text:
Page 1
DNA damage intensity in fibroblasts in a 3-dimensional collagen matrix
correlates with the Bragg curve energy distribution of a high LET
Departments of1Cell Biology,2Internal Medicine,3Hamon Center for Therapeutic Oncology Research, and5Division of
Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas,
Texas, and4Brookhaven National Laboratory, Upton, New York, USA
(Received 9 March 2009; Revised 2 August 2009; Accepted 10 September 2009)
fibroblast cells embedded in a 3-dimensional (3-D) collagen tissue equivalents was investigated using antibodies to the DNA
damage response proteins gamma-histone 2AX (g-H2AX) and phosphorylated DNA-PKcs (p-DNA-PKcs).
Materials and methods:
3-D tissue equivalents were irradiated in positions across the linear distribution of the Bragg curve
profiles of 307.7 MeV/nucleon, 556.9 MeV/nucleon, or 967.0 MeV/nucleon56Fe ions at a dose of 0.30 Gy.
Patterns of discrete DNA damage streaks across nuclei or saturated nuclear damage were observed, with saturated
nuclear damage being more predominant as samples were positioned closer to the physical Bragg peak. Quantification of the
DNA damage signal intensities at each distance for each of the examined energies revealed a biological Bragg curve profile
with a pattern of DNA damage intensity similar to the physical Bragg curve for the particular energy. Deconvolution
microscopy of nuclei with streaked or saturated nuclear damage pattern revealed more details of the damage, with evidence
of double-strand breaks radially distributed from the main particle track as well as multiple discrete tracks within saturated
damage nuclei.
These 3-D culture systems can be used as a biological substrate to better understand the interaction of heavy
charged particles of different energies with tissue and could serve as a basis to model space-radiation-induced cancer
initiation and progression.
The DNA double-strand break (DSB) damage response induced by high energy charged particles on lung
Keywords: heavy ion irradiation, DNA damage, DNA double-strand break repair, 3-D tissue equivalents
Astronauts on prolonged deep-space missions, such
as a voyage to Mars or extended stays on the Moon,
will be traversed by a mixed field of particles
consisting mainly of intermittent radiation from solar
particle events and chronic radiation from high
energy and high mass (HZE) charged particles
(Schimmerling 1992, White and Averner 2001).
Risks to astronauts from exposure to this unique
radiation environment are of great concern to the
National Aeronautics and Space Administration
(NASA) given the potential for protons and HZE
particles to contribute to end organ damage such as
central nervous system degeneration, cataract for-
mation, and carcinogenesis (Cucinotta et al. 2001a,
Rastegar et al. 2002, Raber et al. 2004, Cucinotta
and Durante 2006, Durante and Cucinotta 2008).
Although they are a small percentage of the overall
space radiation spectrum, HZE particles may be the
greatest contributors to the risk for both carcinogenic
and non-carcinogenic effects. This is thought to be a
result of the complex type of DNA damage induced
by HZE particles, in which particles traverse cells
and tissues and deposit their energy not only
through their linear trajectory but also through
Correspondence: Dr Andres I. Roig, MD, Department of Cell Biology, 5323 Harry Hines Boulevard, Dallas, TX 75390-9039, USA. Tel: þ1 214 648 6318.
Fax: þ1 214 648 8694. E-mail: firstname.lastname@example.com
ISSN 0955-3002 print/ISSN 1362-3095 online ? 2010 Informa UK Ltd.
Int. J. Radiat. Biol., Vol. 86, No. 3, March 2010, pp. 194–204
Page 2
secondary low linear energy transfer (LET) delta rays
that cause indirect ionization of the DNA in cells
along the radial distribution to the particle trajectory
(Cucinotta et al. 1998). Therefore, the down-
stream biological effects of an HZE particle can be
spread many cell dimensions away from its linear
An important component in the planning of a
mission to Mars or an extended stay on the Moon is
the design of shields that adequately protect the
astronauts from cosmic radiation. Conventional
aluminum shielding is thought to worsen the
radiation effects inside the spaceship hull and
recently has been shown to produce DNA lesions
that are less amenable to repair compared to more
favourable polyethylene shielding (Wilson et al.
2001, Mukherjee et al. 2008). Ideal shielding would
be hydrogen-rich compounds that fragment heavy
charged particles into less ionising species, therefore
decreasing the DNA damage burden and reducing
cancer risk. Historically, model calculations have
been implemented to design appropriate shielding
but it is becoming increasingly accepted that more
biological data is needed to validate these mathema-
tical models (George et al. 2003, Shavers et al. 2004,
Wilson et al. 2004, Walker et al. 2005). Recent
studies (Desai et al. 2005, Wu et al. 2006) describe
DNA damage or micronuclei induction, respectively,
across the distribution of an iron particle's physical
Bragg curve (300 MeV/nucleon, 600 MeV/nucleon,
1 GeV/nucleon) in order to better understand the
biological effects of particles as they stop and release
their energy in a living substrate. An important
concept described in these studies is that physical
Bragg curves do not necessarily represent the
biological damage caused by a particle across its
trajectory since biological effects are dependent on
the track structure of both primary and secondary
radiation. The Desai et al. and Wu et al. studies were
also performed in monolayers of tissue culture cells,
and while more data such as this is needed,
examining similar biological endpoints in tissues is
warranted. The recent modeling of nuclear fragmen-
tation and HZE particle distributions in 3-dimen-
sional (3-D) simulations of homogenous tissues
attests to this concept (Ponomarev and Cucinotta
2006). One of the main concerns that cannot be
addressed in 2-dimensional (2-D) cellular models is:
Does an HZE particle traversing through a tissue
lead to an increased flux of lighter particles that may
cause chromosomal alterations or mutagenesis rather
than killing a cell outright? This is particularly true
for low fluence exposures such as missions to Mars
where over a three-year mission every cell in the body
would be traversed on average by a single HZE
particle (Cucinotta et al. 2001b). Such knowledge
will help in the design of spacecraft components and
shielding materials to limit such an effect. Further-
more, biological Bragg curves may be more useful to
modeling because there may be differences in the
shapes of Bragg curves that depend upon the
biological endpoint being assayed. This point was
recently raised by Wu et al. (2006) who considered
bystander effects associated with micronuclei induc-
tion after HZE exposure. Modeling bystander effects
or the effects of delta rays in a 2-D tissue culture
setting may not be an adequate model for tissues.
Moreover, the concept of a microlesion, which is a
high LET phenomenon (Koniarek and Worgul
1992, Koniarek et al. 2004), cannot be modelled
adequately in a 2-D tissue culture setting. Having 3-
D tissue models will allow investigators to better
model cell death, cellular transformation and micro-
lesions and build mathematical models to infer HZE
particle effects in humans.
This study was conceived as an initial step in
examining DNA damage induction and repair in a
human tissue. While this 3-D tissue equivalent is not
a tissue per se, it does allow the examination of DNA
double-strand break (DSB) induction and rejoining
through a cross-section of human cells. Another
unique aspect of this study is the use of a dual marker
system for identifying surrogates of DNA DSB.
In prior studies g-H2AX was used to identify DNA
DSB repair foci. Recent publications have shown
that g-H2AX activation responds to chromatin
relaxation and can spread far beyond the initial lesion
in the DNA and is hence, not specific to DNA DSB
(Rogakou et al. 1998, Ward and Chen 2001,
McManus and Hendzel 2005, Han et al. 2006,
Cowell et al. 2007, Asaithamby et al. 2008). Other
studies have demonstrated that antibodies to g-H2AX
can give non-specific binding and have used other co-
localising proteins markers (such as CDKN1A) to
demonstrate the sites of DNA damage after heavy-
charged particle irradiation (Jakob et al. 2003). In this
study an antibody against phosphorylated DNA-
PKcs (pT2609) (p-DNA-PKcs) was used in addition
to g-H2AX to illustrate the DNA damage sites. Co-
localised measures of p-DNA-PKcs and g-H2AX are
considered to be highly specific for DNA DSB (Chan
et al. 2002, Chen et al. 2005).
The DNA damage examined in our experiments
was caused by charged particles impinging upon cells
in 3-D tissue equivalents (contracted fibroblast-
collagen gels) that have been placed at increasing
distance along the Bragg curve profile of56Fe charge
particles of different energies (307.7 MeV/nucleon,
556.9 MeV/nucleon, or 967.0 MeV/nucleon). The
intensity of DNA DSB signal in the form of co-
localised g-H2AX and p-DNA-PKcs (pT2609) were
obtained by measuring the mean grey scale values in
the nuclei of the cells within this 3-D cellular
56Fe particles were chosen because
DNA damage correlation with Bragg curve profiles
Int J Radiat Biol Downloaded from informahealthcare.com by UT Southwestern Medical Centre
For personal use only.
Page 3
while it is a particle with a very low fluence, it is
predicted to have the highest dose equivalence in
deep space and therefore thought to be most
potentially damaging to cells (Hartman et al. 2001)
A dose of 0.30 Gy was used to limit the number of
particle traversals to roughly one or fewer per cell.
Previous studies have focused on the biological
imaging of heavy-charged particle tracks by demon-
strating the various numbers of different proteins
localising at sites of DNA damage and illustrate
specific patterns of distribution for these proteins
(Jakob et al. 2003). In this study, the effect of a tissue
structure on particle trajectories, and thus the pattern
of DNA damage protein distribution in relation to
the particle's position in the Bragg curve, were
studied with finer detail by deconvolving individual
cell nuclei groups.
Materials and methods
Cell culture
Lung fibroblasts from a 61-year-old male (61ML,
American Type Tissue Culture collection [ATTC],
Manassas, VA, USA) were cultured in mono-layers
in X media (4 parts Dulbecco's Modified Eagle
Media [DMEM] to 1 part Medium 199; Hyclone,
Logan, UT, USA) with 10% cosmic calf serum
(Hyclone) until near confluence then sub-cultured,
collected, and mixed with rat tail collagen type 1 (BD
Biosciences, San Jose, CA, USA) to make three-
dimensional fibroblast-collagen gels at a density of
approximately 2.156106cells per gel. For a period
of seven days, the fibroblasts were allowed to
contract the collagen gels in 12-well plastic dishes
(Falcon, BD Biosciences) to a diameter of 5 mm and
thickness of 3–4 mm. These contracted gels (from
here on called plugs because of their shape) were
then transported to the NASA Space Radiation
Laboratory (NSRL) at Brookhaven National Labora-
tory inside tightly capped cryovials filled with serum
supplemented media.
Irradiation logistics
Once at the NSRL, plugs were subsequently placed
inside new sterile freezer vial plastic caps (containing
media and covered with a plastic film to prevent
media leakage and to hold the plugs in place),
positioned atop of a foam support, and fixed in the
path of56Fe charged particles. The beam size was set
to 20620 cm for all irradiations. The foam support
with the plugs was placed in the middle of the beam
path to ensure proper equal coverage of the plug's
cross-section by the beam. Positioning along the
Bragg curve was accomplished by varying the
amount of water-equivalent material (variable filter)
ahead of the plug. Plugs were linearly distributed at
specific positions along the Bragg curve profile for
56Fe of three different energies. These positions
along the Bragg curve were based upon pre-
determined distributions generated by the NSRL
Physics Support Team. Plugs exposed to the 307.7
MeV/nucleon beam were placed behind 0.0, 3.55,
3.80, 3.85, and 4.15 cm of variable filter material, 0,
9.5, 11.0, 11.225, 11.25, and 11.5 cm of material for
the 556.9 MeV/nucleon beam, and 0, 19.775,
20.775, 24.575, 24.925, and 25.175 cm of material
for the 967.0 MeV/nucleon beam. Beam intensity
and dose were monitored for each irradiation by
observing in real-time the progressive amount of
dose being delivered to the target sample. Each plug
sample was fixed with 10% neutral buffered formalin
30 min after irradiation.
Histology and staining
Plug samples were sectioned into 5 micron (m) slices
and attached to positively charged glass slides as
paraffin embedded sections. Sectioning was per-
formed parallel or transverse to the beam path. Slides
were then processed for immunofluorescent staining.
Briefly, slides were deparaffinised with xylene and
then dehydrated with ethanol series. Antigen retrie-
val was achieved by boiling slides in sodium citrate
solution (10 mM, pH of 6.5) for 20 min in a
conventional microwave oven. After a cool-down
period of 30 min in the sodium citrate, samples were
blocked for one hour with 1% bovine serum albumin
(BSA; Sigma, St Louis, MO, USA) for 30 min in a
378C incubator. Following blocking, slides were
incubated for 2 h at ambient temperature in a
primary antibody solution consisting of a phosphate
buffered solution (PBS) and monoclonal gamma-
histone 2AX (g-H2AX) antibody (pS139, ab22551;
Abcam, Cambridge, MA, USA) and polyclonal
phosphorylated DNA-PKcs (p-DNA-PKcs) anti-
body (pT2609, ab4194; Abcam). Species-specific
fluorochrome-labeled secondary antibodies were
used for primary antibody detection (Alexa Fluor
568 [quantum yield¼0.69], Alexa Fluor 488 [quan-
tum yield¼0.92]; Invitrogen, CA, USA). Incubation
was then performed for 1 h at ambient temperature.
40,6 diamidino-2-phenylindole
Laboratories, Inc., Burlingame, CA, USA) was used
for nuclear counterstaining.
200M; Go ¨ttingen, Germany) was used to visualise
the DNA damage and nuclear staining. Images for
relative intensity calculations were taken at 406
objective magnification. All images were taken at the
epifluorescencemicroscope (ZeissAxiovert
A. I. Roig et al.
Int J Radiat Biol Downloaded from informahealthcare.com by UT Southwestern Medical Centre
For personal use only.
Page 4
same exposure interval and were taken at random.
Four fields of view foreach section corresponding to a
sample at a specific position along the Bragg peak
were imaged. Each field of view contained on average
20–25 cell nuclei. Identification of the spatial location
in the Bragg curve for each sample was not made
known to the microscopist in order to decrease bias.
A Deltavision RT microscope and SoftWoRx
software (Applied Precision; Issaquah, WA) were
used to visualise and deconvolve, respectively,
individual nuclear images. Briefly, individual nuclei
were captured at exposures that were below the
saturation limit. Multiple optical sections were
obtained for each nuclei in order to create a three
dimensional distribution of DNA damage. The raw
image was then processed through the deconvolution
software to obtain a focused image of DNA damage
throughout each section of the nucleus. Imaris
software (Bitplane Scientific Solutions; St Paul,
MN, USA) was used to make three-dimensional
reconstructions of the DNA damage response.
Image processing
All 406 objective magnification immunofluorescent
images were processed using Image J (http://rsb.in-
fo.nih.gov). Significant damage signal was classified
as nuclear co-localisation of g-H2AX and p-DNA-
PKcs pT2609 (g-H2AX signal¼red, pT609¼green,
were calculated by carefully circling each damage
focus or streak within each nucleus with the Image J
drawing tool and obtaining the mean grey scale value
average for all the circled damage signals within each
field of view (between 20 and 25 cell nuclei). DAPI
nuclear counterstaining was manually removed from
all images before the damage foci intensity calcula-
tions to eliminate any additional signal. Four separate
intensity measuring experiments (between 80 and
100 total cell nuclei) were performed for each spatial
location in the Bragg curve for each particular energy
as well for non-irradiated controls. The average signal
intensity of the non-irradiated controls was sub-
tracted from the average intensity values correspond-
ing each examined distance. The intensity values for
each distance across the Bragg curve were normalised
to the 0.0 cm distance. Averages and standard errors
of means (SEM) were calculated from the four
separate intensity-measuring experiments for each
spatial location in the Bragg curve for each energy.
Damage signal intensities
Qualitative and quantitative analysis of DNA damage
Figure 1A shows the set-up of the contracted
collagen gels (plugs) and their positioning in the
beam path prior to irradiation, as described in the
methods. With the assistance of the NSRL physics
support team, the plugs were positioned at specific
locations in the particle's physical Bragg curve.
Figure 1B illustrates this process for the 307.7
MeV/nucleon56Fe beam. Individual images in this
panel correspond to media filled plastic support caps
used to find the spatial locations before the peak (0.0
and 3.55 cm), near or at the peak (3.80 and
3.85 cm), and after the peak (4.15 cm). The various
colours observed represent the relative quantity of
energy deposited to the support caps during this
calibration process. Blue colouration is equivalent to
less particle energy deposition and corresponds to
specimens placed at the location where particles
begin to first interact with matter (position 0.0 cm).
As particles traverse space they decrease their
velocity and have more time to interact with matter.
Consequently, the amount of energy being deposited
increases markedly as particles stop in matter. This
larger amount of energy deposition is manifested as
the yellow, white, and red colours (in the case of the
307.7 MeV/nucleon energy positions 3.80 and
3.85 cm) and corresponds to the particles at or very
near the Bragg peak.
Matter placed after the Bragg peak will experience
less energy deposition in general since a significant
number of particles will have already stopped
(position 4.15 cm). Positioning of the tissue plugs
at the desired location in the Bragg curve of the56Fe
particles was accomplished by leaving the location of
the plugs fixed relative to the beam port and placing
increasing amounts of variable filter material in front
of the plugs. This effectively decreases the particle's
velocity and modulates the amount of energy
deposited to the tissue equivalent. Tissue fixation
was performed thirty minutes after irradiating the
tissues at the specific locations of the Bragg curve
and the presence of DSB were subsequently assessed
via the co-localisation of g-H2AX and p-DNA-PKcs.
Fluorescent images were obtained for multiple cells
per distance as detailed in the methods. Figure 2A,
2B and 2C shows sample representative images of
the DNA damage imparted to cells in a 3-D tissue
equivalent at specific locations of the Bragg curve for
the various energies examined. The direction of the
beam is indicated to the left of the figure panels.
Samples corresponding to a position at or near the
Bragg peak (3.85 cm for 307.7 MeV/nucleon,
11.25 cm for 556.9 MeV/nucleon, and 24.575 for
967.0 MeV/nucleon) displayed a more intense DNA
damage response, where a near saturation of over-
lapping signal (yellow) is seen across the entire
nucleus of a number of cells, and less discrete streaks
and foci are observed. Samples located before the
Bragg peak (sample images at 0.0 cm for all energies)
in general displayed less nuclei with near saturating
DNA damage correlation with Bragg curve profiles
Int J Radiat Biol Downloaded from informahealthcare.com by UT Southwestern Medical Centre
For personal use only.
Page 5
signal and instead showed a paucity of damage
within the nuclei, with most of the visible damage
manifesting as DNA damage streaks or discrete foci
(white arrows). Nuclei located after the Bragg peak
continued to show evidence of DNA damage, in the
form of streaks or foci, although the amount of
affected nuclei were less numerous. These findings
are quantitatively illustrated in Figure 2D for the
307.7 MeV/nucleon energy where the percentage of
nuclei with DNA damage in the form of nuclear
saturation or streaks/foci is plotted against the
location of the examined sample in the Bragg curve.
The average mean grey scale values for co-
localised DNA damage were calculated from images
containing multiple nuclei and averaged to represent
the intensity of the acute DNA damage response for
each distance along the Bragg curve. Figure 3 shows
the average intensity of DNA damage as a ratio to
0.0 cm distance (blue dots) in relation to the
corresponding location in the physical Bragg curve
(black curve) forthe
(Figure 3A), 556.9 MeV/nucleon (Figure 3B), or
967.0 MeV/nucleon (Figure 3C)
The calculated intensities for the 967.0 MeV/
nucleon energy closely match the physical curve.
The calculated intensities for the 307.7 MeV/
nucleon and 556.9 MeV/nucleon energies, although
307.7 MeV/nucleon
56Fe particles.
displaying a fairly accurate correlation with their
respective physical curves, exhibit some deviations
with respect to the peak intensity and position of
some of the non-peak intensity values in relation to
the physical curve (generally decreased in compar-
ison to the physical Bragg peak). While several
factors could explain some of these deviations (see
Discussion), the damage intensity data for each of the
examined spatial locations as a particle travels
through a tissue equivalent creates a biological Bragg
curve that closely matches the physical energy profile
for the examined energies since the highest calcu-
lated mean grey scale value for samples in each
particle energy group falls at a distance that is very
near the respective peak of the physical Bragg curve.
Close-up view of the DNA damage by deconvolution
The observed nuclear streaks and saturated damage
observed in the samples placed across the Bragg
curve for the various particle energies examined
contain both in- and out-of-focus light at the focal
plane at which the picture was taken (Figure 4A,
unprocessed image). The out-of-focus light likely
represents DSB residing at different focal planes
within the nucleus per tissue-equivalent section
Figure 1. (A) Collagen-fibroblast plugs are made from lung fibroblast cells grown in conventional monolayer. Once mixed with collagen to
make a three dimensional tissue equivalent, they are properly oriented for exposure to high LET radiation at the NASA Space Radiation
Laboratory (NSRL) at Brookhaven National Laboratory (BNL). (B) Placement of plugs at different positions in the Bragg curve of a 307.7
MeV/nucleon56Fe particle shows varying energies deposited to tissues depending on their location in the Bragg curve.
A. I. Roig et al.
Int J Radiat Biol Downloaded from informahealthcare.com by UT Southwestern Medical Centre
For personal use only.
Page 6
thickness (5 m). Although informative for assessing
the intensity of DNA damage response, the out-of-
focus light makes it difficult to more closely examine
the three-dimensional structure of the various DNA
damage response patterns observed (track vs. nuclear
saturation pattern damage). Visualising the damage
response in all spatial directions can help elucidate
the particle track structure as it traverses tissues. To
correct the problem of the out-of-focus light, cell
nuclei with saturated damage or streaks were imaged
at multiple focal planes (z-stacks) and deconvolved.
Here we show the results for samples in the 307.7
MeV/nucleon energy group. In contrast to the
saturated out of focused damage signal of nuclei at
the Bragg peak (3.80 cm position) as seen by
epifluorescent microscopy (Figure 4A, unprocessed
image), deconvolution brings light into focus at all
examined planes (sample deconvolved image se-
quence, 4A). In this sequence, deconvolution shows
that DNA damage is present all throughout the
nucleus as one proceeds through the microscopic
sections of the sample. The significant amount of
damage encompassing a large amount of the nucleus
likely results from the increased particle-DNA
interaction occurring as an iron particle decreases
in velocity and deposits its energy to the surrounding
matter. Deconvolution of a nucleus at 4.15 cm
(Figure 4B, unprocessed image) showing a combina-
tion of mixed streaking and saturating DNA damage
response after the Bragg peak reveals a particle track
with smaller tangential tracks (white and yellow
arrows, respectively, deconvolved image sequence,
4B). This type of DNA damage pattern (most likely
caused by a combination of primary and secondary
particles as well as spallation products), although
most consistent with DNA damage at the Bragg peak
where the build-up of secondary particles is the
highest, was rarely found in samples placed beyond
the Bragg peak. Such a finding may be due to rare
ions that had not yet stopped in matter or may be a
result of inherent limitations in the experimental
design (see Discussion).
The deconvolved image sequences can also be
used to reconstruct a 3-D surface model of the DNA
damage to better evaluate the DNA damage response
protein distribution in a 3-D architecture. Multiple
deconvolved images of a subset of nuclei with
representative streaked or saturated damage were
Figure 2. (A), (B), (C) DNA damage in nuclei of specimens placed at various locations along the Bragg peak for 307.7 MeV/nucleon, 556.9
MeV/nucleon, or 967.0 MeV/nucleon56Fe ions, respectively. The beam direction for each energy is indicated next to the left of each panel.
The damage is appreciated as either discrete damage streaks or foci (white arrows) or saturated nuclear damage. All images taken at 406
objective magnification. (D) Percentage of nuclei in a 307.7 MeV/nucleon specimen showing streaks/foci or nuclear saturation damage. Co-
localised g-H2AX and DNA-PKcs nuclear streak damage is more prevalent in distances farther away from the Bragg peak while saturated
nuclear damage is more common closer to the peak. Error bars indicate the standard error for the mean (SEM) for three separate counting
DNA damage correlation with Bragg curve profiles
Int J Radiat Biol Downloaded from informahealthcare.com by UT Southwestern Medical Centre
For personal use only.
Page 7
stacked to reconstruct the three dimensional dis-
tribution of DNA damage proteins in both streaked
and saturated damage examples (g-H2AX, red;
p-DNA-PKcs, green) without merging both signals
(Figure 5). With this model the signal from each
protein at areas of DNA damage can be compared.
What becomes immediately apparent is the amount
of g-H2AX response in relation to p-DNA-PKcs:
even though the quantum yield (QY) of the
secondary antibody to g-H2AX (Alexa 568 second-
ary fluorophore, QY 0.69) is lower than the QY of
the secondary antibody to p-DNA-PKcs (Alexa 488
secondary fluorophore, QY 0.92) the streaked
(Figure 5 panel A) and saturated nuclear damage
pattern (Figure 5 panel B) show a predominance of
g-H2AX signal over p-DNA-PKcs at the charged
particle's point of entry into the cell nucleus. This
finding correlates with the pattern of g-H2AX and p-
DNA-PKcs expression in the deconvolved image
sequences of multiple examined nuclei in where we
observe a predominance g-H2AX over p-DNA-PKcs
(data not shown), particularly at the area corre-
sponding to the main particle track. This suggests
that g-H2AX activation can spread beyond the initial
lesion and thus may not be specific for the precise
location of the DNA DSB, consistent with previously
described observations. In a subset of deconvolved
nuclei, we observed isolated p-DNA-PKcs signal (no
co-localisation with g-H2AX). This occurred mainly
at the periphery of the main particle track, as
illustrated in Figure 5 panel B (isolated green p-
DNA-PKcs signal peripheral to the concentrated
DNA damage signal).
This model of a 3-D tissue construct combined with
DNA DSB specific markers represents the first-
generation analysis of the spatial distribution of
DNA damage in a tissue. The data generated suggest
that the biological Bragg curve, in this case for DSB
induction, follows the Bragg curves for56Fe particles
irrespective of the nominal energy ofthe particle. This
is so even for
where the normalised exposure is reduced by roughly
40% as the polyethylene density increases to 20 cm
before increasing again at the Bragg peak (Figure
3C). The density and complexity of ionisations
caused by HZE particles lead to such a deleterious
effect as these high LET radiations leave dense and
complex fields of DNA DSB in the process of linearly
traversing matter (Asaithamby et al. 2008, Paap et al.
2008). Indeed, while the dose used here was averaged
over a cm3, some cells likely absorbed several Gy
when they were positioned in the trajectory line of a
particle that was decreasing in velocity and subse-
quently depositing large amounts of energy. Our
study supports this notion as it shows that the charged
particles cause more intense DNA damage when
tissue equivalents are at or near the Bragg peak
and complements an important concept in the appli-
cation of heavy ions to tumor therapy, that tumors
can be effectively treated when exposed to the
Bragg peak region of a charged particle (an area of
increased relative biological effectiveness [RBE])
56Fe particles accelerated to 1 GeV
Figure 3. (A), (B), (C) Normalised DNA damage intensities
calculated by averaging the mean grey scale values of the DNA
damage intensities corresponding to56Fe ions with 307.7 MeV/
nucleon, 556.9 MeV/nucleon, or 967.0 MeV/nucleon energy
super-imposed over the physical Bragg profiles for each particle
energy. Four separate intensity measuring experiments (between
80 and 100 total cell nuclei) were performed for each spatial
location in the Bragg curve for each particular energy as well for
non-irradiated controls. Error bars indicate the standard error for
the mean (SEM) for these four separate intensity measuring
A. I. Roig et al.
Int J Radiat Biol Downloaded from informahealthcare.com by UT Southwestern Medical Centre
For personal use only.
Page 8
while decreasing the damage in exposed normal
tissues outside of the Bragg peak region (Weyrather
and Kraft 2004, Scholz and Elsasser 2007).
Although our experimental model demonstrates a
correlation of the DNA damage intensity with the
physical Bragg curve generated for a heavy ion of a
Figure 5. 3D reconstructions of deconvolved images from nuclei with streaked (panel A; sample sectioned parallel to beam path, white arrow
illustrating direction of particle path) and nuclear saturation damage (panel B; sample sectioned transverse to the particle path, particle
enters cell head-on). Left-most unprocessed epifluorescent images obtained at 406 objective magnification while right-sided unprocessed
images were obtained at 606 objective magnification. 3-D reconstructions (right-most images, top and side views of the nuclei) demonstrate
a surface rendition of the g-H2AX and DNA-PKcs damage.
Figure 4. Magnified pictures of representative cell nuclei from specimens with saturated nuclear damage or mixed streaked- saturated
nuclear damage for a 307.7 MeV/nucleon56Fe beam. Both images come from samples sectioned parallel to the beam path. (A). Close up
view of the saturated nuclear damage pattern processed with an epifluorescent microscope and corresponding deconvolved image sequences
of the saturated damage revealing distinct particle tracks at multiple optical sections. Unprocessed image obtained at 406 objective
magnification. Deconvolved images obtained at 606 objective magnification. (B). Sample nucleus with mixed streaked-saturated damage.
Deconvolution shows a particle track with small tangential secondary tracks. Unprocessed image obtained at 606 magnification.
Deconvolved images obtained at 606 objective magnification.
DNA damage correlation with Bragg curve profiles
Int J Radiat Biol Downloaded from informahealthcare.com by UT Southwestern Medical Centre
For personal use only.
Page 9
specific energy, not all of the data points fall exactly
in their respective physical Bragg curve profiles
(Figure 3, particularly the 307.7 and 556.9 MeV/
nucleon energies). Several limitations, inherent to
the experimental design, could account for these
discrepancies. When the actual plug samples were
placed inside the caps for irradiation unpredictable
tilting of the plug in the plastic cap could have shifted
the position of a portion of the plug by few or a
fraction of a millimeter and caused the localisation of
the plug in the physical Bragg curve to change
slightly. As a result, the final calculated DNA
damage intensity could deviate from the expected
intensity predicted by the physical profile. Another
limitation could be the sample size used to calculate
the average DNA damage intensity for each exam-
ined distance in the Bragg curve. Images captured for
the damage intensity calculations contain a combi-
nation of in and out of focus damage signals. The
out-of-focus signal for some of the nuclei may have
caused an underestimation of the actual damage
intensity. Including more nuclei in the analysis
(above the 80–100 examined for each distance)
could have compensated for this potential under-
estimation. In addition, the images used to calculate
the damage signal intensities (captured from samples
5 m in thickness) do not contain whole nuclei
(average size up to 10 m) and therefore are not fully
representative of the DNA damage taking place in
each nucleus. Underestimations of the DNA damage
intensity could result from this limitation and thus a
larger sample of nuclei or a thicker sample section
may have conferred more accurate average signal
intensities. Such limitations to the experimental
design, particularly potential tilting of the plug inside
the plastic cap, could also explain why primary and
secondary particle tracks were observed in nuclei
belonging to samples beyond the Bragg peak (such as
in the 4.15 cm nucleus described in the results)
where we would expect a paucity of damage and lack
of particle fragmentation. Any displacement of the
plugs by a fraction of a millimeter before irradiation
could place a small portion of the plug closer to the
Bragg peak where the frequency of primary and
secondary particles and spallation products are at
their highest.
The predominance of g-H2AX over p-DNA-PKcs
signal in areas of particle entry, as observed in the
surface renditions of the DNA damage obtained
from the deconvolution data, seems to support the
observation that g-H2AX activation may not be
specific to DNA damage. Although the presently
described deconvolution microscopy data is qualita-
tive, and not based on large amount of individual
nuclei analysis, it would be a useful to visualise larger
cohorts of cell nuclei at several locations in the
Bragg curve of various HZE particles to quantify the
g-H2AX response in relation to p-DNA-PKcs and
therefore objectively determine if g-H2AX activation
spreads beyond the DSB site marked by the presence
The presence of isolated p-DNA-PKcs signal in
the periphery of the main damage site as observed in
a subset of cells (representative image Figure 5B) is
reminiscent of prior reports demonstrating that
DNA-PKcs does not localise to sites of DNA DSB
(Jakob et al. 2002, Bekker-Jensen et al. 2006). In
these reports, the immunofluorescence analysis for
DNA-PKcs targeted the unphosphorylated form. It
has been established that DNA-PKcs autophosphor-
ylation is a necessary step for DNA repair, and
precise co-localisation with g-H2AX is dependent on
staining for the phosphorylated form of DNA-PKcs
(Chan et al. 2002, Uematsu et al. 2007). Conse-
quently, we infer that the isolated p-DNA-PKcs
signal observed in our experiment may be due to
background signal or potential non-specific binding
of the antibody. To determine with more confidence
that g-H2AX activation is more widespread than
other protein markers of DSB, additional proteins
such as 53BP1 can be examined to determine if
similar patterns arise, and therefore support the
notion of decreased specificity of g-H2AX.
Our experimental method describes an initial step
to examine the induction and repair of DNA damage
in tissues in the context of ionising radiation that
varies in LET. Taking into consideration the limita-
tions described above, and improving on these in
subsequent investigations, recently developed more
complex 3-D tissue equivalents (such as organotypic
cultures (OTC) containing both fibroblasts and
lung epithelial cells) can be implemented in future
experiments to explore the differences in cellular
transformation or cell death with respect to the
position in the Bragg curve. Since it is recognised
that DNA DSB produced by HZE particles in
particular are considered to be more complex as
the LET increases, and subsequently more difficult
to repair (Goodhead 1994, Sutherland et al. 2001),
it is likely that cells receiving large amounts of
DNA damage, as would be the case when they are
located at the Bragg peak, will likely have irreparable
damage and not survive. On the other hand, cells at
either side of the Bragg peak are more prone to
experience non-lethal DNA damage, survive, and
have a higher chance of developing mutations as a
result of inappropriately repaired DNA lesions.
Therefore, using 3-D tissue equivalents models to
explore the intensity of DNA damage depending
on the region of irradiation in the Bragg curve
and correlating these results with the rate of
carcinogenesis has direct application to answer
NASA's concerns of developing estimates of risk
for carcinogenesis after heavy ion exposure. In
A. I. Roig et al.
Int J Radiat Biol Downloaded from informahealthcare.com by UT Southwestern Medical Centre
For personal use only.
Page 10
addition, 3-D tissue equivalents models may have
relevance in the clinical field. Future experiments
can be designed where 3-D tissue equivalents
irradiated with therapeutically relevant ions, such as
carbon at therapeutic dose ranges, can be analysed
for the rate of transformation depending on the
region of irradiation in the Bragg curve. Recent
reports demonstrate an increased chromosomal
aberration frequency observed in cells as they are
irradiated by
LET, i.e., particles closer to the Bragg peak (Manti
et al. 2007). Despite a reduced chromosomal
aberration frequency and increased survival in cells
irradiated at distances far away from the peak, there
are uncertainties with respect to the transformation
rate of the irradiated healthy tissue. Biologically
obtained transformation rates using 3-D culture
models may complement transformation data ob-
tained from biophysical models for optimal treat-
ment planning and reduction of off-target effects
(Scholz and Elsasser 2007).
12C charged particles with a higher
We have shown how biological Bragg curves can be
calculated from the intensity of the DNA damage
and response of cells in tissue-like constructs and
how these closely match the physical Bragg curves
of the tested particle. In addition, with the use of
deconvolution microscopy, particle tracks can be
monitored through the depth of the surrogate tissue
and we can begin to speculate on how HZE
particle-induced DNA damage may be manifested
in tissue. Since this first- generation attempt to
model DNA damage in a tissue was performed,
more realistic 3-D models have subsequently been
genetically modified lung epithelial cells. With
appropriate biomarkers
opportunity to determine the mechanism by which
HZE particles initiate or promote carcinogenesis
may soon follow.
uses normalor
for transformation,the
This research was supported by the Office of Science
(BER), U.S. Department of Energy, Grant No.
DE-AI02-05ER64048 and NASA, NNJ05HD36G
NSCOR. We wish to thank the NSRL physics
support team for assistance in the design and
execution of the irradiation logistics and Oliver
Delgado for transporting and setting up the tissue
equivalents for irradiation.
Declaration of interest: The authors do not have
any conflict of interests or financial arrangements to
Asaithamby A, Uematsu N, Chatterjee A, Story MD, Burma S,
Chen DJ. 2008. Repair of HZE-particle-induced DNA double-
strand breaks in normal human fibroblasts. Radiation Research
Bekker-Jensen S, Lukas C, Kitagawa R, Melander F, Kastan MB,
Bartek J, Lukas J. 2006. Spatial organization of the mammalian
genome surveillance machinery in response to DNA strand
breaks. The Journal of Cell Biology 173:195–206.
Chan DW, Chen BP, Prithivirajsingh S, Kurimasa A, Story MD,
Qin J, Chen DJ. 2002. Autophosphorylation of the DNA-
dependent protein kinase catalytic subunit is required for
rejoining of DNA double-strand breaks. Genes and Develop-
ment 16:2333–2338.
Chen BP, Chan DW, Kobayashi J, Burma S, Asaithamby A,
Morotomi-Yano K, Botvinick E, Qin J, Chen DJ. 2005. Cell
cycle dependence of DNA-dependent protein kinase phos-
phorylation in response to DNA double strand breaks. Journal
of Biological Chemistry 280:14709–14715.
Cowell IG, Sunter NJ, Singh PB, Austin CA, Durkacz BW,
Tilby MJ. 2007. gammaH2AX foci form preferentially in
euchromatin after ionising-radiation. PLoS ONE 2:e1057.
Cucinotta FA, Durante M. 2006. Cancer risk from exposure to
galactic cosmic rays: Implications for space exploration by
human beings. The Lancet Oncology 7:431–435.
Cucinotta FA, Manuel FK, Jones J, Iszard G, Murrey J,
Djojonegro B, Wear M. 2001a. Space radiation and cataracts
in astronauts. Radiation Research 156:460–466.
Cucinotta FA, Nikjoo H, Goodhead DT. 1998. The effects of
delta rays on the number of particle-track traversals per cell in
laboratory and space exposures. Radiation Research 150:
Cucinotta FA, Schimmerling W, Wilson JW, Peterson LE,
Badhwar GD, Saganti PB, Dicello JF. 2001b. Space radiation
cancer risks and uncertainties for Mars missions. Radiation
Research 156:682–688.
Desai N, Durante M, Lin ZW, Cucinotta F, Wu H. 2005. High
LET-induced H2AX phosphorylation around the Bragg curve.
Advances in Space Research 35:236–242.
Durante M, Cucinotta FA. 2008. Heavy ion carcinogenesis
and human space exploration. Nature Reviews Cancer 8:
George K, Durante M, Willingham V, Wu H, Yang TC,
Cucinotta FA. 2003. Biological effectiveness of accelerated
particles for the induction of chromosome damage measured in
metaphase and interphase human lymphocytes. Radiation
Research 160:425–435.
Goodhead DT. 1994. Initial events in the cellular effects of
ionizing radiations: Clustered damage in DNA. International
Journal of Radiation Biology 65:7–17.
Han J, Hendzel MJ, Allalunis-Turner J. 2006. Quantitative
analysis reveals asynchronous and more than DSB-associated
histone H2AX phosphorylation after exposure to ionizing
radiation. Radiation Research 165:283–292.
Hartman PS, Hlavacek A, Wilde H, Lewicki D, Schubert W,
Kern RG, Kazarians GA, Benton EV, Benton ER, Nelson GA.
2001. A comparison of mutations induced by accelerated iron
particles versus those induced by low earth orbit space
radiation in the FEM-3 gene of Caenorhabditis elegans.
Mutation Research 474:47–55.
Jakob B, Scholz M, Taucher-Scholz G. 2002. Characterization of
CDKN1A (p21) binding to sites of heavy-ion-induced
damage: Colocalization with proteins involved in DNA repair.
International Journal of Radiation Biology 78:75–88.
Jakob B, Scholz M, Taucher-Scholz G. 2003. Biological imaging
of heavy charged-particle tracks. Radiation Research 159:
DNA damage correlation with Bragg curve profiles
Int J Radiat Biol Downloaded from informahealthcare.com by UT Southwestern Medical Centre
For personal use only.
Page 11
Koniarek JP, Thomas JL, Vazquez M. 2004. Detection of
microlesions induced by heavy ions using liposomes filled
with fluorescent dye. Advances in Space Research 34:
Koniarek JP, Worgul BV. 1992. Do heavy ions cause microlesions
in cell membranes? Advances in Space Research 12:417–420.
Manti L, Durante M, Grossi G, Pugliese M, Scampoli P,
Gialanella G. 2007. Chromosome aberrations in human
lymphocytes from the plateau region of the Bragg curve for a
carbon-ion beam. Nuclear Instruments & Methods in Physics
Research Section B-Beam Interactions with Materials and
Atoms 259:884–888.
McManus KJ, Hendzel MJ. 2005. ATM-dependent DNA
damage-independent mitotic phosphorylation of H2AX in
normally growing mammalian cells. Molecular Biology of the
Cell 16:5013–5025.
Mukherjee B, Camacho CV, Tomimatsu N, Miller J, Burma S.
2008. Modulation of the DNA-damage response to HZE
particles by shielding. DNA Repair (Amst) 7:1717–1730.
Paap B, Wilson DM 3rd, Sutherland BM. 2008. Human abasic
endonuclease action on multilesion abasic clusters: Implica-
tions for radiation-induced biological damage. Nucleic Acids
Research 36: 2717–2727.
Ponomarev AL, Cucinotta FA. 2006. Nuclear fragmentation and
the number of particle tracks in tissue. Radiation Protection
Dosimetry 122:354–361.
Raber J, Rola R, Lefevour A, Morhardt D, Curley J, Mizumatsu S,
Vandenberg SR, Fike JR. 2004. Radiation-induced cognitive
impairments are associated with changes in indicators of
hippocampal neurogenesis. Radiation Research 162:39–47.
Rastegar N, Eckart P, Mertz M. 2002. Radiation-induced cataract
in astronauts and cosmonauts. Graefes Archive for Clinical and
Experimental Ophthalmology 240:543–547.
Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. 1998.
DNA double-stranded breaks induce histone H2AX phos-
phorylation on serine 139. Journal of Biological Chemistry
Schimmerling W. 1992. Radiobiological problems in space. An
Scholz M, Elsasser T. 2007. Biophysical models in ion beam
radiotherapy. Advances in Space Research 40:1381–1391.
Biophysics 31:
Shavers MR, Zapp N, Barber RE, Wilson JW, Qualls G,
Toupes L, Ramsey S, Vinci V, Smith G, Cucinotta FA.
2004. Implementation of ALARA radiation protection on the
ISS through polyethylene shielding augmentation of the
Service Module Crew Quarters. Advances in Space Research
Sutherland BM, Bennett PV, Schenk H, Sidorkina O, Laval J,
Trunk J, Monteleone D, Sutherland J. 2001. Clustered DNA
damages induced by high and low LET radiation, including
heavy ions. Physica Medica (17 Suppl. 1):202–204.
Uematsu N, Weterings E, Yano K, Morotomi-Yano K, Jakob B,
Taucher-Scholz G, Mari PO, Van Gent DC, Chen BP,
Chen DJ. 2007. Autophosphorylation of DNA-PKCS regu-
lates its dynamics at DNA double-strand breaks. The Journal
of Cell Biology 177:219–229.
Walker SA, Tweed J, Wilson JW, Cucinotta FA, Tripathi RK,
Blattnig S, Zeitlin C, Heilbronn L, Miller J. 2005. Validation of
the HZETRN code for laboratory exposures with 1A GeV iron
ions in several targets. Advances in Space Research 35:
Ward IM, Chen J. 2001. Histone H2AX is phosphorylated in an
ATR-dependent manner in response to replicational stress.
Journal of Biological Chemistry 276:47759–47762.
Weyrather WK, Kraft G. 2004. RBE of carbon ions: Experimental
data and the strategy of RBE calculation for treatment
planning. Radiotherapy and Oncology 73(Suppl. 2):S161–169.
White RJ, Averner M. 2001. Humans in space. Nature 409:
WilsonJW,Shinn JL,Tripathi
Clowdsley MS,Thibeault
Schimmerling W, Cucinotta FA, Badhwar GD, Noor AK,
Kim MY, Badavi FF, Heinbockel JH, Miller J, Zeitlin C,
Heilbronn L. 2001. Issues in deep space radiation protection.
Acta Astronautica 49:289–312.
Wilson JW, Tripathi RK, Qualls GD, Cucinotta FA, Prael RE,
Norbury JW, Heinbockel JH, Tweed J. 2004. A space radiation
transport method development. Advances in Space Research
Wu H, Hada M, Meador J, Hu X, Rusek A, Cucinotta FA. 2006.
Induction of micronuclei in human fibroblasts across the Bragg
curve of energetic heavy ions. Radiation Research 166:
A. I. Roig et al.
Int J Radiat Biol Downloaded from informahealthcare.com by UT Southwestern Medical Centre
For personal use only.


text:
biOrb Life 16 Gallon Aquarium
Transform your home or office into a stylish space with the color, life and contemporary look of the biOrb Life 60 designer aquarium. It's sophisticated lighting system provides a healthier environment for your fish and fascinating night-time viewing for you. With its stylish design, the Life aquarium is unlike anything else on the market. It has two extra thick crystal clear acrylic faces and a seamless, high-gloss, colored acrylic wrap at its sides, top and bottom. Each aquarium includes a unique advanced slim line Intelligent LED Light that provides an automatic 24 hour lighting cycle, including a natural gradual sunrise, sunset and blue moonlight. This sophisticated lighting system provides a healthier environment for your fish and fascinating night-time viewing for you. Just like the biOrb aquarium, this new design continues to use simple maintenance and safe low voltage parts. The biOrb Life Collection uses the same unique multi-stage biOrb filtration, keeping the water cleaner and clearer for longer, providing a healthy habitat for fish. All components to the aquarium are child friendly, working off of only 12 volts. This smaller aquarium to the new collection is the perfect size at only 22" in height and 14.75" in length, holding up to 12 gallons.
Features: 24 Hour Lighting Cycle, Multi-Stage biOrb Filtration, 12V Power
Includes: Aquarium, Bubble Tube, LED Intelligent Light, Ceramic Media, Airstone, Air Pump, Sample Fish Food, Instructions
Tank Size: 16 gallon
Installation Type: Table
Color: Red
Shape: Rectangle
Dimensions: 16.5"L x 11"W x 24.5"H
Material: Acrylic
$ 13.99 to $ 54.99Reg: $ 15.99 to $ 54.99
$ 4.99 to $ 9.99
$ 24.99
$ 24.99
$ 249.99Reg: $ 349.99
$ 159.99Reg: $ 199.99
$ 4.99
$ 449.99Reg: $ 499.99
$ 20.99Reg: $ 24.99
$ 34.99Reg: $ 39.99
$ 24.99
$ 94.99

subset: commoncrawl


text: Benefits Participants Deferring Less Than 10% at Risk in Retirement By Corie Russell firstname.lastname@example.com | August 23, 2012 Page 1 of 2 View Full Article August 23, 2012 (PLANSPONSOR.com) - Households that defer less than 10% of their salary for retirement savings are at risk for not meeting their retirement goals, Putnam Investments' research found. According to Putnam's Lifetime Income Score, households that defer 0% for retirement are on track to replace 54% of their income at retirement with Social Security, but only 16% without it. Households that defer up to 3% are on track to replace 56% of income with Social Security and 23% without it, and those who defer 3% to 10% can replace 84% of income with Social Security, but 50% without it. Putnam also found that households with access to a defined contribution (DC) plan do not always participate. Of the 60% of households eligible for DC plans, 8% do not contribute to a plan. Of that 8%, 3% are not contributing but have a balance (lapsed contributors); and 5% have never enrolled in a workplace plan. Those eligible but never enrolled will rely more heavily on Social Security than the average household (36% versus 32%) and less on retirement plan savings (16% versus 26%). These numbers underscore the importance of plan sponsors and advisers encouraging participants to save at an adequate rate to replace income in retirement, especially with the uncertain future of Social Security. "At the end of the day, the real driver is, 'Are you saving enough?'" Ed Murphy, head of defined contribution at Putnam, told PLANSPONSOR. Prev1 2Next View Full Article Tagged: Retirement Income, Post Retirement, more from plansponsor related articles related research most e-mailed More in news topic from the magazine Asset Class FocusManaged Payout Funds From plansponsor.com Hueler Makes QLACs Available to DC PlansHueler Makes QLACs Available to DC PlansGenworth Survey Highlights Retirement ShortfallsChanging Attitudes Mean a Retirement RebrandDC Consultants Focused on Income QuestionsEBRI Calculates Savings Needs By Age and Gender More in news topic from the magazine Asset Class FocusManaged Payout Funds From plansponsor.com 2014 DC Survey: Plan Benchmarking2014 Defined Contribution Survey2014 Consultant Buyer's Guide2014 403(b) Buyer's Guide2014 Transition Management Survey2014 Defined Benefit Administration Survey More in news topic from the magazine Asset Class FocusManaged Payout Funds From plansponsor.com Employees Leaving Wellness Incentives on the TableNear Retirees Can Expect 400 Thousand in Health Care CostsIncentives Bring Needed Participants to Wellness ProgramsTangible Retirement Messaging Drives Better OutcomesWomen Need Retirement Planning NudgeVanguard Rolling Out Plan Sponsor Workstation go to news topic


text:
Foster v. City of Addis, et al
|Kenneth Dacook, City of Addis and Robert Arrazzattee
|Erika Foster
|Case Number:
|October 25, 2013
|Louisiana Middle District Court
|Baton Rouge Office
|Presiding Judge:
|James J. Brady
|Referring Judge:
|Stephen C. Riedlinger
|Nature of Suit:
|Civil Rights: Other
|Cause of Action:
|28:1331 Fed. Question: Civil Rights Violation
|Jury Demanded By:
Available Case Documents
The following documents for this case are available for you to view or download:
|Date Filed
|Document Text
|November 3, 2014
|RULING granting 23 Motion for Summary Judgment. All claims against Defendants, the Town of Addis, Robert Arrazattee, and Kenneth DeCook, are DISMISSED. Signed by Judge James J. Brady on 11/3/2014. (NLT)
Access additional case information on PACER
Use the links below to access additional information about this case on the US Court's PACER system. A subscription to PACER is required.
Access this case on the Louisiana Middle District Court's Electronic Court Filings (ECF) System
- Search for Party Aliases
- Associated Cases
- Attorneys
- Case File Location
- Case Summary
- Docket Report
- History/Documents
- Parties
- Related Transactions
- Check Status


text:
MJM is the most consistant magic company in the business and that is very important to me. I want to know that Im getting the same service every time...Read More ->
I am so happy that I found this store!! Everything here is top notch including the prices, Fast shipping, and wonderful service. Thanks to all of...Read More ->


text:
Quincy City Council Moves Toward Approval of Marijuana Dispensary
Huckleberry Park Ramp Park Open for Season
Easter Week Expected to Be More Springlike
Duke & Michigan State Complete 2015 Final Four Field
Shooting Victim Identified, Crime Stoppers Offering $1000 Reward
Adams County Coroner James Keller has identified the victim of an early morning fatal shooting as 12-year-old Rayshone D. Humphrey Jr. According to a report from WGEM, officers responded to the shooting scene in the 1100 block of North 5th Street between Cedar and Cherry Streets and found a boy that had been shot outside of a home. He was taken to Blessing Hospital and pronounced dead. Keller revealed an autopsy would be conducted on Sunday.
Cool Things You Can See at This Weekend's Great Outdoors Show
The Great Outdoors Show in Quincy is taking place at the Oakley Lindsay Center through Sunday. Vendors of all sorts have set up displays, and as you might imagine, several have brought some fairly unique items to the show.


text:
(0 holds on 3 copies)
|Call Number
|Item Status
|Due 04-06-15
Description: 4 videodiscs (618 min.) : sd., col. ; 4 3/4 in
Language: English
Based on the novel "Darkly dreaming Dexter" by Jeff Lindsay
Videodisc release of the 2011 television series
Michael C. Hall, Jennifer Carpenter, Desmond Harrington
Back with a vengeance for a sixth season of startling suspense and unexpected twists. It's been a year since last season's shocking and heartbreaking conclusion, and mild mannered criminologist-cum-grisly serial killer Dexter Morgan has come to terms with who he is. But his existence is shattered when he crosses paths with an enemy unlike any he's faced before. A delusional religious zealot with unflinching convictions, this new adversary soon draws Dexter into a dangerous game
Rating: Not rated
DVD, widescreen
English, Spanish or French dialogue
Login to write a review of your own.
Reviews by AHML cardholders
Reviews from other readers
Forensic scientists -- Drama -- Florida -- Miami
Criminal investigation -- Drama -- Florida -- Miami
Serial murderers -- Drama -- Florida -- Miami
Serial murders -- Drama -- Florida -- Miami
Criminals -- Drama -- Florida -- Miami
Psychopaths -- Drama -- Florida -- Miami
Vigilantes -- Drama -- Florida -- Miami
Television programs
Fiction television programs
Television crime shows
Thrillers (Television programs)
Detective and mystery television programs
Mystery television programs
Added--201208 advd
Carpenter, Jennifer, 1979-
Harrington, Desmond, 1976-
Showtime Entertainment
CBS DVD (Firm)
Lists can be used to compile collections of items that you may be interested in checking out at a later date. You may also create public lists and share your favorites with other AHML customers.
Login to add tags.
To create a multiple word tag such as Science Fiction, enclose both words in quotes, like: "Science Fiction"


text:
The International Society of Automation (www.isa.org) is a nonprofit professional association that sets the standard for those who apply engineering and technology to improve the management, safety, and cybersecurity of modern automation and control systems used across industry and critical infrastructure. Founded in 1945, ISA develops widely used global standards; certifies industry professionals; provides education and training; publishes books and technical articles; hosts conferences and exhibits; and provides networking and career development programs for its 36,000 members and 350,000 customers around the world.
ISA owns Automation.com, a leading online publisher of automation-related content, and is the founding sponsor of The Automation Federation (www.automationfederation.org), an association of non-profit organizations serving as "The Voice of Automation." Through a wholly owned subsidiary, ISA bridges the gap between standards and their implementation with the ISA Security Compliance Institute (www.isasecure.org) and the ISA Wireless Compliance Institute (www.isa100wci.org).
Membership Benefits
Full ISA Membership
Automation Affiliate Membership
Student Membership
Earned Membership Grades & Reciprocal Memberships
Saturday, 25 Apr 2015
Galveston, TX,
Sunday, 26 Apr - Thursday, 30 Apr, 2015
Galveston, TX,
Monday, 11 May - Thursday, 14 May, 2015
Huntsville, AL,
Tuesday, 19 May - Thursday, 21 May, 2015
New Orleans, LA,
Sunday, 07 Jun - Thursday, 11 Jun, 2015
Kansas City, MO,
ISA members have access to dozens of unique benefits. Update your member profile, set your communications preferences, find products and services that are right for you, and more – all from the convenience of your ISA website.
Featured Training: ISA Boot Camps
Featured White Paper: Workforce Development
Cover Story: Plant historians
Saturday, 25 Apr 2015
Hilton Hotel,
Sunday, 07 Jun - Thursday, 11 Jun, 2015
Kansas City Marriott Hotel,
Saturday, 13 Jun - Monday, 15 Jun, 2015
Raleigh Marriott City Center,
Thursday, 24 Sep 2015
ISA - International Society of Automation,
Monday, 09 Nov - Thursday, 12 Nov, 2015
Houston Marriott Westchase,
2015 ISA Power Industry Symposium features five acclaimed keynote speakers, an extensive technical program, training opportunities and more
Steve Mustard, industrial cybersecurity expert of the Automation Federation and ISA, selected as keynote speaker at CABA Forum
Automation Federation releases report commissioned by LOGIIC that studies the use of virtualization in IACS environments
Registration opens for 2015 ISA Leak Detection and Repair (LDAR)-Fugitive Emissions Symposium
New ISA book explores evolution of temperature pressure and flow technologies and more
Beamex is pleased to announce its cooperation with Mark Draghicescu Racing at Silverstone race circuit
ISA and Beamex to co host web seminar on how to avoid the pitfalls of calibration system implementation
Honeywell Named Best Industrial Wireless Provider at 2014 Asian Manufacturing Awards
Beamex launches Mikes Calibration Corner
FDT Sponsors Webinar on Changes Needed for Successful Asset Management
ISA has the resources and expert networks that you need – no matter what position you hold in our industries. Browse the products and services designed to meet the needs of these common industry roles:
ISA awarded me a 2013-2014 scholarship to help fund my education. I would not have been able to keep going to school if I had not received it. So, I want to give thanks to the many people of the ISA who work on securing the future of the organization just as the original founders had. ISA was and is forged and founded from true honesty and integrity that you will not find in to many other professions.
-Troy Riddle, Lee College
ISA's products and services are organized by technical topic – a selection of technologies, trends, and areas of focus that are relevant to your on-the-job performance:
The operating logic may be diagrammed to different levels of completeness of information. Whatever the level of completeness, the diagram communicates functional information explicitly to the degree of completeness intended by the diagrammer. The diagram may serve, for example, to improve the interface coordination between a designer of pneumatic circuits, or may simply provide a symbolic functional system description to a plant manager.
To provide a method of logic diagramming of binary interlock and sequencing systems for the start-up, operation, alarm, and shutdown of equipment in the chemical, petroleum, power generation, air conditioning, metal refining, and numerous other industries.
The standard is intended to symbolize the binary control functions of a system in a manner that can be applied to any class of hardware, whether it be electronic, electrical, fluidic, pneumatic, hydraulic, mechanical, manual, optical, or other.
For information about ISA5.2, contact Charley Robinson, ISA Standards.
firstname.lastname@example.com (708) 5321014


text: Home > Medical > Backache > Good posture Updated 17 September 2014 Tips for a healthy back What do teachers, sales assistants, dentists and factory workers have in common? They spend long hours on their feet every day, and could develop back problems. 0 iStock What do teachers, sales assistants, dentists and factory workers have in common? They spend long hours on their feet at work every day. And could all develop serious back problems at some stage in their working lives.What can you do to prevent back problems? The following tips were given by the South African Physiotherapy Association:Tips for your work environmentCounter staffSensible shoes – avoid high-heeled shoes because they increase the curvature of the lumbar spineStand relaxed with top of head held high – chin in, pelvis slightly tilted. Have the weight evenly distributed on both legs.Place one foot on a low stool.Stomach muscles must be pressed towards the spine and the bottom tucked in.Work surface should fit your height.Factory workersWear non-slip shoes. Your back cannot cope well with sudden stress.Wear protective clothing where necessary.Never over reach. Top of work surface should correspond with your arm length.Keep heavy objects on waist height shelves.Painters/overhead workers and shelf packersUse a stepladder if arching the back increases pain.Top of work surface should correspond with your arm length.Remember the danger of slipping on wet floors.Lift always within your capacity. Remember it is better to wait for 5 minutes for assistance with lifting, rather than be off because you have lifted beyond your capacity.Use your whole body when bending and make sure your hips and knees do the heavy part of lifting, keeping your back comfortably still and the weight close to your body.Dentist/oral hygienistsHave a firm chair with back support.Hips should be well back, feet firmly on the ground with support at the small of the back.If chair has wheels, avoid unexpected jarring of your back.Spinal rotation exercise: Turn your head and shoulders to look behind you, holding the back of your chair to turn a little further.Backstretch: Standing up, put hands in the back of the waist and stretch your body up and bend slightly backwards, hold 3 seconds.Exercise tipsDon't ignore pain or push yourself too far.Pain is a warning sign of possible injury.Start exercising slowly and stay within your personal comfort zone.Some muscles may feel sore when you first start exercising. This is normal. Sharp pain however, is not normal. Check it out.With back exercises technique is far more important, than speed. Exercise slowly and keep breathing regularly.It is good idea to stretch after your muscles are warmed up. You can warm them up with gentle movements, such as walking, or by taking a warm shower or bath.- (Health24.com) Read Health24's Comments Policy Comment on this story 0 comments Add your comment Thank you, your comment has been submitted. Logout Comment 0 characters remaining Share on Facebook Logout Comment 0 characters remaining Share on Facebook Loading comments... Ask the Expert Backache expert Susan qualified as a Physiotherapist in 1990, and completed her master's degree in Physiotherapy in 2013 at the University of Pretoria. She has a special interest in human biomechanics, as well as the interaction between domestic and work-related ergonomics. Ask a question Questions posted to Backache expert questions answered by the expert Q: history of back problems posted on 27-03-2015 Q: Nek operasie posted on 25-03-2015 Q: ongeloooflike rugpyn posted on 23-03-2015 Q: Time for a cortisone injection? posted on 21-03-2015 Q: have l3 4 5 lumbar problems posted on 10-03-2015 Q: Kroniese Lae Rug Pyn posted on 10-03-2015 Q: laerug/pyn in bo-bene wat nie aftrek na onder posted on 06-03-2015 Q: Hi, I have sciatica pain -left leg for last 6 months.MRI scan diagnosed disc herniation posted on 02-03-2015 Q: Overloaded backpack posted on 24-02-2015 Q: XR Cervical spine posted on 24-02-2015 Q: cervical osteoarthritis posted on 23-02-2015 Q: Ek het erge lae rugpyn wat af beweeg in my linker been posted on 18-02-2015 Q: Feeling dizzy 3days after back surgery. Is this normal posted on 17-02-2015 Q: lumbale lordose is uitgewis posted on 11-02-2015 Q: Chronic Pain posted on 09-02-2015 load more × Still have a question? Get free advice from our panel of experts Your question More detail Your name (optional) The information provided does not constitute a diagnosis of your condition. You should consult a medical practitioner or other appropriate health care professional for a physical exmanication, diagnosis and formal advice. Health24 and the expert accept no responsibility or liability for any damage or personal harm you may suffer resulting from making use of this content. I agree * You must accept our condition To prevent spam please solve this simple math problem: Forum Rules


text:
Cunnilingus tips-How to lick a girls vagina until she orgasms
This article will show you step by step how to make a girl reach an orgasm with your tongue. Climaxes that are given by cunnilingus are the best a woman can get, far better than an intercourse induced climax.
Learning how to lick a girls vagina until she orgasms is essential if you care about her and want her to have the most pleasure in bed that you can possibly give to her.
Here is a 3 part guide that will make her legs shake.
Cunnilingus tips 1.
The importance of this step on a scale of 1 to 10 is Eleven, if you don't get this one right then forget it. You have to get her mentally and physically relaxed and on top of this you need to make her feel sexy. Relax her with massages and foot rubs, make her feel sexy by complimenting her all day. This step takes a lot of work but it's worth it later when she has an uncontrolable orgasm.
Cunnilingus tips 2.
Ok she is relaxed and you are in bed, now comes the teasing and building the anticipation. Instead of going straight down on her you need to kiss and stroke her whole body and make her want you. Tease her by kissing around her vagina until she can take no more.
Cunnilingus tips 3.
Time for her to reach the big O. Now she is truly aroused start to slowly lick her vagina, you must use a steady pace at first because her nerve endings down there are so sensitive. When she starts getting loud and wet move on to her clitoris and lick this for a few seconds.
That's just a brief guide how to lick a girls vagina until she orgasms.
Discuss this Article
Cunnilingus tips to give her the best orgasm of her life- Learn how to lick a girl and make her scream (with pleasure)
As you can see the key to great cunnilingus is not just licking her, it is as much if not more to do with relaxing her and making her feel special.
So to drive a woman wild in bed forget about vaginal sex and learn how to lick her, master cunnilingus and give her super powerful orgasms, even multiple orgasms every night.
Sex tips can help men improve their sexual encounters. When considering such tips, remember that the condition of the bedroom has an impact on sex.
Uncontrolled penis odor can result in a major date disaster. Men want to make sure they take appropriate steps to eliminate unwanted penis odor before an outing with a potential partner.
Pornography is not everyone's cup of tea, but many couples who view it together find the experience can lead to better sex.
The search for perfect Valentine's Day gifts is on. This year, why not invest in presents that both partners can enjoy together? The following list of sex toys will help guide men and their partners to greater pleasure.
While some causes of penis bumps require treatment, others are natural and harmless. Men can learn about three types of bumps that may affect the penis but are not causes for concern here.
It's hard to fully enjoy sex when one has a numb penis. Restoring lost sensitivity (or preventing it from occurring) enables a man to enjoy sexual pleasure to the fullest.
An itchy penis is a common problem for many men, but it may present a special problem for those with a latex allergy, as it may make using condoms an issue.
Exercise is beneficial to several areas of one's life, including sex. Learn the best exercises for better sex.
Delaying your ejaculation is easy if you use pills or creams but these things don't come cheap and they are a repeat purchase. So the best way to last longer in bed for men is not to use pills but to embark on a natural ejaculation trainer.
This article will focus on natural ways to prevent early ejaculation rather than the products that you can purchase. The reason being that I don't believe that spraying a numbing agent or applying a numbing cream to your penis can have no harmful side effects.
If there is one thing about having sex that most men would like to improve it is their sexual stamina. Early orgasms plaque millions of men but it is quite simple to last longer in bed. Here are two ways to stop early orgasm and last longer in bed for men.
Men love to boast about how good they are in bed and how long they can last for but next time you hear a guy telling someone about his huge amounts of sexual stamina don't take what he says to seriously. You see up to 65% of men lack the ability to stop ejaculating to fast during intercourse, so the chances are that the boasters are liars. If you find that you ejaculate before your partner has a chance to reach her climax then read on for these tips on how to stop ejaculating to fast while...
One of the main causes of men ejaculating too fast is not a hyper sensitive penis but simply an over active brain. Lasting longer during intercourse is not hard as long as your mind is right. An example is that your lover looks hot and you really want sex with her so you keep looking at her and getting yourself all worked up.
There are tons of ways for guys to last longer in bed from delay sprays to natural exercises that give you full ejaculatory control. It's ever more important to be able to outlast a woman in bed because the modern woman demands a guy who can cater for her sexual needs. Here are two tips on having more sexual stamina.
This article will give you 2 tips on how to stop from ejaculating too fast during intercourse. Before we get started though let's get one thing straight and that is no matter how fast you are ejaculating you do not have a problem. This is simply because of the time difference that it takes men and women to reach orgasm.
Stopping ejaculation isn't as hard as you may think, in fact if you follow a plan of action you will be lasting longer within a few days. Here are three ways of stopping ejaculation that will stop you asking, how do I last longer in bed?


text:
Thanks for redeeming.{{else}}
Success! Thanks for repurchasing.{{/if}} {{else}} {{#if Expired}}
Increase my visibility!{{else}} {{#if IsMultiPack}}
{{#if HasMultiPack}} You have {{MultiPackCount}} top spot(s) left. Now loading you into the search listings... {{else}} You have used all top spots. Now loading you into the search listings... {{/if}}{{else}}
We are loading you into the search listing pages...{{/if}} {{/if}} {{/if}} {{/if}}
{{Current}} just saw you!{{/if}} {{#if LastTopSpotPurchaseIsMulti }} {{#if HasAnyTopSpot}}
{{#if HasFreeTopSpot }} {{ UnclaimedTopSpotFree }} Free {{/if}} {{#if HasPaidTopSpot }} {{#if HasFreeTopSpot }} & {{/if}} {{ MultiPackCount }} {{/if}} Top Spot{{#if Plural}}s{{/if}} Left{{else}}
0 Top Spot{{#if Plural}}s{{/if}} Left{{/if}} {{else}} {{#if HasFreeTopSpot }}
{{ UnclaimedTopSpotFree }} Top Spot{{#if Plural}}s{{/if}} Left{{/if}} {{/if}} {{else}} {{#if HasResults}}
{{Current}} see you now!{{else}} {{/if}} {{/if}}
You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
26 year old man | Del Rio, TX, USA | Seeking women 20-30 within 100 miles
Active within 24 hours
Lets have some fun!
I'm from college station and I absolutely love it there. I'm a big AM fan but I didn't actually go to school there. I went up to school in Colorado which is another awesome place to be. I picked up snow boarding there and love it. I enjoy hiking and doing other things in the mountains as well. I'm here now at Laughlin AFB as an instructor pilot. I'm very easy going person who likes to party but also likes to just chill out sometimes. I love chilling by the lake having a few beers while grilling out. Basically anything that is social and outside I love. I love animals especially dogs so if you have one kudos. I like to sing out loud with the music blaring and my windows down while I drive my truck. I enjoy driving in general. Some of my favorite places you need a truck to get there. I enjoy working out. I like running, swimming, lifting, playing sports, and doing crossfit. I like eating healthy (but I also like not to as well lol). I played soccer for most of my life. I've also like playing baseball and football. I'm looking for someone who is smart, funny, caring, and considerate.
Camping , Dining out , Fishing/Hunting , Movies/Videos , Music and concerts , Nightclubs/Dancing , Playing sports , Religion/Spiritual , Travel/Sightseeing , Video games , Volunteering , Watching sports
Baseball, Basketball, Billiards / Pool, Bowling, Football, Golf, Running, Soccer, Swimming, Tennis / Racquet sports, VolleyBall, Walking / Hiking, Weights / Machines
I exercise 3-4 times per week.
I don't believe in astrology
I like Cats, Dogs, Fish
Cowboys, down town, the lake, the river, any place that's fun
United States Air Force Academy, USAF Academy, CO
I love music, movies, Mexican food, working out, and anything outdoors.
I love going to see movies. I enjoy working out, watching tv, chilling with friends, partying, and eating good food.
lone survivor
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|5'11" (180cms)
|4'10" (147cms) to 5'11" (180cms)
|Body type:
|Athletic and toned
|Slender, About average, Curvy, Athletic and toned, Full-figured
|Blue, Brown, Grey, Green, Hazel
|Light brown
|Auburn / Red, Black, Light brown, Dark brown, Blonde, Dark blonde, Grey, Platinum
|No Way, Occasionally
|Social Drinker
|Social Drinker, Regularly
|Law enforcement / Security / Military
|No preference
|I'll tell you later
|No preference
|Never Married
|Never Married, Widow / Widower, Divorced
|Have kids:
|No preference
|Wants kids:
|Definitely, Someday, Not sure
|White / Caucasian
|No preference
|Christian / Protestant
|Christian / Catholic, Christian / Protestant, Christian / Other
|Bachelors degree
|High school, Some college, Associates degree, Bachelors degree, Graduate degree, PhD / Post Doctoral


text:
cummins blowby
Iv got an 01 cummins that between unexpectedly running very low on oil and not knowing it and very possible poor maintenance by previous owners is showing signs of wear. iv got excessive blowby or at least what i consider excessive and was wanting some input if its possibly something "easier" in the cylinder head such as valve guides or something more involved as in piston rings. Im not real familiar with the cummins yet so im not sure what we be the easiest to wipeout due to the previously listed problems. thanks for any input fingers crossed i dont feel like doing a full rebuild any time soon.
does it have a miss? how many miles. what air filter is on it?
could be rings, scored cylinder(s) or a hole in a piston(s) nothing else will cause pressure in the crankcase.
Pull the intercooler hose off of the intercooler and see if you have signs of blowby in there. It could be as simple as you needing a new turbo, without getting into the engine itself. My buddy has an 04 dmax LLY that was "losing oil", so I went under and yanked the boot off and she was dripping oil pretty good. New turbo and has been holding oil ever since. No more blue-ish smoke on start up either..
I have an 2001 first oil leak was the Vacuum pump/power steering pump seal at the crank case it leaked pretty good but I couldn't tell if it was the crank case over flow bottle or the vacuum pump seal so I did the overflow bottle buy pass & Vacuum pump seal problem fixed.
next oil leak was the tappet cover gasket & them I had to re-torque the oil pan bolts these are some the leaks I have had over the years that made a significant loss in oil I now have 301,000 miles on it with no blow-by.
What engine oil are you using? I would do a compression test and a leak down test. That would tell you the condition of the engine. Typically the mains/rod bearings would see the most wear from the lack of oil. There are piston oil cooler nozzles that shoot oil at the bottom of the piston. What oil pressure pressure? Was there any bearing material in the filter and in the oil?
Lots of things cause low cylinder pressure, the cummins VP engine have cracking issues between the exhaust/intake valves. I've replaced my head on my 02 truck a few times.
Usually if you have any piston damage, you can hear it and the truck will be hard starting. I've built a bunch of these engine over the years and they are a very easy engine to build up.
im pretty sure its worn rings cause im pretty sure the 1st owner ran the crap out of the truck. to answer all questions i have a volant oiled air filter, i run royal purple oil, truck holds 50 psi oil pressure pretty much all the time, and it has 192000 miles on it. it doesnt have so much blowby to blow the oil cap off atleast not at idle but it blows enough smoke out the crankcase vent iv had people at stop lights inform me my truck is smoking (-_-) and depending on wind direction you can see a decent cloud rise out from under the truck. Enough oil gets out of the crankcase vent that it leaves about a tennis ball size spot of oil every time i park. no major knocks (other than the normal diesel clatter) no miss fires, and doesnt blow very much blueish smoke that i can tell apart from the puff of black smoke when it starts. i wouldnt be against doing a rebuild if i could do a basic rebuild but i know myself and id want to pour money into better parts.
|All times are GMT -4. The time now is 06:46 AM.
Powered by vBulletin® Version 3.8.7
Copyright ©2000 - 2015, vBulletin Solutions, Inc.
= Copyright RamForumZ.com a Gigathreads.com Network Site =Ad Management by RedTyger


text:
Admission Information for Mirasol Clients
The admission process for Mirasol clients is very easy. When you make your decision to receive help for your eating disorder at one of the Mirasol programs, please call 888-520-1700. Carol Magee, our Admissions Director, will guide you through our simple process.
- Determine Your Insurance and Payment Options. Mirasol's Financial Officer will call you to discuss payment options and insurance coverage (if applicable).
- Medical Exam and Lab Work. We will e-mail you the form that your physician must complete to clear you or your daughter for treatment at Mirasol.
- Telephone Interview. Once we have your medical clearance, our Clinical Director will call you to set up the time and date for the telephone intake. This intake takes approximately 35 to 45 minutes. The date of your arrival at Mirasol can be determined as soon as you complete your medical clearance and the telephone intake. This entire process takes a week or even less depending on the individual circumstances.
For additional information, please call us toll-free at 888.520.1700 or complete the inquiry form below. Note that this is an inquiry form only and does not obligate you to enrollment in any way. All information will be kept strictly confidential.


text:
Atlanta Inn
No availability found through our partners. Please contact the business directly or check some of our recommended alternatives.
- Continental Breakfast
- Continental Breakfast
- Continental Breakfast
- Free High-Speed Internet
- Free High-Speed Internet
- Free High-Speed Internet
TripAdvisor Reviews Atlanta Inn
Travel Blogs from Atlanta
T-Minus 10 days!
Well folks, I am off to India and I could not be more excited! I will be serving on a 15 day trip through Hope Worldwide in New Delhi, India. I will be located at a rehabilitation clinic for individuals with leprosy and at a school/orphanage in one of the largest slums of Asia. I look forward to new friendships, new memories, and of course ...
... to see Iowa but now that I have, I can't believe I've overlooked it. Not because it's a life altering place or experience but that it's another piece to the puzzle of life. Iowa is no Serengeti or Patagonia, but it is worthy of respect. We only have so much time in our lives which is why we are herded to those places of maximum impact. That's if we are lucky enough to afford it. And then, it dawned on me; I finally remembered why I made the decision to move in ...
Ways To Make Your Wheels Road-Safe
It seems an obvious thing to say but the tires on your car are the point of contact between the road and your car or truck. Despite being very important to a car, many people do not take the time to take care of them, thus making your car into a safety hazard. If you have ever encountered a tire blow out or possibly a car in an out of control skid, you will know how frightening this can be, so it makes sense to take some time to ...
It was this big......
So passing through Funks Grove and McLean I have arrived in Atlanta. Home of the second Route 66 giants. It is one of those places like a tidy town winner, clean and brilliantly bright along the main strip with the subtle suburbia hiding in the ...
... tonight, and it wasn't that cold. Oops. At least there's more room in the RV now.
Got some more maps and info on Route 66 from the Joliet Museum. (BTW, we still have 6 cookies, left. Shannon and Rietta, think you could deliver to St. Louis? We should be there in, say, another week – or three. After all, it's all of maybe 200 miles from here.) So now we have about 4 books, several maps, and a bunch of brochures on ...


text:
There are currently no items in your cart.
Do You Love Me
By Kirby Shaw
- Look inside(2) Listen (1)
- Instrument:
- Ensemble:
Do You Love Me arranged by Kirby Shaw. For Choral (SSAB). Pop Choral Series. 12 pages. Published by Hal Leonard (HL.8202086).
This 1962 pop hit by the Contours captures all the sound and energy of the era and makes a perfect dance feature!Available separately: SSAB, SAB, TTB, ePak(C) and ShowTrax CD. Peformance Time: Approx. 2:20.
Buy Together
Do You Love Me
Folder - Legacy Choral
Buy both for $28.69
Customers Who Bought Do You Love Me Also Bought:
Do You Love Me
Irving Berlin's Christmas
$2.95 $2.51
Irving Berlin: From Rags to Ritz
$2.95 $2.51
Christmas Time Is Here (from A Char...
$1.95 $1.66
The Little Drummer Boy / Peace on E...
$1.90 $1.61
$1.95 $1.66
Please Mr. Postman
$1.95 $1.66
$1.90 $1.61
Rock and Roll Is Here to Stay (Medl...
How Sweet It Is to Be Loved by You
$1.90 $1.61
Please Mr. Postman
Save the Last Dance for Me
You Keep Me Hangin' On
$1.90 $1.61
Life Is a Highway
$1.80 $1.53
Unchained Melody
$1.95 $1.66
Unchained Melody
I Heard It Through the Grapevine
$2.25 $1.91
Consider Yourself (from Oliver)
$1.95 $1.66
To play the media you will need to update yourFlash plugin.
Do You Love Me
- Explain exactly why you liked or disliked the product. Do you like the artist? Is the transcription accurate? Is it a good teaching tool?
- Consider writing about your experience and musical tastes. Are you a beginner who started playing last month? Do you usually like this style of music?
- Feel free to recommend similar pieces if you liked this piece, or alternatives if you didn't.
- Be respectful of artists, readers, and your fellow reviewers. Please do not use inappropriate language, including profanity, vulgarity, or obscenity.
- Avoid disclosing contact information (email addresses, phone numbers, etc.), or including URLs, time-sensitive material or alternative ordering information.
- We cannot post your review if it violates these guidelines. If you have any suggestions or comments on the guidelines, please email us.
- All submitted reviews become the licensed property of Sheet Music Plus and are subject to all laws pertaining thereto. If you believe that any review contained on our site infringes upon your copyright, please email us.
- Read Sheet Music Plus's complete Privacy Policy
Do You Love Me
Tell a friend (or remind yourself) about this product. We'll instantly send an email containing product info and a link to it. You may also enter a personal message.
We do not use or store email addresses from this form for any other purpose than sending your share email.
Read our Privacy Policy.
Now you can also view your purchased titles on your iPad with our
free Sheet Music Plus Digital Print Viewer app.
With Digital Print, you can print from your computer immediately after purchase, or wait until its convenient. And our software installation is easy - we'll guide you through the simple steps to make sure you have Adobe Flash Player, Adobe AIR and the Sheet Music Plus AIR application on your computer.
See all System Requirements for Digital Print
Note: Our Digital Print items are purchased solely through our website. By purchasing a Digital Print item, you are authorized by the publisher to print out the purchased number of copies for that particular title. Thus, we are unable to fax, mail or email digital titles in any form or file format (including PDF) as it violates copyright law.
Google Chrome Users: The most recent Google Chrome update is not compatible with Adobe AIR. We recommend using an alternate browser (such as Internet Explorer, Safari, or Firefox) in order to download Adobe AIR and print your music.
Digital Print - Available Instantly!
If you have any questions, comments or concerns, please contact us at firstname.lastname@example.com.
Make a wish list for gifts, suggest standard repertoire, let students know which books to buy, boast about pieces you've mastered: Music Lists are as unique as the musician! Easily share your music lists with friends, students, and the world.
To create a music list, please sign in.Existing Accounts
Sign In and Add to My Music ListNew Accounts
See Similar Sheet Music
More by this Arranger: Kirby Shaw Sheet Music Kirby Shaw Choir Sheet Music Kirby Shaw Choir Sheet Music
Instrument: Choir Sheet Music
Ensemble: Choir Music


text:
Climbing vine under Doug firs
I have a very ugly shed that I am trying to disguise but, it is under several large Doug fir trees. I would love to have a vine clamber over it...any suggestions? It obviously is full shade and very dry...am I looking for the impossible?
Characteristic of Douglas fir/madrona/salal association on bluffs etc. near Puget Sound, where some tall examples can be seen hanging down many yards out of Douglas firs on some sites.
L. ciliosa is more exciting in flower but leaves do not persist as well (if at all, during winter) and is not as much of an evergreen forest species. But since it is a local native and can be seen growing up conifers here and there it might also work out.
As with other climbing honeysuckles a pile of snaking stems is liable to be produced where the specimen ends up perched on top of a structure.


text:
The fact that not all the votes had been counted before Mohammed Morsi, the Muslim Brotherhood's candidate for the Egyptian presidency, declared victory on Monday was of little comfort to the country's sizeable Christian minority, which fears an Islamic takeover.
The uncertainty surrounding the election — by Tuesday, both Morsi and Ahmed Shafiq, the former prime minister and Morsi's old-guard competitor, said they had won — has left Egyptians, and especially the country's 8 million to 10 million Coptic Christians, deeply worried about the future.
The presidential runoff was the final step in an exhausting months-long election process that followed the ouster of President Hosni Mubarak...READ MORE


text:
I am facing a small problem. I have a struct, which has a vector. Note that the vector is dynamic per every iteration. Now, in a particular iteration, how do I store the struct which contains a vector of size n to a binary file?
Also, when retrieving, assume that I know how the size of the vector, how to I retrieve from the binary file, the struct variable containing the vector of all the stored elements?
I am able to store something to the binary file (as I can see the size increasing when writing), but when I am trying to retrieve back the elements, I am getting size of vector to be zero.
Unfortunately, I have to achieve this using the standard STL and not use any third-party libraries.


text:
Turtle Themes
Turtle Themes
Freedom and Confinement
Nobody likes feeling stuck. And Kay Ryan's titular turtle is "often" stuck. All she asks is the freedom to move (see "Mobility Imagery" in "Symbols, Imagery, and Word Play") so she can find food. B...
Here's a riddle for you: In "Turtle," how is Kay Ryan's turtle like Abraham Lincoln? You can make up your own response if you like, but here's one answer: They both understand the power of persever...
Women and Femininity
Few would argue that "Turtle" is an overtly feminist poem, but some readers detect a subtle critique of traditional female roles in society. There's no getting around the fact that Kay Ryan chose t...
As the opposite of arrogance, humility is arguably a positive trait (arrogant hare, humble tortoise). Although the word "humility" does not appear in "Turtle," the turtle clearly qualifies as a hum...
People who Shmooped this also Shmooped...
Noodle's College Search


text:
Christian leaders in America have expressed both concern and hope at news of historic plans to normalize diplomatic relationships between the U.S. and Cuba. Some political leaders have also suggested that Cuba has not shown enough progress in human rights to justify easing economic restrictions.
"First, I am concerned that normalizing diplomatic ties without addressing [Fidel] Castro's horrendous human rights record serves as a defacto endorsement for one of the most oppressive regimes in recent history," said the Rev. Samuel Rodriguez, president of the National Hispanic Christian Leadership Conference, in a statement.
"As a result of Castro's totalitarian rule, millions live in poverty, thousands lie in prisons, and many have lost their lives. In addition, the God-given rights of Cuban citizens are held hostage to governmental persecution." more >>
Pakistan's Supreme Court has ordered the arrest of two Muslim clerics for inciting a violent mob of hundreds of Muslims to brutally beat, torture, burn and murder a married Christian couple in the Punjab Province in early November.
The court also ordered disciplinary action to be levied against five police officials, who were present during the time of the beatings but took no action to protect the two individuals. Additionally, the court ordered a complete investigation into the incident and also ordered compensation payments to be made to the family of the victimized couple.
After being accused of burning pages from the Koran, Shamah Masih, who was a 24-year-old mother of four and four months pregnant at the time, and her husband, Shahzad Masih, were surrounded by an enraged mob, who gathered around the brick kiln in the village of Kot Rodha Kishan where the couple was located. more >>
The Rev. Franklin Graham says Christians should never deny Jesus Christ in order to spare their lives from slaughter by terrorists, such as ISIS, and further asserts that doing so would be a betrayal against martyrs who stood strong for their faith, despite persecution.
"Christians have been martyred going all the way back to the Romans when they fed them to the lions. We would be betraying every person who stood for their faith, who stood for Jesus Christ, if we turn our back on Jesus Christ. … I think we take a stand for our faith," Graham said on Fox News' "The Kelly File" when host Megyn Kelly asked if Christians should lie in order to save their lives when confronted with death at the hands of Islamic terrorists.
Noting that Christians who live in countries where they're the religious minority are the most persecuted people in the world, Kelly commented on a story about four Iraqi Christian children who were beheaded by ISIS militants after they refused to convert to Islam, a story reported by The Christian Post earlier this month. more >>
Lennon Lacy, 17, was found hanged in Aug. and his family insists that he was lynched; the FBI just announced that they would be looking into the case to determine whether Lennon's death was self-inflicted or murder.
"That's all I've ever asked for: what is due, owed rightfully to me, and my family – justice. Prove to me what happened to my child," Claudia Lacy, mother of the deceased, told CNN.
Lennon was last seen on Aug. 28, when he left his home in Bladenboro, North Carolina, to exercise. When he didn't return home, his parents grew concerned, and at 7:30 the following morning, Lennon was found hanging from a swing set in the center of a park. Medical reports stated that he was covered in fire ants, adding to the sadness of the teen's death. more >>
Nobel Peace Prize laureate Malala Yousafzai, who was shot in the head by the Taliban for advocating girls' right to education, expressed heartbreak at the news of the Taliban's attack on a school in Pakistan where at least 130 people, most of them children, were killed.
In a statement released Tuesday, Yousafzai expressed, "I am heartbroken by this senseless and cold blooded act of terror in Peshawar that is unfolding before us. Innocent children in their school have no place in horror such as this."
"I condemn these atrocious and cowardly acts and stand united with the government and armed forces of Pakistan whose efforts so far to address this horrific event are commendable." more >>
Thirteen Christian families in a small village located in the Punjab province of Pakistan are now homeless after the local municipal government demolished their homes because the families refused to work for no pay and become bonded laborers.
Members of the Christian families, who all worked at a local brick kiln in the village of Samundri, refused to continue working unless they received fair compensation for their labor. Pakistan Christian Post reports that the village's municipal government carried out the destruction of their homes last week after being pressured to do so by the "influential" Muslims who owned the brick kiln operation.
"Houses of 13 Christian families have been demolished by the Town Municipal Administration Samundri under the pressure of the influential people of the village just because they refused to bonded labour without any penny," the news report stated. more >>


text:
Maya2581's Mom Answers Activity
Maya2581 is a star in these topics:
Pregnancy > Emotional Health During Pregnancy (1 helpful answer)
Getting Pregnant > Get your life ready (3 helpful answers)
Pregnancy > Giving Birth (2 helpful answers)
Back pain after having an epidural
Most recent answer (02/12/2015):
I have pain in the exact spot. I just reported it to my doctor who literally rolled her eyes at the suggestion. Perhaps I should show her...posted by Gggggg
11 week old goes stiff and rigid for 5 to 8 seconds, then cries afterwards
Most recent answer (11/17/2013):
My lg is 3 months and she has only ever done this twice (so far) and both times were in the bath?! She is in a rigid bath seat. Scares me...posted by MrsSezMac
My 3yr old son complained that his penis hurts. When I check him it?s ?stiff?. Is this normal?
Most recent answer (01/16/2008):
My 4 year old does the same thing. We had him checked, doc found no prob. Changed detergent, bought bigger undies, same prob. He's crying...posted by Maya2581