text:
|Parent Central
|Youth Services
|CYS Sports
|School Support
|Strong Beginning
The Youth Services (Grade 6/7 - 12) program is offered as a supplement
not a substitute for, the family as the primary agent for the care
development of the young adult.
The Youth Center offers various
activities that are intended to provide age appropriate,
supervised activities for the family members of active duty military
The program offers closely monitored activities and programs
Operation Hours
We are committed to providing high quality, comprehensive
Youth Center Summer Events
Summer Teen Camps
Ping Pong Madness camp, 1400-1600, 9-14 AugustYouth will learn the basics as well as tips and tricks for table tennis and then compete with each other
using their new skills
Youth Bagging Opportunities!
To be eligible you must:
Middle School 8th graders will only be allow to bag at the end of their 8th grade school year.
Money Matters Workshop (Youth Center)
The Money Matters Workshops can give you the tools you need to make the right choices in planning for Clollege; Budget living within your means; Credit/Debt; Saving & Investing; and Entrepreneurship. Stop by the Youth Center to sign up for these informational workshops that lead to BGCA Money matters Scholarships.
Must have an active CYS Services Membership to participate (ages 13-18 years)
Camp Walker building #252


text:
Mercedes sales jump 16% in September on China demand
Oct. 6, 2015 12:38 CET
Mercedes brand's global sales increased 16 percent in September, buoyed by a 53 percent jump in China deliveries.... Read More »
Smart ForTwo EV to arrive in U.S. late next year
12:01 am U.S. ET | Oct. 5, 2015
The next-generation electric Smart ForTwo will arrive in the U.S. at the end of 2016. Government incentives likely will determine whether demand will outpace the 25 percent take rate seen for the electric version of the ForTwo last year.... Read More »
Renault will supply Daimler's Smart with electric motors
Sept. 16, 2015 13:00 CET
Renault will supply motors for electric versions of Daimler's Smart city cars under their deepening five-year-old alliance. Renault already uses the motors in its Zoe EV.... Read More »
Mercedes sales rise 18% in August on China surge
Sept. 4, 2015 15:28 CET
Mercedes' brand sales increased 18 percent in August, marking the 30th consecutive month of record sales at the automaker. Demand was particularly robust in China, where sales rose 53 percent. Eight-month brand sales were up 15 percent to 1.18 million.... Read More »
Updated Smart ForTwo convertible is due next summer
12:01 am U.S. ET | Aug. 31, 2015
The convertible version of the redesigned Smart ForTwo will debut next month at the Frankfurt auto show and go on sale in the U.S. early next summer.... Read More »
Smart ForTwo redesigned to better fit U.S. buyers
12:01 am U.S. ET | Aug. 31, 2015
Smart's all-new 2016 ForTwo may have a European flavor to it, but significant portions of the car were specifically designed for the U.S. market.... Read More »
Smart ForTwo takes on more American dimensions
3:34 pm U.S. ET | Aug. 27, 2015
The bite-size Smart ForTwo may have a European flavor, but the third-generation model coming to the U.S. in September was cooked up using plenty of American ingredients. All of the key changes to the 2016 model -- increased width, new transmission choices, and more substantial styling -- were made primarily with the U.S. in mind.... Read More »
How Daimler's Car2go could transform how New Yorkers get around town
3:55 pm U.S. ET | Aug. 20, 2015
As residents of Brooklyn and Queens know all too well, not having a car makes it difficult to get from one borough to the other. But soon there will be another option: hopping into an unnervingly small, but surprisingly roomy, two-person vehicle and motoring to the other borough, where the rental ends once the driver parks.... Read More »
Daimler seeks way around France's 35-hour workweek
Aug. 12, 2015 12:23 CET
Daimler is trying to circumvent France's 35-hour workweek. It wants workers in Hambach, where it makes the Smart ForTwo, to put in more hours and has hinted that it may shift production if it cannot reach an agreement with employees.... Read More »
Smart sticks with single nameplate
12:01 am U.S. ET | Aug. 3, 2015
The Smart microcar will continue to be a niche product in the U.S. with annual sales of about 10,000. Management has chosen to sell only one car here - the miniscule ForTwo and not the four-seat ForFour.... Read More »
Luxury brands draw tech battle lines
12:01 am U.S. ET | Aug. 3, 2015
European luxury brands are leaving few segments untouched, but the new battleground is high-technology and semiautonomous features.... Read More »
Curse of the $16.5 million Ferrari pits tycoon against an auction house
July 11, 2015 06:15 CET
Following 16 years of lawsuits and ownership claims from Paraguay to Switzerland, the fate of a 1954 Ferrari 375 Plus roadster, which Victoria's Secret founder Les Wexner thought he bought last year for 10.7 million pounds ($16.5 million), is finally coasting toward resolution in a London court.... Read More »
Smart has no plans for an SUV, CEO says
12:01 am U.S. ET | June 15, 2015
Daimler's Smart subsidiary will not expand its lineup with an SUV/crossover in the near future, said the brand's CEO, Annette Winkler.... Read More »
Smart has no plans for crossover, CEO Winkler says
June 10, 2015 19:36 CET
Daimler's Smart subsidiary will not expand its lineup with an SUV/crossover in the near future, the brand's CEO, Annette Winkler, said.... Read More »
Mercedes brand sales rise 13% in May on China, U.S demand
June 8, 2015 11:44 CET
Global sales of Mercedes cars rose 13 percent in May, lifted by gains in the U.S. and China, along with strong demand for SUVs, compact models and the C class.... Read More »
Mercedes sales increase 12% in April on China, U.S demand
May 7, 2015 11:45 CET
Daimler said vehicle sales at its Mercedes cars unit, including Smart, rose 12 percent last month, driven by robust demand in the U.S. and China as well as strong sales of the Mercedes C class and GLA.... Read More »
BLOG: Daimler mimics BMW with conservative profit outlook
6:17 am U.S. ET | April 29, 2015
Daimler seems to have taken a lead from BMW in keeping its 2015 profit outlook conservative to avoid any risk whatsoever of potentially giving the financial markets an unpleasant surprise later in the year.... Read More »
BLOG: Daimler mimics BMW with conservative profit outlook
6:17 am U.S. ET | April 29, 2015
Daimler seems to have taken a lead from BMW in keeping its 2015 profit outlook conservative to avoid any risk whatsoever of potentially giving the financial markets an unpleasant surprise later in the year.... Read More »
Smart CEO to speak at Europe Congress
12:01 am U.S. ET | April 13, 2015
Smart CEO Annette Winkler, the first woman to lead an automotive brand in Europe, will speak at the Automotive News Europe Congress on June 10 in Birmingham, England.... Read More »
Mercedes sales increase 16% to monthly record
April 8, 2015 12:03 CET
Mercedes sales rose 16 percent in March to a monthly record, powered by robust demand in Europe, China and the U.S. and strong sales of the automaker's C class, compacts and SUVs.... Read More »
Mini clears Superleggera roadster for production, report says
11:30 am U.S. ET | March 17, 2015
It's not quite as exciting as a Beatles reunion, but it looks like one of Britain's greatest hits -- the affordable sports car -- could finally be returning. Car magazine reported that BMW officials have approved for production the Mini Superleggera concept car, which debuted last year.... Read More »
Mercedes sales rise 15% in Feb. on Europe demand
March 6, 2015 12:43 CET
Mercedes brand sales increased 15 percent in February, driven by robust demand in Europe and strong global sales of its C class, compacts and SUVs.... Read More »
Yamaha plans 2-seat car for Europe, source says
Feb. 27, 2015 10:06 CET
Japanese motorcycle maker Yamaha aims to start making and selling two-seat cars in Europe as early as 2019, a person familiar with the plan told Reuters on Friday.... Read More »
Faurecia helps cut CO2 in new Renault Twingo
12:01 am U.S. ET | Feb. 3, 2015
Faurecia's emissions control systems helps the third-generation Renault Twingo reduce fuel consumption by 26 percent to 4.2 liters per 100km and cuts CO2 emissions by 35 grams per kilometer to 95g/km in variants with the 1.0-liter gasoline engine equipped with stop-start technology.... Read More »
Daimler seeks profitability at Smart with 2 minicar sizes
Jan. 20, 2015 06:01 CET
Daimler hopes to make its Smart small-car unit sustainably profitable by offering the same car in two sizes: the two-seat ForTwo and the four-seat ForFour.... Read More »
- Fiat expects low-cost compact car family to mimic Dacia's success
- Fear and respect: VW's culture under Winterkorn
- Winterkorn to step down from remaining VW Group posts, report says
- A mortifying end to Audi engineering guru's distinguished career?
- Wagons give lift to Europe's severely weakened volume midsize segment
Hillside Honda rewards: Paid vacations, Rolex watches, family atmosphereThirty is a special number at Hillside Honda in New York City. Sales staffers who sell 30 rides a month get a $1,000 paid vacation and three extra days off.
» Watch the Video

subset: commoncrawl


text:
Pakistan's Nawaz Sharif vows to pursue warmer relations with India
- Pakistan elections: Nawaz Sharif set for 3rd term, India welcomes result
- PML-N will revisit Pak foreign policy on India: Nawaz Sharif's aide
- Nawaz Sharif: The Lion of Punjab roars in Pakistan
- Pakistan elections: Nawaz Sharif set for 3rd term, India welcomes result
- PML-N will revisit Pak foreign policy on India: Nawaz Sharif's aide
- Nawaz Sharif: The Lion of Punjab roars in Pakistan
Top Stories
- Ghulam Ali's concert in Mumbai cancelled after Shiv Sena's threat
- PM Modi a sensitive person, don't judge him on social media posts: BJP on Dadri lynching
- Why President Mukherjee hit the right notes on core values of 'diversity', 'plurality' and 'tolerance'
- Barack Obama apologises for air strike on Kunduz hospital: White House
- Commercial vehicles entering Delhi to pay environmental tax: NGT
Nawaz Sharif, poised for a record third term as Pakistan Prime Minister after his party's emphatic win in the landmark general elections, has vowed to pursue better relations with the US while seeking "warmer ties" with India. Opinion: An extraordinary election
In an interview to The Wall Street Journal after the impressive victory of Pakistan Muslim League-N (PML-N) in the polls, Sharif recalled that his country's relationship with the US was quite good when he was in power.
"I'd like to take this relationship further. We need to strengthen the relationship," said 63-year-old Sharif, who has held the top position twice in the past.
"The US may wish to re-examine its drone campaign or risk stoking the anti-Americanism that feeds Pakistani public anger," Nawaz said, referring to the strong opposition to the controversial drone strikes by the CIA inside Pakistan.
Responding to questions on the drone strikes, Sharif said he would discuss these issues of concerns with the US leadership.
"These are the concerns that the Pakistani people have," Sharif said, when asked about the drone operations.
"We'll need to address these concerns. I'm very hopeful and confident about that," he added.
According to The Wall Street Journal, Sharif said he would "seek warmer ties" with India and offered an "olive branch" to Afghanistan.
Sharif who was ousted by the army in 1999 said he expects a smooth relationship with the military this time.
"There is no problem with the military," he said. "I don't think the military is responsible for what Musharraf did, he did it in his personal capacity. The military should not be blamed for this," he told the journal.
Yesterday, both US President Barack Obama and Secretary of State John Kerry said that they were looking forward to working with the new government of Pakistan.
... contd.


text:
Page 1
Inhibition of poly(ADP-ribose) polymerase
suppresses inflammation and promotes recovery
after ischemic injury
Tiina M Kauppinen1,2, Sang Won Suh1,2, Ari E Berman1,2, Aaron M Hamby1,2and
Raymond A Swanson1,2
1Department of Neurology, University of California San Francisco, San Francisco, California, USA;2Veterans
Affairs Medical Center, San Francisco, California, USA
The brain inflammatory response induced by stroke contributes to cell death and impairs
neurogenesis. Poly(ADP-ribose) polymerase-1 (PARP-1) is a coactivator of the transcription factor
NF-jB and required for NF-jB-mediated inflammatory responses. Here we evaluated PARP inhibition
as a means of suppressing post-stroke inflammation and improving outcome after stroke. Rats were
subjected to bilateral carotid occlusion-reperfusion, and treatment with the PARP inhibitor N-(6-oxo-
5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34) was begun 48h later. PJ34 was found
to rapidly suppress the ischemia-induced microglial activation and astrogliosis. Behavioral tests
performed 6 to 8 weeks after ischemia showed deficits in spatial memory and learning that were
lessened by the PJ34 treatment. Immunohistochemical evaluation of hippocampus at 8 weeks after
ischemia showed increased neuronal density in CA1 layer of PJ34-treated animals relative to
vehicle-treated animals. Bromodeoxyuridine labeling showed formation of new neurons in
hippocampal CA1 area in PJ34-treated animals, but not in vehicle-treated animals. Together, these
results suggest that treatment with a PARP inhibitor for several days after ischemia enhances long-
term neuronal survival and neurogenesis by reducing inflammation.
Journal of Cerebral Blood Flow & Metabolism (2009) 29, 820–829; doi:10.1038/jcbfm.2009.9; published online
4 February 2009
Keywords: PARP-1; ischemia; hippocampus; microglia; astrogliosis; neurogenesis
Poly(ADP-ribose) polymerase-1 (PARP-1; also known
as poly(ADP-ribose) synthetase or ADP-ribosyl trans-
ferase) functions as a sensor of DNA damage.
When activated by DNA strand breaks, the enzyme
consumes NAD+to generate ADP-ribose polymers on
several nuclear acceptor proteins involved in DNA
repair and genome integrity (Jagtap and Szabo, 2005).
Extensive activation of PARP-1 can promote cell
death in ischemia and other conditions through a
process involving NAD+depletion and mitochon-
drial release of apoptosis inducing factor (Jagtap and
Szabo, 2005). By an entirely separate mechanism,
PARP-1 may also contribute to injury after ischemia
through its influence on the cellular inflammatory
response (Hamby et al, 2007; Moroni, 2008; Oliver
et al, 1999).
PARP-1 influences inflammation by interacting
with the transcription factor NF-kB. PARP-1 en-
hances the NF-kB activity at promoter sites by
facilitating the binding of NF-kB subunits to DNA
and by promoting transcription complex formation
(Hassa and Hottiger, 2002; Kraus and Lis, 2003). NF-
kB drives expression of several pro-inflammatory
cytokines and proteases including iNOS, ICAM-1,
and TNFa (O'Neill and Kaltschmidt, 1997). The
expression of these proteins is down-regulated by
PARP-1 inhibition (Chiarugi and Moskowitz, 2003;
Oliver et al, 1999; Zingarelli et al, 1998). PARP-1
inhibition has also been shown to suppress NF-kB-
dependent gene transcription in microglia (Kauppinen
et al, 2008) and to suppress the morphologic changes
associated with microglial activation (Chiarugi and
Moskowitz, 2003; Kauppinen and Swanson, 2005;
Ullrich et al, 2001).
Ischemic stroke induces an inflammatory res-
ponse, which contributes to cell death and impairs
neurogenesis (Ekdahl et al, 2003; Hoehn et al, 2005).
Received 17 November 2008; revised 12 January 2009; accepted 13
January 2009; published online 4 February 2009
Correspondence: Dr TM Kauppinen, Department of Neurology,
University of California San Francisco, VAMC, 4150 Clement
Street, San Francisco, CA 94121, USA.
E-mail: firstname.lastname@example.com
Journal of Cerebral Blood Flow & Metabolism (2009) 29, 820–829
& 2009 ISCBFM All rights reserved 0271-678X/09 $32.00
Page 2
Prior studies have identified minocycline as an
effective agent for suppressing brain inflammation
(Koistinaho et al, 2004). The mechanism of this anti-
inflammatory effect has not been established but may
be due to the potent inhibition of PARP-1 by
minocycline (Alano et al, 2006). The aim of the
present study was to determine whether a PARP
inhibitor, N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-
N,N-dimethylacetamide (PJ34), could suppress brain
inflammation and thereby enhance long-term recov-
ery and neurogenesis in a rat model of forebrain
ischemia. The inflammatory response triggered by
brain ischemia takes many hours to develop, thus,
providing a relatively long time window in which to
initiate a therapeutic intervention (Dirnagl et al,
1999). Here we initiated treatment with PJ34 at 48h
after ischemia, a delay that precluded direct neuro-
protective effects. The delayed treatment with PJ34
was found to suppress microglial and astrocyte
activation within 24h, and evaluations performed
at 6 to 8 weeks after ischemia showed increased
neurogenesis, increased pyramidal neuron density in
hippocampal CA1 region, and improved spatial
memory in the rats treated with PJ34.
Materials and methods
Sprague–Dawley rats were obtained from Simonsen La-
boratories and were housed two per cage under conditions
of constant temperature, light from 6:00 AM to 6:00 PM, and
free access to food and water. N-(6-oxo-5,6-dihydrophe-
nanthridin-2-yl)-N,N-dimethylacetamide (PJ34) was ob-
tained from Inotek (Beverly, MA). Other materials were
obtained from Sigma-Aldrich, except where noted.
Transient Forebrain Ischemia
Transient forebrain ischemia was produced with bilateral
common carotid artery occlusion and hypotension as
described by Smith et al (1984), under a protocol approved
by the San Francisco Veterans Affairs Medical Center animal
studies committee. Animals were anesthetized with 2%
isoflurane in closed plastic chamber and maintained with
30% O2/70% N2O delivered via face mask. Rectal tempera-
ture was monitored and maintained at 371C±0.51C through-
out the surgical procedure with thermal blanket. The
common carotid arteries were exposed through a midline
skin incision and carefully freed from the adjacent vagus
nerve. The exposed common carotid arteries were occluded
by microvascular clips and systemic mean arterial pressure
was lowered to 40±5mmHg by withdrawing blood (7 to
10mL) from the femoral artery into a heparinized syringe
maintained at 371C. Ten minutes later, the clips were
removed from the common carotid arteries and the blood
was reintroduced to the femoral artery. The incisions were
closed, the femoral artery catheter was removed, and
anesthesia was discontinued. Animals exhibiting seizures
after reperfusion were killed (21 of 120 rats) and not assigned
to control or drug-treatment groups.
Treatment Schedule
PJ34 was administered at a dose of 15mg/kg by intra-
peritoneal injection, in a volume of 1.0mL of 0.9% saline.
Treatment was initiated at 48h after ischemia and
subsequent injections were given at 12-h intervals for 1 to
7 days (Figure 1). The control group received injections of
saline vehicle alone. Bromodeoxyuridine (BrdU), a syn-
thetic thymidine analog that incorporates into DNA during
the S-phase of the cell cycle, was used to label dividing
cells. Bromodeoxyuridine was administered at a dose of
50mg/kg by intra-peritoneal injection starting at day 5 after
ischemia and subsequent injections were given at 12-h
intervals until day 9. Rats were killed at the designated
time points, and brains were perfusion fixed with 4%
formaldehyde, except the rats designated for hematoxylin-
eosin staining.
Assessment of Neuronal Death
Acute ischemia-induced neuronal death was evaluated 9
days after ischemia. Brains were harvested without perfu-
sion and were frozen with powdered dry ice. Coronal 20mm
frozen sections were prepared, fixed in 70% ethanol, and
stained by hematoxylin-eosin staining, as described pre-
viously (Hamby et al, 2007). Five coronal sections were
analyzed from each animal, beginning 4.0mm posterior to
bregma and spaced 200mm apart. An observer blinded to the
treatment conditions counted the number of eosinophilic
neurons in the hippocampal CA1 area in both hemispheres
of each rat. Data from each animal were expressed as the
total number of eosinophilic neurons in both the right and
the left CA1 cell fields in the five coronal sections.
Behavioral Tests
The spontaneous open field activity test and the Morris
water maze test were performed 6 to 8 weeks after
ischemia, as described previously (Morris, 1984). Rats
Figure 1 Experimental design. PJ34 and saline i.p. injections
were started 48h after 10mins forebrain ischemia (FI) and
continued every 12h for 7 days. Bromodeoxyuridine injections
were given twice daily between days 5 and 9 after ischemia.
Animals were killed 3 days or 8 weeks after ischemia for
immunohistochemistry (IHC), or at 9 days after ischemia for
hematoxylin-eosin staining (H&E). Behavioral testing was
performed at 6 to 8 weeks after ischemia.
PARP inhibition after stroke
TM Kauppinen et al
Journal of Cerebral Blood Flow & Metabolism (2009) 29, 820–829
Page 3
were housed individually in the testing room beginning 3
days before testing to minimize the effects of social
influences on behavior. As a first step, spontaneous open
field activity was assessed to identify any locomotor
differences that could bias the measures of learning and
memory. Rats were individually placed in a plexiglass
enclosure (40?40 inches) equipped with two rows of
infrared photocell panels interfaced with a computer. The
lower row detected horizontal exploratory activity, and the
higher row detected vertical exploratory activity or rearing.
Light path breaks were recorded and assessed for measure-
ments of the number of movements, active times, path
length, and rearing events. On each of three consecutive
days, open field activity was recorded for 10mins after an
initial 1min adaptation period.
Spatial learning and memory were tested by the Morris
Water Maze test, using a circular pool (180cm in diameter,
50cm deep) filled with opaque water of constant tempera-
ture (241C). The rats were trained first to locate a platform
with a visible cue (days 1 to 2), and then to locate a hidden
platform (days 3 to 5) using large spatial cues in the room.
The platform was moved to a new quadrant in each session
during the visible platform cue training. The platform
remained in the same quadrant throughout all the sessions
during hidden platform training. The rats received two
training sessions per day for five consecutive days. Each
session consisted of three 1-min trials with a 10-min inter-
trial interval. The interval between the two daily sessions
was 3h. Once the rats located the platform, they were
allowed to remain on it for 10secs. Rats that failed to find
the platform within 1min were manually placed on the
platform for 15secs. Time to reach the platform (latency),
distance traveled (path length), and swim speed (velocity)
were recorded with an EthoVision video tracking system
(Noldus Information Technology, Leesbug, VA) set to
analyze two samples per second. Two rats (one from the FI
+saline group and one from FI+PJ34 group) were
excluded from the analysis, because they never found the
platform. These animals also exhibited large infarcts in
hippocampus, in contrast to the selective neuronal injury
observed in the other postischemic rats.
The paraformaldehyde-fixed brains were cut in 50mm
cryostat sections through the rostral-caudal extent of the
hippocampus, and 5 to 6 evenly spaced sections were
evaluated from each brain and analyzed for each histologic
'n.' Immunohistochemistry and double-immunofluores-
cence staining were performed in floating sections, as
described previously (Liu et al, 1998). Primary antibodies
used were mouse monoclonal anti-rat CD11b (1:100
dilution) (clone OX-42, Serotec, Oxford, UK), rabbit anti-
glial fibrillary acidic protein polyclonal antibody (1:1000
dilution) (Chemicon, Temencula, CA), mouse anti-NeuN
monoclonal antibody (1:1000 dilution) (Chemicon), sheep
anti-BrdU polyclonal antibody (1:180 dilution) (GeneTex
Inc, San Antonio, TX), and mouse anti-PAR monoclonal
antibody (1:1000 dilution) (Trevigen Inc, Gaithersburg,
MD). Secondary antibodies were mouse, rabbit, or sheep
anti-IgG conjugated with Alexa Fluor 594 or 488 (1:500
dilution each) (Molecular Probes Inc, Eugene, OR). Fluor-
escence signals were visualized by confocal microscopy
with appropriate filter sets. Immunostaining was evaluated
by using the Zeiss LSM 510 confocal image system with a
sequential scanning module by an observer blinded to the
experimental conditions. Controls prepared with primary
or secondary antibodies omitted showed no detectable
fluorescence under the conditions used for confocal
photography. Colocalization of BrdU with NeuN was
confirmed by collecting stacks of images from consecutive
optical slices of 0.77mm thickness to generate 3 dimen-
sional images of the cells of interest.
Evaluation of Brain Microglial and Astrocyte
Microglial activation was scored on the basis of CD11b
expression intensity, density of CD11b-expressing cells,
and microglial morphology, as detailed in Table 1. The
scoring method is slightly modified from 6 that previously
published (Kauppinen et al, 2008). Astrogliosis was
evaluated by quantifying the intensity of the fluorescence
signal from brain sections immunostained for glial fibril-
lary acidic protein (GFAP), which is upregulated in
reactive astrocytes (Eng and Ghirnikar, 1994). All of the
brain sections from the different treatment groups were
stained in parallel to ensure identical staining conditions.
Data were acquired using the NIH Image J program and
normalized to the mean expression level observed in sham-
operated rats.
Table 1 Criteria for evaluating microglial activation status
Score CD11b expressionScore Morphology
0 No cells with continuous CD11b-positive
1–9 cells with continuous CD11b-positive
processes per 100mm2
10–19 cells with continuous CD11b-positive
processes per 100mm2
>20 cells with continuous CD11b-positive
processes per 100mm2
0 No cells with activated morphology (amoeboid shape with
enlarged soma and thickened processes)
1–40% of CD11b-positive cells have activated morphology
2 40–90% of CD11b-positive cells have activated
>90% of CD11b-positive cells have activated morphology
Microglial activation was evaluated by two different criteria, CD11b expression and morphology, which combined give maximum score of 6.
PARP inhibition after stroke
TM Kauppinen et al
Journal of Cerebral Blood Flow & Metabolism (2009) 29, 820–829
Page 4
Statistical Analysis
Results are presented as a mean±standard error. Morris
water maze path-length data were analyzed with linear
mixed model curve fitting using the SAS Proc MIXED
procedure (SAS Institute, Cary NC, Ver 9.1.2). The models
fit allowed separate trajectories during the testing and
training phases as well as animal-specific trends. Immuno-
staining data were assessed by the nonparametric Kruskal–
Wallis one-way analysis of variance test followed by
Dunn's test for multiple group comparison. Cell death
was assessed by one-way analysis of variance followed by
Tukey–Kramer test for multiple comparisons between the
treatment groups.
Delayed-Onset Poly(ADP-ribose) Polymerase Inhibition
Reduces Microglial Activation and Astrogliosis After
Forebrain Ischemia
increase CD11b expression (Kreutzberg, 1996). Brains
harvested at serial time points after forebrain ischemia
showed microglial activation that was evident within
24h and peaked approximately 3 days after ischemia
microglia retracttheir processesand
(Figure 2A), consistent with prior reports (Hamby
et al, 2007; Sugawara et al, 2002). Administration of
the PARP inhibitor, PJ34, beginning 48h after ischemia
reduced microglial activation to basal levels within
24h (Figure 2A and 2B). There was no rebound in
microglial activation either during or after the 7-day
treatment course with PJ34 (not shown). To further
evaluate the role of PARP-1 in microglial activation,
immunostaining for poly(ADP-ribose) (abbreviated as
PAR), the enzymatic product of PARP-1, was evaluated
in PJ34- and vehicle-treated animals at 3 days after
ischemia. Ischemia-induced PAR accumulation was
seen predominantly in microglia, with little PAR
labeling in other cell types (Figure 3), and the PAR
formation was completely suppressed by PJ34. Ische-
mia also induced a robust astrogliosis that peaked at 5
to 7 days (Figure 2C), and treatment with PJ34 also
suppressed this astrocyte response (Figure 2C and 2D).
Prior studies have shown that PARP activation in
hippocampal neurons begins within the first hour
after ischemia and persists for up to 24h, and that
morphologic evidence of neuronal death is not
prominent until after that time (Deshpande et al,
1987; Nagayama et al, 2000). Because PARP inhibi-
tion can block acute ischemic neuronal death in
addition to the postischemic inflammatory response,
Figure 2 Time course of glial activation in CA1 hippocampus after ischemia. (A) Microglia are immunostained for CD11b. (B)
Astrocytes are immunostained for glial fibrillary acidic protein (GFAP). Rats treated with PJ34 beginning 48h after forebrain
ischemia (FI) showed a near-complete reversal of glial activation after ischemia. Photos are representative of two to three animals at
each time point. (C) Quantification of microglial activation at 3 days after ischemia, scored as detailed in Table 1. (D) Astrocyte
activation at day 5 after ischemia as quantified by intensity of GFAP immunofluorescence (*P<0.05; n=3).
PARP inhibition after stroke
TM Kauppinen et al
Journal of Cerebral Blood Flow & Metabolism (2009) 29, 820–829
Page 5
we delayed PJ34 treatment until 48h after ischemia
to selectively target the inflammatory response.
Hippocampi from brains (n=5 for each treatment
group) harvested9 days
evaluated by hematoxylin-eosin staining to confirm
that delayed PJ34 treatment did not affect acute
after ischemiawere
neurodegeneration. There was no decrease in the
number of eosinophilic neurons in the rats treated
with PJ34 (3,823.2±113.3) beginning at day 2 after
ischemia when compared with saline-treated rats
(4,013.2±206.8) (Supplementary Figure 1).
Delayed Poly(ADP-ribose) Polymerase Inhibition
Promotes Neurogenesis in Hippocampal CA1
Bromodeoxyuridine was injected on days 5 through
9 after ischemia to evaluate the influence of PJ34 on
neurogenesis (Liu et al, 1998). Brains harvested 8
weeks after ischemia showed nonneuronal BrdU-
positive cells in all treatment groups (Figure 4A).
Newly produced neurons, as identified by double
staining with BrdU and NeuN, were observed in the
dentate gyrus of all treatment groups, though number
of new neurons was higher in ischemic rats com-
pared with sham animals (not shown). In the CA1
region, BrdU/NeuN double-labeled
observed only in the postischemic rats that had been
treated with PJ34 (Figure 4B). Conversely, the
number of nonneuronal BrdU cells was lower in this
treatment group than in the saline-treated post-
ischemic rats.
Delayed Poly(ADP-ribose) Polymerase Inhibition
Results in Increased CA1 Pyramidal Neuron Density
After Ischemia
NeuN staining in brains harvested 8 weeks after
ischemia showed the neuronal density in the CA1
pyramidal layer to be much greater in the PJ34
treated than in saline treated postischemic rats
(Figure 5). Neuron density in the PJ34-treated group
was 61.2±12.8% of the sham-ischemia group, and
neuron density was 17.7±3.4% of the sham-ische-
mia group (P<0.01; n=4).
Delayed Poly(ADP-ribose) Polymerase Inhibition
Improved Spatial Memory After Forebrain Ischemia
There were no significant differences in spontaneous
locomotor activity between treatment groups in the
open field test, and no significant group differences
in swim speed during the Morris water maze test. By
contrast, analysis of swim path-length showed
significant differences between the treatment groups,
indicating differences in spatial learning and mem-
ory (Figure 6). In the testing phase (no visual cue on
the platform), comparison among all four treatment
groups revealed differences in path-length among the
groups to be significant at the P=0.003 level, and
direct comparison of PJ34-treated ischemia group
with the saline-treated ischemia group showed mean
path-length to be longer in the saline-treated ische-
mia group at P=0.009. The saline-treated ischemia
group also performed worse than the PJ34-treated
ischemia group in the training phase sessions (visual
Figure 3 Ischemia induced PARP-1 activation in microglia. (A)
PAR formation (red) was found predominantly in microglia
(CD11b immunopositive cells, green). Arrows show double-
positive cells. Animals treated with PJ34 (FI+PJ34) did not
exhibit microglial PAR formation. (B) Merged image with
orthogonal projections confirms colocalization of PAR and
CD11b. Arrow head indicates the cell shown in orthogonal
section and high magnification. Images are representative of
three animals in each treatment group at day 3 after ischemia.
PARP inhibition after stroke
TM Kauppinen et al
Journal of Cerebral Blood Flow & Metabolism (2009) 29, 820–829


text:
Amanda Lilley
As an independent designer, my goal is to create approachable, beautiful, functional knits for you and your family. You can get all of my latest updates by joining me on Facebook
Thank you knitters for your valued support!
Happy knitting!
original designs
no photo
no photo
no photo


text:
About سكسي عربي
سكسي عربي Kirby gave Paul and Linda specially drawn illustration after learning of Macca`s tune `Magneto and Titanium Man,` which was inspired by Kirby`s Marvel Comics creations. Definitely a more than pleasant way to wake up! Wet Kiss - Once you have not been kissing enough that both of your lips are not wet, with your mouth barely open, gently rub back and forth, up and down, around and around. The sudden cold blast makes for a sensual explosion, and they will not often try it on you next, as well as get very passionate. سكسي صاخب Good luck, Enders. سكسي عربي Burnout: Spinning the rear tires in water to heat and clean them before a run for better traction. The Beatle photo blog has pics of Paul McCartney`s 1976 meeting with comic book genius Jack Kirby.
سكسي عربي The torque is also what lets your engine pull a huge load up a hill. I never recorded them, but remembered a few jokes here and there that always made me laugh. that should not make him stop begging. Wake Up Kiss - Before your partner awakes lean over and kiss their cheek and move over giving soft kisses until you reach their lips. سكسي عربي Which is why I`m really excited that the other Erica racing is not only driving a Mustang this year too, but has gone with a Black/Pink color scheme for her DragStar Pro Stock ride.
Over time, the width of the hem grew wider and the term `flare-leg` was favored in marketing over the term `bell-bottom`.


text:
Are you a grower or a shower?
Super-Size Me
It's Product Review Friday again and I have a unique presentation for you today. Many months ago our friends at sex toy.com sent me a penis enlargement kit called Andro Penis and asked me to review it. This would certainly be a daunting task because it would take at several months of devoted use to effectively test this product. I would also have to find a willing Dr Dick Review Crew member to join me in this effort.
I put out an SOS message to all the men on the Review Crew asking for volunteers. I explained the extraordinary commitment this review would take. And asked if there might be at least one Review Crew member stalwart enough to subject himself and his cock to at least sixteen weeks of traction in the Andro Penis. You see, this instrument is placed over the penis and worn for four or nine hours a day for four to six months.
Not surprisingly, I got only one positive reply from my inquiry. Dr Dick Review Crew member, Carlos, said he'd be up to the task; all in the name of science and possibly a bigger dick.
Andro Penis —— $203.06
Dr Dick & Carlos
Dr Dick: "Before Carlos and I begin our discussion, I want to direct your attention to three postings I've made concerning penis enlargement. Historical Views On Cock Size, First Penis Enlargement Methods and Devices and Pumps and Pumps Effects On Penis Size. These three articles will hopefully put today's discussion in context."
Carlos: "Yeah, Dr Dick asked me to read through all these columns before I accepted his invitation to join him in reviewing the Andro Penis. I think I should also mention that every since I was a teenager I've been embarrassed about the size of my cock. People tell me that I have nothing to be ashamed about, that my cock is average sized, but that never seemed to make my desire for a bigger penis go away."
Dr Dick: "Yes, I'm afraid it's precisely men like Carlos, here, that often fall prey to the unscrupulous people who market dubious enlargement devices, pills and creams."
Carlos: "Yep, that would be me. I've been gullible enough to plunk down hard-earned money on a half-dozen enlargement schemes. All have been a disappointment. I guess that's why I volunteered for this project. I knew it wouldn't cost me any money, and there was the chance that the Andro Penis, what with all the medical jargon on their site, might actually work."
Dr Dick: "Indeed, the Andro Penis website is loaded with scientific studies touting its efficacy. There's even a page on their site filled with doctors and their endorsements of the product. Yet, upon closer inspection, the physician statements are mostly generic. Each speaks of tissue expansion by way of stretching, or in this case, traction."
Carlos: "I already know about this because I've been stretching my earlobes for a couple of years."
Dr Dick: "That's right! People have been stretching body parts as a means of adornment for just about as long as we've had body parts to stretch."
Carlos: "The Andro Penis is a medical looking apparatus that uses traction to stretch your penis. Ya have to wear on your dick for hours on end, every day, for up to six months. I promised Dr Dick that I would be able to handle this kind of commitment and that I was motivated to give this a try. The enlargement kit comes in a handsome case, which contains the stretching device, loads of extra parts, an instructional DVD, and booklet with written instructions in 27 languages. The booklet also helps you track your progress."
Dr Dick: "Once I was confident Carlos understood the commitment I turned over the kit and sent him on his way. I told him that I wanted to hear from him at least once a month for an update."
Carlos: "Ok, so you should know that the Andro Penis is pretty simple to attach to your cock. There's a ring that fits down around the base of your cock with two metal rods attached to it on either side. These two metal rods attach to another part where your dick-head fits into this kind of noose. And then you just tighten it till there's the desired tension. I know it sounds super uncomfortable, but it wasn't that bad. At least it wasn't at first. And then every seven days you attach these little extenders to the end of each of the metal rods. This is what causes your dick to stretch. The longer you wear the thing and the more extenders you use is supposed to determine how big your unit will get. The kit recommended that I wear the device for nine hours a day. That's a lot! You can take breaks, if you need to, but the whole idea is to keep up the traction for a total of six months."
Dr Dick: "That's precisely the thing I wanted Carlos to report to me about. Would he be able to sustain that kind of commitment and endure that kind of discomfort just to grow his dick bigger?"
Carlos: "Actually, it wasn't all that uncomfortable to start with, but it does feel really weird. Like any novelty, I was gung ho for the first few weeks. I mostly wore the Andro Penis in the evening and at night. I couldn't wear it during the day at work, that's for certain. It creates this very unsightly bulge in your pants. So I was sure I would never go out of the house with it on. Of course, just wearing it in the evening didn't allow for the proper amount of wearing time. I had to start wearing it while I slept. My wife didn't like this at all!"
Dr Dick: "I was pretty certain that finding the required number of hours a day, every day, would be a super challenge. And as Carlos suggests, the Andro Penis will no doubt get in the way of a relationship."
Carlos: "True! And then there's the issue of taking a piss. You have to free your cock from the contraption every time you need to pee. And don't even think about gettin a boner with this thing in place. By the second month I was dreading my daily dick straightjacket. I began to resent having to abuse my cock like this. I cursed myself for not loving the beautiful cock God gave me. To be honest, there was a slight noticeable increase in length, but not in girth. But not so much that made wearing the Andro Penis worth it."
Dr Dick: "That's pretty much what I thought would happen. When Carlos reported in at the beginning of the third month I could tell he was over it. I asked him if he wanted to continue the experiment."
Carlos: "I told him NO! I felt like I was letting Dr Dick down. I made a promise to wear the Andro Penis for four months, but I could barely get past two months. Every day I would find a new excuse to either not wear the thing or wear it for a shorter period than I was supposed to. I suppose if you're serious about enlarging your cock, more serious than I was at the beginning, and you have the stamina for this kind of regiment; then you might dig the Andro Penis. Like I said, I started out with the best of intentions, but I soon developed an intense animosity toward the device. It was a lot more invasive than I ever thought it would be. It was like carrying around a ball and chain."
Full Review HERE!
The Thrill Is Gone
Name: Billy
Gender: Male
Age: 46
I have heard it's normal for sex drive to diminish as you age. I'll run this by you. I'm a 46 year old male and the last time I was at a strip club with bare boobs bouncing around me, you may as well have rolled a grapefruit across the floor. Actually, I can see more use from the grapefruit. I don't recall the last time I did it, and jerking off was almost disgusting. My tool has shrank to nothing. I barely touch it and it just dribbles, it doesn't fire off anymore. I don't even like to touch it to go piss anymore. I've had to shave around it, so I actually find it, to keep from pissing my pants. Is this normal?
No, Billy, this isn't normal. I think you already know that too, right?
Do you know anything about andropause? If not, you ought to. Here's what I suggest. Use this site's search function in the sidebar. Type in the key word: "andropause" and you will come up with a wealth of information about this issue.
You can also use the CATEGORY pull down menu. Look for the subcategory: Sex and Aging, under the main category: Aging. Everything is alphabetized.
But for the time being, here's a typical question and response —
Name: Wilson
Gender: male
Age: 58
Location: Lancing MI
I'm a successful entrepreneur, in decent health (I could stand to lose a few pounds.) I have just about everything a man could want in life, but I'm miserable. I have no energy and I feel like I'm sleepwalking through my life. I have no sex drive at all; my wife thinks I'm having an affair…I wish. Even Viagra doesn't do the trick anymore. Is this just old age, or what?
Old age, at 58? Middle age, perhaps! Regardless what we call it, you sound like you're in the throws of andropause — male menopause — ya know, the change of life!
Never heard of such a thing? You're not alone. It's only been recently has the medical industry has begun to pay attention to the impact changing hormonal levels has on the male mind and body. Most often andropause is misdiagnosed as depression and treated with an antidepressant. WRONG!
Every man will experience a decrease testosterone, the "male" hormone, as he ages. This decline is gradual, often spanning ten to fifteen years on average. While the gradual decrease of testosterone does not display the profound effects that menopause does, the end results are similar.
There is no doubt that a man's sexual response changes with advancing age and the decrease of testosterone. Sexual urges diminish, erections are harder to come by, they're not as rigid, there's less jizz shot with less oomph. And our refractory period (or interval) between erections is more pronounced too.
While most all of us have heard of a mid-life crisis, and it's tragic consequences — red convertible sports cars, comb-overs, and the trophy wife or lover — fewer have heard of andropause. A mid-life crisis is essentially a psycho-social adjustment to aging — bored at work, bored at home, bored with the wife or partner — that sort of thing. Andropause, although it may coincide with a mid-life crisis, is not the same thing. Andropause is a distinct physiological phenomenon that is in many ways akin to female menopause.
Unlike women, men can continue to father children after andropause, but like I said, the production of testosterone diminishes gradually after age 40. I suppose you know that testosterone is the hormone that stimulates sexual development in the male infant, bone and muscle growth in adult males, and is responsible for our sexual drive. But did you know that by the age of 55, the amount of testosterone secreted into our bloodstream is significantly lower than at 45. And by age 80, most male hormone levels have decreased to pre-puberty levels.
Men, are you over 50? Are you feeling weak, lethargic, depressed, and irritable? Do you have mood swings, hot flashes, insomnia, and decreased libido, like our buddy Wilson, here? Then you too may be andropausal. You need to get some lead back in your pencil!
All kidding aside, andropausal men might want to consider Testosterone Replacement Therapy (TRT). Ask your physician about this. Just know that some medical professionals resist testosterone therapy, mistakenly linking Testosterone Replacement Therapy with prostate cancer. Even though recent evidence shows prostatic disease is estrogen-dependent rather than testosterone-dependent. However, before starting a testosterone regiment, insist on a complete physical, including blood work and a rectal examine. Mmmm, rectal exams!
Testosterone is available in many forms — oral, injectable, trans-dermal and by way of implants. The oral form is not recommended because of the high risk of liver damage. But injections, patches, pellets, creams and gels might be just the answer. I encourage you to be informed about TRT before you approach your doctor, because the best medicine is practiced collaboratively — by you and your doctor.
Good luck
Plays Well With Others
Name: Jim & Elaine
Gender: Couple
Age: 42 & 38
Location: Denver
We have been happily married for 15 years. We have a good, but pretty vanilla sex life together. We want to spice things up and are talking about maybe looking for other couples online. We're both in good shape and have very outgoing personalities. Both of us have had one short affair in the past, now we think we want to play together. Thoughts?
You guys want to look for other couples online…for ummm sex? I mean you imply that but you don't really come right out and say it, do you? I know you are new to this and you are just feeling your way through this unfamiliar territory, but unless you want to look like rank amateurs by other consensual non-monogamous couples, like swingers and polyamorous folks…and that's what we're talking about, right? You'd better get comfortable articulating precisely what it is you want, how you want it, and with whom.
If you've already begun your online search, you've probably already discovered that there are several different avenues for you to pursue. There are, of course, dating and profile sites. There are also sites that feature ads from other non-monogamous couples. If swinging is what you are after, there are exclusive swing parties and more inclusive swinger clubs. And each of these outlets may offer special groupings for the fetish-oriented swinger.
Since you don't actually say what kind of consensual non-monogamy you're looking for, let's talk swinging for now. Like I said, this isn't the only kind of consensual non-monogamy, but it's probably the oldest most established variety.
Before you swing, you guys need to decide what type of swing-set you want. If the vocabulary that follows is unfamiliar to you, you have some remedial homework to do before you launch your swing-capade. There is "soft" swinging and "hard" swinging. And bisexually may or may not be an option for you.
If you assume that all swingers are open-minded about sex, consider this; lots of swing outlets prohibit male-on-male sex. Personally, I find this extremely bizarre and off-putting, but I suppose it only reflects the prejudices of the popular culture. There are some swing-sets that allow novice swingers to simply to be voyeurs. I can't fuckin' figure this out either. Maybe it's a heterosexual thing.
If you gravitate toward the club-set there are 3 types to consider:
- SEX clubs — these clubs allow full-on sex, but only in designated areas.
- NO-SEX clubs — allow for lots of exhibitionism and voyeurism, including nudity, but no full-on sex. These clubs are great for meeting other swingers and to set up your own sex dates.
- Swinger parties are NO-SEX events, and are usually held in a nightclub or restaurant. Again, you can meet like-minded folks there and set up your own sex dates.
Whichever outlet you choose; make sure you understand the rules and regulations of the get together before you attend.
Like I said, it's of the utmost importance that you guys decide, in advance, what your limits are. A good number of otherwise healthy marriages flounder at this point. Have a clear and frank exchange with each other on the ground rules of your swinging and then stick to them. Trying to negotiate a change to the rules of engagement during a swing is a very bad idea. That's not to say that your ground rules won't change and evolve over time; just don't attempt to adjust them while they are in play.
Never push your partner into doing something he/she is not ready to do. Be open with each other before, during and especially after a swing. Effective communication is essential. This goes for communicating with your fellow swingers. Be sure to let everyone know that you are newbees to the scene. (Don't worry, everyone will have figured that out already.) Novices stick out like a sore…hard-on.
Most clubs and groupings don't allow single men. Most swing-sets are women oriented, to the degree that women set the tone for the swing. That being said, it's still a man's world. Men generally dictate the type of sexual expression that will be tolerated — thus the prohibition, stated or unstated, against male on male sex. Female on female sex is, of course, encouraged for obvious reasons. How's that for a screwed-up double standard?
Most clubs expect full or partial nudity. My swinger friends advise that if you just want to attend so you can ogle others, stay the fuck home! Novice swingers, like you guys, ought to stay together until you feel comfortable being apart. But for Christ sake, don't glom on to one another like the other swingers have the cooties.
Most of all, take responsibility for your eroticism and your sexuality. Be friendly and good-natured. And don't try to pretend you're a more accomplished sexual athlete than you are.
Be advised, you are about to embark on a sexual journey that will take you to the edges of what society regards as appropriate sexual behavior. Don't be surprised if some of your more traditional friends discriminate against you when they find out about your new activities. Finally, swinging is far less about what you do (sex) and way more about who you are (a lifestyle). To that end, I'd like to turn you on to a fantastic resource. Check out my friends, John and Allie, at SwingerCast. And be sure to listen to my two-part interview with them right here on my site. You'll find Part 1 HERE and Part 2 HERE!
Good luck
Putting A Ring On It
Name: William
Age: 30
Location: UK
Hi There
I am new to this scene, and I have very little experience in anal sex and I am seeking your help and advice. I am a top but I have a problem keeping my dick hard or staying hard during anal sex. I find it harder to fuck an ass compared to fucking a pussy. Here is the problem: Once I get my dick hard, put on a condom and start fucking, my dick sometimes goes soft on me. Is that normal? How can I keep my dick hard long enough in the ass to enjoy the fuck? Sometimes even when my dick is hard, I find it hard to penetrate an ass. I use lube, so what am I doing wrong? People in gay porn can fuck and fuck like there is no tomorrow. I want to enjoy anal sex too!! Any advice? Please let me know if there is anything I can do to improve in this area?
Boy, you're in luck, William! One of my most popular tutorials, Finessing That Ass Fuck — A Tutorial For a Top, is waiting for you. Check it out! It will answer a lot of the questions you have about butt fucking. You should also know that this is the companion piece to my tutorial for ass fuckin' bottoms handsomely titled: Liberating The B.O.B. Within. Don't know what a BOB is? No to worry, all will be explained.
But before you disappear to do your homework, I'd like to address one of the specific issues you raise, about keeping your dick hard while fucking. You are right to point out that fucking an ass (male or female) is different from fucking a pussy. But regardless of what hole you're invading, a nice hard stiffy is essential.
Are you familiar with a cockring, William? If not, I suggest you acquaint yourself with these amazing low-tech wonders. Here's what you should do. Mozie on over to the Dr Dick's Sex Toy Reviews site and search for my tutorial, Cockring Crash Course. (The search function in the sidebar will assist you.) Prepare yourself to be sorely amazed at the variety and functionality of these little devils.
Cock rings can create larger, firmer erections. Since blood flow enters your dick through arteries deep inside your dick, and leaves it through the veins near the surface of your tool; wearing a cock ring can help to keep more blood inside your dick shaft. And as all you rocket scientists know, blood is what causes erections in the first place. Also some men claim that wearing a cock ring intensifies their orgasm.
And while you're on the sex toy review site, use the CATEGORY pull-down menu in the sidebar and look for cockrings. You'll find it under the last heading, Sexual Enrichment. This will bring up all the cockrings we reviewed, and there's a load of 'em, don't cha know.
I recommend the flexible and/or adjustable cockrings. These are generally made of stretchable rubber or leather. For the more daring there are the metal variety. These may look pretty, but they can be a bitch to put on and to take off. Here's how ya do it.
- Pull your ball sack through the ring first.
- Follow this by popping each of your balls through the ring one at a time.
- Now bend your cock down and pull it through the ring.
As you can see, putting on one of these little buggers before you have a raging hardon is gonna make it a whole lot easier. To take the cock ring off, simply reverse these steps, pushing your flaccid cock back through the ring first, followed by each of your balls and finally your ball sack.
It's absolutely essential that you not wear an inflexible (metal) ring for longer than a couple hours. Make sure you don't buy one that is too small either. If your dick is turning an angry red or worse, purple, or it is cold to the touch, you're in trouble. Take that ring off immediately. If you don't you will risk serious injury to your precious johnson. Remember people, play smart with all your toys!
Good luck
Do you like this posting? Your one-time $10 donation will help support this sex positive effort. With your help we can continue to enrich, inform, and educate all who visit here. Join us and help make a difference!


text:
Katherine Belsey has not received any gifts yet
I just posted a list on Bored Panda. Click to add your paper toy or any other paper creation, if it can be made from one piece!…Continue
Posted on August 22, 2015 at 12:54pm
It looks lil a Rubik's cube -- or a calendar -- but how can 12 months fit on one cube, and how can this Rubik's cube be solved? Once you know the trick, it's surprisingly easy! (scroll down to see a gif) I designed this paper toy almost a year ago, and although it is available as a…Continue
Posted on August 19, 2015 at 5:53pm
The fun thing about this 3D pop-up fire truck is that it is printed in one piece from a single sheet of letter sized or A4 paper. And of course it folds flat. …Continue
Posted on July 27, 2015 at 1:30pm
This is a a pop-up 3D school bus with a working stop sign. …Continue
Posted on June 8, 2015 at 6:00pm
© 2015 Created by Castleforte.


text:
The following tags exist in this scene. You can find similar scenes by combining tags in this scene together using the checkboxes, or just click a tag name to browse...
Uploaded by GidgieScott
Suggest Pornstars
Do you recognise pornstars in this scene? Begin typing a pornstar name, then click on the button to suggest them!
White lez with short red hair sucks on the tits of another lesbo and then licks her pussy as well
Lovely hot matured lesbian brunette loves to lick and suck their pussies eachother and then rubbing for orgy
Video Suggestions
- 36:26Two Hot Dykes End Up In Bed Together For Some Hot ActionJan 30, 2012
- 40:18Mature Lesbians Kiss And Trib Each Other In BedroomMar 6, 2012
- 26:31Carpet Cleaning Lesbian Sex Loving WhoresJan 29, 2012
- 35:45Mature Slut And A Young Cuties Has Lesbo FunJan 22, 2012
- 36:47Teen Jennifer And Nora Fucking In There BedroomFeb 2, 2012
- 12:09Two Thick Bitches With Round Asses Lesbian FuckingFeb 3, 2012
- 6:24Lesbian Gypsy Sees Lots Of Excitement In Gal Pal's FutureJan 19, 2012
- 14:57Crucified Or Pleased Lesbians Try Out ReligionJan 28, 2012
- 37:00Two Cops Arrest A Sexy Redhead Who Is Fucked HardFeb 4, 2012
- 42:15Asian Girl Is Used And Seduced By Her MasseuseMar 19, 2012


text:
Many companies prefer their employees to discuss their strengths and weaknesses at work. There are many ways in which one can list their strengths without seeming cocky and list their weaknesses without being perceived as dispensable. It is critical to remember the primary purpose of the exercise, which is to demonstrate that one consistently evaluates their merits and faults in order to improve performance.Know More
When asked to list weaknesses, it is unwise for an employee to leave the question blank or answer that he has no weaknesses. According to About.com, employees should list skills that were previously unpolished and show the steps that have been taken to improve in that direction. Another option is to list weaknesses that are unrelated to your job duties. For example, a person who edits copy for a living could list they are not skilled in accounting or managing money.
Listing strengths is equally difficult, as many people are naturally shy when it comes to bragging about their skills. The most important consideration is ensuring that the strengths listed correlate to the specific job duties for which an employee is responsible. Employees should list positive traits that demonstrate knowledge in a nuanced area or list personality traits that are considered the best for the position sought. Employees should celebrate past successes at work and be sure to mention the amount of dedication put into the project, rather than natural talent.Learn more about Applying & Interviewing
Some academic strengths are focus, command, discipline, self-assurance and positivity. Cultivating these strengths helps students enhance their performance both in the classroom and in their studies outside of the classroom. It also helps in their development as individuals.Full Answer >
Strengths listed on a resume should begin with job-related skills and qualities but should also include highlights that are not typically associated with current or desired employment. Having a well-rounded strengths profile makes candidate come across more human, relatable and real.Full Answer >
Key strengths include being an effective communicator and a solid problem solver, and possessing a strong work ethic. Being a good decision maker, having organizational ability and being flexible are other key strengths for a professional environment.Full Answer >
Figuring out which strengths and weaknesses to list on a resume is a process, with generic traits like capacity for hard work, making the applicant seem bland, while personality aspects, technical and artistic skills related to the workplace and qualities like adaptability seem novel and interesting. Weaknesses should be honest but framed with positivity and preceded by statements about how there is room for improvement and a conscious effort toward that improvement is being made.Full Answer >


text:
Let's-do-it again memories to share for generations
Wisconsin Water Ski Shows
Do you know who owns the title "Show Ski Capital of the World?" Wisconsin of course! With more water ski show teams than any other places in the world, families line Wisconsin lakeshores on summer evenings to watch the action and top it off with a stop for ice cream on the way back to the cabin.
Must Reads
Sit down, pour another cup of coffee and dig into some of our best stories. Read about everything from the best places to taste Wisconsin cheese to where to take the kids tubing this summer.


text:
© Jemella LTD
A little wonder for perfecting your style
Best for... Backcombing; sectioning hair for blow-drying; precision styling; tucking in rogue hairs in updos. The fine toothed design and anti-static properties make this comb perfect for intricate styling. To use... For damage-free backcombing (that's also easy to brush out), take a small section of hair and hold it away from the head. Place the comb in the section a few centimetres away from the root. Push the comb down the hair, against the direction of growth, in two short strokes, stopping short of the roots. For the third stroke, push back right to the roots. Continue with this three-stroke method on every section you need to backcomb. Style tip... Use the tail end, not the teeth, to create the most accurate sections in your hair when styling.
Price Includes
- ghd Tail Comb
- Free Delivery with all Styler or Hairdryer Purchases
Product Features
- Fine-toothed, anti static
- Heat resistant for use with hair dryer
- Hand crafted & finished exclusively for ghd
- Professional quality & design
produkter der matcher
Tail Comb
kr 109,00+
Paddle and Dressing Brushes
Paddle Brush
kr 239,00=
Pris for begge varer
kr 348,00
You may also like ...
Straight & Tame Creamkr 239,00
Heat Protect
Heat Protect Spraykr 199,00
Unfortunately, {0} doesn't want to receive emails from ghd, so we cannot send them your hint. But don't worry, here's a couple of ways to get what you want, including:
- Leave not-so-subtle hints with strategically placed magazines and the ghd adverts circled
- Share our social posts so they #getthehintdarling
You know what you want, and we like that! We are on a missions to make sure you get the gift you really want, and it seems you are too. Unfortunately, sometimes less is more and you have used all of your hints for today. But there is always tomorrow!
Suprises can be great when they're just what you're after. We hope you not-so-subtle hint gets you the gift you really want. Just to make sure, drop more hints so they get the message.


text:
Need Help? No Problem!
On filter orders over $59.
Enter Discount Code:
at checkout! Can not be combined with any other offer.
Shop Our Popular Brands
Have questions about this product?
Click the image above.
Collapsed Dimensions 27"(L) x 6-1/8"(W) x 3"(H)
Expanded Dimensions 27"(L) x 16 x 6(W) 1/8"(H)
Additional Specifications:This product is not manufactured or distributed by Research Products Corporation, owner of the registered trademark Aprilaire®.
Learn more about air qualityand tips on savings.
Enter your email address:


text:
Ramona Baker and Chase McGee plan to marry on May 11, 2013 at Snipe's Farm and Retreat in Chapel Hill, N.C. The bride-to-be is the daughter of Robert and Leslie Baker of Nikiski.
Editor's note: The Clarion this week is taking a look back at some of the people and events in the news in 2010.
Editor's note: This is the final part of a three-part series exploring autism in the community.
Peninsula Clarion ©2013. All Rights Reserved.


text:
Federal Communications Commission
Before the
Federal Communications Commission
Washington, D.C. 20554In the Matter of
Adopted: August 7, 2013Released: August 7, 2013
Federal Communications Commission
DA 13-1725attorney is not involved in Competitive Decision-Making. (In this regard, an In-House Counsel's
Federal Communications Commission
DA 13-1725would be released publicly by the Commission upon a proper request under our rules implementing
Federal Communications Commission
DA 13-1725or (3) a Reviewing Party may request a complete set of the documents at cost, allowing two business days
Federal Communications Commission
DA 13-1725the Confidential Information from the non-confidential information. Two copies of each Confidential
Federal Communications Commission
DA 13-1725other rights and remedies available to the Submitting Party at law or in equity against any person using
Federal Communications Commission
Acknowledgment of Confidentiality
WT Docket No. 13-193I hereby acknowledge that I have received and read a copy of the foregoing Protective Order in
Note: We are currently transitioning our documents into web compatible formats for easier reading. We have done our best to supply this content to you in a presentable form, but there may be some formatting issues while we improve the technology. The original version of the document is available as a PDF [2], Word Document [1], or as plain text [3].
[1] https://apps.fcc.gov/edocs_public/attachmatch/DA-13-1725A1.doc
[2] https://apps.fcc.gov/edocs_public/attachmatch/DA-13-1725A1.pdf
[3] https://apps.fcc.gov/edocs_public/attachmatch/DA-13-1725A1.txt
[4] mailto:firstname.lastname@example.com
[5] mailto:firstname.lastname@example.com
[6] mailto:email@example.com


text: Kate Gosselin Responds to Jon's Comments About Kids by Free Britney at November 7, 2013 10:53 am. Comments Kate Gosselin is responding to Jon Gosselin's comments about their kids, and she's not happy. Share Tweet Email Edit Delete Follow Jon Gosselin? Facebook Disqus Stars: Jon Gosselin, Kate Gosselin Related Videos: Jon Gosselin Videos, Kate Gosselin Videos Related Post: Kate Gosselin SLAMS Jon Gosselin, Defends Kids' Development in Blog Post Uploaded by: Free Britney Uploaded: November 07, 2013 Duration: 86 seconds


text:
Healthcare Providers and Services
Company Overview of Mayo Foundation For Medical Education And Research
Executive Profile
Robert K. Smoldt
|Total Calculated Compensation
|This person is connected to 0 Board Members in 0 different organizations across 1 different industries.
Mr. Robert K. Smoldt serves as Secretary at Mayo Foundation. Mr. Smoldt served as Member of Board of Stewardship Trustees at Catholic Health Initiatives.
Corporate Headquarters
200 First Street SWPhone: 507-284-2511
Rochester, Minnesota 55905
United States
Fax: 507-284-0161
Board Members MembershipsThere is no Board Members Memberships data available.
EducationThere is no Education data available.
Other Affiliations
Annual CompensationThere is no Annual Compensation data available.
Stocks OptionsThere is no Stock Options data available.
Total CompensationThere is no Total Compensation data available.
|There is no Competitor Compensation data available.
Sponsored Financial Commentaries
Sponsored Links
Report Data Issue
To contact Mayo Foundation For Medical Education And Research, please visit . Company data is provided by Capital IQ. Please use this form to report any data issues.
Please enter your information in the following field(s):
Your requested update has been submitted
Our data partners will research the update request and update the information on this page if necessary. Research and follow-up could take several weeks. If you have questions, you can contact them at email@example.com.


text:
Oregon Children's Theatre
1939 NE Sandy Blvd.
United States
(503) 228-9571
Ross McKeen
Dani Baldwin, Jessy Friedt, Kay Spielman, Natasha Kautsky, Marcella Crowson, Ross McKeen, Sharon Martell, Stan Foote, Ticia Evans
Ross McKeen, Stan Foote, Kregg Arntson, Pam Caldwell, C. Andrew Gibson, Vatea Herman, Kelliann Klindtworth, Nichole Martin, Stanley Penkin, Jeanie-Marie Price, Rebecca Semper, Dann Wheeler
Oregon Children's Theatre's mission is to advance growth, development and creativity through exceptional theater experiences.
We presents the highest quality professional theater productions in two world-class venues; work with schools and teachers to ensure our plays directly support classroom learning for students; provide teachers with free services to encourage the continued integration of arts learning activities into classrooms; partner with disadvantaged schools and social service agencies to deliver free services to underserved children; and teach children through year-round acting classes and a mentoring program for teens who are passionate about theater.


text:
Page 1
In vivo neuronal subtype specific targets of Atoh1 (Math1) in
dorsal spinal cord
Helen C. Lai1, Tiemo J. Klisch2,3, Rene Roberts1, Huda Y. Zoghbi2,3, and Jane E. Johnson1
1Department of Neuroscience, 5323 Harry Hines Blvd., UT Southwestern Medical Center at
Dallas, Dallas, TX, 75390, USA
2Department of Molecular and Human Genetics, One Baylor Plaza, MS BCM225, Baylor College
of Medicine, Houston, TX, 77030, USA
3Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA
Neural basic helix-loop-helix (bHLH) transcription factors are crucial in regulating the
differentiation and neuronal subtype specification of neurons. Precisely how these transcription
factors direct such processes is largely unknown due to the lack of bona fide targets in vivo.
Genetic evidence suggests that bHLH factors have shared targets in their common differentiation
role, but unique targets with respect to their distinct roles in neuronal subtype specification.
However, whether neuronal subtype specific targets exist remains an unsolved question. To
address this question, we focused on Atoh1 (Math1), a bHLH transcription factor that specifies
distinct neuronal subtypes of the proprioceptive pathway in mammals including the dorsal
interneuron 1 (dI1) population of the developing spinal cord. We identified transcripts unique to
the Atoh1-derived lineage using microarray analyses of specific bHLH-sorted populations from
mouse. Chromatin immunoprecipitation-sequencing (ChIP-seq) experiments followed by enhancer
reporter analyses identified five direct neuronal subtype specific targets of Atoh1 in vivo along
with their Atoh1-responsive enhancers. These targets, Klf7, Rab15, Rassf4, Selm, and Smad7, have
diverse functions that range from transcription factors to regulators of endocytosis and signaling
pathways. Only Rab15 and Selm are expressed across several different Atoh1-specified neuronal
subtypes including external granule cells (EGL) in the developing cerebellum, hair cells of the
inner ear, and Merkel cells. Our work establishes on a molecular level that neuronal differentiation
bHLH transcription factors have distinct lineage-specific targets.
First discovered in Drosophila, neural specific basic-helix-loop-helix (bHLH) transcription
factors are crucial for determining proper neural cell fates (Jan and Jan, 1994). In
vertebrates, bHLH transcription factors are essential for the general neuronal differentiation
as well as neuronal subtype specification of diverse cell types in the peripheral and central
nervous systems (Bertrand et al., 2002). They are thought to share activity in inducing
neuronal differentiation, but have distinct functions in specifying neuronal subtypes (Parras
et al., 2002; Nakada et al., 2004a). While several studies have found targets of bHLH
transcription factors, they have mostly focused on their common role in neurogenesis
(Bertrand et al., 2002; Castro et al., 2006; Seo et al., 2007).
Corresponding Author: Jane E. Johnson, 5323 Harry Hines Blvd., UT Southwestern Medical Center at Dallas, Dallas, TX 75390,
email@example.com, (214) 648-1870 (tel), (214) 648-1801 (fax).
The authors declare no conflict of interest.
NIH Public Access
Author Manuscript
J Neurosci. Author manuscript; available in PMC 2012 January 27.
Published in final edited form as:
J Neurosci. 2011 July 27; 31(30): 10859–10871. doi:10.1523/JNEUROSCI.0445-11.2011.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 2
Elegant genetic studies in Drosophila and mouse suggest that in addition to shared
downstream transcriptional targets, bHLH transcription factors have unique targets relevant
for the function or development of that specific neuronal subtype. Studies misexpressing
scute or ato (Chien et al., 1996; Jarman and Ahmed, 1998), or substituting Neurog2 with
Ascl1 (Parras et al., 2002) respecified neurons in a context-dependent manner. Similarly,
overexpression of Ascl1 and Atoh1 in the chick spinal cord induces progenitors to
differentiate into specific neuronal subtypes (Gowan et al., 2001; Nakada et al., 2004a).
We focused our study on Atoh1 (mammalian atonal (ato) homolog 1), a bHLH transcription
factor required for the formation of different proprioceptive neuronal subtypes (Bermingham
et al., 2001). Due to its discrete expression in defining progenitors to the dorsal interneuron
1 (dI1) population of the developing spinal cord (Bermingham et al., 2001; Gowan et al.,
2001), Atoh1 was an ideal bHLH to identify neuronal subtype specific targets. In addition to
dI1 neurons, Atoh1 specifies progenitors to the granule layer of the cerebellum (Ben-Arie et
al., 1997), several hindbrain neurons (Ben-Arie et al., 1997; Machold and Fishell, 2005;
Wang et al., 2005; Maricich et al., 2009b; Rose et al., 2009a; Rose et al., 2009b), sensory
hair cells of the inner ear (Bermingham et al., 1999; Zheng and Gao, 2000; Izumikawa et al.,
2005; Raft et al., 2007), and Merkel cells in the skin and vibrissae (Ben-Arie et al., 2000;
Maricich et al., 2009a; Morrison et al., 2009; Van Keymeulen et al., 2009). However,
fundamental mechanistic understanding of how Atoh1 directs specification of these neuronal
subtypes is lacking in the spinal cord since the only known direct Atoh1 targets in vivo
besides Atoh1 itself (Helms et al., 2000) are transcription factor, Barhl2 in dI1 neurons
(Saba et al., 2005). In contrast, in the developing cerebellum a variety of direct Atoh1 targets
were recently identified (Klisch et al., 2011) adding to the previously known targets, Barhl1
and Gli2 (Kawauchi and Saito, 2008; Flora et al., 2009).
In this study, we identified unique targets of Atoh1 by comparing sorted Atoh1-lineage cells
in the developing dorsal neural tube with a neighboring population defined by the expression
of the bHLH factor Neurog1 (Ngn1, neurogenin1). We identified transcripts enriched in
Atoh1-lineage cells and biased against identifying common bHLH targets. Using ChIP-seq
data from a FLAG-tagged Atoh1 knock-in mouse, we identified five new direct lineage-
specific in vivo targets of Atoh1 whose enhancers respond to Atoh1 expression: Klf7,
Rassf4, Rab15, Selm, and Smad7.
Materials and Methods
Experimental Animals and Generation of Transgenic Mice
Atoh1BAC-GFP (Math1GFP-BAC) (Raft et al., 2007) and dNeurog1-GFP (TgN1-13G)
(Nakada et al., 2004b) transgenic mice were generated previously. Atoh1BAC-GFP
transgenic mice contain the bacterial artificial chromosome RPCI-23318G16 with the Atoh1
coding sequence replaced with nuclear localized GFP. dNeurog1-GFP transgenic mice
contain an enhancer from the Neurog1 gene that directs GFP reporter expression primarily
to the dP2/dI2 domains in the dorsal neural tube. Transgenic embryos were identified by
GFP fluorescence. Tail clips and yolks sacs of transgenic mice were PCR genotyped for
Transgenic mice of Klf7 site A and Rassf4 site A enhancers cloned into the BgnEGFP
vector (Lumpkin et al., 2003) were generated using standard procedures (Brinster et al.,
1985). Each transgene was isolated from recombinant plasmid on a standard agarose gel and
microinjected at 1–3 ng/μL into pronuclei of fertilized eggs from B6SJLF1 (C57BL/6JxSJL)
crosses by the Transgenic Core Facility of University of Texas Southwestern Medical
Center at Dallas. All animal experiments were approved by the Institutional Animal Care
and Use Committee at UT Southwestern.
Lai et al.Page 2
J Neurosci. Author manuscript; available in PMC 2012 January 27.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 3
Fluorescence Activated Cell Sorting (FACS) and mRNA isolation
Atoh1BAC-GFP and dNeurog1-GFP transgenic mouse E10.5 neural tubes from forelimb to
hindlimb were dissected in DMEM/F12 (Gibco) and dissociated with 0.25% Trypsin-EDTA
(Gibco). Fluorescence activated cell sorting (FACS) was performed by the UT Southwestern
Flow Cytometry Core Facility. Cells were sorted into GFP − and + cells with a MoFlo
(Dako/Beckman Coulter, 100 μm nozzle) or a FACSAria (BD Biosciences) cell sorter
directly into ZR RNA buffer (Zymo Research). RNA was extracted using the Mini RNA
Isolation II Kit (Zymo Research).
cDNA Preparation, Reverse-Transcription PCR (RT-PCR), and quantitative RT-PCR
cDNA for RT-PCR was prepared from TURBO DNase (Ambion)-treated GFP+ and − cell
sorted RNA from E10.5 neural tubes using Omniscript or Sensiscript reverse transcription
kits (Qiagen) with oligo dT primer. Quality of GFP sorted cells was verified by RT-PCR
(Qiagen Taq) using primers to Atoh1 (5′-GCT GGT AAG GAG AAG CGG CTG TG-3′ and
5′-TGT ACC CCA TTC ACC TGT TTG C-3′) or Neurog1 (5′-CCA CTG TGG CAT CAC
CAC TC-3′ and 5′-GCG TCG TGT GGA GCA GGT CTT TG-3′), GFP (5′-CAG AAG
AAC GGC ATC AAG GTG AAC-3′ and 5′-GGG TGC TCA GGT AGT GGT TG-3′), and
GAPDH (5′-ACC ACA GTC CAT GCC ATC AC-3′ and 5′-CAG CTC TGG GAT GAC
CTT GC-3′) mRNA and visualized on a 2% agarose gel with ethidium bromide.
cDNA for quantitative PCR (qPCR) was prepared from TURBO DNase (Ambion)-treated
pooled GFP+ and GFP− cell sorted RNA from E10.5 mouse neural tubes using the
Superscript III First Strand Synthesis System (Invitrogen) with random hexamers. qPCR
was carried out with Fast SYBR Green Master Mix (ABI) on the 7500 Fast Real-Time PCR
System (ABI) using the following primers: Atoh1 (5′-GTA AGG AGA AGC GGC TGT
G-3′ and 5′-AGC CAA GCT CGT CCA CTA-3′), Rab15 (5′-GGC TTG GGC TGT GTC
ATT G-3′ and 5′-GGC AGA CAG GCC AGG AAA-3′), Selm (5′-TCG TGC TGT TAA
GCC GAA ATT-3′ and 5′-CCG GGT CAT TTG GCT GAG T-3′), Smad7 (5′-CGA AGA
CAG GAA ACG AGA GTC A-3′ and 5′-GGT GGT GCC CAC TTT CAC A-3′), Neurog1
(5′-CTG CGC TTC GCC TAC AAC TAC-3′ and 5′-ATC TGC CAG GCG CAG TGT-3′),
and Ppib (Peptidylprolyl isomerase B, formerly known as Cyclophilin B) (5′-GGA GAT
GGC ACA GGA GGA A-3′ and 5′-GCC CGT AGT GCT TCA GCT T-3′). The ΔΔCt
method was used to determine the mRNA fold change of transcripts in the Atoh1BAC-GFP
+ to dNeurog1-GFP+ cells (Figure 1D) and the Atoh1BAC-GFP+ to GFP− cells (Figure 2B)
using Ppib as the endogenous control gene. Graphs were made using Prism 5 software.
Microarray Analysis
Integrity of the isolated total RNA was analyzed using a 2100 Bioanalyzer (Agilent). One
microgram of RNA pooled from Atoh1BAC-GFP+ or dNeurog1-GFP+ sorted cells from
multiple embryos from multiple litters was processed for Affymetrix Mouse 430 2.0
microarrays by the UT Southwestern Microarray Core Facility using standard protocols.
Two biological samples per transgenic mouse type were processed for microarrays,
preprocessed with MAS 5.0 to generate normalized signal intensity data, and analyzed using
GeneSpring Agilent GX 7.3 Expression Analysis software. Signals <0.01 were set to 0.01
and each chip was normalized to the 50th percentile and analyzed for probes with over two-
fold change in signal where the Affymetrix probes had to be present or marginal in at least
one of the Atoh1BAC-GFP or dNeurog1-GFP samples. Individual arrays from Atoh1BAC-
GFP+ cells versus the dNeurog1-GFP+ cells were compared. Fold differences in transcript
levels are presented separately since reliable statistical analyses cannot be performed on
only two arrays. The raw microarray data has been deposited in NCBI's Gene Expression
Omnibus database (Edgar et al., 2002) (GEO Series accession number GSE23089)
(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE23089). The processed data will
Lai et al.Page 3
J Neurosci. Author manuscript; available in PMC 2012 January 27.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 4
be provided upon request or can be found at
Tissue Preparation, Immunostaining, and Microscopy
Whole mouse male and female embryos from wildtype or Atoh1 knockout mice (Ben-Arie et
al., 1997) were dissected at E10.5 where E0 was timed as halfway through the dark cycle of
the morning that the vaginal plug was detected. Mouse and chick embryos were fixed in 4%
paraformaldehyde for 1.5–2 hours, washed, cryoprotected, embedded, and cryosectioned
(transverse, 20–30 μm). Heads of E16.5 embryos were fixed overnight. The noses were cut
and embedded for coronal sections while the rest of the head was cut at a 45 degree angle
between coronal and transverse sections to obtain the appropriate visualization for the
developing inner ear. P0 pups were perfused with 2 mL PBS, brains dissected out, fixed
overnight, and embedded for sagittal sections.
Mouse or chick sections were incubated in primary antibody and the appropriate secondary
fluorophore conjugated antibodies (Alexa fluorophores 488, 568, 594, 647, Molecular
Probes/Invitrogen). Primary antibodies used were: 1:100 rabbit anti-Atoh1 (Helms and
Johnson, 1998), 1:4000 rabbit anti-Lhx2/9 (Liem et al., 1997), 1:500 rabbit anti-Neurog1
(Gowan et al., 2001), 1:500 chick anti-GFP (Aves), 1:500 mouse anti-BOSS (Kramer et al.,
1991), 1:1000 mouse anti-c-myc (Santa Cruz Biotechnology), 1:100 mouse anti-Islet1/2
(40.2D6)(DSHB and Ericson et al., 1992), 1:100 mouse-anti Lhx1/5 (4F2)(DSHB and
Tsuchida et al., 1994), and 1:200 goat anti-Neurod (Santa Cruz Biotechnology). Sections
were imaged using a Zeiss LSM510 confocal microscope. For Figures 5G, J, and K, the
endogenous GFP fluorescence gain and offset was kept constant and 16-bit tiff images taken
on the same day within one set of chick electroporation experiments of an enhancer-GFP
construct plus control, BOSSAtoh1, or mycAscl1. Mean GFP pixel intensity/cell of
colocalized GFP and BOSS- or myc-tagged bHLH was processed using ImageJ (Abramoff,
2004). The threshold for colocalization was optimized for each image. In the cases where
the GFP fluorescence was below detectable levels (Figure 5D, D″, F), the GFP fluorescence
of the entire image was calculated. Insets in Figure 5 were manipulated with Photoshop to
show that GFP fluorescence was detectable in some cases and that the neural tube was
adequately injected. S.E.M. are given and p-values were determined in Excel using a two-
tailed two-sample unequal variance (heteroscedastic) t-test.
In situ Probe and Enhancer Cloning
In situ probes were cloned from E10.5 neural tubes or P0 cerebella cDNA. Enhancers were
cloned from whole genomic DNA of ICR wildtype mice into the BgnEGFP vector
(Lumpkin et al., 2003). Primer sets, cloning sites, and parent plasmid vector used for each
probe or enhancer construct will be provided upon request or at
http://www8.utsouthwestern.edu/utsw/cda/dept120915/files/150735.html. PCR products
were obtained using standard protocols from the iProof kit (Bio-Rad), KOD Xtreme
HotStart DNA Polymerase (Novagen) kit, or Expand High Fidelity (Roche) kit.
In situ Hybridization (ISH)
ISH was performed as per standard protocols. A detailed protocol is available upon request.
Digoxygenin (DIG)-labeled antisense RNA probes (1–5 mg/mL) were hybridized overnight
at 65°C, incubated with anti-digoxygenin AP antibody (Roche), and then incubated with
NBT/BCIP (Roche). The sections were immediately imaged or counterstained with eosin
before imaging with a Zeiss Discovery Stereomicroscope V12.
Lai et al.Page 4
J Neurosci. Author manuscript; available in PMC 2012 January 27.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Page 5
Chromatin Immunoprecipitation and Sequencing (ChIP-seq)
Chromatin immunoprecipitation of p300, a transcriptional co-activator, followed by high
throughput sequencing (ChIP-seq) of E11.5 neural tubes from the posterior hindbrain to the
hindlimb was performed as previously described for E11.5 forebrain, midbrain, and limb
(Visel et al., 2009). The Atoh1-FLAG ChIP-seq from P5 cerebella was performed as
described (Flora et al., 2009). The recovered DNA was submitted for Solexa sequencing at
the Center for Cancer Epigenetics Solexa Sequencing Core at MD Anderson (Houston, TX),
where the sequencing libraries were prepared and sequenced on the Illumina Solexa GAII
pipeline according their standard operating procedures (Klisch et al., 2011). ChIP regions
along with 30 vertebrate species conservation are viewed in UCSC browser, NCBI37/mm9
mouse build (Kent et al., 2002).
The following enhancer sequences of Atoh1 regulated genes in the dorsal neural tube were
analyzed with MEME (Bailey and Elkan, 1994). Atoh1 enhancer A and B (Chr6: 64683369–
64684709), Barhl1 enhancer, Barhl2 enhancer, Klf7 subset of site A, Rassf4 subset of site A
(Chr6: 116620232–116621468), Selm site B, Smad7 subset of site A (Chr18: 75594185–
75594975), and Rab15 site A (see Table 2 for coordinates). MEME was asked to find ten,
eight base pair motifs that occurred in every sequence (one per sequence, min and max
width = 8). As a control, sequences 2000 base pairs upstream of the enhancers were subject
to the same analysis.
Chick in ovo Electroporation
Fertilized White Leghorn chick eggs were obtained from the Texas A&M Poultry
Department. The neural tube of chick embryos staged HH14-17 (Hamburger and Hamilton,
1992) were injected with 1 μg/μL of the enhancer-GFP plasmid construct and 1 μg/μL of the
appropriate bHLH transcription factor plasmid and electroporated as previously described
(Nakada et al., 2004a). Embryos were harvested after 36–48 hours at 38°C. bHLH plasmids
used for this study were made in the pMiWIII expression vector: myc-tagged rat Ascl1-AQ
(Mash1 NR-AQ, myccontrol) inactive mutant control (Nakada et al., 2004a), BOSS-tagged
mouse Atoh1 (BOSS-Math1, BOSSAtoh1) (Helms et al., 2000), and myc-tagged rat Ascl1
where five myc tags were inserted at the N-terminus of Ascl1 (Mash1, mycAscl1) (Nakada et
al., 2004a).
Microarray analyses identified transcripts enriched in the Atoh1-specific population of the
dorsal neural tube relative to the neighboring Neurog1-specific population
To determine downstream targets of Atoh1 unique to the Atoh1 lineage, we identified
transcripts enriched specifically in the dorsal progenitor and interneuron 1 (dP1/dI1)
populations located adjacent to the roof plate in the developing neural tube. The dP1 domain
begins expressing Atoh1 and differentiates into the dorsal interneuron 1 (dI1) population
marked by LIM-HD transcription factors, Lhx2 and Lhx9 (Bermingham et al., 2001; Gowan
et al., 2001). Similarly, the neighboring progenitor population (dP2) is marked by Neurog1
and differentiates into the dorsal interneuron 2 (dI2) population as marked by Lhx1 and
Lhx5. To identify transcripts present in the Atoh1-derived (dP1/dI1) domains that are distinct
from the Neurog1-derived (dP2/dI2) domains, we compared transcripts in these two related,
but discrete, cell populations.
Two transgenic mouse lines, Atoh1BAC-GFP (Raft et al., 2007) and dNeurog1-GFP
(Nakada et al., 2004b), drive GFP either to the dP1/dI1 domains or the dP2/dI2 domains,
respectively. Immunostaining with an antibody to each neighboring population, Neurog1
Lai et al.Page 5
J Neurosci. Author manuscript; available in PMC 2012 January 27.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript


text:
New technology meant for industrial applications.
Eco-friendly diaper brand unveils two new throwback diaper designs.
Brings 20 years of experience in China.
Zintra on Zintra panels offer design options, increased sound absorption
Feminine care products and baby diapers will benefit thousands of refugees.
Sign up now to receive the free weekly newsletter, and more!


text:
Peaceful place ,good photo,Sonja
Thank you sonjabgd& for your visit and vote!
Very beautiful composition and point of view!!
ciao Eleni
Thanky ou eleni. for your visits!
Nice composition, Luca!! Nice soft colours!! Greetings , Isabel Contest March
Thank you:
Isabel R.Marques for your visit and comment. Luca.
Great landscape.....greetings.....M Y c o N T e s T.....
Very nice composition.
Greetings from Turkey, Nuri.
Welcome to; <> my March contest...<> my number one
Thank you:
for your visit and comments.
That river was better of this.
Nice winter shot.
I like snow on the mountains.
Warm regards , Mahdi
Thank you Mahdi Kalhor for your visit and kind comment. Luca from Italy.
this was a great landscape and a nice photo.
Thank you Strausberger for your visit and comment.
Photo taken in Latisana UD, Italy


text:
5th TOPANGA Charity CUP Results
ResultsTournaments July 4, 2015 0
Today TOPANGA of Japan is putting on one of the biggest fighting game charity events ever. The 5th TOPANGA... Read more
TOPANGA Lends a Hand This Weekend with Their Fifth Street Fighter IV Charity Cup
NewsTournaments July 1, 2015 0
While the United States will be celebrating its Independence Day on July 4, TOPANGA of Japan is set to... Read more
Before there was BROKENTIER, before the Underground Syndicate Asked "When's Mahvel?", before we had shirts complaining about behavior on... Read more
Arc System Works to Donate Part of March Earnings Towards Ongoing Tohoku Earthquake Relief Efforts
Four years ago, Japan suffered one of its deadliest earthquakes to date. The 2011 Tohoku Earthquake and Tsunami left... Read more
Focus Attack, Donate Cans Charity Food Drive Tournament Streaming Live from Lakewood, Colorado
NewsTournaments November 15, 2014 0
UPDATE – This tournament is now live. You'll find the official broadcast below. Watch live video from StrikeFirstCO on... Read more
Extra Life Charity Kick Off feat. Super Smash Bros. Melee, Ultra Street Fighter IV Streaming Live from San Francisco, California
StreamTournaments October 23, 2014 0
Earlier today, the folks behind Northern California competitive gaming organization ShowDown, along with Humble Bundle, launched a 24-hour charity... Read more
The Art of Fighting Charity Tournament Streaming Live from Albuquerque, New Mexico
Tournaments September 27, 2014 0
The fine folks of the New Mexican fighting game community will be coming together this weekend to put on... Read more
Edmonton Super Smash Bros. Community Holding Third Annual Jarrett's Tournament to Raise Autism Awareness
NewsTournaments September 26, 2014 0
This weekend will see the third installment of Jarrett's Tournament take place at the University of Alberta in Edmonton, Canada.... Read more
The Art of Fighting Charity Event Returns to New Mexico Later This Month
NewsTournaments September 12, 2014 0
Last year, a special charity project was put together in New Mexico by a dedicated group of fighting game players.... Read more
TOPANGA has announced that the 4th edition of their Charity Cup tournament series will be taking place on July... Read more


text:
Select Local Merchants
Chopsticks HouseRead MoreIf you're craving Chinese food, try Cutler Bay's Chopsticks House. Chopsticks House serves up the finest gluten-free and healthy eats in the area. Tots and tykes will be right at home at Chopsticks House with its kid-approved food and ambience. Sometimes you need food fast, and Chopsticks House totally gets it, offering both takeout and delivery. Catering from Chopsticks House will take your party to the next level. Store your bike at one of the many racks outside of Chopsticks House. You can take it easy on your wallet at Chopsticks House — prices are generally less than $30 per person. Breakfast, lunch, and dinner are all served at the restaurant, but the dinner menu is the real standout.20553 Old Cutler RdCutler Bay, FL US
Harvey's Smoke House BBQRead MoreChow down on ribs, slaw and more at Harvey's Smoke House BBQ, a down-home barbecue joint in Cutler Bay. If slimming down is on the agenda, note that the food here is anything but low in fat. Little guys and gals will also love dining at Harvey's Smoke House BBQ, which offers a family-friendly environment (and menu). Harvey's Smoke House BBQ caters to all party sizes, both large and small. Diners who appreciate a no-frills environment come to Harvey's Smoke House BBQ in jeans and a hoodie. Grab your meal to go at Harvey's Smoke House BBQ if you're in a hurry — or better yet, have them bring it to you through their delivery service! You can also have Harvey's Smoke House BBQ cater your next event. The neighboring lot provides free parking to guests. You won't break the bank at Harvey's Smoke House BBQ, with a meal typically hovering below the $15 mark.20218 Old Cutler RdCutler Bay, FL US
Pollo TropicalRead MoreOne of the more popular Mexican restaurants in Cutler Bay, Pollo Tropical delivers wonderful chicken. It's a culinary destination for guests looking for awesome food. Thanks to its outdoor seating, the restaurant is a fantastic option when you want to soak up a bit of that Florida sun. It should be noted that business casual is the recommended attire. Also, while the prices may be low, you can bet that the ingredients will be fresh. In fact, you should easily be able to enjoy a good meal for $9 or $10 here, and can probably get in and out for $5 if you try. If you're searching for the perfect spot for a family gathering (or a birthday meal), it's rated as a nice local option for big groups and families with children. WiFi's available if you're trying to get some work done, and in addition to its quick service (both drive-thru and take-out are available), the restaurant also caters for events in the area. One of the better fast-food chains in the area, this Pollo Tropical definitely stands out from the competition. Don't worry about trying to find a spot on the street, as visitors to the restaurant do have access to a private parking lot nearby. Prefer to pedal your way there? Bike parking is also provided.18710 S Dixie HwyCutler Bay, FL US
Cutler Bay Sports Bar and GrillRead MoreIf you have yet to eat at Cutler Bay, it's the perfect time to visit the hugely-popular bar. It's a culinary destination for customers interested in delicious food. If you're wanting to plan ahead, most fans will be quick to point out that Wednesdays, Fridays, and Saturdays are the best nights to visit. No specific attire is required, so feel free to dress casually and comfortably. Also, if you're in the mood for tunes, it's one of the better places for live music. It's recommended that you should try the shrimp, the grouper, or the onion rings, as all three are great. In terms of the restaurant itself, it's a good option for families with children, large groups, and dancing. In addition to its convenient take-out offerings, the restaurant even provides catering for events around town. Or, if you're looking for a good spot to hit up after work, it's got a popular happy hour and pretty decent selection at its bar. Specializing in lunch, Cutler Bay definitely stands out from the rest. Don't worry about trying to find a spot on the street, as visitors to the restaurant do have access to a private parking lot nearby.20305 Old Cutler RdCutler Bay, FL US
T and W SubstantialRead MoreOne of the best-rated delis in Cutler Bay, T and W Substantial has a familiar atmosphere with a tantalizing menu that features subs. It's an ideal choice for guests seeking good food. Try to remember that business casual is the suggested attire. Also, though the overall price can be a bit on the low side, you won't be sacrificing any quality. In fact, you should be able to enjoy a good meal for $11 or $12, and can probably get in and out for $8 if you try. If you're searching for the perfect spot for a family gathering, it's been reviewed as a great local option for big groups and families with kids. WiFi is available if you want to get some work done, and if you're in a hurry, you can always take advantage of the convenient take-out menu. When you add everything up, T and W Substantial is a solid choice when you're in the mood for Latin cuisine, and want to expand your horizons beyond the typical Mexican fare.19100 SW 106th AveCutler Bay, FL US
Rovira Restaurant Latin FoodRead MoreRovira Restaurant Latin Food in Cutler Bay is a popular Cuban restaurant that features a phenomenal menu. The familiar atmosphere and reasonable prices allow customers to focus on what matters: the impeccable quality. No specific attire is required, so feel free to dress casually and comfortably. Also, though the prices are considered to be lower than average, you aren't going to sacrifice any quality. Most local parents will tell you that it's a good place to bring the kids, as well. Specializing in lunch, Rovira Restaurant Latin Food is a terrific choice when you're in the mood for Latin cuisine, and want to break away from the typical Mexican fare. Don't worry about trying to find a spot on the street, as visitors to the restaurant do have access to a private parking lot nearby. Prefer to pedal your way there? Bicycle parking is also offered.10389 SW 186th StCutler Bay, FL US