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Swapping single-stranded DNA sequence specificities
of relaxases from conjugative plasmids F and R100
Matthew J. Harley* and Joel F. Schildbach†
Department of Biology, The Johns Hopkins University, Baltimore, MD 21218
Communicated by Robert T. Sauer, Massachusetts Institute of Technology, Cambridge, MA, August 6, 2003 (received for review May 25, 2003)
Conjugative plasmid transfer is an important mechanism for diversi-
fying prokaryotic genomes and disseminating antibiotic resistance.
Relaxases are conjugative plasmid-encoded proteins essential for
plasmid transfer. Relaxases bind and cleave one plasmid strand site-
and sequence-specifically before transfer of the cleaved strand.
TraI36, a domain of F plasmid TraI that contains relaxase activity,
KDand high sequence specificity. Despite 91% amino acid sequence
identity, TraI36 domains from plasmids F and R100 discriminate
between binding sites. The binding sites differ by 2 of 11 bases, but
both proteins bind their cognate site with three orders of magnitude
higher affinity than the other site. To identify specificity determi-
nants, we generated variants having R100 amino acids in the F TraI36
background. Although most retain F specificity, the Q193R?R201Q
The reverse switch (R193Q?Q201R) in R100 TraI36 confers a wild-type
base contributions to recognition suggests that the specificity differ-
ence derives from multiple interactions. The F TraI36 crystal structure
shows positions 193 and 201 form opposite sides of a pocket within
the binding cleft, suggesting binding involves knob-into-hole inter-
actions. Specificity is presumably modulated by altering the compo-
sition of the pocket. Our results demonstrate that F-like relaxases can
switch between highly sequence-specific recognition of different
sequences with minimal amino acid substitution.
gative plasmid directs transfer of a copy of itself, in single-stranded
form, to a recipient cell (reviewed in refs. 1–3). Despite the
sequence diversity of conjugative plasmids that have been studied,
many elements of the transfer process are conserved (3), including
the involvement of relaxases. Relaxases, also called mobilization
proteins or nickases, are essential for conjugation (4–6). These
proteins bind and cleave one DNA strand at the plasmid origin of
transfer (oriT). The cleaved strand is transferred to the recipient.
Relaxases act as part of the relaxosome, a complex of multiple
proteins and plasmid DNA (6–8). The relaxosome may serve to
generate the single-stranded DNA (ssDNA) substrate that the
relaxase targets. Relaxases cleave ssDNA in a Mg2?-dependent
transesterification reaction that proceeds through a stable phos-
photyrosyl intermediate (9–15). At the end of conjugative transfer,
Relaxases exhibit sequence specificity for their cognate oriT
sequences (9, 11, 12, 20–26). We previously demonstrated, by using
the 36-kDa relaxase domain of F TraI (TraI36), an N-terminal
330-aa fragment of F factor TraI (27), that this specificity can be
remarkable. F TraI36 binds a single-stranded F oriT sequence with
a subnanomolar KD, and some single-base substitutions over a
10-base region can reduce affinity by between 10- and 10,000-fold
(24). In vivo, this specificity is reflected in the poor efficiency with
which F TraI mobilizes plasmids containing the TraI-binding site
for the highly homologous R100 plasmid, despite a difference of
only two bases (24, 28). To better understand the basis of ssDNA
that contribute to ssDNA recognition. Here we demonstrate that
despite the 91% amino acid sequence identity shared by F factor
between prokaryotes. During bacterial conjugation, a conju-
and R100 TraI36, the two proteins have easily detected differences
in specificity. We then describe the results from a panel of TraI36
variants that identify specificity determinants. We found that just
two amino acid residue differences account for the two base
difference in ssDNA binding and cleavage specificity.
Materials and Methods
Protein Engineering and Purification. Primersequencesareavailable
on request. All cloned gene segments and mutations were con-
firmed by DNA sequencing. The F TraI36 expression construct
(pET24a-traI36) was engineered previously (27). For R100 TraI36
expression, the N-terminal 331-codon region of R100 traI was PCR
amplified. Primers encoded an NdeI site overlapping the start
codon and an EcoRI site after a stop codon engineered at codon
332. The NdeI?EcoRI-digested PCR product was ligated into
NdeI?EcoRI-digested pNEB193 (New England Biolabs), trans-
traI36 gene was excised from pNEB193, ligated into NdeI?EcoRI-
digested pET24a(?) (Novagen), and transformed into strain XL10
Gold. The 3?-most codon was removed by PCR as described (27).
Gene fragments for chimera R100?F?F were generated by
NdeI?PstI digestion of the TraI36 expression vectors, purified by
electrophoresis through low melt agarose, isolated by using the
Wizard PCR Preps DNA Purification System kit (Promega), and
ligated. The R100 insert fragment for chimera F?F?R100 was
PCR-amplified by using R100 traI36 as template, and cloned into
purified StuI?EcoRI-digested pET24a-traI36. Constructs were
transformed into strain TB1. An unintended M278T mutation in
chimera F?F?R100 was corrected by using the QuikChange site-
directed mutagenesis kit (Stratagene).
Single, double, and triple mutations were generated by using the
QuikChange kit. An ''F'' or ''R'' preceding the variant description
indicates F or R100 background, respectively. The F-Q193R?
R201Q variant was PCR-generated by using the F-Q193R traI36
variant as the template. Variants F-E153D?Q193R?R201Q and
F-I185S?Q193R?R201Q were PCR-generated from F-Q193R?
R201Q, R-R193Q?Q201R from R-Q201R, and R-D153E?
R193Q?Q201R from R-R193Q?Q201R.
Expression plasmids were transformed into strain BL21(DE3).
Proteins were expressed and purified as described for wild type F
TraI36 (27), and concentrated by using CentriCon10 or Centricon
Plus-20 filters (Amicon).
Oligonucleotide Synthesis and Purification. Sequences of oligonucle-
otides used in binding and cleavage assays are shown in Fig. 1.
Oligonucleotides were purchased from Integrated DNA Technol-
incorporated by using TAMRA-CPG columns. Labeled oligonu-
Abbreviations: TraI36, 36-kDa relaxase domain of TraI; ssDNA, single-stranded DNA;
dsDNA, double-stranded DNA; oriT, plasmid origin of transfer; TAMRA, carboxytetra-
*Present address: Department of Plant Pathology, University of California, One Shields
Avenue, Davis, CA 95616.
†To whom correspondence should be addressed. E-mail: email@example.com.
© 2003 by The National Academy of Sciences of the USA
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cleotides and some PCR primers were purified by polyacrylamide
gel electrophoresis as described (29).
performed as described (29).
32P 5?-end labeling was
DNA Binding and Cleavage. Affinity measurements were performed
by titrating protein into 4 nM solutions of fluorophore-labeled
oligonucleotide and following changes in fluorescence anisotropy
and emission intensity as described (24, 30), or, for highest affinity
interactions, in a buffer with 250 mM NaCl replacing the 100 mM
NaCl normally used. Data were fit by using the global fitting
algorithm SPECTRABIND (31, 32) as described (30). Cleavage of
single-stranded, 22-base oligonucleotide with F factor or R100
sequence by various TraI36 proteins was examined by using a
nicking assay (18) as described (24). Despite repeated purification
attempts, some variants copurified with a small amount of a
nonspecific nuclease activity. Binding and cleavage assays were
repeated in the presence of 1 ?M nonspecific oligonucleotide for
these variants and the wild-type F TraI36 with no differences in
CD. CD experiments were performed by using 2.5 or 5 ?M protein
in 20 mM sodium phosphate (pH 7.5), 100 mM NaCl, and 0.1 mM
EDTA on an AVIV215 CD Spectrometer. Wavelength scans were
performed at 25°C with 1-nm steps and 1-s signal averaging at each
wavelength. Thermal denaturations were observed at 222 nm by
using 1°C steps from 25°C to 75°C, 60-s temperature equilibrations,
and 30-s signal averaging.
Results and Discussion
Wild-Type F and R100 TraI36 Binding. In vivo plasmid nicking and
transfer experiments have demonstrated that TraI proteins from F
and R100 can distinguish the two-base difference between their
cognate TraI binding site (Fig. 1) and the site from the other
plasmid (20, 24, 26, 28). To quantify the binding differences, an
expression construct for the R100 TraI relaxase domain (TraI36)
compared with that of the previously generated F TraI36 (27). The
share 91% amino acid sequence identity.
Dissociation constants for the interactions between F and R100
TraI36 and the TraI-binding sites from F and R100 oriT regions
changes of TAMRA-labeled single-stranded oligonucleotides on
the binding assays are performed in absence of Mg2?, the binding
is reversible and the assay measures an equilibrium binding con-
stant. Dissociation constants are listed in Table 1, and representa-
tive binding curves are shown in Fig. 2. F TraI36 binds to the F oriT
with a 0.55 nM KD, in agreement with previous measurements (24,
3.3 ?M KD, correlating well with previous competition studies (24).
R100 TraI36 is also specific for its cognate site, binding the R100
oriT oligonucleotide with a 2.4 nM KDand the F oriT oligonucle-
otide with an estimated 3.2 ?M KD. (In experiments yielding KD
values ?1 ?M, binding frequently did not reach saturation. How-
ever, because both fluorescence intensity and anisotropy data were
5 ?M. KDvalues above this range are listed as ?5 ?M.)
Binding of Chimeras and F TraI36 Variants. To identify specificity
generated. Chimera R100?F?F contained R100 sequence for the
N-terminal 44 aa, including 6 amino acid differences between the
F and R100 proteins. The affinity of this protein for F oriT is
reduced 10-fold but its affinity for R100 oriT is unchanged, relative
to wild-type F TraI36 (Table 1). Chimera F?F?R100 contained
R100 sequence for the C-terminal 98 aa, including 9 amino acid
differences. This chimera has wild-type F affinity and specificity
(Table 1).
Table 1. Binding constants of wild-type and chimeric
TraI36 proteins
TraI36 protein
F oriT
R100 oriT
KD(nM) ? SD
KD(nM) ? SD
F factor
0.55 ? 0.07
3,200 ? 1,400
6.3 ? 0.2
0.52 ? 0.3
3,300 ? 650
2.4 ? 0.15
*Chimera R100?F?F contains amino acids 1–44 from R100 TraI36.
†Chimera F?F?R100 contains amino acids 232–330 from R100 TraI36.
Fig. 1.
sequence starts at 154?, where the numbering is according to Frost et al. (2).
The prime indicates the ssDNA sequence bound by TraI is on the ''bottom''
between F and R100, are in bold, and bases differing from the F sequence are
underscored. The vertical line indicates nic, the site of DNA cleavage by TraI.
Sequences of the binding site oligonucleotides. The 5? end of the F
Fig. 2.
Emission intensity of 3? 22-base TAMRA-labeled binding site oligonucleotides
increased on binding wild-type F (circles) and R100 (squares) TraI36. Oligonu-
cleotides include TraI binding sites from F (Upper) and R100 (Lower) oriT.
Intensity data were adjusted for fluorophore dilution and normalized for
initial intensity (arbitrary units) relative to each other. Solid lines indicate
results of the simultaneous fit of intensity and anisotropy (not shown) data to
a 1:1 binding model by using SPECTRABIND (31, 32).
Differing specificities of the wild-type F and R100 TraI36 proteins.
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The results from the chimeric proteins suggested that the major
specificity determinants are located between amino acids 44 and
having one to three R100 amino acids in the F background
(designated by an F preceding the amino acid substitution) were
generated and examined. To help speed identification of specificity
determinants, multiple substitutions were combined in a single
variant when the amino acid positions were close enough to make
it experimentally feasible. The KDvalues for the variants are shown
in Table 2. Variants F-A160V, F-R197K?L198I, F-E202S,
F-K203E, and F-K205R?E206Q?Q207R retained F specificity,
with KDvalues for both binding sites within 2-fold of the values for
wild-type F protein.
Variants F-E153D and F-R75K show a general increase in
affinity. F-E153D binds the R100 oriT oligonucleotide with 8-fold
higher affinity, and F-R75K with 3-fold higher affinity, than wild-
type F TraI36. The variants also bind the F oriT oligonucleotide
with increased affinity. Assay conditions, specifically the 4 nM
concentration of oligonucletide required for sufficient fluorescent
signal, make it difficult to measure affinities significantly higher
than that of wild-type F. Therefore, relative affinities of F,
F-E153D, and F-R75K TraI36 for F oriT were determined by using
higher salt concentrations (250 mM vs. 100 mM NaCl), at which F
TraI36 has reduced affinity for ssDNA (J. C. Stern and J.F.S.,
unpublished data). These results (not shown) confirm that the two
variants have a severalfold increased affinity for F oriT, indicating
that these variants have increased affinity but essentially unaltered
Four single amino acid substitutions cause distinct changes in
binding specificity. F-I185S shows a 2-fold reduction in affinity for
F oriT but a 16-fold increased affinity for the R100 oriT sequence,
relative to F TraI36. F-Y190L and F-Q193R have 35-fold and
1,500-fold decreased affinity, respectively, for the F oriT oligonu-
cleotide, but unchanged affinity for the R100 oriT, relative to F
TraI36. F-R201Q has an 870-fold reduction in affinity for F oriT.
The F-R201Q variant also appears to have reduced affinity for
R100, although the affinity is low and difficult to accurately
Because the substitutions at 193 and 201 cause the most signif-
icant changes in binding, we combined them in the F-Q193R?
R201Q variant. This protein (Table 2 and Fig. 3) has a switched
acid substitutions are sufficient to change the specificity from F to
an ''R100-like'' specificity, although the affinity of the variant for
the R100 site is still considerably reduced relative to that of the
R100 TraI36 protein. Clearly, the altered specificity results from a
combinatorial effect of the two substitutions.
Variants with three and four amino acid substitutions were
engineered and tested to determine the effect of altering amino
acids within the context of the Q193R?R201Q substitutions.
F-E153D?Q193R?R201Q has a 6-fold, and F-I185S?Q193R?
R201Q a 3-fold, enhanced affinity for the R100 site, relative to the
has improved binding to the F site, relative to the double variant,
whereas F-I185S?Q193R?R201Q binding to F oriT is too low to
accurately measure. Combining both the I185S and E153D substi-
tutions with Q193R?R201Q fails to improve the binding to the
R100 oriT sequence. Consistent with the results from the F-Y190L
variant, F-Y190L?Q193R?R201Q has poor binding to both the F
and R100 oriT sequences.
The Q193R?R201Q switch is the key to changing the ssDNA-
binding specificity from F to R100-like. Adding other amino acid
substitutions improve binding, but the F TraI36 variants still dem-
onstrate only a 20-fold higher affinity for the R100 site relative to
the F site compared with the 1,400-fold difference exhibited by
R100 TraI36. Clearly, additional substitutions are required to fully
convert the F TraI36 specificity to that of R100 TraI36. Some of
these substitutions may, like I185S, exert different effects in differ-
ent contexts, making them more difficult to identify through
individual amino acid changes.
Binding of R100 Variants. The results for F TraI36 indicate that
between F and R100. Given this, placing the residues from F into
Table 2. Binding constants of F TraI36 variants
TraI36 protein
F oriT site R100 oriT site
KD(nM) ? SD
KD(nM) ? SD
F factor
0.55 ? 0.067
3,200 ? 1400
1.0 ? 0.22
1.2 ? 0.19
19 ? 0.60
860 ? 30
0.42 ? 0.021
480 ? 37
0.49 ? 0.27
0.99 ? 0.25
0.33 ? 0.13
1,500 ? 39
3,300 ? 650
2.4 ? 0.15
1,600 ? 660
390 ? 3.9
200 ? 0.71
3,600 ? 490
460 ? 6.4
160 ? 8.5
72 ? 7.5
120 ? 11
Fig. 3.
intensity of a 3? 22-base TAMRA-labeled binding site oligonucleotides was
followed during titration of wild-type F (circles), R100 (squares), F-Q193R?
R201Q (triangles), or R-R193Q?Q201R (diamonds) TraI36 proteins. Oligonu-
cleotides contained F (Upper) or R100 (Lower) oriT sequences. Intensity data
were adjusted for fluorophore dilution and normalized for initial intensity
(arbitrary units) relative to each other. Solid lines indicate results of the
simultaneous fit of intensity and anisotropy (not shown) data to a 1:1 binding
model by using SPECTRABIND (31, 32).
Variant TraI36 proteins demonstrate altered specificity. Emission
Harley and SchildbachPNAS ?
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the R100 background might shift the specificity of the protein from
R100 to F. Results for a series of variants having F amino acids
substituted into the R100 background, which are designated by an
''R'' preceding the amino acid substitutions, are listed in Table 3.
Variants R-R193Q and R-Q201R both bind F oriT with at least
5-fold higher affinity than R100 oriT. Each of these single substi-
tutions reduce binding to the R100 oriT site by at least 150-fold and
increase binding to the F oriT site by at least 25-fold, relative to
wild-type R100. The variants R-R193Q?Q201R (Fig. 3) and
R-D153E?R193Q?Q201R both display essentially F specificity and
affinity, each binding at least 3,000-fold better to the F sequence
than the R100 sequence. The high affinities of these R100 variants
for the F sequence were confirmed by measuring binding at high
salt conditions (data not shown). The conversion from the R100 to
the F specificity is therefore more easily accomplished than the
conversion from F to R100.
Anisotropy Differences. In addition to displaying altered binding,
variants F-Q193R?R201Q and R-R193Q?Q201R also cause dif-
ferent effects on fluorescence anisotropy on binding the TAMRA-
labeled oligonucleotides. As shown in Fig. 4 for the R100 oriT
oligonucleotide, binding by R100 TraI36 causes a relatively small
increase (from 0.14 to 0.15) in fluorophore anisotropy. In contrast,
binding by F TraI36 causes a considerably larger increase in
fluorescence anisotropy (from 0.14 to 0.23). Variant R-R193Q?
Q201R, which has the F-binding specificity, also causes the F-like
large increase in fluorescence anisotropy on binding. The
F-Q193R?R201Q variant, which preferentially binds the R100 site,
shows an R100-like small relative increase in anisotropy. A similar
effect is seen with the F oriT oligonucleotide, with F TraI36 and
R-R193Q?Q201R causing small increases in fluorescence aniso-
tropy of the oligonucleotide and R100 TraI36 and F-Q193R?
R201Q causing larger increases (not shown). This phenomenon
appears to be related to specificity rather than absolute affinity
because F-Q193R?R201Q and R-R193Q?Q201R bind R100 oriT
with only a 4-fold difference in affinity but cause different effects
on anisotropy.
These results indicate that, in this system, anisotropic differences
not only distinguish between bound and unbound states, but also
report on a difference between the physical properties of the
protein?ssDNA complexes. The basis of this phenomenon is un-
clear. There may be differences in the conformations of the bound
oligonucleotides that could affect local motion of the fluorophore
and, thus, anisotropy. There may also be different interactions
between the fluorophore and the DNA and?or protein in the
different complexes, altering anisotropic behavior by altering the
motion or fluorescent lifetime of the fluorophore. Analogous
results were obtained for the F TraI36 variant F-R150A, which
causes an increase in fluorescence intensity but not steady-state
anisotropy on binding the 22-base 3?-TAMRA labeled oriT oligo-
nucleotide (30). It is also possible that there is an oligomerization
no other evidence consistent with this model.
Fine Specificity of ssDNA Recognition. Binding of the variants to
oligonucleotides having single base substitutions was examined
(Table 4). The base numbering is for the F sequence according to
Frost and colleagues (2), with a prime indicating the ''bottom''
(cleaved) DNA strand.
The strongest correlation observed was between the amino acid
at position 201 and the base at position 145?, suggesting a direct
interaction between the two. Those proteins with R201 (F,
R-Q201R, R-R193Q?Q201R) bind sequences with 145?G at least
proteins with Q201 (R100; R-R193Q; F-E153D?Q193R?R201Q)
bind oligonucleotides with 145?T at least 9-fold better than other-
wise identical oligonucletides with 145?G. Consistent with these
findings, DNA-binding proteins often contact G bases through Arg
Table 3. Binding constants of R100 TraI36 variants
TraI36 protein
F oriT
R100 oriT
KD(nM) ? SD
NKD(nM) ? SD
F factor
0.55 ? 0.067
3,200 ? 1,400
46 ? 0.3
120 ? 29
3,300 ? 650
2.4 ? 0.15
350 ? 20
600 ? 5.7
1,750 ? 230
Table 4. Binding constants of F and R100 TraI36 variants for F and R100 oriT sites
TraI36 protein
acid at
145?G?147?G (F oriT),
145?T?147?A (R100 oriT),
F factor
0.55 ? 0.067
46 ? 0.3
120 ? 29
1,500 ? 39
3,200 ? 1,400
2,500 ? 330
620 ? 80
3.4 ? 0.4
1,400 ? 540
170 ? 28
2.8 ? 1.1
120 ? 6
11 ? 5
32 ? 14
3,900 ? 440
3,300 ? 650
1,750 ? 230
350 ? 20
600 ? 5.7
72 ? 7.5
2.4 ? 0.15
Boldface indicates bases that differ from the F oriT sequence.
*Values an average of at least two measurements.
Fig. 4.
ent specificities. Shown are changes in fluorescence anisotropy of 3? 22-base
TAMRA-labeled R100 oriT-binding site oligonucleotide on binding of wild-
(diamonds) TraI36 proteins. Variants F-Q193R?R201Q and R-R193Q?Q201R
lines indicate results of the simultaneous fit of intensity and anisotropy (not
shown) data to a 1:1 binding model by using SPECTRABIND (31, 32).
Differences in fluorescence anisotropy changes correspond to differ-
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side chains, and T bases are more frequently contacted by Gln than
Arg side chains (33).
Other correlations between amino acids and specificity are
relatively weak. Variant R-R193Q, which has Gln at both 193 and
201, exhibits a 100-fold preference in binding to sequences with
147?G versus 147?A. In contrast, those proteins with Q193 and
the F oriT (145?G?147?G) over the 145?G?147?A sequence, but
context of 145?T. Those proteins having R193 and Q201 do not
distinguish significantly between 147?G and 147?A. R-Q201R,
which has Arg at both 193 and 201, shows a small (2- to 4-fold)
preference for 147?A over 147?G.
These data indicate that F TraI36 has more specific recognition
affinity than the F site, yet this specificity is due largely to the
identity of the base at position 145?. In contrast, F TraI36 discrim-
inates between the F and R100 oriT bases at both 145? and 147?.
The sum of the data suggest that the specificity is influenced by
the amino acids at 193 and 201, and can also be influenced by the
bases at 145? and 147?. This suggests that these bases and amino
acids interact, directly or indirectly, to determine specificity.
CD Studies of Variants. The stability of the proteins was assessed to
examine the possibility that the altered recognition was related to
altered stability. CD wavelength spectra for wild-type F and R100
TraI36, and variants F-Q193R?R201Q, F-E153D?Q193R?R201Q,
and R-R193Q?Q201R are similar (not shown). These proteins also
have similar transitions in thermal denaturation experiments (not
shown), indicating similar stabilities and suggesting that the altered
specificities are not explained by altered protein stabilities. Both F
(27) and R100 (not shown) TraI36 show considerable hysteresis
during thermal and chemical denaturation and renaturation, pre-
cluding calculation of the Gibbs free energy of unfolding.
Oligonucleotide Nicking. The correlation between oligonucleotide
binding and nicking was assessed because F and R100 TraI36
exhibit different levels of specificity in oligonucleotide cleavage,
making cleavage a further measure of the specificity of these
enzymes. F TraI36 cleaves its cognate oriT sequence well, but
cleaves the R100 sequence poorly (Figs. 5 and 6). R100 TraI36
cleaves its cognate sequence preferentially, but can also cleave the
F sequence relatively well. The ssDNA cleavage specificity results
for F and R100 TraI36 are consistent with the results of in vivo
nicking and transfer experiments using the full-length proteins.
R100 TraI nicks and efficiently transfers chimeric plasmids that
in its ssDNA cleavage and plasmid mobilization activities, and
interacts less effectively with the R100 sequence (24, 28).
All variants tested retain overall specificity for the TraI binding
site as demonstrated by the presence of a single cleavage product
in each assay, with the cleavage products having equivalent elec-
trophoretic mobilities (Figs. 5 and 6). Variants F-Q193R?R201Q
and F-E153D?Q193R?R201Q have reduced apparent cleavage
activity on the F oriT oligonucleotide, relative to the F parent, to a
level near that of R100 TraI36 (Fig. 5 Upper). These variants also
show greatly increased apparent cleavage activity on the R100 oriT
oligonucleotide, to the level of R100 TraI36 (Fig. 5 Lower). The F
single variants show reduced apparent cleavage activity on the F
oriT oligonucleotide and slight (F-R201Q) or no (F-Q193R) ap-
preciable gain of apparent cleavage activity on the R100 oriT
oligonucleotide (data not shown).
R100 background variants R-R193Q?Q201R and R-D153E?
Fig. 5.
relates with binding specificity. A 5? radiolabeled oligonucleotide was incu-
bated with protein in a Mg2?-containing buffer, the reaction was stopped
with SDS, and the products were electrophoresed through a denaturing gel.
Cleavage is indicated by conversion of the oligonucleotide from a 22-base to
a 14-base fragment. Shown are F (Upper) and R100 (Lower) oriT oligonucle-
otides cleaved by wild-type F, F-Q193R?R201Q (F-QR?RQ), F-E153D?Q193R?
R201Q (F-ED?EQ?RQ), and wild-type R100 in the presence of 1 ?M unlabeled,
Oligonucleotide cleavage specificity of variant F-Q193R?R201Q cor-
Fig. 6.
relates with binding specificity. Shown are F (Upper) and R100 (Lower) oriT
oligonucleotides cleaved by wild-type F, R-R193Q?Q201R (R-RQ?QR),
R-D153E?R193Q?Q201R (R-DE?RQ?QR), and wild-type R100 TraI36. The left
lane is the oligonucleotide in the absence of protein. The protein concentra-
F TraI36 in Lower, which do not contain a 10 nM sample.
Oligonucleotide cleavage specificity of variant R-R193Q?Q201R cor-
Fig. 7.
within the DNA-binding surface, as shown by the x-ray crystal structure of F
TraI36 (ref. 40; PDB code 1P4D). Shown is a portion of the structure with the
main chain shown as a ribbon diagram. The constituent atoms of the pocket
sulfur in orange. Side chains are labeled. Also forming the pocket are Gly-197
and the main chain atoms of Met-2, Ala-195, and Phe-196.
Amino acids Gln-193 and Arg-201 line pockets within the TraI36-
Harley and SchildbachPNAS ?
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text:
Pierre Boulez: 20th Century Recordings on DG - works by Bartok, Berg, Birtwistle, Debussy, Ligeti, Messiaen et al. / Krystian Zimerman, Leif Ove Andsnes, Hélène Grimaud [Limited Edition, 44 CDs]
44 CD / Deutsche Grammophon / 2015-02-10Works: Béla Bartók: Pieces (4) for Orchestra, Op. 12/Sz 51; Concerto for Orchestra, Sz 116; Dance Suite for Orchestra, Sz 77; Pictures (2) (Két kép), for orchestra, Sz. 46, BB 59...
Performers: Aimard, Pierre-Laurent [Piano]; Aler, John [Tenor Vocal]; Andsnes, Leif Ove [Piano/Conductor]; Arrignon, Michel; Barenboim, Daniel [Conductor, Piano]; Bashmet, Yuri [Conductor/Viola]; Bauer, Vincent;...
Conductors: Boulez, Pierre
Ensemble: ; Berlin Philharmonic; Chicago Symphony Orchestra; Ensemble de Violoncelles de l'Orchestre de Paris; Ensemble InterContemporain; Ensemble Modern (Frankfurt); London Symphony Orchestra; Members of the...
On March 26, 2015 Pierre Boulez reaches his 90th year. This great master of music - composer, conductor and writer - has been an exclusive...continue...
Recording information: Abbey Road Studios (01/2001-02/2001); Concertgebouw, Amsterdam, The Netherlands (01/2001-02/2001); Groáer Saal, Alte Oper, Frankfurt, Germany (01/2001-02/2001); Gro&aacut...continue...
K. Szymanowski: Symphony Nos. 2 & 3; Violin Concerto / Ryszard Karczykowsk, tenor; Chantal Juillet, violin; Kenneth Jewell Chorale; Detroit SO; Doráti; Montréal SO; Dutoit
1 CD / Decca / 2014-09-16Works: Karol Szymanowski: Symphony no 2 in B flat major, Op. 19, M24; Symphony no 3, Op. 27 "Song of the Night"; Concerto for Violin no 2, Op. 61
Performers: Juillet, Chantal
Conductors: Dorati, Antal; Dutoit, Charles
Ensemble: Detroit Symphony Orchestra; Kenneth Jewell Chorale; Montréal Symphony Orchestra
Designed to serve as the perfect introduction to the music of a turbulent century, DG/Decca's '20C' series covers a wide range of key works by...continue...
Szymanowski: Symphonies Nos. 3 & 4; Stabat Mater / Sally Matthews, soprano; Ekaterina Gubanov, mz; Toby Spence, tenor
1 Super Audio CD / LSO Live (London SO) / 2013-09-10Works: Karol Szymanowski: Symphony no 3, Op. 27 "Song of the Night"; Stabat mater, Op. 53; Symphony no 4, Op. 60 "Symphonie concertante"
Performers: Gubanova, Ekaterina; Matsuev, Denis; Matthews, Sally; Smoriginas, Kostas [Baritone Vocal]; Spence, Toby
Conductors: Gergiev, Valery
Ensemble: ; London Symphony Chorus
This second, star-studded installment of Valery Gergiev's Szymanowski symphony cycle features tenor Toby Spence as tenor soloist in the Symphony No.3, subtitled Song of the...continue...
Recording information: Barbican, London (2012-12-11&2012-12-18&2012-)....continue...
Song of the Night: Szymanowski: Violin Concerto no 1; Symphony no 3 / Christian Tetzlaff, violin - Boulez
1 CD / Deutsche Grammophon / 2012-01-24Works: Karol Szymanowski: Concerto for Violin no 1, Op. 35, M37; Symphony no 3, Op. 27 "Song of the Night"
Performers: Davislim, Steve; Tetzlaff, Christian [Violin]
Conductors: Boulez, Pierre
Ensemble: ; Vienna Philharmonic Orchestra
Pierre Boulez honors singular Polish composer Karol Szymanowski by recording - live - the Violin Concerto no. 1 and the Symphony no. 3 "Song of...continue...
Recording information: Groáer Musikvereinssaal, Vienna....continue...
Mariss Jansons conducts Lutoslawski, Szymanowski & Tchaikovsky
1 CD / BR Klassik / 2011-06-28Works: Witold Lutoslawski: Concerto for Orchestra; Karol Szymanowski: Symphony no 3, Op. 27 "Song of the Night"; Alexander Tchaikovsky: Symphony no 4, for orchestra, choir & solo viola, Op. 78
Performers: Bartminski, Rafal; Guez, Nimrod; Rohn, Andreas
Conductors: Jansons, Mariss
Ensemble: Bavarian Radio Symphony Orchestra
Three works that are very close to his heart were brought together by Mariss Jansons for this new CD release. Karol Szymanowski's Third Symphony depicts...continue...
Recording information: Philharmonie im Gasteig, München....continue...
Szymanowski: Symphonies 3 & 4 / Wit/Polish National Orchestra [DVD]
1 DVD Video / ICA Classics / 2011-02-22Works: Karol Szymanowski: Symphony no 3, Op. 27 "Song of the Night"; Symphony no 4, Op. 60 "Symphonie concertante"
Performers: Bartminski, Rafal; Broja, Jan Krzysztof
Conductors: Wit, Antoni
Ensemble: Warsaw Philharmonic Chorus; Warsaw Philharmonic Orchestra
This recording, filmed live from the Warsaw Philharmonic Hall, benefits from DTS 5.1 surround sound. Superbly filmed for Polish Television, these two performances embody the...continue...
Run Time: 55 min.
Region: All
Picture Format: NTSC, 16:9
Sound Format(s): LPCM Stereo, DTS 5.1 Surround
Szymanowski: Symphonies no 2 and 3 / Wit, et al
1 CD / Naxos (USA) / 2008-03-25Works: Karol Szymanowski: Symphony no 2 in B flat major, Op. 19, M24; Symphony no 3, Op. 27 "Song of the Night"
Performers: Marczyk, Ewa; Minkiewicz, Ryszard
Conductors: Wit, Antoni
Ensemble: Warsaw Philharmonic Orchestra
Composed in 1910, at a time when Szymanowski was influenced by Richard Strauss, Reger and Scriabin, the unusually structured 'Symphony No. 2' is a work...continue...
Szymanowski: Symphonies no 3 and 4 / Stryja, Polish State PO
1 CD / Naxos (USA) / 1997-03-14Works: Karol Szymanowski: Symphony no 3, Op. 27 "Song of the Night"; Symphony no 4, Op. 60 "Symphonie concertante"; Concert Overture, for orchestra in E major, Op. 12, M12
Performers: Ochman, Wieslaw; Zmudzinski, Tadeusz
Conductors: Stryja, Karol
Ensemble: Polish National Philharmonic Chorus Katowice; Polish National Philharmonic Orchestra Katowice

subset: commoncrawl


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Bourbon Blue Send to Phone
OpenDinner nightly, Brunch Sat.-Sun.
Open late
- Valet parking
- Dress code: Casual
- Entertainment: Entertainment
- Full bar
- Kid-friendly
- Outdoor dining
- Reservations suggested
* Click here for rating key
Text data on Bourbon Blue to your phone, or a friend's cell phone.
Check out the 2015 edition of GAYOT's Annual Restaurant Issue, which features the Top 40 Restaurants in the US and more.


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I'm using the Fruitful theme for a website I'm working on for my husband. When I publish a page on the site, all formatting seems to be lost on the live site. Spacing, font etc. all looks great on the back end, but once published, all text is squished together and bold/italicized/underline text options are all lost. Anyone know what's happening here?
For example, on this page: http://www.commissionapainting.com/contact/
Text in the left column should have a line of space before the "Please contact us", and the heading "Email us" in the right column is not bolded.
And on this page: http://www.commissionapainting.com/pricing/ we had to get creative using bullet points in order to create new paragraphs.
Anyone know what's going on here? I'm not great with html or style sheets, so if possible, please make replies as detailed as possible! Any help is greatly appreciated!


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DPAP News
November 13
SPA's Langbein Receives Award at APPAM for Comparative Research Analysis
Professor Laura Lanbein and PhD alumnus Pablo Sanabria win award at APPAM's Fall Research Conference for work examining relationship between laws and their effects on bribe-seeking behavior.
#SPA80for80: Gwen Sykes, SPA/MPA '01
As CFO of the U.S. Secret Service, Gwendolyn Sykes brings to SPA students several years of experience managing financial operations for federal agencies.
January 8, 2015
#SPA80for80: Ambassador Kenneth Merten, SPA/MPA '86
Ambassador Kenneth Merten was inspired for a career in diplomacy by a strong interest in the world beyond our borders.
December 23, 2014
#SPA80for80: Congressman Jim McGovern, CAS/BA '81 & SPA/MPA '84
Congressman Jim McGovern (D - MA) got his introduction to politics while at SPA. Today he is a champion for progressive causes in the U.S. House of Representatives.
December 22, 2014
#SPA80for80: Mark Sylvia, SPA/BA '96 & SPA/MPA '98
Massachusetts has been at the forefront of the clean energy revolution. Directly involved in the state's progressive movement is School of Public Affairs alumnus Mark Sylvia.
December 22, 2014
#SPA80for80: Andy MacCracken, SPA/BA '11 and SPA/MPA '14
When President Barack Obama decided to move forward on executive actions to support federal student loan borrowers, he reached out to SPA alumnus Andy MacCracken.
December 22, 2014
#SPA80for80: Brianne Nadeau, SPA/MPP '06
Recently elected as Washington DC's Ward 1 councilmember, Brianne Nadeau brings a commitment to community activism and expertise in communications and advocacy.
December 19, 2014
Success Story
Legally Blind MPA Student Supports Disabled Workers
Duilio Correa, SPA/MPA '13, credits his AU education with helping him become an analyst at the Department of Health and Human Services.More Success Stories


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LAS VEGAS — The 27th annual Scouting for Food Drive kicks off November 14. An estimated 7,000 Boy Scouts and volunteers, serving over 30,000 hours, will fan out across Las Vegas neighborhoods distributing their signature yellow bags door-to-door for collection on November 21. "We hope to collect more than 200 tons of food so we can feed Southern Nevada families in need … [Read more...]
Industry Focus: Non-Profit
As the state continues its steady economic recovery, Nevada's non-profit organizations have gotten creative in order to fulfill their mission. Collaborative efforts move forward making industry leaders optimistic about the coming years. Executives representing several non-profits throughout the state recently met at the Las Vegas offices of City National Bank to discuss the … [Read more...]
Clark County School District Superintendent Pat Skorkowsky to Speak at Las Vegas Global Economic Alliance Special Meeting
(Las Vegas) – The Las Vegas Global Economic Alliance announced that Clark County School District Superintendent Pat Skorkowsky will be the keynote speaker at a special meeting its holding on Feb. 11 at the Four Seasons Hotel. Superintendent Skorkowsky will speak about his efforts to change the way that the nation's 5th largest school district does business in a talk entitled … [Read more...]
What has the greatest potential to improve Nevada's economy?
"Quality education and healthcare are key to the diversification of our employment base, which will bring businesses and individuals to Southern Nevada and positively impact our economy." Joselyn Cousins • Vice President, Community Development, United Way of Southern Nevada "Not a single event or government program will solve Nevada's economic problems, but understanding … [Read more...]
United Way Oversees $1.3 Million Grant from Emergency Food and Shelter Program to Local Nonprofits
United Way of Southern Nevada receives $1.3 million grant from Emergency Food and Shelter Program (EFSP) Phase 30 funding to distribute to local nonprofits. Allocated to 12 organizations across the Valley, these funds must be distributed to the community by March 31, 2013. "United Way is committed to meeting the emergency and immediate needs of the community, helping them to … [Read more...]
What was your very first job?
"My first job was traveling around the intermountain west refinishing gym floors at high schools, junior high schools and churches. I probably lost a few brain cells breathing the toxic fumes but it gave me the desire to get my college degree as fast as possible. Hopefully the education made up for the lost brain cells." Richard Bird • Regional Manager, Marcus & … [Read more...]


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Box Car Coffee Roasters
Coffee Buzz: Steam Espresso Bar, Black Black Coffee & More
If you spend a lot of time in coffee shops and if you appreciate the art of a perfectly pulled shot, you will love Steam Espresso Bar. And I do mean love. The month-old spot, which took over an old photography studio on South Pearl Street, is a stunner. The sun-filled space pulls together a...
May 24 2013, 10:30 AM
The Buzz: Black Eye Coffee Shop and Roostercat Coffeehouse
Denver's coffee scene just continues to grow. Case in point: In the last two weeks, Black Eye Coffee Shop (pictured) and Roostercat Coffeehouse have opened their doors. Both spots have distinct appeal. Black Eye is the newest entry in the hip, coffee-geek circuit (think ...
September 25 2012, 10:00 AM


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Are You Ready to be a Gold Award School?
The HealthierUS School Challenge was established to recognize schools that are creating healthier school environments through their promotion of good nutrition and physical activity. Four levels of superior performance are awarded: Bronze, Silver, Gold, and Gold of Distinction for both elementary and secondary schools.
The overarching goal of the HealthierUS School Challenge is to improve the health of the Nation's children by promoting healthier school environments. To help meet the goal, the Challenge identifies schools that have made changes to the school nutrition environment to:
The Idaho Child Nutrition Programs encourages you to try for the Bronze or Silver award if this is your first time applying for this award. You may find that your school is already doing many of the requirements. If your school is a repeat award winner, aim for Gold or Gold Award of Distinction during the next award cycle.
NEW CRITERIA for 2014-15
Are you doing some innovative things in your school cafeteria? Do you know about the Smarter Lunchroom Movement? The new HUSSC criteria has a big focus on little changes that help students make healthy choices using the Smarter Lunchrooms self-assessment scorecard. Learn more at USDA's HUSSC website.
|HealthierUS School Challenge Criteria Chart
USDA will be offering webinars on the HealthierUS School Challenge, check back regularly for scheduled webinars and archived webinar recordings. The HUSSC Criteria was updated August 31,2014. These webinars may still contain useful information, but they refer to the 2012 criteria.
|Submitting an Application Webinar
|Reviewing Applications Webinar (Insight on how your application will be reviewed)
|HUSSC School Wellness Webinar
|Nutrition Education, Physical Activity, and Physical Education Webinar
|Other Criteria For Excellence, including Farm to School Presentation
If you are interested in applying for the HealthierUS School Challenge contact:
Dianne Esplin, RD, LD, SNS
Coordinator, Child Nutrition Programs
State Department of Education
650 West State Street
Boise, ID 83702
Phone: 1-208-332-6827
E-mail: email@example.com
Kimberly School District (4/14 - 4/18)
Kimberly Elementary School (Bronze)
Marsing Joint School District #363 (4/14 - 4/18)
Marsing Elementary School (Silver)
Marsing Middle School (Silver)
Marsh Valley School District (12/12 – 12/16)
Inkom Elementary School (Silver)
Middleton School District #134 (6/12 – 6/16)
Middleton Middle School(Gold Award of Distinction)
Payette School District #371 (6/12 – 6/16)
Payette High School (Silver)
Middleton School District #134 (5/12 – 5/16)
Middleton High School (Silver)
Middleton School District #134 (3/12 – 3/16)
Purple Sage Elementary School (Silver)
Middleton School District #134 (1/12 – 1/16)
Heights Elementary School (Silver)
Middleton School District #134 (12/11 – 12/15)
Mill Creek Elementary School (Silver)
Kootenai School District (9/11 – 9/15)
Harrison Elementary School (Silver)
Lakeland Joint School District #272 (9/11 – 9/15)
Spirit Lake Elementary School (Silver) (5/11 - 5/15)
Moscow School District (5/11 - 5/15)
A.B. McDonald Elementary School (Silver)
Gooding School District (9/09 - 8/13)
Gooding Elementary School (Gold Award of Distinction)


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More Star Wars films announced
View Single Post
February 15 2013, 02:35 AM
Location: "Who are you?"
Re: More Star Wars films announced
I think Vader's floating jump after disarming Luke in
The Empire Strikes Back
is the creepiest move he ever did. Part of its effectiveness as a surprise move comes from us never having seen him do anything like it. As we learn about telekinetic Force powers in that sequel, it makes sense in that context. But Luke's duel with Vader is a revelation to Luke and to us that there is much that Yoda never got a chance to show him. We are still learning during the duel, with Luke, just how far Force powers go.
This is yet another reason why the films should be watched—and the story told—in the order 4, 5, 6, 1, 2, 3, the order in which the ideas were developed in the real world. Change that order, and the suspense in
The Empire Strikes Back
is lost, beyond just the climax being spoiled.
A life is like a garden. Perfect moments can be had, but not preserved, except in memory. LLAP
Leonard Nimoy (1931-2015)
View Public Profile
Send a private message to CorporalCaptain
Find More Posts by CorporalCaptain


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Winning ticket for Mega Millions sold in Calif.
ATLANTA A single winner has taken the $46 million jackpot from the latest drawing of the multistate Mega Millions lottery.
The only ticket to match all the winning numbers and Mega Ball from Friday night's drawing was sold in California.
The next Mega Millions drawing Tuesday night will have a jackpot of $12 million.
One player won the second prize of $250,000 and one player won the third prize of $10,000.
The winning numbers from Friday night's drawing were: 2, 11, 19, 21 and 34. The Mega Ball number is 38.


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For lunch, dinner, late night and everything in between, Wendy's flavorful menu is chock-full of good food, served fresh. Choose from burgers loaded with your favorite toppings, chicken wraps and salads, chicken nuggets and fresh, delicious salads for a meal on the go or a dine-in experience.More information and Specs
No cash valu;e no cash/credit back. Expires 90 days from date of purchase. Valid at these four locations: Corunna Road, Bristol Road, Vienna Road, and Pierson Road.
Standard terms and conditions apply. Please visit our Terms of Service Page for more details.
Seize the Deal offers hundreds of discounts daily from local businesses – from restaurants, theaters and spas to golf courses, family fun and much, much more. The Value Vault opens twice a week with exciting new products. Click the bright blue ADD TO CART button to Seize the Deal. Hurry, before they're gone.
© 2014 Seize the Deal. All rights reserved


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See our store section for gifts by different stores
See our top gift ideas section for most popular items.
Create your own personal gift registry. Register for any item, from any store, for any occasion. Find the gift registries of your family and friends.
Ever find the perfect gift idea for someone, but their birthday was months away? We make it easy to remember your gift ideas.
Provides timely email reminders for a birthday, anniversary, and other special occasions.
Get advice from friends and family on which gifts to buy.
Five Photo Picture Perfect Personalized Mouse Pad
Above and Beyond Jets Mousepad
Personally Yours Mouse Pad
Make It Happen Mousepad
You Make a Difference Mouse Pad
Photo Sentiments for Him Personalized Mouse Pad
Little Monster Photo Mouse Pad
Personally Yours Personalized Mouse Pad
Design Your Own Photo Mouse Pad
Leather Mouse Pad with Wrist Rest
Inspirational Quote Personalized Mouse Pad
Dot to Dot Personalized Mouse Pad
Personalized Irish Coat of Arms Mouse Pad
Excellence Eagle Mousepad
The Greatest Gift Dad Mouse Pad
Teaching Professions Personalized Mouse Pad
Personalized New Calendar Mouse Pad
Bucky Badger Computer Mouse Pad
Personalized Photo Mouse Pads for Guys
Photo Fun Personalized Mouse Pad
Personalized Marvel Superhero Mouse Pad
Take Your Breath Away Mouse Pad
Personalized Photo and Calendar Mouse Pad
Personalized Vertical Photo Montage Mouse Pad


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Thanks for checking out our Accessories Department. Here you'll find a great selection of Accessories from top brands like Independent, Alkr & Volcom. To help you find exactly what you are looking for you can filter products by Brand, Price and several other useful criteria.
Products in the Accessories category have been rated stars based on 0 reviews


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Choose St. Joseph Health.
Are you confused about insurance, networks, PPOs and HMOs? Let us help
you decide what is the best plan for you, while ensuring your plan and
physician is in our network.
Help with Choosing Insurance
Already a member of the St. Joseph Health network? Congratulations! These
resources below are available for you:
All members of St. Joseph Health have access to their medical records online.
Log-in to the personal hub.
Our hospitals offer a variety of classes and events to support you in living
the healthiest life possible. For classes in your areas, please select
the link of the hospital below:
Contributor: HealthCalling Team
It's important to have a nutritious lunch because the daily food and drink habits that you make or break at noon can blow your entire eating plan for the day. People too often eat lunch ...
Stay in touch with our experts for the latest wellness topics and news.


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Back Talk: A Conversation with Sebastian MasudaJuxtapoz // Wednesday, November 23, 2011
An icon of Harajuku Kawaii culture and the founder of %6DOKIDOKI, Sebastian Masuda is one of the most recognized figures of cutting-edge Japanese pop culture. In 2009, he created and directed the "Harajuku Kawaii Experience," a world-tour fashion show and talk show event, drawing more than 40,000 fans to the event in San Francisco. This December in Los Angeles, he will launch the world debut of RUNE BOUTIQUE with MEDICOM TOY.


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Richard J. Aldrich
Email: r dot j dot aldrich at warwick dot ac dot uk
Tel: 02476 523523
Room: E1.11
Advice and feedback hours: Wednesdays 1.00 & Thursday 2.00
Richard J. Aldrich is a Professor of International Security at the University of Warwick and joined PAIS in September 2007. His main research interests lie in the area of intelligence and security communities. His most recent book is Spying on the World: The Declassified Documents of the Joint Intelligence Committee, a co-edited volume of documents published by Edinburgh University Press in 2014. Currently he is on research leave completing a co-authored book on intelligence and the UK core executive, 1909-1989.
He recently led a related AHRC project entitled "Landscapes of Secrecy: The Central Intelligence Agency and the Contested Record of US Foreign Policy, 1947-2001" which received follow on funding. This involved a team of eight scholars at the universities of Nottingham and Warwick who examined the creation of the public record of the CIA in realms such as history, memoirs, novels and film. The end of project conference took place at the University of Nottingham in the East Midlands Conference Centre and the full conference is now available as a podcast.
Warwick Intelligent Futures
Warwick Intelligent Futures (WIF) is a foresight project. Our main purpose is to distil the predictive content of the latest academic research for a series of futurology and foresight conferences each year around an important emerging theme. We focus interdisciplinary research on a wide range of issues in an effort to inform the current decisions that will effect our tomorrow.
Our main vehicle is themed foresight conferences which are high-quality one day events held in Birmingham, Brussels, London and Washington. These are high profile "research road shows" with renowned speakers from Warwick and elsewhere. Our next events are:
"Human Futures and Cyber Futures"
Friday 15th May 2015
RUSI, Whitehall, London
"Science, Security and the Future of Freedom"
Wednesday 3rd June 2015
University of Warwick, Coventry
Research interests
He also maintains related interests in issues of cyber security, liberty and privacy, set against a background of accelerating globalization. These wider interests extend to developments in information technology, including public key cryptography. The technology of secrecy - particularly secure communications - has exerted an important but largely neglected influence upon the conduct of international relations. He has recently completed a history of GCHQ which was published by Harper Collins in an updated paperback format in 2011.
Additional interests include contemporary history and how the past connects to the present. He advises a number of UK government departments on issues of records management, declassification and corporate memory.
He was co-editor of the journal Intelligence and National Security for eight years and now co-edits the companion book series Studies in Intelligence with Christopher Andrew.
Resources for Secret Intelligence: A Reader
Teaching and supervision
Please note that I am currently on study leave during the period 2014-2015.
Normally I teach on the following modules:
Nine Ideas of International Security: PO135 - BA Year 1 -term one. This is co-taught with a total of nine colleagues
Vigilant State: The Politics of Intelligence: PO382 - BA Year 3 - both terms; This is co-taught with Dina Rezk.
Insurgency and Counter-Insurgency; PO972 - MA - term one
Terrorism and Counter-Terrorism; PO973 - MA - term two


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The Soda Group represented the sellers:
•7041 Portmarnock
•488 Partridge Circle
•7335 Edenmore
•6360 Watercrest Way #404
•6423 Moorings Pt. Cir.
•6305 Tanager
•7712 US Open Loop
•6639 Pebble Beach
•6710 Pirate Perch
•3913 Solymar
The Soda Group represented the buyers:
•13939 Wood Duck Circle
•6903 Dominion Lane
•6319 Yellowtop Drive
•14716 Amberjack Terrace
•7025 Brier Creek
•6505 Meandering Way
•4512 Whitemoss Terrace
•436 Canal Road
•12017 Thornhill Court
•6403 Meandering Way
The Soda Group is experienced in working with short sales, foreclosures and first-time home buyers, as well as new construction, waterfront condos and golf course development, serving the greater Sarasota, Bradenton and Lakewood Ranch areas.
The Soda Group can be reached at the Keller Williams Lakewood Ranch office, 6710 Professional Parkway, Suite 301, Sarasota, Florida 34240. For a free consultation, contact (941) 961-5857. Website:


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November 27, 2015
NASHVILLE, Tenn. (AP) -- A new state study shows Tennessee must improve its response to victims of human trafficking for recent changes in law to be effective.
The state legislature passed a series of changes in law aimed at harsher punishment of people convicted of coercing adults and children into sexual servitude. Effective July 1, a dozen anti-trafficking statutes took effect. There are stronger penalties for traffickers and a longer period of time during which offenders can be charged.
But The Tennessean reports the study finds services to victims of trafficking are disjointed. The 97-page report assigns responsibility for responding to victims to the Department of Human Services and the Department of Children's Services.
Click Here to access the online Public Inspection File
Viewers with disabilities can get assistance accessing this station's FCC Public Inspection File by contacting the station with the information listed below. Questions or concerns relating to the accessibility of the FCC's online public file system should be directed to the FCC at 888-225-5322, 888-835-5322 (TTY), or email@example.com.


text:
Help and Support forum is here:
Server advertising is forbidden as well.
Thanks :) :eng101:
*IMPORTANT* when finished, only gona make a server if we have alot of people typing in the comments saying they will join (i will release the gamemode no matter what)...
Hello, I started this thread for any one that is interested in posting their scripts, add-ons or any modifications that they have done to their Stranded server, also please feel free to post any advice that may help others.
I was shown where to edit...
I'm making a pretty big scenebuild with all the 'classical' contraptions in them. I have all the classics: The bathtubs with wheels and balloons, the bigass catapult, the airboat plane, the large metal container with giant cannon wheels, the big red...
Hello. I am trying to make an ESC Menu Example
Please give me useful feedback.
- Scaly
I need some help with lua code
I Got a perp server and i need help with add some new cars and fix some small bugs.
Add me on STEAM: DaRk F!R3
Joined my friends server to help him test his new addon and my chatbox goes really small, I'd like to fix as I don't speak with many ants.
plox help!
what do you think about this one https://nitrous-networks.com ?
is that good and worth the money?
So I have different vip ranks like
Bronze VIP ,silver gold etc
When i make a VIP Job after the normal jobs, the normal jobs will disappear?
Why does this happen?
None of the normal jobs will work when i put the VIP Job on...
Where can i get this model of Alyx?
I don't mind if it has bugs or issues, it looks so much like the 2008 concepts and i'd really appreciate it if i could get this model.
But if it turns out that the model isn't out yet, could someone make a...
garrysmods.org is a new repository of Garry's Mod downloads, created as a successor to garrysmod.org and a great alternative to the Workshop for those who like customisation, visibility, and tinkering.
The garrysmod.org site was accidentally...
Hey guys, I've been using some time trying to get a video as an background on my Garry's Mod servers loading screen.
Apparently when joining the server, the Screen is black. But when I visit the site to my servers sv_something url, it's just as I want...
Hi guys!
I've created a cool little subreddit for Garry's Mod RP servers, where players and server owners
can discuss their ideas and experience in Gmod RP.
If you would like to advertize, just follow the guide in the sidebar.
Our community is hosting a small mapping contest that is accepting public submissions (we only have 2-3 Hammer-capable members) and we're bringing it to FacePunch. The maps won't be meant to be exclusive to our community, aside from a bit of branding...
So I was thinking about getting my own VPS and running about 3-5 servers on it, with slots 10-20. Just how much RAM would I need in total?
Please get back with some USEFUL information.
- Scaly
Guys i made a mistake and changed the bind of 'R'. I wanna unfreeze somethings but when i press 'R' my binding runs, i can't unfreeze anything. I tryed to make USE DEFAULTS but it didn't worked. How can i fix it? Thanks
I had an idea while strolling in game (rust) during the night, I was wondering if it could be possible to include lightning into the game? Some high definition lightning, Purple, Violet and blue It would look absolutely amazing against the pitch black...
This isn't me asking for any particular reason other than the shear curiosity out of what people have said to me.
Many people that I have talked with have stated there concerns about the Garry's Mod "community or population" of the game has began to...
Me and my friend loves roleplaying on my server (its private :buckteeth:) and we cant find any good roleplay maps the only onces we know is rp_hometown1999 rp_hometown2000 rp_downtown. does anyone know any good maps for rp?
Since the version of Wenli's tools I use has been giving me tons of script errors for a while now I figured I will turn to the community with this question; does anyone know of an updated Wenli's Tools, made either by Wenli himself or someone else that is...
My name is Tyler or ToXiic_Tgod and I would like to offer help to anyone who needs a Dark-RP Moderator, or Admin, even a trial Mod/Admin. I am posting this in hopes to gain more experience with staff positions. I currently have 457 hrs on...
Hello, I'm looking for some people to make a server whit, build a community whit, grow it up together.
I dont know if this is the right place to write it here but now I try,
If u want to build a community whit me, comment.
and comment for more...
We at Nuclear Disaster Network are looking for a new developer.
We currently have 3 servers (DarkRP, Modified DarkRP, and TTT, More coming soon!) And we have a domain that is under Construction.
But to run all this we need some people with some...
GMod National Rally 2 ! - Now with less drama.
The Garry's Mod National Rally is back! For a second run, but this time in 2015.
Server information will be listed below.
Connection (Home hosted)
Download: 150mbps (Usually hangs around 80-120)
Upload: 12 mbps
Internet 10/100/1000
Computer hosted on (I got an old server from a friend for free, might as well put it to use)
4GB DDR2 (Upgrading to 8GB of DDR2)
So guys, since I'm well educated on the world of 3 D, I need some ideas on what types of models or props I should import into garrysmod. I literally know how to import any 3D model into the game with support for specular, bump and normals maps and support...
Anyone know a way to basically return everyone to "user" on my servers through Fadmin?
I'm going to start looking for new staff, lol.
Does anybody else agree with this? Every server is the same. People have to code in "configLua" for it to be a good script.I understand a business is a business, but in my opinion it killed or help kill Garry's Mod.
No hate on any of the...
Are there any solid statistics on the amount of CPU, RAM and bandwidth each slot uses for GMod?
I'm trying to make an informed decision on which dedicated server I should buy, but with no statistics available without just getting a dedicated server, it...
I've looked everywhere and can't find a face posing walking dead game season 2 Clementine. Anyone??
Hello, I am new to lua and had the unoriginal idea of making a MOTD with a blurred base texture. I'm not sure if this is possible but it would be much obliged if you could help me out in one way or the other.
(User was banned for this post ("DEVELOPER...
If anyone has the gamemode they would like to share with me, anywhere I can get the gamemode or are willing to code the gamemode.
Any of this, add me on steam or just reply! (Steam name: basicallymental).
Moved to help sorry :(
I'm interested in star wars RP and I'm looking for Havoc squadron player models for use on a server. If you know where I can find them please post in replies. Thank you, greatly appreciated.
Some Pictures of Havoc
Havoc standard troop
Hey, i was wandering how to make the server only accept people if they have clocked over 250 hours on gmod. I know its possible because ive seen it on many servers.
To be honest, there are so many bad servers. But I just want to see what you guys think are the worst!
My number one worst, is Firestorm Gaming. The Owner cannot properly run a server. He is too afraid to admit it. When I posted all of the things wrong...
Hello, i am currently having a problem with Garry's mod and most source games, the problem is i cannot join some/most servers because my game says "server is not responding" like i have no internet connection.
but, the server IS online and my friends are...
Hey guys, so a long time ago I played on this DarkRP server called "Exorsus gaming." Crappy admins, crappy owner, but the redeeming factor was that it had a mod where there were trains that would go past all of the tracks on the map every once in a while...
Hi I've been looking for a train addon for darkrp where the train has shipments on it and the players have to jump on the train take a shipment off of it and get off in time. I've seen this addon on codehire a while back but, now that codehire is down I...
So I have problem with changing pain and death sounds.
In terrortown/gamemode/init.lua (line 219-225) I wrote this:
resource.AddSingleFile( "deathsounds/animedeathsound1.wav")
When I went to open one of my builds I noticed that a lot of my props are missing. Why is this?
Say, does anyone remember back when garry's mod had tower defence severs?
What happened to it and the servers running it?
I just can't seem to find them anymore and i have no way of making one because i have 0 coding experience.
So today i was modding GTA V with my brother and then suddenly a person adds me, everything seems legit, and their a real person, how ever, what they asked me for was funny as fuck, they have about 900 hours on gmod, now how ever their account is private...
I have a Garrys Mod Server and I have some addons like ULX and Ulib.
But I can go through some blocks like crates.
What should I do?
Please HELP!
So I uploaded a model on the Gmod workshop and want to turn it into a simple NPC not SNPC. The NPC base or whatever you want to call it is "npc_cscanner". The NPC I am trying to make is pretty much a re-modeled city scanner. When I launch the game and...
I recently saw that Garry's Mod 2 was in development, and I haven't found any answer on Google so I wanted to ask you some informations.
Here are my questions :
1. Will the server run on multi-core processes, unlike...
Bored and want a new gamemode to play.
Hi guys so me and some friends were playing on the questionable ethics TTT server and here are my overall impressions on it.
As we entered the server and started to play we all noticed that a younger player was being bullied because of his voice, this...
Hey i got a server and a perp mod when i joining the server i cant see keys,fists and also the F1 and F2 menu is not working (i uploaded the sql file) and the ncp's is not working to, when i running the mod on my pc its working fine!
anyone know why its...
So its been basically socially chosen that 80 unites is the "Standard" guage for rails in gmod
In source engine 1 unit == one inch and this is reflected by the players dimensions, 6 feet high by a foot and ~a half.
However the dimensions of the rails...
Old : http://facepunch.com/showthread.php?t=1235980
Old one got closed for reaching 5000 posts.
What shit gets you mad in Garry's Mod?
I need help alot with my GMOD Server!
There is a few things done to it but some things need doing.
If anyone can help me make this server then comment below!
Thank You!
GMod National Motorsport
GMod National Motorsport is another event in the 'GMod National' line.
We will be doing laps on a circuit, and we'll be doing a hillclimb....
I thought this would fit here better than fast threads.
I may have the most play time ever as I just hit. 1300 hours.
1300 hours = 54.1666667 days. Oh lord
Steam Workshop
darkrp admins
shitty bhop/surf servers
dead sassilization
no tower defense
long load times
Hey guys, do you have an ideas on what gamemode I should host on my gmod community? (We have 3 servers)
Thanks in advance
Basically every bhop server I go to nowadays has either hacked/configured times and/or people spamming the N word and trolling.
I just want a bhop server that has GOOD maps (Don't care if they're easy or hard, just not bad maps *cough* *cough* frankerz...
Hello there! I wanted to talk about the future of Gmod and things like Source 2 and whether or not there will be a retail version.
To start i would like to know your thoughts about a retail release and if you think Valve might make some sort of...
I've looked everywhere for a spider-Man 2099 ragdoll. The one listed on steam has a broken download link and I can't find one anywhere else. Does anybody know where I can download one?
This is the broken workshop download page...
Hey there!
Recently I installed Windows 10, and installed some upgrades on my PC. Ever since then my Garry's Mod has been having weird frame drops. Dips from 180 - 45 in a matter of seconds.
Here is my current PC:
Intel Core 2 Quad Q6600 2.40GHz
List your favorite and least favorite gamemodes, and explain why you like/hate them.
(Sorry if this has been done before.)
(Please do not say their username just make up one like idk, bob1234) So i've seen alot and I mean alot of horrible admins on gmod servers. But i want to hear about horrible admins you have encountered, please tell me your story.
I mean i have been playing Garry's Mod for a very long time, Long enough to see Coderhire go offline :'( and Scriptfodder come online. Personally i didn't make another Coderhire like website because i saw scriptfodder come online. But since it came online...
This thread is for posting of ACF beta content, and general ACF chat.
Problems? Ideas? Gripes? Put 'em here.
Kafouille's been gone, and he's the only one who can update the svn- so this thread will be used to post ACF releases that...


text:
November 26, 2015
DECATUR, Tenn. (AP) -- A woman who called the Meigs County Sheriff's Office to report she had shot her boyfriend has been charged with voluntary manslaughter.
Sheriff's Lt. Scotty Wiggins told the Chattanooga Times Free Press Dede Neely fatally shot Brian Cofer Wednesday morning at a home in the Georgetown community.
Neely told police Cofer had pulled a gun the night before and threatened to kill her if she left. Neely said Cofer tried to attack her with a rifle the next morning, but put the weapon down. Neely said she picked it up and shot him.
Authorities say at the time of the shooting a 3-year-old daughter Neely had with Cofer was in the home as were Cofer's father and teenage brother.
Neely was held in jail in lieu of $25,000 bond.
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Welcome to the access law website
IMPORTANT - legal aid
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text:
Originally Posted by polyglot
Do not open them! They are packed under nitrogen in the factory and far better sealed and protected from oxygen than you can manage at home. Glass, marbles, butane and all that bullshit is useful only once you have opened containers..
A glass container would be diffusion-tight in contrast to an original plastic container.
It is possible (though a hassle) to refill chemistry from one container to another without losing a nitrogen topping.


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Yirmin, "squeezed from the right scrotum"! LMAO how true.
A.S. Winning these little American racing series means little in F1, look at Bourdais, 4 times Champ Car champion and useless in F1.
Or Michael Andretti, a complete tool in an F1 car, McLaren would have been better letting a spanner drive the car!


text:
Fantastic Four reboot-- Casting, Rumors, Pix, ect;
View Single Post
February 24 2013, 03:45 AM
Location: Arizona, USA
Re: Fantastic Four reboot-- Casting, Rumors, Pix, ect;
Nick Ryder wrote:
Well frankly the idea that the FF were the "only" super heroes around or the X-Men as the only super powered beings around always struck me as somewhat odd in that they all came from the same "universe". I can understand the whole "well you can't mention Iron Man in a Fox movie" and I can take it to a point. Sure, they don't have the rights to the character, BUT that doesn't mean you can't name drop or casually mention that "Oh and Stark's been working on some exo-armor concepts and needs my input I'll be home before dinner Sue darling"
It's a name - big deal.
Well, to Marvel it is. I'm pretty sure in situations like this you can't even mention a character without having some kind of a license to use them.
They say a little knowledge is a dangerous thing, but it is not one half so bad as a lot of ignorance. - Terry Pratchett,
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Secrets of Success< Secrets of Success Main Page
All Secrets submitted by MOMMACASSEY
Please note these are member-submitted secrets and are not reviewed by SparkPeople's health experts for accuracy. If you feel a secret is inappropriate, please click the "Report Inappropriate Secret" on the secret's page and our staff will review it.
Cereal portions are a lot smaller than the bowls we eat out of! When I have actually measured out the serving sizes, they are so small I end up deciding to double it--not like I need the calories! An easy fix is to pour the milk into the bowl first. The cereal seems to settle into an empty bowl, but if the milk is already there, the bowl appears more full, and that makes it easier to accept the portion size as a decent meal.
- Submitted by MOMMACASSEY 5/29/2013 in Healthy Eating | 0 Comments
While I am at work, I will have an occasional cup of coffee. However, I keep a tall mug of water on my desk at all times so that I can sip on it throughout the day. As soon as I have swallowed the last, I get up and go get more water. If I did not do this, I would not drink nearly as much water as I do throughout the day. A bonus is that the stainless steel inside of the mug I use adds no flavors to the water I'm drinking--I hate when my drink tastes like some other, absorbed flavor!
- Submitted by MOMMACASSEY 4/20/2011 in Healthy Eating | 1 Comment
Kids Play With Their Food and Eat Less
My child has to be coerced to eat--there are certain foods she enjoys eating herself, but she runs off & plays between bites. I stretch out my meals so I'm taking bites when she is. I eat less & feel more full. My portions are bigger than hers, but my bites are, too. Plus, I've learned the value of making finger foods fun, healthy, & kid-friendly at the same time. I want her to eat healthy, & it's impossible for me to cook healthy for her but not for me, because she wants whatever I have!
- Submitted by MOMMACASSEY 4/14/2011 in Healthy Eating | 0 Comments
Page: (1 total)


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Enhancement Details
Select one or more of the following search criteria to get information on particular product enhancements.
Items found: 6
If you don't have the codec already, you should be prompted to install it. If not, you can download it here.
Analyzing Effects of Changed Nominal Dimensions
Edit nominal dimensions in the Tolerance Analysis Powered by CETOL Technology dialog box, to analyze the effects of changes.
Closing Gaps in a Model
You can automatically close gaps in the model.
Enhanced Workflow in Tolerance Analysis
You can delete and substitute components and dimensions in an existing analysis.
Tolerance Analysis Measurement Saved as a Feature
You can save a Tolerance Analysis measurement as a feature with the feature parameters representing Tolerance Analysis results.
User-Defined Dimensions
You can create annotation dimension on the fly if there are no existing dimensions or annotations.
Virtual Components
You can create placeholder components to simulate dimension properties that are not accounted for in the CAD model.


text:
Are you Asae Oura?Claim your profile
Publications (17)25.97 Total impact
Article: A Prospective Study of Factors Associated with Risk of Turnover among Care Workers in Group Homes for Elderly Individuals with Dementia[Show abstract] [Hide abstract]
ABSTRACT: Objectives: A prospective study was conducted to assess factors associated with risk of turnover among care workers in group homes (GHs) for elderly individuals with dementia. Methods: In January, 2010, 51 out of 238 GHs in Sapporo City responded to our request for participation in a survey. During February and March of 2010, 438 out of 700 care workers (62.6%) in the GHs returned a completed questionnaire to us. They were followed up with until March of 2012, and 395 subjects (90.1%) responded to our survey, which was conducted twice. Over the course of 2 years, 91 subjects left their jobs. Cox's proportional hazards model was used for analysis, adjusting for gender and age. Results: Less provision of social support by supervisors, colleagues, family or friends was significantly associated with increased risk of turnover. Financial aids for off-the-job training was marginally significantly associated with reduced risk of turnover. Longer duration of working for frail elderly care was significantly associated with decreased risk of turnover. Habitual smoking was significantly associated with increased risk of turnover. Conclusions: Social support in the workplace may be important to reduce the risk of turnover in care workers at GHs. Further prospective studies are required in other areas in Japan.Journal of Occupational Health 10/2013; 55(6). DOI:10.1539/joh.12-0265-OA · 1.11 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Objectives The aim of this study is to clarify risk factors of urinary incontinence (UI) in elderly Japanese women. Methods We randomly selected 1600 women, aged between 65 and 74 years, from the resident registration of Sapporo City and we analyzed 746 women, who responded twice on surveys in 2010 and 2011. UI was defined as frequent UI when it occurred at least once a week in both 2010 and 2011. ResultsMultivariate logistic regression analysis revealed that the past maximum body weight, smoking index, past history of bladder disease or hemorrhoids, and the participant's mother's history of UI were significantly associated with an increased risk of UI. Conclusion Lifestyle habits such as weight gain and smoking habits were associated with an increased risk of UI in Japanese women. Further study is needed, because modification of these risk factors may possibly result in reducing risk of UI.Lower urinary tract symptoms 07/2013; 6(2). DOI:10.1111/luts.12026 · 0.30 Impact Factor
Article: A Cross-sectional Study on Association of Work Environment, Coping Style, and Other Risk Factors with Depression among Caregivers in Group Homes in Japan.[Show abstract] [Hide abstract]
ABSTRACT: A cross-sectional study was conducted to explore the potential association between work environment and/or stress coping ability, and depressive status among caregivers working for "group homes (GHs)" in Japan. In January 2010, 438 out of 700 caregivers working at GHs in Sapporo City returned completed questionnaires to us. The questionnaires consisted of the Center of Epidemiological Scales-Depression, items about worker's attributions, Ozeki's coping scale, and so on. An analysis using a logistic regression model was used to find the associations adjusting for gender and age. Subjects who were 45 yr or older, had a spouse, had job training, a standard workload and scored high in emotion-oriented coping were significantly associated with a decreased risk of depression. Subjects who were less proud of their job, less willing to continue care for the frail elderly and had fewer acceptances by their supervisors or colleagues for consultation were significantly associated with an increased risk of depression. This study supports our hypothesis that there can be possible variables among individual factors, work environment and/or coping style for stress which may modulate a risk on the depressive status of caregivers.Industrial Health 05/2013; 51(4). DOI:10.2486/indhealth.2012-0204 · 1.12 Impact Factor
Article: [Assessing weight and other factors associated with the urinary incontinence of elderly women: a cross-sectional study][Show abstract] [Hide abstract]
ABSTRACT: Urination diseases--particularly urinary incontinence (UI)--strongly affect individuals' quality of life (QOL). It is important to maintain overall QOL in societies with an increasing number of long-living elderly people, such as in Japan. Thus, this study aims to clarify the risk factors concerning UI in elderly women. For this study, we obtained the approval of the Ethical Committee of Sapporo Medical University. In October 2010, we randomly selected 1,600 women, aged between 65 and 74 years, from the registry of Sapporo city residents and mailed out a self-administered questionnaire. Our questionnaire consisted of five subsections (24 items in total), including fundamental attributes, health condition, past history, present illness, and the International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF). The self-reported prevalence of UI was defined as frequent UI being at least once a week. A logistic regression analysis was used to assess the risk factors for UI. Among those who received the questionnaire, 802 women (response rate: 50.1%) returned a completed questionnaire with written informed consent. Their mean age (+/- standard deviation) was 69.8 +/- 2.6 years, and the prevalence of UI was found to be 29.7%. The mean scores (+/- standard deviation) of ICIQ-SF were 1.7 +/- 2.9 for all participating subjects, and 5.55 +/- 2.50 and 0.09 +/- 0.53 for the groups with or without UI, respectively. The results of the multiple logistic regression analysis were as follows: the odds ratio (95% confidence interval) was 1.94 (1.32, 2.85) for a past maximum weight heavier than or equal to 60 kg; 1.98 (1.18, 3.32) for a smoking index more than or equal to 300; 2.54 (1.47, 4.39) for a poor self-perceived health status; 1.62 (1.09, 2.40) for having a past history of bladder diseases; 1.72 (1.11, 2.69) for having a past history of hemorrhoidal disease; and 2.05 (1.36, 3.10) for a history of UI in one's mother. The self-reported prevalence rate of UI was 29.7% in women aged 65-74 years. In the future, we plan to conduct a follow-up survey to further clarify the risk factors of UI that have been implicated in this cross-sectional study.[Nippon kōshū eisei zasshi] Japanese journal of public health 02/2013; 60(2):79-86.
Article: A demographic description of caregivers in Sapporo Japan : male caregivers are here to stayJapanese Journal of Health and Human Ecology 01/2013; 79(1):3-9. DOI:10.3861/jshhe.79.3
Article: Relationship of Hobby Activities With Mortality and Frailty Among Community-Dwelling Elderly Adults: Results of a Follow-up Study in Japan[Show abstract] [Hide abstract]
ABSTRACT: The proportion of Japanese adults aged 65 years or older is predicted to increase; thus, it is becoming more important to identify factors that influence health status among elderly adults in Japan. We conducted a follow-up study of community-dwelling elderly adults to assess the relationship of hobby activities with mortality and incident frailty. We randomly selected 3583 individuals aged 65 to 84 years from the residential registries of 7 study areas in Hokkaido, Japan in August 2007. Among them, 1955 (54.6%) returned completed questionnaires with written informed consent by mail. The baseline assessment questionnaire comprised questions on current and past involvement in hobby activities, self-perceived health status, smoking and drinking habits, and body height and weight. Questions on hobby activities were from 4 categories: solitary physical, group physical, solitary cultural, and group cultural activities. We later conducted a follow-up survey of the participants to ascertain all-cause mortality and incident frailty. A Cox proportional hazards model was used for analysis of data from September 2007 to May 2010. After adjusting for potential confounders, the risk of incident frailty among respondents participating in solitary physical activities was significantly lower than in those who did not participate in such activities (hazard ratio = 0.57; 95% CI 0.33, 0.99). Furthermore, the risk of incident frailty among respondents taking part in group cultural activities was significantly lower than in those who did not participate in such activities (0.41; 0.19, 0.87). These findings may be important for programs that seek to promote good health among elderly adults.Journal of Epidemiology 05/2012; 22(4):340-7. DOI:10.2188/jea.JE20110057 · 3.02 Impact Factor
Article: What is a common factor in different areas?: The case of depression among caregivers of the frail elderly in Sapporo and Kochi, Japan[Show abstract] [Hide abstract]
ABSTRACT: Depression affecting caregivers is a risk factor for discontinuing at-home care. The present cross-sectional study was conducted to evaluate factors related to depression among family caregivers. A cross-sectional study was conducted to evaluate factors related to depression among family caregivers of the frail elderly. The study included caregivers whose relatives received periodic visits from a nurse from one of seven Sapporo-based home-visiting nursing service stations in October 2008 or from one of nine Kochi-based home-visiting nursing service stations between June 2009 and March 2010. Caregivers were asked to answer a self-administered questionnaire about various factors that might affect their depression and to complete a Center for Epidemiologic Studies Depression Scale evaluation. We analyzed 127 pairs of responses in total. After we adjusted the results for the caregivers' gender and age, the factors that decreased the risk of depression in the Sapporo group related to relieving the stress that results from being a caregiver (OR = 0.12, 95%CI = [0.03, 0.42]) and the ability to independently pay more than ¥10 000 for part of care service costs (OR = 0.17, 95%CI = [0.05, 0.58]). In contrast, feeling ill was a factor that increased caregivers' risk of depression in the Kochi group (OR = 4.23, 95%CI = [1.29, 13.9]), but not in Sapporo group (OR = 2.53, 95%CI = [0.91, 7.08]). Caregivers in the Sapporo group were more likely to feel ill (P < 0.01) than their counterparts in Kochi. The Japanese government should take measures to reduce caregivers' depression. Otherwise family members will increasingly discontinue to provide at-home care.Geriatrics & Gerontology International 10/2011; 12(2):230-7. DOI:10.1111/j.1447-0594.2011.00754.x · 2.19 Impact Factor
Chapter: Intervention Programme for Promoting Physical Activities in the Citizens of Sapporo City, Japan[Show abstract] [Hide abstract]
ABSTRACT: In accordance with nutritional improvement, the environmental sanitation, and advance in medical technology after World War II, Japan has achieved almost the longest lifespan in the world. This change has two implications. Firstly, this has led to a drastic increase in elderly population in Japan. Secondly, a part of Japanese has faced the problem of over-nutrition due to e.g. an inappropriately increased intake of total energy and total fat, a more sedentary lifestyle or insufficient physical activity in association with use of various mechanic devices or transportation including a car in their life. Their lifestyles are not only associated with an increased risk of lifestyle-related morbidity, but also with increased medical expenditure. Some studies reported higher prevalences of obesity, glucose intolerance (Kawamori 2002), hypertension (Ueshima et al. 2000), and/or hyperlipidemia (Koba and Sasaki 2006) in recent years as compared to past. These lifestyles factors and morbidity are suggested as possible risk factors for a higher mortality related to cardiovascular diseases and certain types of cancer.Health in Megacities and Urban Areas, First Edition edited by Alexander Kraemer, MMH Khan, Frauke Kraas, 01/2011: chapter 5: pages 75-86; Springer., ISBN: 978-3-7908-2732-3
- [Show abstract] [Hide abstract]
ABSTRACT: To investigate the relationship between dietary intake of minerals and the risk of chronic obstructive pulmonary disease (COPD), a case-control study was conducted in central Japan. A total of 278 referred patients (244 men and 34 women) aged 50-75 years with COPD diagnosed within the past four years and 340 community-based controls undertook spirometric measurements of lung function. A structured questionnaire was administered face-to-face to obtain information on demographics and habitual food consumption. Dietary intakes of six major minerals were derived from the Japanese food composition tables. The COPD patients had lower habitual energy-adjusted intakes of calcium, phosphorus and iron than controls, but not sodium. A significant reduction in prevalence of COPD was observed for calcium, with adjusted odds ratio 0.65 (95% confidence interval: 0.37- 0.98) for the highest level versus lowest level of intake. A high iron intake was also inversely associated with the COPD risk. In conclusion, an inverse association was evident between dietary calcium intake and the risk of COPD for Japanese adults.Asia Pacific Journal of Clinical Nutrition 12/2010; 19(4):572-7. · 1.70 Impact Factor
Article: Factors Related to Hospitalization or Institutionalization among the Frail Elderly in Sapporo, Japan01/2010; 11(1):65-68. DOI:10.5939/sjws.10008
Article: Ecological studies on influenza infection and the effect of vaccination: Their advantages and limitations[Show abstract] [Hide abstract]
ABSTRACT: Ecological studies lack the ability to control for the effects of confounding factors. The findings of a linear relationship between average exposure and disease frequency in ecological studies do not imply that such a linear relationship will be present at the individual levels. This is known as the 'ecological fallacy'. Despite these limitations, ecological studies may be the best approach to studying exposures that are easier to measure at the group rather than the individual level because most ecological studies make use of routinely collected data. They are also useful for monitoring the effectiveness of population interventions such as vaccination programs, health education campaigns and mass screening programs. Thus, ecological studies are useful epidemiologic tools for public health surveillance if we know their limitations and interpret their results carefully. Ecological studies often help to generate hypotheses, although they rarely provide a strong test of a causal hypothesis.Vaccine 07/2008; 26(50):6470-2. DOI:10.1016/j.vaccine.2008.06.037 · 3.62 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Confounding is a kind of bias which occurs in a research. Confounding is less frequent in randomized controlled trials (RCT) for evaluation of influenza vaccines. However, there are obstacles or difficulties in conducting RCT for evaluation of influenza vaccines, particularly, in the elderly people. Therefore, a retrospective or prospective cohort study has been primarily performed to evaluate effectiveness of influenza vaccine in elderly people. Confounding by indication or other confounding exist in most observational studies. Accordingly, at the stage of designing or analyzing a study, confounding should be controlled with a restriction, matching, stratified or multivariate analysis technique.Vaccine 07/2008; 26(50):6459-61. DOI:10.1016/j.vaccine.2008.06.040 · 3.62 Impact Factor
Article: Comparison of the Clinical Features of Japanese Patients with Primary Biliary Cirrhosis in 1999 and 2004: Utilization of Clinical Data When Patients Applied to Receive Public Financial Aid[Show abstract] [Hide abstract]
ABSTRACT: In Asia there are few reports considering time intervals in the examination of clinical features of primary biliary cirrhosis (PBC). Therefore, we tried to compare the characteristics of patients with PBC in two different years. In two fiscal years (1999 and 2004), 9,761 and 13,142 patients with symptomatic PBC were registered to receive public financial aid from the Ministry of Health, Labour and Welfare of Japan, respectively. For the present study, clinical data from 2,127 patients in 1999 and 6,423 ones in 2004 were available. We compared the data in the two different years, including sex, age, major symptoms, and laboratory data. Male/female ratios were the same figure (0.13 for 1999 and 2004). The median age was significantly older in 2004 than in 1999 (59 years for 1999, 63 years for 2004, respectively, p<0.01). Jaundice and esophageal varices were found significantly less frequent in 2004 than in 1999 (p<0.01 for each item). Levels of total bilirubin,gamma-glutamyl transpeptidase (gamma-GTP), total cholesterol, and immunoglobulin M were significantly lower in 2004 than in 1999 (p< 0.02 for total bilirubin, and p<0.01 for other each item). The positive rate of antimitochondrial antibodies was significantly higher in 1999 than in 2004 (87.0% for 1999, 83.5% for 2004, respectively, p<0.01)). Complicated autoimmune diseases such as Sjögren's syndrome, rheumatoid arthritis, and chronic thyroiditis were found significantly more frequent in 2004 than in 1999 (p<0.01 for each item). Among the patients with PBC in 2004, an increase in median age, and lower levels of laboratory data such as gamma-GTP have been found compared to 1999. These results may show an accumulation of patients with better prognosis and the recent medical progress in controlling patients with PBC.J Epidemiol 2007; 17: 210-214.Journal of Epidemiology 11/2007; 17(6):210-4. DOI:10.2188/jea.17.210 · 3.02 Impact Factor
Article: Depression among caregivers of the frail elderly in Japan before and after the introduction of the Public Long Term Care Insurance System[Show abstract] [Hide abstract]
ABSTRACT: Cross sectional studies were conducted in five towns in Japan before and after the introduction of the Long-term Care Insurance System (LTCIS), in order to evaluate the factors relating to depression among family caregivers for the frail elderly. Depressive caregivers were more likely to consult with their doctors, to be in poor health, to care for demented elderly with behavioral disturbances than the non-depressive caregivers both before and after the LTCIS. Before LTCIS, depressive caregivers were more likely to attend to the elderly for more than 16 hours per day than their counterparts. After the LTCIS, depressive caregivers were more likely to be a spouse, to care for a frail elderly male, and less likely to be able to go out without accompanying the elderly than their counterparts. Even after the introduction of LTCIS, half of the caregivers were depressive. It is suggested that a government agency should be created to support not only the frail elderly but also their caregivers.Zeitschrift für Gerontologie + Geriatrie 05/2007; 40(2):112-8. DOI:10.1007/s00391-007-0412-z · 0.81 Impact Factor
Article: Factors Related to Institutionalization among the Frail Elderly with Home-Visiting Nursing Service in Japan[Show abstract] [Hide abstract]
ABSTRACT: A dramatic increase in the number of elderly people in Japan has led to a concurrent increase in the number of frail elderly in need of care. It is estimated that the number of frail elderly will reach 3.9 million by the year 2010. Family members are often both physically and mentally burdened with caring for the frail elderly. The present study was conducted to identify the risk factors for institutionalization among the frail elderly receiving in-home care in Japan. Copyright (c) 2006 S. Karger AG, Basel.Gerontology 02/2006; 52(1):66-8. DOI:10.1159/000089828 · 3.06 Impact Factor
Article: Smoking, drinking, sleeping habits, and other lifestyle factors and the risk of systemic lupus erythematosus in Japanese females: Findings from the KYSS study[Show abstract] [Hide abstract]
ABSTRACT: Many risk factors have been proposed for systemic lupus erythematosus (SLE). However, there is little information about the relationship between lifestyles and SLE in Japan. Two case control studies were conducted in Kyushu, southern Japan, and in Hokkaido, northern Japan, to examine the relationship between lifestyles and development of SLE in females. The participants were 78 patients and 329 controls in Kyushu and 35 patients and 188 controls in Hokkaido. Smoking was associated with an increased risk of SLE after adjusting for age in both regions. However, in Hokkaido, this association between smoking and SLE did not reach statistical significance after adjusting for alcohol drinking. The present study suggests that smoking may increase the risk of SLE among Japanese females.Modern Rheumatology 02/2006; 16(3):143-50. DOI:10.1007/s10165-006-0474-6 · 2.40 Impact Factor
Article: Burden among caregivers of the frail elderly and its correlation with the introduction of a long-term care insurance system for the elderly in fourth year[Show abstract] [Hide abstract]
ABSTRACT: The present study was conducted in October 2003 to investigate the factors related to psychological stress and the heavy burden of caregivers taking care of frail elderly persons in the northern part of the Onga district, Fukuoka Prefecture, Kyushu, Japan. A total of 40 caregivers were assessed using a self-administered questionnaire involving the Japanese version of the Zarit Caregiver Burden Interview (J-ZBI) and thus described their own caregiving situation. J-ZBI research had been done on a yearly basis for the previous 5 years and the results of each investigation regarding Center for Epidemiologic Studies Depression Scale (CES-D), and incidence of depression, ADL and dementia were compared prior to and after initiation of public long term care insurance for the elderly (LTCI). Compared to caregivers with a middle or light burden, heavily burdened caregivers were more likely to have a higher J-ZBI, and CES-D. Compared to caregivers with a light burden, heavily burdened caregivers were more likely to 1) be in poor condition, 2) complain of their economical situation, 3) spend a longer time with care talker. Compared to caregivers with a middle burden, heavily burdened caregivers more frequently used short-stay services (i.e., temporary nursing home assistance). J-ZBI and ADL were compared to before LTCI, and 4 years after LTCI had been started. Scores for both were lower after 4 years. Compared to before LTCI, day-care and day service were used less frequently 3 and 4 years after LTCI. After LTCI, caregivers showed a lower J-ZBI score, but the incidence of depression among caregivers was higher, compared to the general public. It is suggested that a government agency should be created to support not only the frail elderly but also their caregivers.Nippon Ronen Igakkai Zasshi Japanese Journal of Geriatrics 08/2005; 42(4):411-6. DOI:10.3143/geriatrics.42.411
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