text: I'm 19 and I recently started a lawn care business with a buddy. I like mowing lawns and am considering switching my major to turf management. I'm a real estate major right now and was considering minoring in business. Anyway my question is has anyone majored in turf management? What kind of career opportunities do you have in the future? Is there a lot to learn in turf management or would i be better off majoring in business and just start my own business? Any help would be awesome! I consider myself a blue collar guy with a business state of mind because I want the money!


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Advertising by Design: Generating and Designing Creative Ideas Across Media (2nd edition)
Category: E-Book
Advertising by Design: Generating and Designing Creative Ideas Across Media (2nd edition) By Robin Landa
English | 2010 | 288 Pages | ISBN: 0470362685 | PDF | 26 MB
Don′t miss the new updated edition of the complete guide to the creative processes behind successful advertising design. The second edition of Advertising by Design has been developed and greatly expanded. Sill the most comprehensive text on creative concept generation and designing for advertising, the book includes a number of features that make it an effective tool for instructors, students, or anyone interested in this field. This includes a practical approach to generating and designing creative integrated-media advertising for brands, organizations, and causes that encompasses brand-building through engagement, community building, added value, and entertainment.Fully supplemented with interviews from esteemed creative directors, along with real-world examples, Advertising by Design is both a perfect text for courses that incorporate advertising concepts and design, and a valuable reference for anyone interested in the creative side of advertising.
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Lampion Center is known for highly skilled therapy, adoption services and innovative community services. We are also on the cutting edge of brain development information and how ACEs inform and positively impact the prevention services, resilience and wellness of our community.
What is an "ACE" (Adverse Childhood Experience)?
An "ACE" is an experience that causes toxic stress in a child. This stress can impair a child's development and effect their ability to function and make healthy relationships / life choices as an adult.
- Physical, Emotional or Sexual Abuse or Neglect
- Witnessing Domestic Violence (Emotional or Physical)
- Living with Someone with Chronic Mental or Physical Illness
- Living with Someone with Alcohol/Drug Abuse
- Absent, Separated or Divorced Parents
- A Family Member involved with Criminal Activity or In Jail
What does the "ACE" score mean?
- When a doctor takes a health measurement & family history it is used to help them assess risk and the likelihood of future health problems. ACE scores are similar but they focus on childhood events or situations that cause significant stress. The more stressful events that are experienced, the more risk there is for health issues and toxic stress levels as an adult.
- There are 10 questions on the ACE questionnaire. A high ACE score doesn't mean that physical and mental health issues will absolutely develop, it means the higher the score, the more at risk the person is for health implications into adulthood.
What can Parents and Caregivers do to better build a child's brain?
- Set devices aside and play / interact with your children daily in a focused, heartfelt way, in child-friendly activities. Quality time together, talking and listening over dinner, playing games, walking, or other activities that bring all together. Ask about their day, their friends, their feelings, their dreams/day dreams. Follow your child's lead. For infants, back and forth interactive games like peek-a-boo and patty cake are perfect for interaction.
- Create Optimism. Compliment your child and encourage his/her strengths on a daily basis. By repeatedly having positive experiences and thinking positive thoughts, brain science shows that we train our brains to be optimistic and hopeful about the future.
- Provide empathy and help children learn that all of their emotions are OK. Children learn to manage their emotions in a healthier way when we help them recognize and name their emotions.
- If your child is upset, stay with them and help them return to a calmer state ("time with", rather than "time out"). Find fun ways of involving kids in taking a breath and calming themselves. This will help them lower their stress and anxiety and learn how to decompress (as an added benefit: adults get calmer while helping their kids).
- Attend a free "Stewards of Children" child sexual abuse prevention training. Contact Lampion Center: 812.471.1776.
- To best meet a child's needs, Caregivers/Parents need to be healthy, including getting help if there is a mental health, relationship, or substance abuse issue.
- In cases of separation / divorce, help children maintain healthy contact with parents, grandparents, and extended family whenever possible.
- Protect/remove children from exposure to aggression, violence and criminal activity.


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2004-03-05 at 10:35:00 am #6355
"Honey, can you pick up milk, bread and a cartridge?" Mass Merchandisers Cashing in on Printing Supplies
Recent CAP Ventures research indicates that the strongest source for purchasing printing supplies after office superstores is Mass Merchandisers, which represent 35% of all purchases. Lower prices, packaging, and marketing efforts were the main reasons for the upward growth of this channel.
Source: Trends in Home Printing, CAP Ventures
Traditional channels continue to be the main push behind supplies, but alternative channels are catching the eyes of many print manufacturers. The price of replacement inkjet cartridges is sometimes more than the initial cost of the printer, and in the hurried lifestyles of today's consumer, convenience and price are often the most important factors when purchasing supplies.
Alternative channels can accommodate consumers' demands. With so many retail formats to choose from, many consumers are opting to try them all. Nearly one- third (31%) of households shop in 29 or more retailers or retail channels per year. This opens up many opportunities for manufacturers.
In CAP Ventures' new white paper entitled Chains & Channels: Supplies Presence in Alternative Channels, analysts focus in on the top chains in each alternative channel category and evaluate the sensibility and effectiveness of these channels in selling printing supplies. The alternative channels discussed in this report are:
- Computer superstores
- Consumer electronic stores
- Convenience stores
- Drugstores
- Mass merchandisers
- Warehouse clubs
- Craft Stores
- Grocery Stores
"Depending on the willingness of a vendor/manufacturer to participate in alternative retail channels, the success will be commensurate with the commitment and flexibility of the participant," commented Cathy Martin, an Associate Consultant for CAP Ventures. "Trial and error is a definite part when choosing an alternative channel, and what once didn't work may become an exciting new opportunity."


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This café and gourmet market serves Stumptown coffee and pastries, delivered fresh every day from Patisserie Colson. Linger at the coffee bar up front, or shop for a wide variety of organic grocery staples and local specialty products—like McClure's Pickles and Fine & Raw chocolate—in the rear.
|Venue name:
|Mott 156 (CLOSED)
156 Mott St
New York
|Cross street:
|between Broome and Grand Sts
|Opening hours:
|Mon–Thu 7am–9pm; Fri 7–10pm; Sat 7:30am–10pm; Sun 7:30am–9pm
|Subway: J, Z to Bowery; B, D to Grand St (Chrystie St)
|Average coffee: $4. Disc, MC, V
|Do you own this business?
To improve this listing email: email@example.com


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Lessons in Liberty is a series of lectures and courses sponsored by The Foundation for American Christian Education (FACE) to help citizens better understand the source of liberty in our American Christian Constitutional Republic. The speakers are authoritative and the lectures topical. Join like-minded patriots who love God's Word, and together, master the "lessons of liberty" so you can be used of God to help bring every aspect of culture "captive to the obedience of Christ."
Max Lyons
How to Really Make America Great Again: Why Education is the Key to Restoring America
Monday, January 16, 7:00-9:00 p.m. EST
"Make America great again" has rung in our ears for the last year or more, but what will truly make America great? It has been a rallying cry to restore America to what it once was and how much of the world sees us—as great. Some of the words Webster's 1828 uses to define "great" are excellence, admirable, elevated, dignified, noble all of these qualities can only come from God. Making America great again is only achieved through teaching a Biblical worldview, understanding what is means to have a Christian form of government instead of just Christians who formed our government. It is teaching internal Biblical principles that will really create change. "Really" means with actual existence, in truth, not in appearance only. If you are looking for real change, true change, then come and listen to How to Really Make America Great Again. Education is the key to restoring America.
Max Lyons served as teacher and administrator in three Christian schools since 1979. He holds a Bachelor of Arts in Mathematics from Virginia Wesleyan College, a Masters in Christian School Administration from Regent University, and a Ph.D. in Christian Education from Whitefield Theological Seminary. He currently serves as Director of Teaching Services at The Foundation for American Christian Education. He is the author or co-author of five books. Max and his wife Margie have 4 children and 7 grandchildren.
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A modest charge of $10 per lecture (individual or family) includes the live presentation, video-taped session and handouts. Or register for the entire 2016-2017 lecture series for $99 and save 25%. Class size is limited to 40 classroom participants and 100 online connections.
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Modern historians and media have always portrayed Ancient Romans and Roman emperors as being cruel and savage people. However, compared to modern 20th century governments, we can see that this picture of the Romans is a distorted one. The Ancient Roman culture did not produce ruling monsters the size of Stalin, Mao Tse-tung, Hitler, and Islamic fundamentalists who caused the death of millions upon millions of their own people as well as minorities in the name of an ideology and a religion. They also did not have presidents like Harry Truman and Lyndon B Johnson, who ordered the firebombing of Tokyo, Osaka, Bremen and Vietnamese cities, respectively, and the nuclear bombing of Hiroshima and Nagasaki, killing millions of women and children just because they wanted to increase their popularity by putting World War II and the Vietnam War to an end, taking the soldiers out of the battlefield and be elected presidents on their own (they had succeeded incumbent presidents). They also did not have Islamic terrorists, whose only objective is mass-murder of civilian population because the fundamentals of their religion say so.
I know you are going to say that Emperor like Caligula or Nero would have used atomic weapons if they had that technology, but these monsters were killed by the Praetorian Guards when things started to get out of hands, which paved the way for emperors such as Trajan, Adrian, Antoninus Pius and Marcus Aurelius.
We must not get actual human events out of its historical and cultural context, for the Romans and the Greeks were the products of their time and had completely different cultural mores (the values that constitute the cultural fabric of a given civilization) than human beings of today. And one of their moral values was "courage", and, in order to get social acceptance, a military had to fight face to face with the enemy on a given battlefield, and the enemy had the form of an armed soldier, not women and children. We must judge the Romans in their historical circumstances; in ancient times, an ordinary Roman citizen enjoyed the highest standard of living and political and social freedom in antiquity; Rome was also the only City-State to grant citizenship to foreigners. The Romans were vicious only with those nations that tried to destroy Rome, like Carthage.
In Ancient Times, specially among the Romans, the cultural values revolved around survival of the individual and of the group (clan, tribe, nation), never around monotheistic religious doctrines or political ideologies. The Romans would never waste their money, time and energy building pyramids to honor a tyrant, for example, for it was useless for survival; they would rather build bridges, aqueducts, irrigation systems, and a strata via (roads), because these were useful for the Roman citizens, who were living human beings. The Soviet Union, Communist China, and Cuba squandered away precious government income to deify and worship their tyrants, like Mao and Stalin; the result, terrible famines that wiped out entire populations.
In time of war, the objective of the Roman consuls and emperors were the conquest of a hostile nation to ensure the survival of Rome, which was completed through a process called romanization of that region.


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The Mail is reporting that Newcastle have held a meeting today to plan for how they can get Loic Remy on loan from Chelsea this month – since Loic seems our last good option to sign a proven Premier League striker in this transfer window – a player we desperately need.
Newcastle have reportedly discussed a plan of how to get Remy to Newcastle by next Monday, but they may be disappointed with news that Chelsea would prefer to allow on-loan striker Radamel Falcao leave the club rather than allow Remy to leave.
Loic Remy – while on loan at Newcastle
Loic Remy has looked a better player than Falcao this season and certainly his wages are a lot less – so it's not too surprising if Chelsea take that stance.
Loic had a very good loan season with us two years ago in the 2013-2014 season – when he scored 14 goals in 26 Premier League games and he could hit the ground running on Tyneside – so to speak.
Chelsea are in the process of signing Alexandre Pato from Corinthians on loan for the rest of the season and he is currently reported to be undergoing a medical at the London club.
Pato arrived in England yesterday although Chelsea have made no comment on the Pato situation at all.
Singing Pato should help Chelsea allow a least one striker to leave this month – so let's hope Loic is made available and that Newcatsle can sign him.
That would go a very long way to securing our safety in the Premier League this season.
Comments welcome.


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Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate; a comparative study
Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)
All of the yeast PH domains that have been previously shown to bind PtdIns(4,5)
These results showed that both the PLCδ1PH-GFP and the GFP-Tubby domain are useful reporters of PtdIns(4,5)
Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)
The PLCδ1PH-GFP reporter has since been widely used successfully to monitor PtdIns(4,5)
Research in the last 10 years has clearly demonstrated that phosphoinositides may not be the sole determinants of the cellular distribution of phosphoinositide-binding protein-modules
In the present study we evaluated several yeast PH-domains characterized in
Localization responses of lipid binding domains during manipulation of PM PtdIn(4,5)P2
Several yeast PH domains showed PM localization in spite of varying
Additional file 1
Figure S1. This is a Figure showing the distribution of some of the GFP-tagged PH domain constructs
Click here for file
Table 1
Localization responses of selected yeast PH domains and the human Tubby domain expressed in HEK293-AT1 or COS-7 cells.
PH domain
Response to
AngII stimulation
Response to
PtdIns(4,5)P 2
elimination by
Presumptive inositide
dependence of
PM localization
no change
no change
inositide independent
no change
no change
inositide independent
no change
no change
inositide independent
Tubby domain
Figure 1
Translocation responses of selected PH domains after PLC activation or phosphoinositide 5-phosphatase action
Translocation responses of selected PH domains after PLC activation or phosphoinositide 5-phosphatase action. The GFP-tagged versions of the indicated PH domains were expressed either in HEK293-AT1 cells for AngII (100 nM) stimulation (panel A) or in COS-7 cells for 5-phosphatase action (panel B). AngII stimulation causes rapid PLC activation with a substantial decrease in PtdIns(4,5)P2 levels (~80%
There were several other issues that made some of these domains less than optimal. The Num1-PH domain - which has the highest specificity of PtdIns(4,5)P2 binding
Comparative analysis of the Tubby-domain and the PLCδ1-PH domain
1. The Tubby domain binds to the PM with higher affinity
When expressed in HEK293-AT1 cells, a significantly higher fraction of PLCδ1-PH was found cytosolic compared to the Tubby-domain. Fig
Figure 2
Cellular distribution of the Tubby-domain and PLCδ1PH domain expressed in HEK293-AT1 cells
Cellular distribution of the Tubby-domain and PLCδ1PH domain expressed in HEK293-AT1 cells. The GFP-Tubby-domain and PLCδ1PH-GFP were expressed either separately (panel A) or were co-expressed (here the PLCd1PH was tagged with mRFP, panel B) in HEK293-AT1 cells. Confocal images show a stronger localization of the Tubby domain to the PM. The PM
To further elucidate the difference in the membrane binding properties, namely, the association/dissociation rates and mobility of the two probes, we performed fluorescence recovery after photobleaching (FRAP) analysis in HEK293-AT1 cells expressing the GFP-tagged versions of the respective reporters. Selected areas of the PM were bleached and the recovery of fluorescence was recorded (see additional file
Additional file 2
Figure S2. This is a Figure showing the FRAP analysis performed with the PLCδ1PH and the Tubby domain.
Click here for file
Additional file 3
Legend to Fig. S1. This is the legend describing what is shown in Fig. S1
Click here for file
Additional file 4
Legend to Fig. S2. This is the legend describing what is shown in Fig. S2
Click here for file
2. Response to agonist-induced PLC activation
Initial experiments showed that while PLCδ1PH-GFP showed a full translocation in > 90% of cells from the membrane to the cytosol after 100 nM AngII stimulation, the Tubby domain-GFP construct had much more variable responses ranging from no detectable change to a full translocation. For a better comparison of the two probes during PLC activation, the GFP-tagged Tubby-domain and the mRFP-tagged PLCδ1-PH domain were co-expressed in HEK293-AT1 cells. The dynamics of the redistribution of the two probes during this process was then followed after stimulation with different concentrations of AngII. At high concentrations of AngII (0.1-1 μM) both reporters translocated rapidly from the membrane to the cytosol. However, while the translocation of PLCδ1PH-GFP was almost always complete, that of the Tubby domain was often partial. When the AngII concentration was lowered to 30 nM, there was a significantly longer delay in the translocation of the Tubby-domain compared to that of the PLCδ1-PH domain (Fig.
Figure 3
Translocation responses of Tubby-domain and PLCδ1PH domain in AngII and ionomycin stimulated HEK293-AT1 cells
Translocation responses of Tubby-domain and PLCδ1PH domain in AngII and ionomycin stimulated HEK293-AT1 cells. HEK293-AT1 cells were co-transfected with the GFP-Tubby-domain and the PLCδ1PH-mRFP constructs. After 24 h, cells were analyzed by confocal microscopy. Cells were stimulated with 30 nM AngII (at 16 s) followed by 10 μM ionomycin (at 117 s) as indicated. Panel A shows a representative image series recorded at the indicated time points. Panel B shows the average responses (cytoplasmic fluorescence increase) of 50-54 cells (mean ± S.E.M) (left) and the average curve normalized to the maximal cytoplasmic fluorescence attained after ionomycin treatment (full translocation) (Panel B, right). Note the significantly slower and only partial response of the Tubby domain after AngII stimulation.
To determine the extent of maximum translocation of the reporters, high concentration of ionomycin (10 μM) was added after AngII stimulation. The high Ca2+ concentration attained with this amount of ionomycin activates endogenous PLC enzymes even without G-protein activation, completely eliminating both PtdIns(4,5)
In a subsequent set of experiments, we examined the response of the reporters to overexpresssion of a constitutively active Gq (Gq*). Gq mediated PLC activation has been the proposed mechanism by which the transcription factor Tubby is released from the PM allowing its diffusion into the nucleus
Figure 4
Distribution of the Tubby-domain and PLCδ1PH domain in cells expressing a constitutively active Gq protein
Distribution of the Tubby-domain and PLCδ1PH domain in cells expressing a constitutively active Gq protein. HEK293-AT1 cells were co-transfected with the GFP-Tubby-domain, PLCδ1-PH-GFP plasmids together with a constitutively active Gq α-subunit (Q209L). Panel A shows cells in which the PLCδ1-PH localization is completely lost while that of the Tubby domain is still quite substantial (upper) while in some cells both construct completely lose membrane localization (lower). Panel B shows an example where a cell that still shows substantial localization of the Tubby domain loses its PM localization after stimulation with even a low concentration (10 nM) of AngII.
3. Response to PtdIns(4,5)P2 dephosphorylation
Next, we studied the translocation of the probes when PtdIns(4,5)P2 was eliminated with the help of a polyphosphoinositide 5-phosphatase (5-ptase). For this, we used the rapamycin-induced recruitment of the type IV 5-ptase to the PM that can acutely eliminate PtdIns(4,5)P2
Figure 5
Translocation responses of the Tubby domain and PLCδ1PH domain after rapid dephosphorylation of PtdIns(4,5)P2 at the PM
Translocation responses of the Tubby domain and PLCδ1PH domain after rapid dephosphorylation of PtdIns(4,5)P2 at the PM. COS-7 cells were co-transfected with the PM-targeted FRB tagged with CFP (not shown), the type-IV 5-ptase domain tagged with cerulean (not shown), the GFP-Tubby-domain and PLCδ1PH-mRFP. After 24 h, live cells were analyzed by confocal microscopy. Addition of 100 nM rapamycin induces translocation of the 5-ptase to the PM, causing a complete loss of both PtdIns(4,5)P2 reporter localization. Panel A shows representative images before and after rapamycin addition. Panel B shows a full time course of cytoplasmic fluorescence intensity changes based on the average of 10 cells (Mean ± S.E.M.) expressed as a -fold increase over initial values (upper) or as a percent of the maximal response (lower panel, means are shown only).
Ins(1,4,5)P3 and PtdIns(4,5)P2 binding properties of the Tubby domain
Previous reports have suggested that the Tubby domain does not bind InsP3 although no direct binding data have been presented
Figure 6
InsP3 and PtdIns(4,5)P2 binding of recombinant PLCδ1PH-GFP and GFP-Tubby-domains
InsP3 and PtdIns(4,5)P2 binding of recombinant PLCδ1PH-GFP and GFP-Tubby-domains. Panel A: InsP3 binding assays were performed at room temperature using [3H]-Ins(1,4,5)P3 in the presence or absence of the indicated concentrations of unlabeled InsP3 as detailed under "Experimental Procedures". Means ± range of 2 experiments performed in duplicate are shown. Panel B shows the binding of recombinant proteins to lipid vesicles containing phosphatidyletanolamine in the absence or presence of PtdIns(4,5)P2 and InsP3. Vesicles were pelleted with ultracentrifugation and ''S'' and ''P'' represent the soluble (unbound) and pellet-associated (bound) GFP fusion protein, respectively, analyzed by a PhosphorImager after SDS-PAGE. The result of one of two experiments with identical results is shown. Note the negligible InsP3 yet very good PtdIns(4,5)P2 binding of the Tubby domain. The latter shows only a minor sensitivity to the presence of InsP3 in contrast to PLCδ1PH-GFP.
Inhibitory effects of the PLCδ1PH-GFP and Tubby-domain-GFP on Ca2+ signaling
Expression of protein domains that bind to PtdIns(4,5)P2 and/or InsP3 can exert an inhibitory effect on Ca2+ signaling. Binding to PtdIns(4,5)P2 can sequester a fraction of the lipid in the membrane and also hamper its availability to PLC enzymes. Binding and buffering of InsP3, on the other hand, will delay the onset of the Ca2+ release and also inhibit coupling between the individual Ca2+ release events causing a slower and smaller cytoplasmic Ca2+ increase
Recorded cells were grouped into four categories based on their expression level of the respective constructs (mRFP) and their averaged Ca2+ responses were calculated. As shown in Fig.
Figure 7
Inhibition of agonist-induced Ca2+ signaling by expressed mRFP-Tubby-domain, PLCδ1PH-mRFP and p130-PH-mRFP
Inhibition of agonist-induced Ca2+ signaling by expressed mRFP-Tubby-domain, PLCδ1PH-mRFP and p130-PH-mRFP. COS-7 cells were transfected with the indicated mRFP fusion constructs, and their cytosolic Ca2+ responses were analyzed by ratiometric Ca2+ imaging using Fura2. The amount of fluorescent protein was determined after the release of all membrane bound constructs by the addition of high concentraction of ionomycin at the end of each experiment. ATP-induced Ca2+ responses were grouped into four categories according to expression levels of the red fluorescence and the responses were averaged (see Table 2 for these values in each group in arbitrary units). Note that each construct interferes with the Ca2+ signal in these COS-7 cells where the ATP-induced PLC activation and InsP3 increase is relatively modest. Such inhibitory effects are substantially smaller in HEK293-AT1 cells that show robust PLC activation (see Fig. 8). Table 2 shows the numerical values of the Ca2+ signaling parameters.
Figure 8
Effects of InsP3 buffering on AngII-induced Tubby-domain and PLCδ1PH translocation responses
Effects of InsP3 buffering on AngII-induced Tubby-domain and PLCδ1PH translocation responses. HEK293-AT1 cells were transfected with the indicated GFP reporter constructs alone or together with p130PH-mRFP. After 24 hours, cells were examined in a Zeiss Live 5 DuoScan confocal microscope at room temperature. Cells were stimulated with 100 nM AngII at 60 second followed by 10 μM ionomycin at 360 sec. Data were collected at 1 fps rate and the cytoplasmic fluorescence intensity was analyzed in selected regions of interest outside the nucleus. Values were normalized for minimum and maximum fluorescence values for each run and these values were averaged. Means ± S.E.M. are shown obtained from: n = 10 (Tubby domain alone), n = 12 (PLCδ1PH alone), n = 21 (Tubby domain + p130PH-mRFP) and n = 20 (PLCδ1PH + p130PH-mRFP) cells. The traces were truncated at 420 sec to better illustrate the AngII-induced changes.
Table 2
Ca2+ signaling parameters in ATP-stimulated COS-7 cells expressing either one of the mRFP-Tubby-domain, PLCδ1PH-mRFP and p130-PH-mRFP proteins
mRFP fluorescence
T 1/2 activation*
< 100
6.0 ± 0.3**
6.0 ± 1.8
13.5 ± 3.5
12.0 ± 1.5
< 100
3.5 ± 0.4
6.5 ± 1.1
12.5 ± 3.9
18.0 ± 6.1
< 100
3.5 ± 0.4
12.0 ± 4.9
19.5 ± 5.3
> 100
* The lag time of the Ca2+ response was defined as the time it takes for a cell to reach half-maximum of its Ca2+ peak after addition of 50 μM ATP.
**Values are calculated from cells expressing the mRFP-tagged proteins at the indicated intensities. Note the largest effects of the p130-PH-mRFP protein that does not bind to the membranes but binds InsP3 with comparable affinity to the PLCδ1PH-mRFP
Effects of Ins(1,4,5)P3 buffering on the translocation responses of the two domains
Since the PLCδ1PH domain has significant InsP3 affinity, large InsP3 increases can promote the translocation of the PLCδ1PH-GFP fusion protein from the membrane to the cytosol
As shown in Fig.
The present study was designed to widen the repertoire of phosphoinositide binding modules capable of providing information on PtdIns(4,5)P2 changes in mammalian cells. So far most such studies have utilized the PLCδ1PH-GFP construct, which does not detect PtdIns(4,5)P2in cellular location other than the PM and also could suffer from overestimating PtdIns(4,5)P2 decreases, due to a displacing effect of InsP3. The extent of this distortion may vary from cell-type to cell-type and also depends on expression levels
In a thorough recent study, several PH domains of
The discrepancy between the probes behavior in the yeast and mammalian cells is not unprecedented. For example, we did not observe Golgi localization of the PtdIns4
Another PtdIns(4,5)P2 reporter characterized in this study was the Tubby domain of the mammalian transcription factor Tubby protein. The Tubby domain was described as a high affinity PtdIns(4,5)P2 binding module found at the C-terminus of the Tubby protein and being responsible for its lipid binding and membrane localization
Firstly, both studies found that the Tubby protein as well as the Tubby domain has higher affinity to the PM than PLCδ1PH-GFP. In fact, the wild-type Tubby domain was found to have high enough affinity that it did not show agonist-induced responses in many cells prompting the authors to create a mutant (R332H) with a reduced affinity that was found useful in their studies
Nevertheless, a higher affinity of the full-length Tubby protein (fused to eGFP) to the membrane was also shown in the Nelson study and manifested as a rightward shift (compared to the PLCδ1PH-GFP response) in the dose-response curve with muscarinic agonist measuring the translocation of the Tubby protein from the membrane to the cytosol. Our results are in good agreement with these studies as we also found a significantly higher fraction of the Tubby domain at the PM than with PLCδ1PH, and documented a substantial difference between the agonist-sensitivities of the Tubby domain and PLCδ1PH-GFP translocation responses. Moreover, our FRAP analysis showed that the dissociation of the Tubby domain from the membrane is significantly slower than that of the PLCδ1PH-GFP.
All three studies showed that Tubby domain (or the full-length Tubby protein) displayed no sensitivity to InsP3 in agreement with the original claims
In contrast to these findings, a striking reduction was observed in neuroblastoma cells in the translocation responses of the PLCδ1PH domain, but not those of the Tubby protein, when InsP3 3-kinase was expressed, with a strong reduction in the cytoplasmic Ca2+ response
The question remains whether the tighter binding of the Tubby domain to the membrane is related to its lower affinity to InsP3. A significant amount of free InsP3 present in the cytosol could indeed partially displace the PLCδ1PH-GFP from the membrane while not affecting the binding of the Tubby domain. However, overexpression of either the InsP3 kinase
An important observation of the present study was the delayed Tubby response relative to that of the PLCδ1PH domain during PLC activation but not when the 5-phosphatase was recruited to the PM. This finding may simply suggest that the InsP3 increase does indeed contribute to PLCδ1PH translocation. However, it is also possible that the endogenous PLC activation mechanism is more sensitive to masking of PtdIns(4,5)P2 by the Tubby domain than the catalysis by the recruited 5-phosphatase. The fact that the Tubby domain does not bind the soluble headgroup, InsP3, even though it binds PtdIns(4,5)P2 with higher affinity, already suggests that the binding force of the Tubby domain must receive significant contributions from interactions with the glycerol backbone. If this were indeed the case, the Tubby domain would obscure the phosphodiester group more and with that could block the access of PLC to this site.
Lastly, these studies have shown a perfect example of how the same lipid, PtdIns(4,5)P2 can regulate several effectors in the same cell simultaneously and yet differently. Just by having two different apparent affinities of two reporters used in the present study - mimicking two endogenous binding partners - is sufficient to elicit a differential response to the same PLC activation. This was also convincingly demonstrated in the experiments where the Kir6.2 KATP channel
There are several other conclusions highlighted by these studies. Firstly, all probes based on binding to PtdIns(4,5)P2 (or to any other lipid) show some bias depending on its affinity to the lipid. Secondly, the experimental system may also determine the extent to which the probe manifests its limitation. Thirdly, even the fluorescent protein used a fusion partner can modify the utility of a bioprobe. These should be all reminders that we need to be cautious of selecting a probe and consider it as "the" gold standard.
The present study analyzed a set of PtdIns(4,5)P2 reporters some selected from yeast PH domains and one comprising of the Tubby domain of the transcription factor, Tubby. All of these fluorescent probes confirmed that PtdIns(4,5)P2 shows highest abundance in the PM and that this lipid is undetectable in intracellular membranes. Most of the yeast-derived PH domains were found less useful, some expressing at low levels or showing weak membrane localization, others causing membrane blebbing, Detailed comparison of the PLCδ1PH-GFP and the Tubby domain showed that both reporters are suitable to follow PtdIns(4,5)P2 changes in living cells, but the Tubby domain is less sensitive because of its higher affinity to PtdIns(4,5)P2. PLCδ1PH domain was found to be the most sensitive reporter but because of its binding to InsP3 some of its translocation is caused by InsP3 increases that can become a problem especially in certain cell types. The differential responses of the two domains to PtdIns(4,5)P2 changes helps us understand and envision the principles of inositide regulation of multiple effectors.
AngiotensinII (human octapeptide) was from Bachem (Torrance, CA), rapamycin was from Calbiochem (San Diego, CA) or LC Biochemicals (Woburn, MA). All other chemicals were of the highest purity grades.
DNA constructs
The PLCδ1-PH-GFP and its color variants and the p130-PH-mRFP constructs have been described previously
Transfection of cells for confocal microscopy and intracellular Ca2+ measurements
For single cell analysis HEK293-AT1 (a HEK293 cell line stably expressing the rat AT1a angiotensin receptor) or COS-7 cells were used. Cells were plated onto 25-mm-diameter circular glass cover slips at a density of 3 × 105 cells/dish one day before transfection with plasmid DNAs (0.5-1 μg/dish) using the Lipofectamine2000 reagent (Invitrogen) and OPTI-MEM (Invitrogen). One day after transfection, cells were washed twice with a modified Krebs-Ringer solution, containing 120 mM NaCl, 4.7 mM KCl, 1.2 mM CaCl2, 0.7 mM MgSO4, 10 mM glucose, 10 mM Na-Hepes, pH 7.4 and the coverslip was placed into a metal chamber that was mounted on a heated stage with the medium temperature kept at 33°C. Note that some experiments were performed at room temperature. Cells were incubated in 1 ml of the Krebs-Ringer buffer and the stimuli were added in 200 μl warm buffer removed from the cells. Cells were examined in an inverted microscope under a 60× oil-immersion objective (LSM 510-META; Carl Zeiss MicroImaging, Inc.) equipped with an objective heater (Bioptech).
FRAP analysis
FRAP experiments were performed in a Zeiss Live5 DuoScan Confocal Microscope system in which continuous fast scanning is possible while photobleaching with another laser. Images were collected at 0.1-0.5 s intervals and selected regions in the PM were photobleached at 100% power of the bleaching laser. After background subtraction, two parameters were calculated from the intensity curves obtained in the center of a larger bleached area: the mobile fraction (MF), which represents the percent recovery after photobleaching compared to the initial fluorescence value before photobleaching; and a T1/2 value, which is the time required to reach half maximal recovery.
Cytoplasmic Ca2+ studies
For Ca2+ measurements, cells were loaded with 3 μM fura-2/AM (45 min, room temperature) in a Hepes-based M-199, containing 200 μM sulfinpyrazone and 0.06% pluronic acid. After loading, Ca2+ measurements were performed at room temperature in the same modified Krebs-Ringer solution containing no Ca2+. An inverted microscope (IX70; Olympus) equipped with a Lambda DG-4 illuminator (Sutter Instruments) and a Micromax 1024 BFT camera (Roper Scientific) and the appropriate filter sets were used for Ca2+ analysis. Data acquisition and processing were performed by the MetaFluor software (Molecular Devices).
In vitro Ins(1,4,5)P3 and PtdIns(4,5)P2 binding assays
Recombinant proteins were produced in the BL-21-DE3 strain of Escherichia coli (Invitrogen). Bacterial cells were grown to A600: 0.6 at 37°C and induced with 300 μM isopropyl-1-thio-β-
The in vitro InsP3 binding assay and the InsP3 displacement from PtdIns(4,5)P2 binding were performed as described previously
Binding to phospholipids was performed with mixed lipid vesicles. 110 μg of PtdIns(4,5)P2 and 1.4 mg of phosphatidylethanolamine (bovine liver; Avanti) were mixed and dried under a nitrogen stream followed by high vacuum, and the dried mixtures were suspended to a final total lipid concentration of 2 mM (PE) in 20 mM Hepes, pH 7.2, 100 mM NaCl, 2 mM EGTA, 0.1 μg/ml bovine serum albumin by bath sonication. 5 μl of the purified GFP fusion protein (~1 μg) and 5 μl of buffer or Ins(1,4,5)P3were added to 90 μl of phospholipid vesicles. The reaction mixture was incubated at 30°C for 10 min followed by ultracentrifugation at 85,000 rpm for 20 min at 4°C. The 100-μl supernatant was mixed with 30 μl of 5× Laemmli buffer, and the pellet was resuspended in 100 μl of incubation buffer followed by the addition of 30 μl of 5× Laemmli buffer. After vortexing, 40 μl of each fraction was loaded onto a 12% Tris glycine gel without boiling and separated by SDS-PAGE at 4°C. After electrophoresis, gels were analyzed in a Storm 860 PhosphorImager (Molecular Dynamics) using blue fluorescence screening for quantitation of the GFP fusion protein band in the gel. Western blot analysis was also performed on parallel samples using the purified polyclonal antibody against GFP (Clontech).
GFP: Green fluorescent protein; InsP3: Inositol 1,4,5-trisphosphate; PtdIns: Phosphatidylinositol; PtdIns(4,5)P2: Phosphatidylinositol 4,5-bisphosphate; PLC: Phospholipase C; PM: Plasma membrane.
Authors' contributions
ZS has created constructs, run most of the experiments, analyzed data, participated in experimental planning, wrote draft of the manuscript, and prepared raw figures. AB created constructs, prepared recombinant proteins and run the InsP3 and lipid binding assays. YJK has run some of the experiments and analyzed the corresponding data. ML has provided all yeast constructs, discussed data and their implications, read the manuscript. TB has designed experiments, trained ZS to run microscopy experiments, participated in microscopy sessions, analyzed data, completed the writing of the manuscript and prepared the final figures. All authors have seen and approved the final version of the manuscript.
We are grateful to Dr. Roger Y. Tsien for the monomeric red fluorescent protein and to Dr Philip W. Majerus for the human type-IV 5-ptase clone. Confocal imaging was performed at the Microscopy & Imaging Core of the National Institute of Child Health and Human Development, NIH with the kind assistance of Drs. Vincent Schram and James T. Russell. This research was supported in part by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health.
Inositol trisphosphate and diacylglycerol as intracellular messengers
Phosphoinositides in cell regulation and membrane dynamics
Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate
The complex and intriguing lives of PIP2 with ion channels and transporters
The regulation of cell motility and chemotaxis by phospholipid signaling
Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion
Nuclear inositide signalling -- expansion, structures and clarification
Receptor-induced transient reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in living cells
Visualization of phosphoinositides that bind pleckstrin homology domains: calcium-and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools
Live cell imaging of phosphoinositide dynamics with fluorescent protein domains
Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic
Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1
Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns
Single-cell imaging of graded Ins(1,4,5)P3 production following G-protein-coupled-receptor activation
Pleckstrin homology domains: not just for phosphoinositides
Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains
Tubby proteins: the plot thickens
G-protein signaling through tubby proteins
PtdIns(4,5)P2 functions at the cleavage furrow during cytokinesis
Temporal profiling of changes in phosphatidylinositol 4,5-bisphosphate, inositol 1,4,5-trisphosphate and diacylglycerol allows comprehensive analysis of phospholipase C-initiated signalling in single neurons
Monitoring changes in membrane phosphatidylinositol 4,5-bisphosphate in living cells using a domain from the transcription factor tubby
Maintenance of Hormone-sensitive Phosphoinositide Pools in the Plasma Membrane Requires Phosphatidylinositol 4-Kinase III{alpha}
Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells
Monitoring Phospholipase C activation kinetics in live cells by FRET
Reversible binding and rapid diffusion of proteins in complex with inositol lipids serves to coordinate free movement with spatial information
A plasma membrane pool of phosphatidylinositol 4-phosphate is generated by phosphatidylinositol 4-kinase type-III alpha: studies with the PH domains of the oxysterol binding protein and FAPP1
Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C delta 1 and p130
A novel recombinant hyper-affinity Inositol 1,4,5-trisphosphate (IP3) absorbent traps IP3, resulting in specific inhibition of IP3-mediated calcium signaling
Targeted expression of the inositol 1,4,5-triphosphate receptor (IP3R) ligand-binding domain releases Ca2+ via endogenous IP3R channels
Kinetic analysis of receptor-activated phosphoinositide turnover
Single cell analysis and temporal profiling of agonist-mediated inositol 1,4,5-trisphosphate, Ca2+, diacylglycerol, and protein kinase C signaling using fluorescent biosensors
Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p
Receptor activation alters inner surface potential during phagocytosis
Anomalous negative fluorescence anisotropy in yellow fluorescent protein (YFP 10C): quantitative analysis of FRET in YFP dimers
Synaptic activity augments muscarinic acetylcholine receptor-stimulated inositol 1,4,5-trisphosphate production to facilitate Ca2+ release in hippocampal neurons
Determinants of metabotropic glutamate receptor-5-mediated Ca2+ and inositol 1,4,5-trisphosphate oscillation frequency. Receptor density versus agonist concentration
Endogenous inhibitors of InsP3-induced Ca2+ release in neuroblastoma cells
Phospholipase C in living cells: activation, inhibition, Ca2+ requirement, and regulation of M current


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Discovering Evolutionary Explanations for Disease
The Center for Evolution & Medicine supports interdisciplinary research by bridging the many different disciplines (e.g. infectious disease, genetics, clinical medicine, veterinary medicine, anthropology, psychology, etc.) where relevant research takes place. Research at the Center demonstrates the power of evolutionary biology to address problems in medicine and public health.
ASU will recruit 8 new faculty researchers for the Center in the next 3 years to augment existing expertise at ASU and the Mayo Clinic.
Priority areas include cancer, preventing antibiotic resistance, the microbiome, evolutionary genetics, and diseases of modern environments.
Some Center sponsored research is about human disease, but other projects are about disease in other species, or about basic questions such as the origins of mechanisms that control mutation rates.
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Zimmerman's jail calls discussed moving money
George Zimmerman instructed his wife, Shellie, on how to transfer money from his bank account into hers and his sister's while he was in jail, according to recordings of jail calls released Monday by the Florida State Attorney's Office.
In the six calls, Zimmerman spent considerable time instructing his wife on changing account passwords and moving money out of his account every day.
The calls, which were made from April 12 through April 17, were part of the evidence the state introduced in its request that Zimmerman's bond be revoked. The court was prepared to release all 151 phone calls Zimmerman had in jail, but his attorney requested a hearing to stop the rest.
STORY: George Zimmerman's wife charged with perjury
PHOTOS: Death of Trayvon Martin continues to raise questions
Zimmerman faces second-degree murder charges over the shooting of unarmed teenager Trayvon Martin in Sanford, Fla., in February. He has pleaded not guilty, claiming self-defense.
Prosecutors say the calls show that Zimmerman tried to hide his financial assets from the court. Before Judge Kenneth Lester set bail at $150,000, Shellie Zimmerman testifed that she didn't know how much money had been raised from a website created for her husband's legal defense. He had raised at least $135,000 before his arrest.
After the calls were revealed, Lester revoked Zimmerman's bond on June 1 and sent him back to jail. He will have another bond hearing June 29. Shellie Zimmerman was charged with perjury and released on $1,000 bond.
In one conversation, Zimmerman details how his wife should move money into her account and help his sister open an account so they can move money into that one, too. In another, he waits on the phone while she transfers money into her account to make sure it works.
In another, he tells his wife, "If you have more than $10, then you can, maybe that same day, put 10 in hers and she can take the 10 out … to keep in your pocket, … And then, at the institution, see if you can put 10 into hers right away … And see if she can take 10, that 10 out and put it in the, in the box there."
Another call referred to a safety deposit box.
Zimmerman instructs his wife to set a reminder on her phone to make the daily transfers.
Prosecutors allege that Zimmerman and his wife talked about $10, $20 and $100 to mask the fact that they were referring to thousands of dollars.
"In my account, do I have at least $100?" Zimmerman asks his wife in one conversation.
"There's, like, $8," she tells him.
"Okay, so total, everything, how much are we looking at?" he asks.
"Um, like, $155," she answers.
Other parts of their conversations highlight security concerns for Zimmerman and his family. His lawyer has said Zimmerman has received death threats.
In one exchange, he asked his wife if she's looked into getting a protective vest. He asked her to get three — one for him, one for her and one for his attorney, Mark O'Mara.
"We need to get on it ASAP, 'cause you know the, the — personally, I want you wearing one," he tells her. "As uncomfortable as it is, I want you wearing one."
They talk about mundane things, including paying off their Sam's Club credit card and American Express bills. Zimmerman refers several times to "shower day" when he's able to take a shower in jail.
They talk about the logistics of him leaving jail and about renting two cars, including an SUV, in case they are followed. Shellie Zimmerman wonders, "I don't know if they black out SUVs, though." Then she says, "You could always, like, lay down or something."
"Yeah, exactly. Well, I have my hoodie," he responds.
Shellie Zimmerman tells her husband in one conversation, "All morning online we have been reading so many positive, um, websites dedicated to a — a more true narrative to you."
In another call, she reassures him, "This is only temporary."
"I know," he replies. "Um, my dad's really confident about everything. I don't know what was said in that meeting, but my dad seemed very, very confident."
Posted | Updated


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Pete McLeod of Canada finished his rookie season in the Red Bull Air Race World Championship on a high note with a promising 12th place finish in Barcelona on Sunday in front of a record-breaking crowd of 800,000 spectators.McLeod was able to advance through the Wild Card competition into the Top 12 round in the high-speed, low-altitude race for only the second time in six races this year by taking advantage of the skills he learned flying float planes in northern Canada.McLeod, at 25 the youngest pilot in race history, could have finished a career-high 10th on the difficult 6.4-km race track through 20-meter high Air Gate obstacles set up in the Mediterranean off the beaches of Barcelona. But he just hit a pylon, bursting the Air Gate as he passed through at nearly 370 km/h and being assessed a six-second penalty for the infraction. "I didn't even feel the pylon," said McLeod, who had impressed the veterans with his smooth flying on the challenging Barcelona track with its "quadro" gate made especially difficult by the variable coastal winds."I was pleased with both my runs so it was a great end to the season for me. It's been full of excitement and challenges at the same time." McLeod, who had the heaviest plane with the least powerful engine, did well this year on the more difficult tracks because several pilots with faster planes made mistakes but struggled on the easier tracks.
Pete McLeod, the first Canadian pilot in the Red Bull Air Race World Championship, has been trying to absorb as much wisdom and acquire as much experience as possible during his rookie season flying against the world's top pilots.The 25-year-old newcomer from London, Ontario has handled the pressures of the high-speed, low-altitude precision flying race with aplomb in front of enthusiastic crowds as large as 720,000 - the number that watched the penultimate race of the season last week (Sept. 12/13) in Porto.But the youngest pilot in the championship's history has discovered the hard way that the 11 veterans are not quite as forthcoming with tips and suggestions for McLeod and his three fellow rookies from Australia, Germany and Japan as they had been in the past - for good reason after Austria's Hannes Arch managed to win the championship in his second season after picking their brains when he was a rookie."The veterans can give you tips but they can also deliberately mislead you if you're not careful," McLeod said before the final race of the season in Barcelona, Spain on October 3/4. "The really good tips are few and far between. But if you ask the right question at the right time, there is some good information out there. You always have to get a second opinion. And usually the best tips are the ones they don't repeat. You get the feeling it was something that just slipped out by accident."Championship leader Paul Bonhomme of Britain and the 10 other veterans all have reason to worry about McLeod. The 6-feet (1.85 cm) tall McLeod not only towers over most of his more senior rivals physically. But the other pilots - all of whom are in their late 30s to early 50s - fear it is probably only a matter of time before the former hockey player with the jet black hair ends up towering over the championship as well.
Landing in Porto for the 5th stop on the Red Bull Air Race World Championship, Pete McLeod and his team were up for the big challenge. The Porto track, basically a straight line, would give a great advantage to the faster aircraft.It's no secret that PMR has one of the heaviest and under powered race planes on the 2009 tour. Due to this fact, Pete had to fly the best lines with the fastest vertical turns to keep within seconds of the other pilots. The September weather brought unpredictable sea fog into Porto, creating a problem with the TRW operations, which was located right on the beach.Both days of scheduled training ended up being very quiet with no flying due to low fog on the runway. At the end of the day on Friday, RBAR decided to relocate all 15 race teams, while there was a window in the fog, to a nearby inland airport.With no training track time entering Qualification Day, Pete was not feeling overly confident for the race. "It's been frustrating with the foggy weather keeping us on the ground during scheduled training. As with the whole season my major objective is to learn as much as possible and develop. I will try to get the most out of this by staying focused for qualification."Qualification Day proved to be a new challenge for the race team as we operated away from the elaborate support structure of the TRW. Three track sessions were allotted to each team: one training session and two qualification runs.
The fourth stop of the 2009 RBARWC landed in Budapest Aug 19-20 2009. Coming off a successful race on home turf in Canada, where PMR earned their first championship point, the team was excited to back on the race track."Budapest is a race I've really been looking forward to. Budapest is to RBAR what Monaco is to F1. I can remember following this race every year and dreaming to compete in this venue." said Pete ahead of the race. With 650,000 fans on race day and the water level just low enough to fly under the famous Chain Bridge, the race lived up to all expectations.Unique to Budapest is that the Chain Bridge is actually part of the race track. There is about 10.2 meters of space between the water and the bottom of the bridge. When the clock starts after passing under the bridge racers actually have to climb into the already low track to avoid a DSC for low flying.Heading into qualifying day, Pete and the team were putting up improving times, but it was clear the horsepower friendly track would hurt #84 with its stock engine and 30kg of extra weight. "I'm running a few seconds behind the pack, but the focus is still on putting down clean runs. It's a busy track and penalties are easy to come by" said Pete after an average Q1 run.


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Doggy Day Care in Strawberry Valley, CA
Drop off your dog at your sitter's home in the morning, and pick them up in the evening.
Lucky Dog Camp ~ A Fun Dog Getaway!
Nevada City, CA, 95959
Dog Boarding, Doggy Day Care
"Michael was great! Super easy to connect with and totally professional. He sent me a pic of my dog during his stay without my even asking and I loved that!... (more)"
Your pets can be my best friends!
Nevada City, CA, 95959
Dog Boarding, House Sitting, Drop-In Visits, Doggy Day Care, Dog Walking
About: I have been around dogs all of my life and find their company a blessing. I am very comfortable with animals and see their presence as stimulating ... (more)
Very experienced shelter volunteer!
Grass Valley, CA, 95945
House Sitting, Drop-In Visits, Doggy Day Care, Dog Walking
About: I have had many dogs since I was a child. I have always been an active dog owner. I don't sit around much, so dog walking is an ideal job for me! I... (more)
Experience exceptional service and a personalized match
From start to finish, we'll handle all the details for you.Learn About Premier
Rover also offers doggy day care in:
|Brandy City, CA
|Cal-Ida, CA
|Camp Eighteen, CA
|Clipper Mills, CA
|Lumpkin, CA
|North Star, CA
|Oak Valley, CA
|Oroleve, CA
|Poverty Hill, CA
|Weeds Point, CA
We've been there, cancelling meetings or plans with friends to make sure you can be there for your dog. Good news: You can easily find and book doggy day care in Strawberry Valley on Rover. Whether you need a one-time daytime pet sitter or someone to take care of your beloved pet every weekday, we've got you covered. Care for your dog and peace of mind for you are just a few clicks away. If you already have a favorite overnight sitter, ask them if they offer day care too. Or, if you've never used Rover, this is the perfect opportunity to find a new best friend for your dog when you're at work. We can help you find a sitter who'd love to watch your dog.


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1260 St Ives Place, Los Angeles (City), California
$60,0005 Beds8 BathsActive
$60,0005 Beds8 BathsActive
1260 St Ives Place, Los Angeles (City), California 90069 - Modern Mediterranean Mansion above Sunset and Doheny with panoramic views of Century City, Ocean and Downtown. Estate has 5 bedrooms and 8 baths with movie theatre and elevator. With New modern furnishings, the Mansion sits on its private promontory at the end of a private cul-de-sac. 18 ft foot ceilings, sweeping staircase, with grand entry, gourmet chef's kitchen, family room, living room, & library with built ins. Luxurious large master suite offers balconies, FP, sitting room, dual baths w/ steam showers & 2 walk-in closets. Gourmet kitchen includes granite countertops, Viking 6 burner stove, stainless steel appliances, butler's pantry & breakfast area. Marble entry with main living areas on 1st floor that easily lead out to bright backyard & showcase remarkable grounds which feature numerous dining/ entertaining terraces, more panoramic city views, swimmers pool & spa. Designer details and furnishings including vintage Steinway piano. Walking distance to Sunset Strip.
Listing Courtesy of Keller Williams Beverly Hills


text: We are bidding on two installs. Both have retaining walls that we will have to get by. One is regular retaining wall blocks and one is 8' landscape timbers. Both are about 4' high. The POC is on one side and the area to be irrigated on the other. Homeowners will not allow any surface pipe over the walls. What are our options to get from one side to the other? A first for us. Thank you for the help. John


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Gosaikunda Trekking towards north of Kathmandu valley takes you around the excellent Langtang town, investigate Kyangin Gompa, visit hallowed pools of Gosaikunda, cross Laurebina go and dive through Shivapuri National Park towards Kathmandu valley. You will be honored with unmatched Himalayan highlights with Tibetan Buddhism culture investigation opportunity at Kyangin gompa and follow our means back towards Lama Hotel to move east towards Singompa, Gosaikunda, defeat nature and culture amid our Langtang Gosaikunda Trekking. This astonishing Langtang Gosaikunda trek is particularly planned with the combin of the remote and terrific valley of Langtang. With the high mountain lakes at Gosainkund of the langtang is populated by Sherpa and Tamang families in the North West of Kathmandu valley and well known for its way of life and religion with unbelievable perspectives of Himalayas.
The Gosaikunda zone has been depicted as a religious site. Hindu mythology traits Gosaikunda as the home Hindu divinities Lord Shiva and Goddess Gauri. The Trekking trails of Langtang Gosaikunda are testing which incorporates long, soak and plunges, calm sub-high profound timberland, sky blue gentians, epiphytic orchids and primroses or crosswise over ice sheet mountain sees and fascinating towns.
Langtang Gosaikunda climbing gives you the chance to get ideal in among the Himalayan pinnacles and to get off-the-beaten-track, strolling through some fascinatingly remote and moderately uninhabited territories. Gosaikunda Trekking experience starts as you touch base at socially princely capital city of Kathmandu; go for touring around world legacy destinations and following day drive towards Syabrubesi on a grand drive. At first, we trek through delightful woods, loaded with rhododendron and oak trees, bamboos and interface with nearby occupants of Tamangs and Sherpas. We cross the Lama Hotel, and advance towards glorious Kyangin Gompa; the place from where delightful all encompassing perspectives of Langtang Lirung, Dorje Lakpa, Ganesh Himal and other encompassing pinnacles is seen. From here, we come back to Lama Hotel and turn east towards journey site of Gosaikunda lake that has numerous religious convictions. Alongside Gosaikunda, there are different kunda(ponds or little lakes) like Saraswatikunda, Bhairavkunda and others.
Encourage we cross Laurebina Pass and advance through Helambu towns and Shivapuri National Park before returning back to Kathmandu. Trip to gosaikunda is a reasonably appraised trekking enterprise at focal northern Nepal. You will have the best of trekking encounters in the event that you are physically fit and have energy towards strolling, investigating and voyaging. .


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The Damascus Apparel Launchpad Hoodie
With time comes change, it's un-avoidable and will happen for the rest of our consciousness. Change of the seasons brings us change of temperature and with San Diego being a pretty balanced city for it's weather, for this years winter we developed an all-around middle range 10oz jacket that brings warmth when needed, but also maintains its style and functionality. We hand produce them from start to finish from our warehouse in San Diego, CA.
Gorilla Disko in San Diego, CA shot by Kevin Longwell.
Gorilla Disko in San Diego, CA shot by Kevin Longwell.
The Launchpad zip hood is made with premium heavyweight fabric, generous spec and quality construction. It boasts a Fleece lined hood, heavy gauge round drawcord with nickel eyelets, #5 nickel kissing zipper, 1×1 ribbing at cuffs and waistband.
10 oz 80% cotton 20% polyester blend fleece
100% cotton 32 singles face yarn for supreme print-ability and softness
Split stitch double needle sewing on all seams
Twill neck tape


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Flea infestations of your pet and home MP3
fleas are the mos common pests. this year is the worst year for fleas in at least 25 years. they can cause other serious disease to both your pet and your family.
Home Pest Control : How to Kill Fleas in 24 Hours MP3
Killing fleas in a 24-hour period is unrealistic due to the four stages of a flea. Find the fastest way to kill fleas with help from a professional pest control technician ...
Worst Case Of Fleas MP3
This is not the worst case of fleas I've seen on a dog but it is close. These people needed help with their flea problem so they turned to a professional.
TNR - Too many Fleas and Ticks! MP3
TNR in full force atm. We have to catch up after Fatou's long holidays! This guy from Sukuta needed a treatment for his external parasites, needed it badly.
Flea Control : How to Get Rid of Fleas With Household Items MP3
Some household items that can be used to get rid of fleas include hand soap, water and lemons. Use homemade supplies to get rid of fleas with tips from a pet ...
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How to get rid of fleas fast. Professional advice for natural ways to rid your home of fleas.
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Animal Care Technologies offers online client education resources that can maximize your opportunity to increase online visibility, compliance and overall client ...
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I don't know if these are fleas or not. Found them in my basement. The cat was playing with them...There must be 50000 or so. Never seen anything like it. Maybe ...
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This quick video demonstrates how thousands of fleas can live undetected on a black dog that is not allergic to the flea bite. Since the dog wasn't itching and ...
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Getting rid of fleas in your home can be a daunting task. Don't worry, we're here to make it simple and easy for you. Join Agatha from Parktown Vet as she shows ...
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New MESSAGE: Sept 21 AFTER FLEA'S NEXT COMES WORMS ...Next video coming soon HOW TO RID YOUR CAT OF WORMS (Please visit my playlist for ...
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Forty-four years after the debate about how fleas jump began, researchers say they've solved the mystery thanks to high-speed cameras that show the insects ...
✔ DiResta Lord of the Fleas MP3
This TV show concept was created by my brother John DiResta. John and my self made this pitch movie in the summer of 2004. This is a lifestyle we led on and ...
Home Pest Control : How to Use Salt to Kill Fleas MP3
Salt can be used to kill fleas because it is a desiccant, which dries things out, such as the eggs and pupa. Try using salt to kill fleas with advice from a ...
How to kill Fleas on your Dog Naturally MP3
I am killing Fleas on my Dog Puffy with my HOMEMADE SOAP made with Cedar Wood and Tea Tree Essential Oil. see my dog gets a bath and how the soap ...
How to Make a Homemade Flea Trap MP3
Pets and fleas sometimes go hand in hand. See how to make a homemade flea trap that will kill the fleas & discover more tips for keeping your pets happy and ...
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ST.LOUIS PEST CONTROL owner Rick talks about what people can do to get rid of fleas in their yard. Contact US St.Louis Pest Control www.
How to get rid of fleas forever. Easy DIY flea / pest control. nylar IGR. Insect growth regulator. MP3
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Tips on how to kill fleas and ticks on your pets and in your house in this free online video series.
Does my dog have fleas? MP3
This video will show you what fleas look like and what flea dirt looks like. We have also shown the sores left on a dog after they have been bitten by the fleas and ...
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Getting rid of fleas on kitten with dawn dish soap.
How to Get Rid of Fleas, Gnats and Fruit Flies with Apple Cider Vinegar - SUPER FAST! MP3
Make a homemade trap to get rid of those nasty gnats and fruit flies. Make a simple cheap do-it-yourself gnat trap using a sugar shaker and apple cider vinegar.
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Andrew's Jigger Treatment, Part One Parasitic Fleas Removed in Remote Kenya MP3
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Cedar wood essential oil is a great natural flea repellent for your dog. Orange oil is an awesome tick repellant too. Frontline and Advantix are poisonous!


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So, You bought one of our Cardboard Virtual Reality Viewers?
Use the below images as a guide to assemble your Cardboard Virtual Reality Viewer.
Step Seven:
Place the two (2) plastic lenses into the openings in the center section of the tri-fold cardboard piece. You want to make sure the curved side of the lens is pointing towards your eyes. In the photo below the curved side of the lens is pointing up. You can always pop out the lenses later if you put them in incorrectly.
Step Eight:
Next, fold over the other section of the tri-fold piece over the installed plastic lenses. Using a paper clip or small binder clip can be helpful holding this section together until you need it. Once you have this section assembled, you can set this to the side and grab the other section of folded cardboard.
Step Nine:
This section of cardboard is the main body of the Virtual Reality Viewer. This might take a couple of times to get this correct but just take your time and you should have no problem assembling the viewer correctly. There are black markers on both of the cardboard sections to show what tabs go into which holes. The first step is to fold the far right flap in the photo below over until the #1 tab fits into the #1 slot.
Step Ten:
Using a binder clip on this part of the assembly is also helpful to hold each section together. You don't have to install the magnets on this step but you can see where one of the magnets will be placed in the circular cutout. In the below photo you can also see numbers 2 & 3 on this section of the VR viewer. These slots will be used in the next step when you install the section with the installed plastic lenses.
Step Eleven:
Now take the section you assembled in Step 8 and match the corresponding numbers up with each other. These two cardboard sections will probably not want to stay together just by themselves but that is no problem! Just keep them in place long enough to fold the other sections around the tri-fold lens section. Follow the next steps and you will have a Virtual Reality Viewer in just a few more minutes!
Step Twelve:
Next, continue folding over the outer section around the tri-fold lens section. Make sure the corresponding numbers match up on both cardboard sections like the photo below. A binder clip is handy in this section also. Notice the small section of cardboard to the right of this VR viewer below? This is an important part of the VR viewer.
Step Fifteen:
Next, install both magnets into your VR viewer. One magnet will go into the circular cut out shown in Step 10 and the other magnet will go on the other side of the cardboard side. Take note of the RED TAPE in the photo below. This is the double sided take that was included with your VR viewer. You want to place a piece or two on the flap shown below to ensure your VR viewer doesn't fall apart and you have to do this all over again!
Step Seventeen:
The center divider piece that you installed in Step 13 isn't held in place with anything. You really want to try to secure this with a piece of masking tape or something that will keep this piece from falling out. This piece is very important for the VR viewer to work correctly. I placed 4 small pieces of tape on all four sides of where it mounts into the viewer.
You are done with the assembly! It is almost time for the fun stuff!
Now take your phone and download the following apps:
Google Street View
Google Cardboard
Open the Google Street View App on your Smartphone.
It should look similar to the image below on your phone:
Search for a location you would like to view in the Cardboard VR Viewer (Red Arrow in image above).
Try Searching for Baxter Cycle in Google Street View.
Your search results will look similar to the image below:
Click on the image that comes up at the bottom of your screen (The Red Arrow in the above image).
This will bring up Baxter Cycle's Google Street View location.
Next, click on the Cardboard Icon located in the upper right corner of the below image (Red Arrow in image below).
This will change your phone screen into a Stereographic image (Split Screen) like the image below.
All you have to do now is carefully place your phone into the Cardboard Virtual Reality Viewer and have fun!
You can move to the arrows you see on the ground by flicking down the magnet on the side of the VR viewer.