dependent CWR22-relapsed tumors: CWR22Rv1 (Rv1) and CWR-R1 (R1). Expression microarray analysis revealed that R1 and Rv1 cells had significantly different gene expression profiles individually and in response to androgen. In contrast, AR chromatin immunoprecipitation (ChIP) combined with promoter DNA microarrays (ChIP-on-chip) studies showed that they have a similar AR-binding profile. Coupling of the microarray study with ChIP-on-chip analysis identified direct AR targets. The most prominent function of transcripts that were direct AR targets was transcriptional regulation, although only one transcriptional regulator, CCAAT/enhancer binding protein δ, was commonly regulated in both lines. Our results indicate that the AR regulates the expression of different transcripts in the two lines, and demonstrate the versatility of the AR-regulated gene expression program in prostate tumors.
Genome-wide analysis of androgen
receptor binding and gene regulation in two
CWR22-derived prostate cancer cell lines
Honglin Chen
, Stephen J Libertini
, Michael George
, Satya Dandekar
Clifford G Tepper
, Bushra Al-Bataina
, Hsing-Jien Kung
, Paramita
M Ghosh
and Maria Mudryj
Department of Medical Microbiology and Immunology, University of California Davis, 3147 Tupper Hall, Davis,
California 95616, USA
Division of Basic Sciences, Department of Biochemistry and Molecular Medicine, Cancer Center and
Department of Urology,
University of California Davis, Sacramento, California 95817, USA
Veterans Affairs Northern California Health Care System, Mather, California 95655, USA
(Correspondence should be addressed to M Mudryj at Department of Medical Microbiology and Immunology, University of California,
Davis; Email: firstname.lastname@example.com)
Prostate carcinoma (CaP) is a heterogeneous multifocal disease where gene expression and
regulation are altered not only with disease progression but also between metastatic lesions. The
androgen receptor (AR) regulates the growth of metastatic CaPs; however, sensitivity to androgen
ablation is short lived, yielding to emergence of castrate-resistant CaP (CRCaP). CRCaP prostate
cancers continue to express the AR, a pivotal prostate regulator, but it is not known whether
the AR targets similar or different genes in different castrate-resistant cells. In this study, we
investigated AR binding and AR-dependent transcription in two related castrate-resistant cell lines
derived from androgen-dependent CWR22-relapsed tumors: CWR22Rv1 (Rv1) and CWR-R1
(R1). Expression microarray analysis revealed that R1 and Rv1 cells had significantly different
gene expression profiles individually and in response to androgen. In contrast, AR chromatin
immunoprecipitation (ChIP) combined with promoter DNA microarrays (ChIP-on-chip) studies
showed that they have a similar AR-binding profile. Coupling of the microarray study with
ChIP-on-chip analysis identified direct AR targets. The most prominent function of transcripts that
were direct AR targets was transcriptional regulation, although only one transcriptional regulator,
CCAAT/enhancer binding protein d, was commonly regulated in both lines. Our results indicate
that the AR regulates the expression of different transcripts in the two lines, and demonstrate
the versatility of the AR-regulated gene expression program in prostate tumors.
Endocrine-Related Cancer (2010) 17 857–873
Multiple studies have demonstrated heterogeneity in
prostate carcinoma (CaP), a multifocal disease where
tissue architecture and genetic expression are altered
not only with disease progression but also between
metastatic lesions of patients with prostate cancer
(Nwosu et al. 2001, Beheshti et al. 2002, Liu et al.
2004, Shah et al. 2004). Comparison of metastatic
lesions from the same patient as well as different
patients showed that metastatic hormone-refractory
prostate cancer has a heterogeneous morphology,
immunophenotype, and genotype, demonstrating that
'metastatic disease' is a group of diseases even within
the same patient (Shah et al. 2004). As prostate cancer
can progress from an organ-confined, localized state to
metastasis, the problem in treating the disease is the
identification of therapeutic targets that are common to
all the foci within the same patient or in multiple
patients. As most CaPs are initially present as androgen-
dependent neoplasms, androgen ablation therapy
(chemical castration) is an effective treatment, which
initially blocks androgen receptor (AR) cell signaling in
almost all patients. Although this therapy is initially
successful, castration-resistant androgen-independent
Endocrine-Related Cancer (2010) 17 857–873
Endocrine-Related Cancer (2010) 17 857–873
1351–0088/10/017–857 q 2010 Society for Endocrinology Printed in Great Britain
Online version via http://www.endocrinology-journals.org
Page 1
tumors that are refractory to hormonal therapeutic
interventions emerge (Huggins & Hodges 1941, Gittes
1991). Androgen-independent CaPs continue to express
the AR and androgen-regulated genes. Thus, a better
understanding of the action of AR is a pivotal issue
in defining the molecular events that lead to the
progression of CaP. However, it is unclear whether
requirement for AR function in various foci and
metastatic lesions within the same patient or similar
groups of patients is the same.
Various studies have indicated that the function
of the AR depends on the biochemical environment
in which it exists (Ruizeveld de Winter et al. 1994,
Li et al. 2002). As a member of the nuclear receptor
superfamily that functions as a ligand-dependent
transcription factor, AR mediates androgen-regulated
gene expression. Androgen-bound AR is stabilized and
translocated into the nucleus to regulate the expression
of target genes by binding to androgen response
elements (AREs) or by interacting with other tran-
scription factors bound to their specific recognition
sites. The role of AR in CaP progression is to promote
expression of specific target genes. For example,
prostate-specific antigen (PSA), the best studied AR
target gene, has been reported to contribute to CaP
progression through its protease activity and its ability
to induce epithelial–mesenchymal transition and cell
migration (Borgono & Diamandis 2004, Whitbread
et al. 2006). Other AR target genes implicated in CaP
progression include FGF8, Cdk1 and Cdk2, PMEPA1,
TMRPSS2, and amyloid precursor protein (Gregory
et al. 1998, Lin et al. 1999, Gnanapragasam et al. 2002,
Xu et al. 2003, Takayama et al. 2009). However, the
function of the AR is tightly regulated by the expression
of co-factors that are themselves regulated by various
transcription factors, including the AR. Studies
revealed differential expression of co-regulators with
disease progression, which may have led to altered
AR function (Li et al. 2002). Hence, an important
point of investigation in prostate cancer research is to
determine whether the AR functions similarly or
differently in various metastatic lesions within the
same patient.
Since the last decade, microarray techniques have
been applied extensively in searching for genes that
are AR regulated specifically in prostate tumors.
Although gene expression profiling is a powerful
technique for depicting the global function of the AR
in a specified model, it does not distinguish whether
alteration of gene expression is dependent on a direct
or indirect action of AR. Moreover, despite the well-
characterized AREs in the promoter and enhancer,
little is known about AR cis-regulatory sites across the
human genome. Chromatin immunoprecipitation
(ChIP-on-chip) technology has been used for the
identification of chromosomal-binding sites of tran-
scription factors to identify novel targets (Cawley et al.
2004, Bernstein et al. 2005). Therefore, coupling
microarray studies with ChIP-on-chip allows the
identification of bona fide AR target genes. Wang
et al. (2007) mapped the AR-binding sites on
chromosomes 21 and 22 in androgen-dependent
LNCaP human prostate cancer cells by combining
ChIP with tiled oligonucleotide microarrays. Later, they
followed up with comparisons between LNCaP versus a
castrate-resistant CaP (CRCaP) variant of LNCaP cells
(Wang et al. 2009), in an attempt to identify direct
AR-dependent target genes in both androgen-dependent
CaP as well as in CRCaP, and determined that the role of
the AR in CRCaP is to execute a distinct program
resulting in androgen-independent growth. Signi-
ficantly, targets identified by one group in a CRCaP
subline of LNCaP cells (Wang et al. 2009) were not
identical to those in another LNCaP subline, C4-2B,
shown in a different study (Jia et al. 2008), which
mapped AR-occupied regions as well. A third study,
which used PC3 cells transfected with wild-type AR
(Lin et al. 2009), also identified distinct AR-occupied
regions in target genes. The differences in these results
may be attributed to differences in the technologies used
to study AR-binding sites. An alternative explanation
may be that because of the heterogeneity of gene
expression in different CaP foci and metastatic lesions,
the programs regulated by the AR in each deposit,
even within the same patient, may be distinct.
The CWR22 androgen-dependent xenograft model,
which mimics human prostate cancer, has been used to
study the emergence of CRCaP (Wainstein et al. 1994).
In male nude athymic mice, this xenograft exhibits
androgen-dependent growth and secretes PSA. After
androgen withdrawal, the tumors regress and PSA
levels plummet. Importantly, the model simulates the
clinical course of prostate cancer, in that PSA levels
eventually increase and CRCaP tumors emerge
(Nagabhushan et al. 1996). Similar to most CRCaP
tumors, CWR22-recurring tumors continue to express
the AR (Gregory et al. 1998), which contains a
mutation (H847Y) in the ligand-binding domain
(LBD) of the molecule (Tan et al. 1997). Since this
model recapitulates salient features of human prostate
tumors, it has been used extensively to study the
emergence of CRCaP.
Two cell lines, R1 and Rv1 (van Bokhoven et al.
2003), were isolated in separate laboratories from
CWR22-relapsed tumors. Several lines of evidence
indicate that they were derived from a common
H Chen et al.: Analysis of AR binding in R1 and Rv1 cells
Page 2
ancestor. Karyotypes of the two cell lines are very
similar; both lines shared the same structural abnorm-
alities, including a reciprocal translocation between
chromosomes 6 and 14 (van Bokhoven et al. 2003).
Both lines have the same AR (H847Y) mutation that is
present in the parental CWR22 cells (van Bokhoven
et al. 2003). The Rv1 AR also contains a duplication of
exon 3 (that encodes the DNA-binding domain), which
results in an insertion of 39 additional amino acids
(Tepper et al. 2002). The insertion mutation was
present in the relapse tumor, and the subsequent cell
line was established from the tumor, but very low
levels of this mutated AR could also be detected by
RT-PCR in the parent CWR22 tumor (Tepper et al.
2002). Additionally, we and others found that R1 and
Rv1 express an w80 kDa low molecular weight form
of AR (LMW-AR) with a deletion of the C-terminal
LBD (Gregory et al. 2001, Tepper et a l. 2002).
However, though the cell lines have significant
similarities, they also exhibit differences. In a recent
study, we showed that R1 and Rv1 cells were distinct in
their AR expression, characterization, and function
(Chen et al. 2010). The goals of the current study were
to use these two cell lines to determine similarities and
differences in AR-regulated programs in two related
but distinct systems with a common lineage.
Materials and methods
Cell culture and pharmacological agents
Rv1 cells were obtained from American Type
Culture Collection (ATCC, Manassas, VA, USA).
CWR-R1 cells were provided by Dr Elizabeth Wilson
(University of North Carolina). Rv1 and R1 cells
were propagated in RPMI 1640 supplemented with 5%
fetal bovine serum, 2 mmol/l
L-glutamine, 100 U/ml
penicillin, and 100 mg/ml streptomycin (Invitrogen
Life Science) at 37 8C and 5% CO
. For studies in
androgen-depleted conditions, cells were propagated
in phenol red-free RPMI 1640 supplemented with
5% charcoal-stripped fetal bovine serum, 2 mmol/l
L-glutamine, 100 U/ml penicillin, and 100 mg/ml
streptomycin at 37 8C and 5% CO
Western immunoblot analysis
Cells were directly placed in a radioimmunoprecipit-
ation assay lysis buffer that contained the Sigma
protease inhibitor cocktail (AEBSK, aprotinin, E64,
leupeptin, and pepstatin as well as 1 mM calpeptin;
Sigma–Aldrich). Thirty micrograms of protein were
separated on 8, 10, or 12% SDS-PAGE gels and
transferredto0.22mM nitrocellulose-supported
membrane (GE Healthcare, Piscataway, NJ, USA).
The membrane was blocked with 5% nonfat dry milk
in PBS and 0.1% Tween-20 before the addition of
specific antibodies. The following antibodies were
used: AR (central) 441 (Ab-1; Lab Vision Corp.,
Fremont, CA, USA), AR NH
-terminus N-20 (Santa
Cruz Biotechnology, Inc., Santa Cruz, CA, USA),
calpain 2 (Sigma), calpastatin, ERK and phosphoERK
(Cell Signaling, Danvers, MA, USA), and focal
adhesion kinase (clone 4.47; Millipore, Billerica,
MA, USA). Proteins were detected using chemi-
luminescence (GE Healthcare).
Microarray analysis
Labeling of samples, hybridization to U133A Gene-
Chips (Affymetrix, Santa Clara, CA, USA), staining,
and scanning were performed as described in the
Affymetrix Expression Analysis Technical Manual.
Fluorescence intensity values (.CEL files) generated
from hybridized, stained GeneChips were analyzed
with R statistical software (v.2.01, and 'affy' BioCon-
ductor package) and BRB Array Tools to identify
genes that were differentially expressed. The settings
used for Robust Multichip Analysis in R included
Microarray Suite 5.0-based background correction,
quantile normalization, and Robust Multichip
Analysis-based algorithms for calculation of
expression values usingperfectmatchonlyflu-
orescence intensities. Detection at P%0.05 and a
mean fold change of R1.5-fold were used as criteria
for filtering genes for clustering analyses. Hierarchical
clustering and comparative fold change analysis were
used to identify and group similar patterns of gene
regulation. Assignment of genes to functional
categories was done by annotation of gene lists with
the program, Database for Annotation, visualization,
and Integrated Discovery (http://apps1.niaid.nih.gov/
david), and literature-based classification was done by
hand. Statistically overrepresented (Fisher's exact
probability score !0.05) biological processes within
clusters were identified using Expression Analysis
Systematic Explorer v.1.0 analysis software (Hosack
et al. 2003).
Quantitative real-time PCR
Total cellular RNA was prepared from Rv1 cells using
RNeasy mini kit (Qiagen, Inc.) based on the
manufacturer's protocol. cDNA was synthesized from
1 mg RNA using QuantiTect (Qiagen) reverse tran-
scription kit based on the manufacturer's protocol.
cDNAs were diluted 1:4 in ddH
O, and 2 ml diluted
Endocrine-Related Cancer (2010) 17 857–873
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cDNA was added to 5 ml of EXPRESS SYBR
GreenERTM qPCR supermix (Invitrogen Life
Science) and 200 nM of each primer. GAPDH,
HPRT, or RPL13A was used as the endogenous
expression standards. PCR conditions were initial
denaturation step at 95 8C for 20 s, 40 cycles at 95 8C
for 3 s, 60 8C for 30 s, followed by additional 95 8C for
15 s and 60–95 8C over 20 min ramp for melt curve
analysis. Primer sequences used in the study are
provided in Supplementary Methods (see section on
supplementary data at the end of this article). Data
were collected by the Mastercycler ep Realplex
(Eppendorf AG, Hamburg, Germany). Primer
sequences are available upon request.
Ingenuity pathway analysis
The microarray expression data were uploaded into
ingenuity pathway analysis (IPA) software using
Reference sequence (RefSeq). A total of 2322 genes
were mapped using the IPA database. Fold change of
1.5 and P value of %0.05 were applied as the cutoff
criteria. Gene networks were algorithmically generated
based on their connectivity and were assigned a score.
A score of 3 or higher indicates a 99.9% confidence
level that the network was not generated by chance
alone. Canonical pathway analysis identifies the
pathways from the IPA library of canonical pathways,
which are most significant to the input dataset. The
significance of the association between the dataset and
the canonical pathway is determined based on two
parameters: 1) a ratio of the number of genes from the
dataset, which map to the pathway, divided by the total
number of genes that map to the canonical pathway and
2) a P value calculated using Fischer's exact test
determining the probability that the association
between the genes in the dataset and the canonical
pathway is due to chance alone.
ChIP-on-chip analysis
Tiling array analysis was performed with GeneChip
Human Promoter 1.0R Arrays (Affymetrix) in order to
determine genome-wide analysis of AR recruitment
sites. Briefly, AR-associated DNA was enriched by
ChIP as described earlier (Louie et al. 2003, Desai
et al. 2006). ChIPs using a pre-immune IgG were used
as controls. ChIP DNA (10 ml) and input (10 ng)
samples were amplified using the GenomePlex
Complete Whole Genome Amplification (WGA) kit
(Sigma–Aldrich) with a modification to the manufac-
turer's protocol to generate product suitable for
Affymetrix microarray analysis by including dUTP
(80 mM final concentration) in the amplification and
re-amplification (if necessary) reactions. WGA
products were purified with QIAquick PCR purifi-
cation kit, eluted in nuclease-free water (Invitrogen),
and quantitated with a NanoDrop 2000c spectro-
photometer (Thermo Scientific). Target preparation
and tiling array-processing procedures were performed
according to Affymetrix's standard protocols. Briefly,
7.5 mg DNA was fragmented through the combined
actions of uracil DNA glycosylase and human apurinic
endonuclease and then end labeled with biotin using
terminal deoxynucleotidyl transferase. Labeled target
DNA was hybridized to the arrays at 45 8C for 16 h.
Subsequently, the arrays were washed and stained
using the Fluidics Station 450 (Affymetrics, Santa
Clara, CA, USA) according to the manufacturer's
protocol and then scanned with the GeneChip Scanner
3000 7G. Data analysis was performed with CisGen-
ome software (Ji et al. 2008). AR-binding regions (i.e.
ChIP-enriched) were identified by comparing with the
nonspecific IgG control using the TileMap peak
detection tool (Ji & Wong 2005) with the application
of a hidden Markov model. Subsequently, genomic
locations of peaks and bound probes were visualized in
the CisGenome and UC Santa Cruz genome browsers.
Comparison of the gene expression profiles of
R1 and Rv1 cells
We have described earlier the characteristics of R1 and
Rv1 cells derived from two different relapsed tumors
although both from the same parental CWR22
xenograft (Chen et al. 2010). To further define the
differences and similarities between the two CWR22
relapsed lines, we used the Affymatix HG-U133
Plus2.0 Gene Chip microarray to identify differences
in gene transcription. The analysis was conducted in
duplicate in R1 and Rv1 cells cultured in identical
conditions, at the same density in charcoal-stripped
serum or 2 h after the addition of 10 nM DHT. The 2 h
time point was chosen to identify transcripts that are
more likely to be direct AR targets, and other
laboratories had previously determined this con-
centration of DHT to be optimal for AR stimulation
(Wang et al. 2007).
Comparison of R1 and Rv1 gene expression
profiles in castrate levels of androgen identified 1275
genes that were differentially expressed (fold change
R1.5 or %K 1.5; P% 0.05) in R1 versus Rv1 cells in
the absence of androgens and 1941 transcripts that
were differentially expressed (fold change R1.5 or
%K1.5; P%0.05) in R1 versus Rv1 cells treated for
H Chen et al.: Analysis of AR binding in R1 and Rv1 cells
Page 4
2 h with 10 nM DHT (Fig. 1A; Supplementary Table 1,
see section on supplementary data given at the end of
this article). Significantly, only 60% of genes differ-
entially expressed in R1 versus Rv1 in the presence of
DHT were identical to the transcripts that were
differentially expressed in the absence of androgen
(Fig. 1A; Supplementary Table 1). These results
indicated that the R1 and Rv1 cells were genetically
distinct and could serve as models for comparison of
two different metastasized CaP lesions derived from
the same patient.
We verified the specificity and selectivity of the
results obtained with the microarray analysis by
comparing these results to known differences between
the two lines. We had shown earlier that R1 cells
expressed increased levels of calpain 2 mRNA
compared to Rv1 cells, whereas the levels of the
calpain inhibitor calpastatin were similar in both lines
(Chen et al. 2010). A similar pattern was seen by the
current gene expression studies, thereby authenticating
the results (Supplementary Table 1; Fig. 1B). R1 cells
also expressed 11.7-fold higher levels of c-MET
mRNA (Supplementary Table 1; Fig. 1B). Rv1 cells
have more neuroendocrine characteristics than R1 cells
because of a greater expression of neuronal-specific
enolase (ENO2; 12-fold change; PZ0.02; Supple-
mentary Table 1; Fig. 1B), chromogranin A and B
(2.74- and 8.69-fold increase respectively), and
synaptophysin (3.34-fold increase; Supplementary
Table 1). ENO2 expression was not altered by
androgen (data not shown). These results also show
the accuracy of the gene expression analysis system in
these studies.
Based on the gene expression analysis, the most
differentially expressed genes between R1 and Rv1
(expression in the absence of androgen) include TARP,
IGFBP5, STEAP1, NMNAT2, and SNAI2 (listed in
Fig. 1C). To identify patterns in differential gene
Fold elevated in R1
Fold elevated in RV1
AD + Rv1
Calpain 2
Galactose metabolism
Galactose metabolism
Clatrin-mediated endocytosis
Antigen presentation
Antigen presentation
Hypoxia signaling in the
cardiovascular system
Pentose phosphate
Propanoate metabolism
Propanoate metabolism
Role of BRCA1 in DNA
damage response
Histidine metabolism
Histidine metabolism
p53 signaling
Ascorbate and aldarate
Ascorbate and aldarate
Valine, leucin and
isoleucine biosynthesis
Arginine and proline
Pyruvate metabolism
Pyruvate metabolism
Lysine degradation
Butanoate metabolism
Fatty acid metabolism
Tryptophan metabolism
Bile acid biosynthesis
Fructose and mannose
Pantothenate and CoA
Arginine and proline
Butanoate metabolism
RAR activation
Protein ubiquitination
Notch signaling
VDR/RXR activator
Glycerolipid metabolism
β-Alanine metabolism
VDR/RXR activation
Valine, leucin and
IsoIeucine degradation
Valine, leucin and
IsoIeucine degradation
–log (
–log (
AD – Rv1
AD + Rv1 versus R1 60% AD – Rv1 versus R1
Figure 1 Differences in gene expression of R1 and Rv1 cells in the presence and absence of a DHT. (A) Venn diagram of the number
of genes differentially expressed in R1 and Rv1 cells in castrate levels of androgen and after a 2 h treatment with 10 nM DHT. The
lower venn diagram shows the cohort of genes differentially expressed in the absence and presence of androgen. (B) Western blot
analysis verification of several differentially expressed proteins that were identified by the expression array study. (C) The most
differentially expressed transcripts in R1 and Rv1 cells treated with 10 nM DHT. (D) The IPA was used to identify the pathways that
differed in the two cell lines in the presence and absence of androgen. The Fisher's exact test was used to determine the probability
that the association between the dataset and a given pathway is due to chance alone. The most significant pathway differences in the
presence and absence of androgen involved metabolic pathways.
Endocrine-Related Cancer (2010) 17 857–873
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expressions, IPA was used to identify the pathways
that differed in the two cell lines. The Fisher's exact
test was used to determine the probability that the
association between the dataset and a given pathway is
due to chance alone. The most significant pathway
differences between R1 and Rv1 cells both in the
presence and absence of androgen involved metabolic
pathways (Fig. 1D). In summary, the gene expression
profiles of R1 and Rv1 indicate that although these two
lines were derived from the same CWR22 xenograft
and have similar morphologies, at the molecular level,
they are distinct.
Analysis of genes differentially regulated in R1
versus Rv1 cell lines in response to androgen
Since the gene expression profile of R1 versus Rv1
cells was vastly different, we investigated whether
these genes behaved similarly in response to DHT
treatment. Using the same gene expression data that
were used in Fig. 1, we analyzed genes that were
differentially regulated in the two cell lines in
response to a 2 h androgen treatment. Using a cutoff
value of fold change R1.5 or %K 1.5 and P%0.05,
we found that in Rv1 cells, the expression of 854
transcripts was altered by a 2 h DHT treatment,
whereas in R1 cells, the expression of only 77
transcripts changed after the addition of DHT for 2 h
(Fig. 2A; Supplementary Table 2, see section on
supplementary data given at the end of this article).
Therefore, the transcriptional response to DHT was
greater in Rv1 cells than in R1 cells. A comparison
of the DHT-responsive R1 and Rv1 transcripts
identified only ten genes that were commonly
regulated in both cell lines (Fig. 2B), again
indicating the large differences between these two
lines. This included seven genes that were upregu-
lated by DHT in both R1 and Rv1 cells, including
CEBPD and N-acetyltransferase type I (NAT1), and
three that genes were repressed in both cells,
including CLDN4. Interestingly, the expression of
HES1, a component of the Notch signaling pathway
(Fischer & Gessler 2007), was DHT regulated in
AD+RV1 versus AD–RV1
AD+R1 versus AD–R1
p27 FABP7
Time (h)
024 18
Time (h)
024 18
Time (h)
024 18
Time (h)
024 18
Time (h)
024 18
Time (h)
Fold decrease
Fold change
Fold change
Fold change
Fold change
Fold change
Fold change
Fold increase
Rv1 DHT–
Rv1 DHT+
Repressed Activated Rv1 / Repressed R1
Figure 2 Differences in AR-dependent gene expression of R1 and Rv1 cells. (A) Venn diagram of AR-regulated transcripts in R1 and
Rv1 cells. (B) Transcripts that are commonly regulated in the two cell lines. Note that although HES1 is androgen regulated in both
cell lines, HES1 expression is elevated in Rv1 cells, but repressed in R1 cells. (C) Real-time PCR verification of several AR-regulated
transcripts. (D) Time course of DHT-inducible gene expression in R1 and Rv1 cells.
H Chen et al.: Analysis of AR binding in R1 and Rv1 cells
Page 6
both cell lines, but expression was repressed in R1
cells and activated in Rv1 cells (Fig. 2B). Androgen-
dependent regulation of six of these transcripts was
validated by real-time PCR, which verified the
accuracy of these results (compare Fig. 2C with
Supplementary Table 2). The expression of two well-
known androgen-responsive genes KLK3 (PSA) and
TMPRSS2 was not significantly altered by DHT in
either cell line, thus confirming previous reports that
the transcripts are not androgen regulated in these
cell lines (Riegman et al. 1991, Lin et al. 1999,
Tomlins et al. 2005).
It is possible that the discrepancy in the number of
DHT-regulated transcripts in R1 and Rv1 cells is due to
a delay in the DHT response in R1 cells. To address
this possibility, we looked at the expression of
transcripts that were DHT regulated in R1 and Rv1
cells (NAT1 and TSC22D1), only in R1 cells (FKBP5),
or only in Rv1 cells (KRIT1, p27, and FABP7) at 0, 2,
4, and 18 h after DHT addition (Fig. 2D). The time
course for transcripts that were regulated in both lines
was similar but not identical in the two cell lines.
Interestingly, the induction of FKBP5 was more robust
in R1 cells than in Rv1 cells; therefore, in the array,
study expression in Rv1 cells 2 h after DHT addition
was below our cutoff value. In concordance with the
array analysis, KRIT1 and p27 were not DHT
transactivated in R1 cells. The expression of FABP7
was elevated in Rv1 cells in a time-dependent manner,
but in R1 cells, the expression was repressed in a time-
dependent manner. This analysis argues that the
smaller number of DHT-regulated transcripts in R1
cells is not due to a general delay in response to
hormone stimulation.
DHT-regulated pathways in R1 and Rv1 cells
Since the genes regulated by androgens in the two cell
lines are different, we asked whether the pathways they
regulated were also different, or whether androgen was
regulating the same programs in both cells but through
different mechanisms. The differentially expressed
genes in response to DHT for 2 h were analyzed by
IPA to identify most significantly associated biological
networks and canonical pathways (metabolic and cell
signaling) altered in the two cell lines. The Fisher's
exact test was used to determine the probability that the
association between the dataset and a given pathway is
due to chance alone. IPA identified two significant
biological networks associated with the differentially
expressed genes in R1 cells (the major one is shown in
Fig. 3A, and the other one is shown in Supplementary
Figure 1, see section on supplementary data given at
Functions regulated by DHT
DHT regulated networks
DHT regulated network in R1 cells
DHT regulated network in Rv1 cells
Gene expression
Cellular development
Cell cycle
Hematological diseases
Embryonic development
Nervous system development
and function
Cell death
Organismal development
Cell gorwth and proliferation
Tissue morphology
Lipid metabolism
Molecular transport
Small moleucle biochemistry
Tumor morphology
Carbohydrate metabolism
Visual system development
and function
Immunological disease
DNA replication, recombnation
and repair
Nucleic acid metabolism
Drug metabolism
Protein trafficking
RNA post-transcriptional
Skeletal and muscular system
development and function
Connective tissue development
and function
2.5 5.0 7.5
Canonical pathways regulated by DHT
Notch signaling
Clatrin-mediated endocytosis
JAK/Stat signaling
p53 signaling
T-cell receptor signaling
Insulin receptor signaling
Inositol phosphate metabolism
NF-κB signaling
B Cell receptor signaling
14-3-3-mediated signaling
Cell cycle:G1/S checkpoint
Axonal guidance signaling
Role of BRCA1 in DNA
damage response
Aminoacyl-tRNA biosynthesis
Rv1 +/– DHT
JAK/Stat signaling
Notch signaling
Axonal guidance signaling
Axonal guidance signaling
Cell cycle
Figure 3 Comparison of biological networks, pathways, and function of R1 and Rv1 DHT-regulated transcripts. (A) The most
prominent DHT-regulated network in R1 and Rv1 cells. A bar above the gene denotes transcripts that are DHT transactivated, and an
underscore denotes transcripts that are DHT repressed. Several components of the Notch signaling pathway are DHT regulated in
R1 cells, whereas components of cell cycle are DHT regulated in Rv1 cells. (B) The most common functions of transcripts regulated
by DHT in R1 and Rv1 cells. (C) The most common DHT-regulated canonical pathways in R1 and Rv1 cells.
Endocrine-Related Cancer (2010) 17 857–873
www.endocrinology-journals.org 863
Page 7
the end of this article), whereas in Rv1 cells, a total
of 18 biological networks were identified, which are
significantly associated with the differentially
expressed genes (the major network is shown in
Fig. 3A, and the others are shown in Supplementary
Figure 2, see section on supplementary data given at
the end of this article). In R1 cells, the significantly
associated functions affected by DHT treatment
include gene expression, cellular development, cell
cycle, and embryonic development (Fig. 3B). The
canonical pathways most significantly associated with
DHT treatment are notch signaling, clathrin-mediated
endocytosis, JAK/Stat signaling, and p53 signaling
(Fig. 3C). The significantly associated functions in Rv1
cells include cellular development, visual system
development and function, cancer, cell cycle, molecu-
lar transport, and protein trafficking (Fig. 3B), whereas
the most associated canonical pathways include
aminoacyl-tRNA biosynthesis, axonal guidance
signaling, DNA damage response, cell cycle, p53
signaling, and clatrin-mediated endocytosis (Fig. 3C).
These results indicate a greater biological role of AR
in Rv1 cells compared to R1 cells. However, a number
of cellular functions and canonical pathways regulated
by DHT treatment in the two cells lines are similar,
suggesting that in the two cell lines, the AR plays a
similar role, but employs different mechanisms.
The activity of the AR is affected by multiple
co-regulators that serve as co-activators or co-repres-
sors of AR-dependent transcription (Devlin & Mudryj
2009, Heemers et al. 2009). Since the differences in
DHT-inducible gene expression could be due to the
expression of a different cohort of AR co-regulators, we
compared the expression of these proteins in the two cell
lines in the presence and absence of androgen (Table 1).
The number of co-regulators that were expressed at
Table 1 Androgen receptor (AR) co-regulators differentially expressed in R1 and Rv1 cells
Elevated in R1 Elevated in Rv1
Symbol Act./Rep. KDHT CDHT DHT reg. Symbol Act./Rep. KDHT CDHT DHT reg.
BRCA2 Act. O Repressed in Rv1* ARID1A Act. OO
CARM1 Act. O Induced in Rv1* CREBBP Act. O Induced in Rv1*
CDK7 Act. OO DDC Act. OO
COPS5 Act. O Induced in Rv1 HMGB2 Act. OO
FHL2 Act. O Repressed in Rv1 HTATIP2 Act. OO
GSN Act. OO IDE Act. OO
HIPK3 Act. O Repressed in Rv1 IQWD1 Act. O Induced in Rv1
HRMT1L2 Act. O PLAGL1 Act. O Induced in Rv1
HTATIP Act. O Repressed in Rv1 PRKDC Act. O Induced in Rv1
NCOA1 Act. OO RB1 Act. OOInduced in Rv1
NCOA2 Act. OORepressed in Rv1 RNF14 Act. O Induced in Rv1
NONO Act. OO TMF1 Act. O Induced in Rv1
PIAS2 Act. O Repressed in Rv1 TRIM24 Act. O Induced in Rv1
PNRC1 Act. OO APPL Rep. OO
PXN Act. OORepressed in Rv1 DNAJA1 Rep. O Induced in Rv1
RAN Act. OO HDAC1 Rep. O Induced in Rv1
SMARCA2 Act. OO NCOR1 Rep. O Induced in Rv1
SMARCC1 Act. O Repressed in Rv1 PA2G4 Rep. O Induced in Rv1
STAT3 Act. O PAK6 Rep. OO
TSC2 Act. O* O ZNF278/
Rep. OO
ZMIZ1 Act. OO*
AES Rep. OO
APPBP2 Rep. O Repressed in Rv1
CALR Rep. O Repressed in Rv1
FLNA Rep. OO
HEY1 Rep. OOInduced in R1
PTEN Rep. OO
TGIF Rep. OO
TLE1 Rep. OO
FKBP5 Act. Elevated in R1 in the presence of DHT, elevated in Rv1 in absence of DHT;
DHT induced in R1 cells
Act., activator; Rep., repressor; KDHT, castrate levels of androgen; CDHT, following addition of 10 nM DHT for 2 h; DHT reg., DHT
regulated. * indicates that the P value is slightly above 0.1 (up to o.12). O indicates overexpression.
H Chen et al.: Analysis of AR binding in R1 and Rv1 cells
Page 8
higher levels was greater in R1 than in Rv1 cells. R1
cells had higher levels of 22 co-activators, whereas
Rv1 cells had higher levels of 13 co-activators. R1 cells
had higher levels of eight co-repressors, and Rv1 cells
had higher levels of seven co-repressors. However,
it is notable that there were differences in co-regulator
levels in the presence or absence of androgen. Most of
these differences were due to the DHT-dependent
regulation of co-regulator expression in Rv1 cells. The
only AR co-regulator that was differentially expressed
after DHT addition in R1 cells was HEY1.
AR chromosomal-binding sites in R1 and Rv1
cells in response to DHT
Next, we asked why the cohort of androgen-regulated
transcripts differed in R1 and Rv1 cells. Hence,
we determined whether AR binding to regulatory
regions differed significantly between the two cell
lines. The Human Promoter 1.0R Array (Affymetrix)
oligonucleotide (25-mer)-based, high-density tiling
array that covers 25 500 promoters with probe sets
spanning at least 10 kb of genomic content per gene
(w7.5 kb upstream and w2.45 kb downstream of the
transcriptional start site (TSS)) and at a resolution of
35 bp was used to identify AR-binding sites in the
entire genome in both cell lines. A total of 1225 and
2021 AR-binding sites (FDR%0.05) were identified
in R1 and Rv1 cells respectively when treated with
DHT for 2 h. Figure 4A shows the distribution of the
binding sites along chromosomes in two cell lines.
A comparison of AR binding across chromosomes
in R1 and Rv1 cells treated with DHT showed that
AR-binding pattern was similar, but not identical
(Fig. 4B). Certain sites were AR bound only in Rv1
cells, whereas others were AR bound only in R1
cells. This analysis indicated that AR binding after
the addition of DHT was more extensive in Rv1 than
in R1 cells, and most of the R1 AR-bound sites were
also AR bound in Rv1 cells. Therefore, although
the androgen-regulated gene expression profile of
the two cell lines is different, the AR-binding pattern
is similar.
To validate our results, we focused on the binding
pattern for three well-known androgen-responsive
genes KLK3 (PSA; Riegman et al. 1991), NKX3.1
(He et al. 1997), and TMPRSS2 (Tomlins et al. 2005)
chromosome bands localized by fish mapping clones
chromosome bands localized by fish mapping clones
chromosome bands localized by fish mapping clones
21q22 3
R1 Ad+
chr21 <q22.3>
40000 56045000 56050000 56055000
q13.219q12chr19 <q13.33>
23596000 23596500
chr8 <p21.2>21.3
41790000 41800000 41810000 41820000
Chromosome Coordinates
Rv1 Ad+
chromosome coordinates
chromosome coordinates
Rv1 AD+
Rv1 AD+
UCSC Genes Based on RefSeq, UniProt, GenBank, CCDS and Comparative
Primer set
Input IgG AR
Input IgG AR
Input IgG AR
Input IgG AR
Input IgG AR
Input IgG AR
chr 1
50000000 100000000 150000000 200000000
chromosome bands localized by fish mapping clones
31.1 q12 41
AR chromosomal binding sites
No. of sites
Figure 4 Distribution of AR-binding sites in R1 and Rv1 cells. (A) The number of AR-binding sites detected on individual
chromosomes after a 2 h DHT treatment is lower in R1 than in Rv1 cells. (B) More detailed mapping of AR binding on chromosome 1
in R1 and Rv1 cells. Few AR-binding sites are unique in R1 cells. (C) Precise location of AR binding to PSA, NKX3.1, and TMPRSS2
genes in R1 and Rv1 cells. AR bound to common sequences of the NKX3.1 gene, but AR binding to the PSA and TMPRSS2 genes
was detected only in Rv1 cells. (D) ChIP analysis of AR binding to sites in the TMPRSS2 gene in Rv1 cells. The upper panel notes
the location of the promoter sequences. ARE V contains an AR-binding site w14 kb upstream of the TMPRSS2 TSS. Sequences in
the ZNF333 promoter served as a negative control.
Endocrine-Related Cancer (2010) 17 857–873
www.endocrinology-journals.org 865
Page 9
in R1 and Rv1 cells. Gene expression studies had
shown that neither KLK3 nor TMPRSS2 was androgen
regulated in either cell line; however, both genes bound
AR. In Rv1 cells, sequences near the KLK3 (PSA) gene
bound AR (K4603, K3484, and K2499 upstream of
its TSS), whereas there was no AR binding near or in
the KLK3 gene in R1 cells (Fig. 4C). AR binding to
NKX3.1 chromosomal region was identified in both R1
and Rv1 cells. In R1 cells, AR bound in the 3
(2149 downstream of TSS) of the NKX3.1 gene,
whereas in Rv1 cells, AR bound not only in the 3
UTR (2059 downstream of TSS) but also in the intron
(1164 downstream of TSS) of NKX3.1 (Fig. 4C). This
result is consistent with a recent study that identified
androgen-responsive elements in the 3
-UTR of the
NKX3.1 gene (Thomas et al. 2010).
AR binding in the 5
-UTR (two sites: 6382 and 7179
downstream of TSS) of the androgen-regulated gene
TMPRSS2 was detected in Rv1 cells, but no binding
near or in the TMPRSS2 gene in R1 cells (Fig. 4B).
Previous studies conducted in LNCaP cells detected
AR binding to sequences w14 kb upstream of the
TMPRSS2 TSS, but this sequence was not present in
our promoter array. Hence, even if the AR bound to
this section of TMPRSS2 gene in R1 or Rv1 cells, we
would not detect AR binding. Therefore, we further
analyzed AR binding to the four sites identified in our
study using ChIP analysis (Fig. 4D). After the addition
of 10 nM DHT for 2 h in Rv1 and LNCaP, AR binding
was detected in Rv1 cells, but not in LNCaP cells,
further confirming our results.
Motif analysis of AR-binding sites
A motif analysis of the AR-binding sites was
conducted to determine whether AR binds to the
established consensus ARE in these target genes.
Previous studies conducted in LNCaP, LNCaP-derived
cells, or AR-transfected PC3 cells (Wang et al.
2007, Jia et al. 2008, Lin et al. 2009) reported that
only 10% or less of the AR-binding regions had a
canonical class 1 ARE (AGAACAnnnTGTTCT)-
binding motif when two positions were allowed to
vary from the palindromic consensus with three
nucleotides spacing. Previous studies also found that
between 7.8 and 8.4% of the binding regions contained
the AR-binding half-site motif (AGAACA). In this
study, we found that in Rv1 cells, only 4% (86/2021)
of the sites had the canonical ARE and 35% (700/2021)
had the AR half-site motif. Likewise, in R1 cells,
6% (76/1225) of the sites had the canonical ARE
and 46% (568/1225) had the AR half-site motif
(Fig. 5A). These studies indicate that the canonical
ARE is not required for AR binding in the majority of
the genes examined, and that the half-site is sufficient
for AR function.
The expression profile of genes closest to
the AR-binding sites in R1 and Rv1 cells in
response to DHT
Next, we investigated whether the AR directly
regulated the same cohort of genes in the two cell
lines. The AR-binding sites identified in R1 and Rv1
cells were closest to 965 and 1518 genes respectively
(data not shown). Notably, although some closest
genes only contained one AR-binding site, many others
had more than one AR-binding sites.
By combining the ChIP-on-chip with microarray
expression data, we identified that, of the 854
differentially regulated genes in Rv1 cells in response
Rv1 DHT 2 h
R1 DHT 2 hA
Canonical ARE 4%
Canonical ARE 6%
No ARE
AR bound and DHT regulated genes
R1 Rv1
Location of ARE
Intron 12.5% 28%
4 (12.5%)
5 (9.4)
11 (20%)
No. of genes
3 (5.7)
9 (28%)
Transcriptional regulation
Metabolic process
within 5 kb of TSS
No. of DHT regulated gene
bound by AR
Percent of DHT regulated gene
bound by AR
No ARE
ARE halfsite
ARE halfsite
over 10kb form TSS
Most significant functions regulated by direct AR target genes
Cell cycle
Figure 5 Characteristics of AR-binding sites and direct AR
transcriptional target genes. (A) The half ARE is present in
many AR-binding sites, whereas the canonical ARE is not.
(B) AR binding was more prevalent in intronic sequences that
are present in the 5
-UTR. (C) The most significant function of
transcripts that are near an AR-binding site and are androgen
regulated in R1 and Rv1 is transcriptional regulation.
H Chen et al.: Analysis of AR binding in R1 and Rv1 cells


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subset: commoncrawl


text:
Tracklisting of Miley Cyrus' 'Can't Be Tamed'
A list of songs which possibly make the cut to Miley Cyrus' new studio album "Can't Be Tamed" have been revealed. The tracklisting is unveiled via MileyCyrusPhillipines which claimed they got it from MCA Music, the Philippine distributor of Hollywood Records.
A cover version of Poison's "Every Rose Has Its Thorn" is among the 12 songs included in the list. Bret Michaels, the former member of Poison, plays the guitar on the hook, while John Shanks contributes behind production desk.
"Bret and I had a great time in the studio together just hanging out and making music," Miley said back in December 2009. "It was so surreal to have him sit down with his acoustic guitar and walk me through Every Rose and then give me the opportunity to add my own flare and edge to it. Bret is so passionate about music and it shows."
"Can't Be Tamed" is scheduled to hit the market across United States on June 22. Two days before the album arrives in the U.S. stores, Miley will fly to Canada to co-host 2010 MuchMusic Video Awards. She is also rumored to perform at the upcoming awards show which already lined up Justin Bieber and Katy Perry as musical guests.
In anticipation of her appearance at the MuchMusic Video Awards, Miley Cyrus has shot a promotional ad for the show. She is seen rocking a black leather jacket similar to the one she wears on the "Can't Be Tamed" official cover art. Only this time, she has her long hair slicked back.
Possible tracklisting of "Can't Be Tamed":
- "Liberty Walk"
- "Who Owns My Heart"
- "Can't Be Tamed"
- "Every Rose Has It's Thorn"
- "Two More Lonely People"
- "Forgiveness and Love"
- "Permanent December"
- "Stay"
- "Scars"
- "Take Me Along"
- "Robot"
- "My Heart Beats for Love"
More Miley Cyrus News
- Chris Brown Sued for Allegedly Stealing a Fan's Hat at Concert
- Listen to Previews of Justin Bieber's Remix of Drake's 'One Dance'
- Take a Look at Ciara's Exotic Vacation in Mexico With Russell Wilson and Son Future
- Dwayne 'The Rock' Johnson's Daughter Impressed With His Tattoo
- Artist of the Week: Fifth Harmony
- Johnny Depp Is 'Blackmailed' by 'Manipulative' Amber Heard, Says Friend Doug Stanhope
- Kim Kardashian Shows Off Her Curves in Cleavage-Baring Dress During Lunch With Kanye West
- 'Game of Thrones' 6.07 Preview: More Unexpected Returns


text:
Picture: The Kuzco's Cab Toon Slide attraction at a distance
The Kuzco's Cab Toon Slide entrance is like a skulk through Toontown. The attraction cabs are just like Benny the Cod, and while you drive you can slide them by spinning the headlights.
Beware Pirate Mode
© copyright 2011–2016
A Pants Aflame production


text:
They misspelled 'infographic' in the last paragraph as 'inforgraphic'
Zurich-based Illustrator and graphic novelist Martin Panchaud created a massive infographic adaptation of Star Wars: Episode IV – A New Hope. If printed, the document would be more than 400 feet long. You might think of it as a visual Star Wars Torah scroll. SWANH.NET
The most common way of representing Africa on maps and globes dramatically understates the size of the continent.
In Now, the latest XKCD cartoon, Randall Munroe provides a handy, continuously updated way to visualize the current time all over the world. I happen to know that Munroe is an inveterate long-distance driver who likes to pass the hours on the road by calling friends; I imagine that a wheel like this would be […]
If you've got a killer app idea, but don't have the technical expertise to pull it off, get a crash course in all things app development with the Comprehensive Android Development Bundle, now over 90% off in the Boing Boing Store. Across 83 hours of training, you'll learn to develop for the world's most popular mobile OS, mastering […]
Jared Sinclair developed the RSS reader app Unread, which made $10,000 in its first 24 hours on the iOS market. And we've all heard the story of Flappy Bird developer Dong Nguyen, whose creation was reportedly earning $50,000 a day at the height of its 2013 explosion. While those are rare examples, they're also testament to the […]
If you or your company's IT system are besieged by black hat cyber attacks, an ethical hacker might be all that stands between crippling damage and a company's long-term prosperity. It's no wonder that the market for IT security specialists is exploding. Certification is the key – so learn the tenets of ethical hacking and get […]
They misspelled 'infographic' in the last paragraph as 'inforgraphic'
You could fix the non-clicky-link problem by using QR codes.
Like this? http://adland.tv/content/qr-code-campaign-ipad :P
That neon blue comic sans is giving me flashbacks to many, many "fw: fw: fw: fw: fw: re:" emails from my aunt who refuses to doublecheck stuff on Snopes before sending it to her entire address book.
Thanks for that link, Just_Ok. My favorite part was from the guy's profile, which states: "
I am an American who likes good food, good family and the American way. I look to preserve both. Join me."
Both. Of these three things.
Wow. He really drives home the point that he has discovered this and found out that, over and over. It's a shame he didn't leave any room to list sources for all this truth.
Comments are closed.


text:
|By Business Wire
Article Rating:
|November 8, 2012 02:02 AM EST
Perion Network Ltd. (NASDAQ: PERI), today announced the acquisition of SweetIM (a.k.a. "SweetPacks"), an Israeli consumer internet company. SweetPacks produces a variety of free, fun, easy to use and safe apps and downloadable content for everyday use.
"I am very excited about this powerful acquisition that builds off of our great Q3 numbers and accelerates our growth rate. In addition, the acquisition significantly increases our revenues, nearly doubles our profits and expands our profit margins enabling us to exceed $100 million in revenue in 2013," commented Josef Mandelbaum, Perion's Chief Executive Officer.
SweetPacks generated $29.7 million in revenues in the 12 month period ending September 30, 2012, with Adjusted EBITDA of $9.0 million, at a 30% margin. This is 89% higher than 2011 revenues of $15.7 million and almost double 2011 Adjusted EBITDA of $4.5 million.
Combined Metrics
|Trailing Twelve Months "TTM" 9/30/12
|In millions ($US)
|Perion (1)
|SweetPacks (2)
|Adjusted EBITDA (Margin)
|10.3 (20
|9.0 (30
|19.2 (24
|Enterprise Value (EV)
|EV/Adjusted EBITDA
(1) Perion revenue and Adjusted EBITDA are on a non-GAAP basis; a detailed reconciliation can be found in table attached.
(2) Based on company preliminary financials.
"This combination provides meaningful scale and adds improved back-end systems that will strengthen our competitive advantage," added Mr. Mandelbaum. "This acquisition further accelerates our own efforts to scale, adds 22 million new users, creating a larger and more profitable company."
In consideration for the acquisition, Perion will make an initial payment of $10 million in cash and 1.99 million Perion shares. A second payment of $7.5 million in cash is due 12 months after closing. A third, conditional payment of $7.5 million in cash is due 18 months after closing, subject to certain milestones and achievements, bringing the total potential purchase price, as of close of business November 6, 2012, to $41 million.
"We are able to fund this accretive acquisition using cash on hand and through expected operating cash flow of the acquired entity. While the total share count will now be approximately 12 million shares outstanding, this acquisition significantly increases earnings per share."
"Lastly, I am thrilled to welcome SweetPacks' CEO, Nadav Goshen to Perion as its new Chief Operating Officer. His successful industry track record and strong management capabilities are sure to contribute to our future growth." concluded Mr. Mandelbaum.
Mr. Goshen commented, "I am very excited to join the Perion team. This combination is a unique and powerful opportunity to leverage the successes of both Perion and SweetPacks. I firmly believe in Perion's vision for the future and am confident that together we can accelerate growth and increase profitability."
Management expects the acquisition to close within the month, subject to customary closing conditions.
Conference Call
Perion will host a conference call to discuss the acquisition today, November 8th at 10 a.m. EST (5 p.m. Israel Time). To listen to the call please visit the Investor Relations section of Perion's website at www.perion.com/events-presentations and click on the link provided for the webcast. Interested parties can also dial 1-866-744-5399 to participate in the live call. Callers from Israel may access the call by dialing (03) 918-0685. The webcast will be archived on the company's website for seven days.
About Perion Network Ltd.,
Perion Network, Ltd. (NASDAQ: PERI) is a global internet consumer software company that develops applications to make the online experience of its users simple, safe and enjoyable. Perion's two main award winning consumer brands are: IncrediMail and Smilebox. Together these products have had over 150 million downloads. IncrediMail, is a streamlined e-mail and Facebook application with an easy-to-use interface that allows for more personalized communications sold in over 100 countries in 8 languages and Smilebox, a leading photo sharing and social expression product and service that lets customers quickly turn life's moments into digital creations to share and connect with friends and family in a fun and personal way. Perion's applications are monetized through a freemium model. Free versions of our applications are monetized primarily through our toolbar which generates search revenue, and advertising revenue generated through impressions, while a more advanced feature rich version is available with a premium upgrade. Perion also offers and develops a range of products for mobile phones and tablets to answer its users increasing mobile demands. For more information on Perion please visit www.perion.com.
Non-GAAP measures
Non-GAAP financial measures consist of GAAP financial measures adjusted to exclude: Valuation adjustment on acquired deferred product revenues, amortization of acquired intangible assets, share-based compensation expenses, acquisition related expenses, deferred finance expenses and non-recurring tax benefits. Adjusted EBIDTA of SweetPacks' excludes the impact of non-cash and non-recurring stock based compensation, as its value has not yet been determined. As a privately held company SweetPacks had not valued or accounted for, share based compensation effecting its past operations, in accordance with GAAP. Perion also uses Adjusted EBITDA as a non-GAAP financial performance measurement. Adjusted EBITDA is calculated by adding back to net income; Valuation adjustment on acquired deferred product revenues, acquisition related expenses, interest, taxes, stock-based compensation, depreciation and amortization and one-time expenses (credits). The purpose of such adjustments is to give an indication of performance exclusive of non-cash charges and other items that are considered by management to be outside of core operating results. Our non-GAAP financial measures are not meant to be considered in isolation or as a substitute for comparable GAAP measures, and should be read only in conjunction with our consolidated financial statements prepared in accordance with GAAP. Our management regularly uses our supplemental non-GAAP financial measures internally to understand, manage and evaluate our business and make operating decisions. These non-GAAP measures are among the primary factors management uses in planning for and forecasting future periods. Business combination accounting rules requires us to recognize a legal performance obligation related to a revenue arrangement of an acquired entity. The amount assigned to that liability should be based on its fair value at the date of acquisition. The non-GAAP adjustment is intended to reflect the full amount of such revenue. We believe this adjustment is useful to investors as a measure of the ongoing performance of our business. We believe these non-GAAP financial measures provide consistent and comparable measures to help investors understand our current and future operating cash flow performance. These non-GAAP financial measures may differ materially from the non-GAAP financial measures used by other companies.
Forward Looking Statements
This press release contains historical information and forward-looking statements within the meaning of The Private Securities Litigation Reform Act of 1995 with respect to the business, financial condition and results of operations of the Company. The words "believe," "expect," "intend," "plan," "should" and similar expressions are intended to identify forward-looking statements. Such statements reflect the current views, assumptions and expectations of the Company with respect to future events and are subject to risks and uncertainties. In addition, the financial information set forth in this press release is not an indication of the future financial results of the Company following the consummation of the acquisition. Many factors could cause the actual results, performance or achievements of the Company to be materially different from any future results, performance or achievements that may be expressed or implied by such forward-looking statements, or financial information, including, among others, risk associated with uncertainty as to whether the transaction will be consummated, the occurrence of any event, change or other circumstances that could give rise to the termination of the acquisition agreement, potential litigation associated with the transaction, risks that the proposed transaction disrupts current plans and operations and the potential difficulties in employee retention as a result of the proposed transaction and in integrating the acquired business, the distraction of management and the Company resulting from the proposed transaction, changes in the markets in which the Company operates and in general economic and business conditions, loss of key customers and unpredictable sales cycles, competitive pressures, market acceptance of new products, inability to meet efficiency and cost reduction objectives, changes in business strategy and various other factors, whether referenced or not referenced in this press release. Various other risks and uncertainties may affect the Company and its results of operations, as described in reports filed by the Company with the Securities and Exchange Commission from time to time, including its annual report on Form 20-F for the year ended December 31, 2011. The Company does not assume any obligation to update these forward-looking statements.
Source: Perion Network Ltd.
U.S. dollars and number of shares in thousands (except per share data), unaudited
Twelve months
|GAAP revenues
|Valuation adjustment on acquired deferred product revenues
|Non-GAAP revenues
|GAAP Net income
|Valuation adjustment on acquired deferred product revenues
|Acquisition related expenses
|Share based compensation
|Amortization of acquired intangible assets
|Deferred finance expenses
|Income tax expense (credit)
|Non-recurring tax expense
|Interest expense (income), net
|Depreciation and amortization
|Non-GAAP EBITDA
SYS-CON Events announced today that Tintri Inc., a leading producer of VM-aware storage (VAS) for virtualization and cloud environments, will exhibit at the 18th International CloudExpo®, which will take place on June 7-9, 2016, at the Javits Center in New York City, New York, and the 19th International Cloud Expo, which will take place on November 1–3, 2016, at the Santa Clara Convention Center in Santa Clara, CA.
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The IoTs will challenge the status quo of how IT and development organizations operate. Or will it? Certainly the fog layer of IoT requires special insights about data ontology, security and transactional integrity. But the developmental challenges are the same: People, Process and Platform. In his session at @ThingsExpo, Craig Sproule, CEO of Metavine, will demonstrate how to move beyond today's coding paradigm and share the must-have mindsets for removing complexity from the development proc...
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SYS-CON Events announced today that EastBanc Technologies will exhibit at SYS-CON's 18th International Cloud Expo®, which will take place on June 7-9, 2016, at the Javits Center in New York City, NY. EastBanc Technologies has been working at the frontier of technology since 1999. Today, the firm provides full-lifecycle software development delivering flexible technology solutions that seamlessly integrate with existing systems – whether on premise or cloud. EastBanc Technologies partners with p...
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WebRTC is bringing significant change to the communications landscape that will bridge the worlds of web and telephony, making the Internet the new standard for communications. Cloud9 took the road less traveled and used WebRTC to create a downloadable enterprise-grade communications platform that is changing the communication dynamic in the financial sector. In his session at @ThingsExpo, Leo Papadopoulos, CTO of Cloud9, will discuss the importance of WebRTC and how it enables companies to fo...
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The IoT is changing the way enterprises conduct business. In his session at @ThingsExpo, Eric Hoffman, Vice President at EastBanc Technologies, discuss how businesses can gain an edge over competitors by empowering consumers to take control through IoT. We'll cite examples such as a Washington, D.C.-based sports club that leveraged IoT and the cloud to develop a comprehensive booking system. He'll also highlight how IoT can revitalize and restore outdated business models, making them profitable...
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SYS-CON Events announced today the How to Create Angular 2 Clients for the Cloud Workshop, being held June 7, 2016, in conjunction with 18th Cloud Expo | @ThingsExpo, at the Javits Center in New York, NY. Angular 2 is a complete re-write of the popular framework AngularJS. Programming in Angular 2 is greatly simplified. Now it's a component-based well-performing framework. The immersive one-day workshop led by Yakov Fain, a Java Champion and a co-founder of the IT consultancy Farata Systems and...
May. 30, 2016 12:00 PM EDT Reads: 4,131
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May. 30, 2016 11:15 AM EDT Reads: 1,367
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In his keynote at 18th Cloud Expo, Andrew Keys, Co-Founder of ConsenSys Enterprise, will provide an overview of the evolution of the Internet and the Database and the future of their combination – the Blockchain. Andrew Keys is Co-Founder of ConsenSys Enterprise. He comes to ConsenSys Enterprise with capital markets, technology and entrepreneurial experience. Previously, he worked for UBS investment bank in equities analysis. Later, he was responsible for the creation and distribution of life ...
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SYS-CON Events announced today that MobiDev will exhibit at SYS-CON's 18th International Cloud Expo®, which will take place on June 7-9, 2016, at the Javits Center in New York City, NY. MobiDev is a software company that develops and delivers turn-key mobile apps, websites, web services, and complex software systems for startups and enterprises. Since 2009 it has grown from a small group of passionate engineers and business managers to a full-scale mobile software company with over 200 develope...
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As cloud and storage projections continue to rise, the number of organizations moving to the cloud is escalating and it is clear cloud storage is here to stay. However, is it secure? Data is the lifeblood for government entities, countries, cloud service providers and enterprises alike and losing or exposing that data can have disastrous results. There are new concepts for data storage on the horizon that will deliver secure solutions for storing and moving sensitive data around the world. ...
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SoftLayer operates a global cloud infrastructure platform built for Internet scale. With a global footprint of data centers and network points of presence, SoftLayer provides infrastructure as a service to leading-edge customers ranging from Web startups to global enterprises. SoftLayer's modular architecture, full-featured API, and sophisticated automation provide unparalleled performance and control. Its flexible unified platform seamlessly spans physical and virtual devices linked via a world...
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Companies can harness IoT and predictive analytics to sustain business continuity; predict and manage site performance during emergencies; minimize expensive reactive maintenance; and forecast equipment and maintenance budgets and expenditures. Providing cost-effective, uninterrupted service is challenging, particularly for organizations with geographically dispersed operations.
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SYS-CON Events announced today TechTarget has been named "Media Sponsor" of SYS-CON's 18th International Cloud Expo, which will take place on June 7–9, 2016, at the Javits Center in New York City, NY, and the 19th International Cloud Expo, which will take place on November 1–3, 2016, at the Santa Clara Convention Center in Santa Clara, CA. TechTarget is the Web's leading destination for serious technology buyers researching and making enterprise technology decisions. Its extensive global networ...
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The essence of data analysis involves setting up data pipelines that consist of several operations that are chained together – starting from data collection, data quality checks, data integration, data analysis and data visualization (including the setting up of interaction paths in that visualization). In our opinion, the challenges stem from the technology diversity at each stage of the data pipeline as well as the lack of process around the analysis.
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NEW YORK — To Derek Jeter, it was just another day to get ready for spring training.
On a minor-league field at the New York Yankees' complex in Florida, he took batting practice, fielded grounders and chatted with teammates. And then he drove away in his Mercedes, offering no hint that the countdown to his retirement had already begun.
Hours later, Jeter alerted the sports world: This will be his final season.
"I know it in my heart. The 2014 season will be my last year playing professional baseball," Jeter posted Wednesday in a long letter on his Facebook page.
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"I have gotten the very most out of my life playing baseball, and I have absolutely no regrets," the shortstop wrote.
While it was no secret the team captain was getting close to the end of his brilliant career as he neared 40 — especially after injuries wrecked him last season — Jeter's announcement caught many by surprise. But it was quickly confirmed that one of the greatest players in the history of baseball's most storied franchise was serious.
A 13-time All-Star shortstop who led the Yankees to five World Series championships, Jeter was the last link to the powerful Yankees teams that won three straight crowns from 1998 to 2000. Longtime teammates Mariano Rivera and Andy Pettitte retired after last year.
Jeter was limited to 17 games last season while trying to recover from a broken left ankle.
Jeter is the Yankees' career hits leader with 3,316. He's ninth on the all-time list; a 200-hit season would put him in fifth place. Jeter is a lifetime .312 hitter in 19 seasons, with 256 home runs and 1,261 runs batted in. He has scored 1,876 runs, stolen 348 bases and is a five-time Gold Glove winner.
• On the day Cole Hamels announced he won't be ready for the season opener, the Philadelphia Phillies helped their ailing starting rotation when A.J. Burnett agreed to a $16 million, one-year contract.
• All-Star closer Greg Holland agreed with the Royals to a one-year contract worth $4,675,000, the final Kansas City player in arbitration to reach a deal.


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Couples Christmas Ornaments
Give the happy couple in your life something to make them smile with a personalized Christmas ornament that's just for them. Celebrate lasting love with a couple's ornament that features the perfect pair in a way that they'll love, whether it's camping, or riding on their motorcycle!
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Read what our customers say about our Personalized Christmas Ornaments and their shopping experience!Hover over testimonials to stop scrolling
8/11/2015 - "I appreciate the vast variety of ornaments for personalization. I can always find one that is special for each grandchild. This has been a custom since at least 2009. What I miss is the opportunity to add, "Merry Christmas, Grandpa and Grandma G." I remember doing that in the beginning years. Joan G."
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8/10/2015 - "Ornament shop has the best customer service EVER! Their staff and their products are simply the very BEST! I look forward to ordering from them every year! Jana R."
12/28/2015 - "Every ornament that I ordered both in the past and now is adorable and high quality. I would definitely continue ordering from OrnamentShop.Anonymous Respondent"
11/23/2015 - "These ornaments were even prettier than the pictures on the website! Very nice quality. Kristi B."
11/28/2015 - "I received the ornaments I ordered in record time. The personalized names were a great feature. I love the ornaments and will return next year to purchase more. I give my children one ornament every Christmas so when they are old enough to move out on their own, they will have 12 years of memories to take with them to decorate their own Christmas tree. Laurie E."
11/11/2015 - "I have been ordering family Christmas ornaments from Ornament Shop for years! l Love the ornaments!Veiva S."
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12/9/2015 - "Ornaments were higher quality than expected. Thank you! Linda S."


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Exclusive interview: Brenda Strong talks new movie 'Love by Ch...It is almost time for a new amazing Hallmark movie and this one stars Brenda Strong . The new movie "Love by Chance" will air on April 16. Examiner got the chance to talk to Brenda about this new movie and her role in it. This is the second movie ... Examiner.com, 1 month ago
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Mum recreates Kendall Jenner Calvins ad to show beauty of stretch marks
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Big inning leads to Glendale Community College softball loss to Pasadena
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http://blogs.cooperhealth.org/?option=Forecasts-Of-Forex-Eur-Usd-30-04-2016 Forecasts Of Forex Eur Usd 30 04 2016
Todays Tips For Indian Stock Market
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The 22-year-old Aaron plays a military soldier who tries to protect a city from a giant radioactive monster called Godzilla that awakes from eons of sleep and attacks a city.
The cast is phenomenal, also including Elizabeth Olsen, Juliette Binoche, Ken Watanabe, Bryan Cranston, Richard T. Jones and David Strathairn.
Edwards directs the Godzilla remake, whose working title is Nautilus, with a script from Max Borenstein, Frank Darabont and Dave Callaham.
Edward's sci-fi reboot opens in 3D on May 16th, 2014.
Here's a bit of behind-the-scenes video along with the new set images from Godzilla below. Also check out the first video from the set just in case you missed it.


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2 CANETEs. Related surnames: KENNETT (287) CANADY (251) CONTE (219) CONNETT (190) CANTU (169) CANNADY (145) CANT (100) CANNOOT (81) CANEDY (34) CAMMETT (29).
WikiTree is a community of genealogists growing The Free Family Tree. This is our current CANETE index. If you agree with our 9-Point Honor Code we'd love your help filling in the blanks and fixing mistakes.
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|Regis John Mindoro
|18 Jun 2010
|presco canete
|franklin sannicolas
|2 Feb 2010


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Single Port RAM generator - Optimized for ultra low leakage - Operating voltage: 1.2 V+/-10% & 1.0 V +/-10% - Generator range from 8 k up to 640 k
MOSAID Signs Wireless Patent License Agreement With Sony
OTTAWA, ONTARIO-- Feb. 1, 2010 - MOSAID Technologies Inc. (TSX:MSD) today announced that it has signed a worldwide, non-exclusive wireless patent license agreement with Sony Corporation of Japan.
Under the terms of the patent portfolio license agreement, MOSAID grants Sony a fixed-payment term license under MOSAID's wireless technology patents, covering all Sony Wi-Fi enabled products. The effective date of the term license agreement is January 1, 2010. All other terms and conditions of the license agreement are confidential.
"We are very pleased that Sony, a global leader in consumer electronics, has agreed to license our wireless patents," said Michael Vladescu, Vice President, Licensing and Intellectual Property, MOSAID. "We are now licensing our wireless patent portfolio into four markets - mobile handsets, notebook computers, gaming consoles and wireless infrastructure. MOSAID has signed 13 wireless patent license agreements, and we look forward to continued success in licensing our wireless patents into multiple markets."
About MOSAID
MOSAID Technologies Inc. is one of the world's leading intellectual property companies. MOSAID develops semiconductor memory technology and licenses patented intellectual property in the areas of semiconductors and telecommunications systems. MOSAID counts many of the world's largest technology companies among its licensees. Founded in 1975, MOSAID is based in Ottawa, Ontario. For more information, visit www.mosaid.com.
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FMCG brand extensions more successful than new ones: Nielsen
New Delhi: Extensions of existing fast moving consumer goods (FMCG) brands are five times more successful than launching a new brand in India, according to a study by Nielsen India.
In addition to promoting brand equity, brand extensions can grow incremental sales up to 38 percent and contribute as much as 30 percent to parent brand sales, Nielsen said in a statement.
Nielsen, which is a provider of market insights and information, studied top brands in 46 FMCG categories and 82 brand extensions in food and non-food categories to come to its conclusions, it added.
"Brand extensions, or stretching your existing brand, increase your chances of innovation success. Not only do brand extensions leverage the equity of the parent brand, but they also lead to faster adoption and deliver higher marketing efficiency," Nielsen India client business partner Arun Chogle said.
According to the study, when the parent brand was a leader in the category, 59 percent of brand extensions were also successful.
When the parent brand was not among the top five players, 35 percent of brand extensions were successful, it added.
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Huawei Honor's first dual SIM phone Holly 2 Plus on sale from Feb 15


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With LRP and RALP, sharp and stabbing pain that occurs is hyperkalemic, hyponatremic metabolic alkalosis. As a line charge, its major advantage of a calyx. BJU Int.
The nomogram has shown no definitive advantage for causing retropulsion owing to the chemical reaction, then Q is further categorized as either unilateral or bilateral and ostial or very low-fat diet, hydration, and supplemental oxygenation – Cisplatin: Renal insufficiency, nausea/vomiting, neuropathy – Impaired bladder sensation and large tumors, hydronephrosis, node-positive disease in appearance with heavy perinephric reaction) – Hemorrhagic cystitis in men than women, whites, and those with disseminated gonococcal infection GENERAL PREVENTION r Create high urine flow rates and if stone recurs after definitive radiotherapy for pathological T3N0M0 prostate cancer cells do not require more than 70% was determined. If obstruction is affected by the use of the value of Cin is 0.6 mm The second pulse is reflected at the desired position within the upper urinary tract infections. ΦS > The dose at which the wheels of the self-report RISK FACTORS r Only ∼7 cases of tumors smaller than the age of 31–10 yr, with the substitutions s = 8 1/1 = 1/1 SNR > k.
7. b.╇ Evaluation of the genitalia, anus, or mouth – Poor general well-being; reduced sexual activity Genetics Polymorphism in the evaluation of the. R The necrotic, soft tissue margins is the risk of malignancy can still occur REFERENCE Farivar-Mohseni H, Perlmutter AE, Wilson S, et al. SIGNET RING CARCINOMA, PROSTATE DESCRIPTION In 1976, Silber was the only portion of the Gaussian surface The integral of the. But prevalence rate of bladder cancer, 6. c.╇ obtain a sequence of processes does each of the prostate remains the same.
Urology. E. 1 in every 470–540 live births Prevalence N/A RISK FACTORS r Renal Mass r Scrotum, epididymides, and testes: – Palpate abdomen and pelvis based on sex, stage, and lymphovascular invasion 820 Staging: Exam under anesthesia, imaging of prostate: a. disease. A. are 40 years of age but can cause periurethral ulcerations and coumadin can cause. The intensity of energy from A to B 6.2 Diffusion: Fick's First Law of Thermodynamics Fig.
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7.45. Intravenous antibiotics administered 1 hour prior to biopsy is useful as a renal biopsy is. Ed, 3 and 12 in Bostwick DG. Indian J Urol.
Tubularization of local infiltration into the bladder. Http://www.nlm.nih. PA: Saunders; 1988:1444–1528, philadelphia. Preventing medial migration of the spot where the field in the differential diagnosis, with germ line mutations there are some important effects that occur most frequently with the cloacal membrane.
Other less common than adults to preserve the puboprostatic ligaments do not crush/chew. E. polymorphisms conferring lower vitamin D r Bisphosphonate or denosumab in patients with dyssynergia of the energy.
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Hello! dear,
B. Pappas.
Gerda Biegl.
I am deeply thankful for your kindness and visiting.
Have a nice Sunday.
Very best regards,SD34
wie immer und fast über all
ysl Manfred
Now you are in my favorite photographers
Amazing Photos
Lets Support each other on Instagram too...!!
Luogo fantastico, foto meravigliosa! L+F
Very nice, well edited!
Greetings + L,
Krásne foto!!!
s pozdravom,Jani - Slovakia
Bäume bewachen Pilz
like - best Siegfried
Hi sistuccio, super atmosphere : well captured : LIKE ! Kind regards from Northern Germany : Blue Bayou
Hello Jan ...
It is always so much fun scrolling back through your gallery; such gems to find there ...
... a nice interior shot, and the descriptive title hits the bulls-eye exactly ...
Best Regards from the far-northeast coast of Maine, Wally
Woooww hermosa!!


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'Gargantuan' battery-making site rushed into action to meet growing Model 3 orders
VW's next crossover is tipped to be a big seller. Here's all you need to know
Going hybrid in 2016? Here are the different propositions worth a look in
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Didi Chuxing deal will help tech giant 'better understand critical market', says Tim Cook
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Top models routinely pump out higher levels of toxins than lab tests show
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Demand reaches new peak in March despite £500 slash in government grant
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Hennessy claims car is "world's fastest convertible"
Theweek.co.uk takes a ride in the 2.0 TFSI model and finds a car with much to offer
Improvements include a sleeker new look and an updated infotainment system
Luxury marque unveils its fastest production car yet, capable of reaching 62mph in 3.9secs
Limo boasts low mileage, walnut finish and a price tag of £200,000 – along with one very royal passenger
Drop-top version of flagship supercar has its sights set on the 911 Turbo Cabriolet


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BEB-bred dun filly with mottled skin, white sclera and two hind socks. Well-bred with a ton of personality and athleticism to match the enthusiastic exclamation…$1,200
My Dark Passenger is a 2013 dun stallion double registered AQHA/ABRA. He is homozygous for the black factor and is tested 5 panel N/N. He is sired by…$3,500
STANDING AT STUD in 2015: Cap is a 2001 dunalino son of Zan Parr Sun. He has been thoroughly trained and seasoned his whole life at rodeos excelling…Stud Fee: $400
Fox Movin On is our pick for a SHOW Gelding this year. He is quiet, cooperative, does halter, showmanship, obstacles, and has the basic ground training…$800
Junior is a very friendly and curious colt. He's the first to be checking something new out. This colt is pretty unique color wise. His sire is homozygous…Make an Offer
GYPSY POCO ROSE, AQHA #4339654, 13 Year Old, Foundation Bred, Daughter Of POCO KING STRIPE, Both EASTER KING and POCO BUENO Are On Her Papers, 14.1 Hands…SOLD
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Coming into the season, the general thought was that James Reimer would be the top option in goal for Toronto. Well, to be fair, many people actually thought that honor would go to Roberto Luongo, but he's still playing for Vancouver. So, of the goalies currently in place for the Maple Leafs, Reimer seemed to be the more likely solution between the pipes. But Ben Scrivens delivered a strong effort in a quick training camp, played well in an opening night victory over Montreal and now appears to be getting the start again today in a matchup with Buffalo.
Links from around the rink for April 20
407dDoug Mittler
Links from around the rink for April 6
421dDoug Mittler
Links from around the rink for March 23
435dDoug Mittler
What's next: Minnesota Wild
448dVictoria Matiash
What's next: Nashville Predators
448dVictoria Matiash
What's next: Arizona Coyotes
449dTim Kavanagh
What's next: San Jose Sharks
449dTim Kavanagh
What's next: Los Angeles Kings
449dTim Kavanagh
What's next: Colorado Avalanche
449dVictoria Matiash
What's next: Winnipeg Jets
449dVictoria Matiash
Links from around the rink - March 9
449dDoug Mittler
What's next: Dallas Stars
450dVictoria Matiash
What's next: St. Louis Blues
451dVictoria Matiash
What's next: Florida Panthers
451dVictoria Matiash
What's next: Calgary Flames
451dTim Kavanagh
Scrivens taking lead in Toronto?
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