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The Asphalt Jungle (1950)[DVDRip][big dad e™]
- Category Movies
- Type Divx/Xvid
- Language English
- Total size 1.4 GB
- Uploaded By BigDadE
- Downloads 904
- Last checked 8 minutes ago
- Date uploaded 9 years ago
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The Asphalt Jungle
Recently paroled from prison, legendary burglar "Doc" Riedenschneider, with funding from Alonzo Emmerich, a crooked lawyer, gathers a small group of veteran criminals together in the Midwest for a big jewel heist.
Infohash : 2ACEA8A934D1B973375D1CD36E9B9522D63D190F
The Asphalt Jungle (1950) B & W
Director: John Huston
John Huston was a filmmaker of quite exceptional ability. A versatile talent, he turned his hand to a wide range of subjects and concerns. Over the years his work has received massive critical and commercial approval. Among his cinematic triumphs are The Maltese Falcon, The African Queen and The Misfits. To this list can be justifiably added his 1950 thriller, The Asphalt Jungle.
The film concentrates on the (largely unsympathetic) characters involved in a large-scale diamond heist. Heading the cast is Sam Jaffe, playing a high class German jewel thief recently out of prison. He has an apparently fool-proof plan for a robbery. Five assorted individuals soon join forces with him to fulfil his dream. Nothing can go wrong, can it?
The Asphalt Jungle is a very well constructed film. Tension builds minute by minute. The characters (principal and secondary) are well drawn and strongly delineated. The ensemble acting is terrific. The superlative cast includes such stalwart character actors as Steriing Hayden, Loius Calhern and James Whitmore. The director uses the camera remarkably assuredly, creating a tense atmosphere which perfectly complements the nature of the plot. Watch out for a young Marlyn Monroe as Calhern's mistress!
In 2008, The Asphalt Jungle was selected for preservation in the United States National Film Registry by the Library of Congress as being "culturally, historically, or aesthetically significant".
Media Info:
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mbam-log-2011-02-03 (22-28-29).txt
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- The Asphalt Jungle (1950)[DVDRip][big_dad_e™].AVI (1.4 GB)
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Towngate sponsors local theatre group
29 August 2012
Stagecraft Productions have now returned from the Edinburgh Fringe Festival. Performer Katie Mahon sent us this lovely message to tell us how they got on:
Thank you so much to Towngate Fisheries for supporting and sponsoring a generous £200 to the local drama group I am apart of, in our journey to the Edinburgh Fringe Festival. We travelled up to Edinburgh in the first week of August and did a show every day for eight days while receiving a four star review from broadway baby. Thank You!:D
18 July 2012
Towngate Fisheries are really excited to be sponsoring local theatre group Stagecraft Productions. Their next production, 'Be My Baby', will be performed at the Edinburgh Fringe Festival in August.
It's 1964, and 'Be My Baby' follows the fortunes of Mary Adams, aged 19 and pregnant. Forcibly sent to a mother and baby home, Mary and her fellow inmates have to cope with the shame of their pregnancy and the dawning realisation they must give up their babies for adoption. Despite the daily battles with an overbearing matron, the girls' effervescence shines through. As they escape into the girl groups' songs they love, this bittersweet, touching and funny drama unfolds.
Stagecraft Productions are really looking forward to go to the Edinburgh Fringe Festival, but they still need more help with sponsorship. If you think you can help please call 07870 180165 or email firstname.lastname@example.com
For more information about 'Be My Baby' follow the production on Twitter.

subset: commoncrawl


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By far one of the hardest materials to procure. This is a quarter ounce denim. A Bandana that over time will just get better and better.
Care: Hand wash with cold water and hang dry. Material: Quarter-Ounce Denim / Approx Size: 16 in. x 16 in. All of our pieces are hand-cut, no two exactly the same (kinda like us).
Tips on how to wear your scarf
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Concessions used Juice cart mobile food cart for sale
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Want to know how much a juice cart price? Contact us now and we will sent you more detailed information on our carts.


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Marvel Studios Countdown: Will 'Loki: Agent of Asgard' Mean More Tom Hiddleston?
A few weeks ago we opined about the potential of a Loki solo movie, wondering if the people at Marvel felt the same way as Tom Hiddleston's many eager fans. Surprise -- they do! While not exactly a hard word on a Loki-led film, the groundwork is certainly being laid down by an all-new monthly Marvel comic titled Loki: Agent of Asgard. The trickster god will be the protagonist in the series, which reenvisions him as Asgard's ultimate protector while Thor plays superhero on Midgard (Earth).
For those wondering how a god of mischief can work in a more heroic light, writer Al Ewing told Marvel.com that he's not throwing out Loki's established characterization. "He's not told what to do exactly; he's given an objective and left to accomplish it in his own way," says Ewing. "There may be a larger plan at work that he's being asked to follow, but then Loki always has his own plans going on. And plans within those plans." In the 2014 series, Loki will answer to the All-Mother, and his first issue will see him in conflict with the core Avengers from the film (Captain America, Iron Man, Thor, Black Widow, Hulk and Hawkeye), obviously hoping to attract the film's fans into reading a monthly comic starring a character they seem to love. Ewing describes the move as "shameless, shameless, shameless."
They even acknowledge Hiddleston's popularity in his career-making role as creative inspiration for the new book. Ewing said he kept Hiddleston in mind while writing Loki, and artist Lee Garbett elaborated, "Tom Hiddleston's performance is so nuanced and attractively flawed it'd be crazy to not try and add that to the mix and I've definitely got his performance in mind when drawing him but at the same time I don't want it to be too restricted by likenesses, etc.; it's more the vibe I'm after. That magnetism and dark charm. Those are the touchstones. That and keeping him sexy." That's something that Tumblr will be glad to hear. The series will bow in early 2014.
Loki: Agent of Asgard was just one of Marvel's big Avengers-related announcements at New York Comic Con this year. Avengers World, a book featuring a globally focused Avengers team; Avengers Undercover, with the teen stars of Avengers Arena going deep cover in the villain group the Masters of Evil; and Secret Avengers, the covert-ops "secret agent" Avengers book, will all see new first issues in 2014. Black Widow will also be getting her own solo series in the upcoming year, with a focus on espionage over superheroism. That's a lot for Avengers fans to look forward to, and, if Guardians of the Galaxy is any indication, you never know when the next Marvel IP will end up the next Marvel film.
Catching Up with Agents of S.H.I.E.L.D.
I've seen a few lists this week on how to improve Marvel's Agents of S.H.I.E.L.D., and I have to say -- three episodes in and I'm far from hooked. All shows having growing pains, but since this was spinning off of a series of films, from experienced TV creators like the Whedon Brothers, I think we all expected can't-miss experience. Instead, S.H.I.E.L.D. is relatively low stakes, with precious, snarky dialogue and a cast of mercurial characters. Some less kind would call the cast of rookie agents cyphers. In the third episode, witheringly sarcastic Skye slows down some techno babble, citing that she's a high school drop out, yet she's also supposed to be a high-tech computer genius. Which is it? Couldn't they have had Skye ask to explain the science in layman's terms without citing herself as uneducated? Do we still wholly believe she's techno smart when she tells us she's ignorant? In the same episode, man-meat Agent Ward mumbles his character's defining moment as a youth (punished by an older brother for sneaking some birthday cake ) with all the conviction of a wet rag. The cake bit was weak and the delivery was openly reluctant of the writing. This kind of scripting undermines whatever character work they're trying to do here.
Clark Gregg's Coulson and Ming Na Wen's Agent May continue to be standouts simply through quiet strength and underplayed determination. I don't expect every character to behave like them, though I do want to see the other members of the ensemble perform with their conviction of character. Glibness seems to be the name of the game with S.H.I.E.L.D., so if that's what they're aiming for, then they've hit the bull's-eye. I'd prefer something that's fun without being weirdly dismissive of the fact that they're all supposed to be incredibly talented secret agents.
Tone down Skye's eye-rolling quips for every situation. Put the whole team in uniforms when they're on a mission (still unsure why Agent May is the only person who wears a S.H.I.E.L.D. uniform). I appreciate that there's a new family-friendly action series on television, but family friendly doesn't mean it can't have a little more punch. Right now, it's Marvel's version of TV's A-Team -- dopey, quick, one-off, low-rent adventures that see a ragtag unit of conflicting personalities dropping in to save the day, then loving it when their plan comes together. While there's still time for the show to grow and change, my frustration is that this is exactly what the show wants to be -- there's no reason to change. If that's the case, it's just not the show for me.
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First of all thanks for visiting MattCarstens.com. This site is where I am going to take on some independent reporting projects, integrate my thoughts into those projects and post stories I've written for other news outlets. Make sure to comment and enjoy!
As most of you probably know Mariner color commentator Mike Blowers was arrested a few months ago for a DUI in Peirce County. The reports came out nearly a month after the incident occurred with KING 5 getting the scoop.
The next day Geoff Baker did his due diligence with a little tidbit at the bottom of his Figgins to 3B story where the Mariners media relations staff told him they had no plans to punish Blowers and that he had learned his lesson.
About 20 days after that the M's were hit with another DUI case, this time non-roster invite Adam Kennedy. Kennedy, however didn't hide from the arrest.
Kennedy called nearly every Mariner beat reporter and gave them all individual interviews. He came surprisingly forward which I think a lot of people appreciated. That is the kind of behavior I would have expected from someone that is a long time part of the Mariner family, not a non-roster invitee.
Since there wasn't much looked into Blowers' case I thought I'd order the police report and find out some of the details.
Blowers was arrested Dec. 2 at 12:55 a.m. According to the police report he was traveling eastbound on SR 512.
"I observed a vehicle ahead of me (approximately 1/2 mile) traveling in lane two of three, suddenly began swerving into lane three then back into lane two," Officer Sherman J. Fox said in his police report. "As I closed the distance between his vehicle and mine, I observed the vehicle (I was now close enough to identify the vehicle as a black Chevy Tahoe) continue to swerve into lane three. As we approached the Golden Given underpass, I observed the Tahoe swerve almost completely onto the right shoulder (the driver's side wheels were on the fog line), and traveled there for approximately 1/2 mile before drifting back into lane one. At [12:57 a.m.], I activated my emergency lights and the Tahoe pulled over on the right shoulder at the Golden Given Underpass."
According to the pre-arrest observations Fox noticed Blowers had bloodshot, watery eyes with an obvious odor of intoxicants on his breath.
After admitting that he had been drinking, Blowers was placed under arrest 1:08 a.m. and was taken to the State Patrol office in Tacoma.
At the office Blowers called his lawyer and his lawyer told the police that he would not be answering to any more questions, but would submit to the blood alcohol content (BAC) test. Blowers was administered the two tests at 2:03 a.m., one registering at .140 and the other at .139. He was then picked up by a friend 40 minutes later.
According to Ellensburg Police Captain Dan Hansberry the typical range for BAC during a DUI arrest is between .08 and .16. As a quick reference here are the affects of a .13 to .15 BAC courtesy of Oklahoma State University:
"Gross motor impairment and lack of physical control. Blurred vision and major loss of balance. Euphoria is reduced and dysphoria."
As mentioned above Blowers has not issued a statement. The Mariners have just mentioned that he expressed regret.
Details on Mike Blowers DUI
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May check in:
Free $20 now, Free pound of silver later!
I'm in debt, I shouldn't be buying silver right? For most intents and purposes yes, BUT
1: I bought the silver intending to give it away at Christmas/birthdays/to extraordinary people.
2: It was FREE. Didn't cost me a dime because of all the cash back and interest I have earned from the Workthepig system. 99% of it is purely cash back this year, I didn't set up my Netspend suite until the last few months so $0.14 is interest from Netspend it will be $250 this time next year because I have five cards loaded with $1000 each in their respective savings accounts.
I have scheduled ACH deposits from my free Discover Bank Account to each Netspend card to waive the "inactivity fee" associated with each card. I do $25 per month so I know when the account says there is $75 on the Netspend card, interest has posted! It's not much, and you can withdraw $75 every 3 months back to your discover savings account so you don't have to keep messing with the money. Set it and watch it grow for you. You could transfer all of the interest out once per year and get $50 per year per account, $250 in my case. So next year I could buy ½ pound MORE SILVER because of the interest from the Netspend accounts. Assuming silver stays the same price, which it won't.
So let's take a look at the heavy hitters this year. According to Credit Karma, I have had my Capital One Journey Card for 1 year now I got it with a 486 credit score as a "rebuilder" that gets me 1.25% cash back! (NOTE: 1.25% doesn't seem like much, but thats an extra $250 for every $1000 in cash back compared to a 1% card.)
Here is a review of my cash back this last year:
Here is a review of my cash back this last year:
April 2016 I had a 486 credit score. I had failed to pay my student loans to the tune of $14,000 and because I didn't pay them, the interest compounded until I owed $20,000. Double Whammy. AVOID AT ALL COSTS, This applies for CREDIT CARDS TOO: you end up PAYING INTEREST ON your INTEREST. It's the worst.
So I was determined to get back on track:
I had a $1500 limit on my Capital one Journey card so I spent less than $150 each month and paid off my balance. By staying/utilizing under 10% of my credit limit (Called "Credit Utilization") I got the BIGGEST CREDIT SCORE BOOST that you can get. Under 30% is ok for your credit, under 20% is better and under 10% is best.
After paying my bill on time for 4 months with low utilization I applied for my next two cards on the same day. Literally back to back.
I did this because it will only show up as 1 hard pull on your credit score (lots of hard pulls are bad, applying two cards at a time saves you HALF [50%] of the hard pulls on your credit.)
Those cards were the American Express Blue Cash Everyday card that gets me 3% cash back at grocery stores and the Citi Double Cash card that gets me 2% back anywhere. My Citi card is a Mastercard, but I am not sure if they are all mastercards.
All three of these cards have $0 annual fee AND get cash back, meaning I can sock drawer them if I end up not needing them anymore and use them once a year. This is different from a card with a $75 annual fee. Because you have to pay to own it every year, and if you stop paying, your average credit age will go down, which is bad.
So total cash back for April 2016-April 2017
Capital One Journey card: $63.08
Citi Double Cash card: $158.06
American Express Blue Cash Everyday Card: $142.43
Plenti: $53
Ibotta: $54.75
Netspend Interest: $0.14* (One day's interest on one account before the quarter closed)
Total FEES: $0.00
Total Interest PAID on the Credit Cards $0.00
Total FREE MONEY: $471.46
But you don't have to buy silver, you can buy whatever you want. Rent? Diamonds? A fine suit? Groceries? Pay off debt? It's your money. YOU DECIDE.
You work hard to fill your piggy bank's belly, it should work just as hard for you.
MoneyFarmer.org Growing Money Farmers since 2017!
CLICK HERE, JOIN US (there's $20 in it for you now, and...just look at all this silver 1 year later!)Stop paying full price for life! You work hard to fill your piggy bank's belly, it should work just as hard for you.
!!!!!UPDATE!!!!! Since posting this I have elected to pay for the silver in cash and use the cash back combined with the interest from my high interest savings accounts towards a 5 day trip to Puerto Rico! It was the most amazing experience that I have ever had, HANDS DOWN!
It's not like I returned the silver and bought it again with cash. The cash back is in my account, so if I have accrued $500 in cash back then something worth $500 would be free to me. It doesn't mater what was free, it's that you got $500+ in value back from the spending and saving you are already doing anyways.


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Don't miss out on future posts like this – you can receive updates directly to your inbox by email by adding your email address to the box on the top right of this page and hitting subscribe. You can also follow NorthHantsMum onTwitter, Facebook Profile, LinkedIn and Feedly. I hope to see you there!
These classes look so much fun! I just wish I didn't work Monday to Thursday otherwise I would totally be taking up Shelley's offer for NHM Readers! See further down the post for the exclusive offer for NHM Readers.
My name is Shelley and I run Gym-Minis (www.gym-minis.co.uk) which holds fun balancing, climbing and agility sessions for children from crawling to 5 years all over North Hampshire and has done since 1992 (gosh that's a long time) !
We currently run classes during school term time in Basingstoke, Hook, Crondall and Alton. There are three classes each morning to cater for children from 'crawling' to 5 year old, each class lasts approximately 45 minutes.
The classes begin and end with a song or rhyme and the children move around the equipment in small groups forming 'trains' to move from one station to the next. At each station they encounter a new challenge, learn new skills and will have the opportunity to explore balancing, climbing and agility using our colourful and safe equipment. The 'floor equipment' section helps with co-ordination skills and introduces them to simple games.
All of the classes encompass tasks to encourage Agility, Climbing and Balance, Co-ordination and Self-confidence
The equipment used allows your child to experience all manner of actions, such as rolling, climbing, balancing, jumping and a variety of floor exercises. The layout is altered regularly to provide new challenges.
Over the years thousands of children have enjoyed our fun filled classes helping them to develop lifeskills, agility, social skills and confidence. If you have a look at our facebook page we have got lots of lovely testimonials from our past and present Mum's and some photos of the action at the classes! http://www.facebook.com/pages/Gym-Minis/439707346074833#!/pages/Gym-Minis/439707346074833
We run classes on the following days/locations:
Mondays – Chineham Village Hall, Thornhill Way, Chineham, Basingstoke, Hants, RG24 8YE
Tuesdays – Hook Community Centre, Ravenscroft, Hook, Hants, RG27 9NN
Wednesdays – Four Marks Village Hall, Lymington Bottom, Four Marks, Nr. Alton, GU34 5AA
Thursdays – Crondall Village Hall, Croft Lane, Crondall, Nr Farnham, GU10 5QG
We have two classes each morning with the equipment arranged to suit the age groups:
- Early years' (crawling to 2 yrs) class at 9.40 am
- 2 to 3 year class at 10.30 am
Each class is set up for specific age groups and the child's parent is expected to work with their child until the child is three years old.
- The 'early years' class which caters for children from crawling to 2 yrs allows the children to explore the equipment in an informal session and includes group rhymes and songs.
- The 2 to 3 year class is a little more structured and the tasks a little more adventurous. The children work and play in small groups (max 6) with their parent and a trainer to help and assist.
We also offer wonderful exclusive children's birthday parties during the school holidays or at weekends where we will come out to a location of your choice and setup the equipment for the party for your child. Please let me know if you would like any further details on these.
So why not come and give us a try…we guarantee they (and you) will enjoy it and make lots of new friends! We would like to offer all northhantsmum readers their 1st session with us completely FREE (you must be a new mum that has not been to Gym-Minis before, sorry)!
Spaces for all classes are limited and we do have waiting lists for some of them so please get in touch ASAP if you would your child to be put down (even if they are too little at the moment we can put them down for future ones).
Please feel free to email or call me with any enquiries:
01420 562461
or visit www.gym-minis.co.uk for further information on us.
I look forward to hopefully seeing some of you soon 🙂
Shelley x


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The yellow fever mosquito, Aedes aegypti, vectors disease-causing agents that adversely affect human health, most notably the viruses causing dengue and yellow fever. The efficacy of current mosquito control programs is challenged by the emergence of insecticide-resistant mosquito populations, suggesting an urgent need for the development of chemical insecticides with new mechanisms of action. One recently identified potential insecticide target is the A. aegypti D1-like dopamine receptor, AaDOP2. The focus of the present study was to evaluate AaDOP2 antagonism both in vitro and in vivo using assay technologies with increased throughput. The in vitro assays revealed AaDOP2 antagonism by four distinct chemical scaffolds from tricyclic antidepressant or antipsychotic chemical classes, and elucidated several structure-activity relationship trends that contributed to enhanced antagonist potency, including lipophilicity, halide substitution on the tricyclic core, and conformational rigidity. Six compounds displayed previously unparalleled potency for in vitro AaDOP2 antagonism, and among these, asenapine, methiothepin, and cis-(Z)-flupenthixol displayed subnanomolar IC50 values and caused rapid toxicity to A. aegypti larvae and/or adults in vivo. Our study revealed a significant correlation between in vitro potency for AaDOP2 antagonism and in vivo toxicity, suggesting viability of AaDOP2 as an insecticidal target. Taken together, this study expanded the repertoire of known AaDOP2 antagonists, enhanced our understanding of AaDOP2 pharmacology, provided further support for rational targeting of AaDOP2, and demonstrated the utility of efficiency-enhancing in vitro and in vivo assay technologies within our genome-to-lead pipeline for the discovery of next-generation insecticides.
Mosquitoes transmit pathogens and parasites that cause diseases that adversely affect human health worldwide, including malaria, yellow fever, and dengue. Existing approaches for mosquito control have demonstrated efficacy in reducing incidences of such diseases, but are becoming inadequate due to the emergence of insecticide-resistant mosquito populations (Hemingway and Ranson, 2000; Hemingway, 2014). The need for novel mode-of-action compounds to control mosquitoes is further emphasized by the fact that it has been several decades since a new public health insecticide has been deployed to reduce the spread of vector-borne diseases (Hemingway et al., 2006).
Arthropod G protein–coupled receptors (GPCRs) mediate critical biologic processes (Hauser et al., 2006) and have emerged as potential insecticide targets (Hill et al., 2013). Molecular approaches, including genome sequencing efforts, have identified more than 100 GPCRs within the genomes of several arthropod vector species (Hill et al., 2002; Nene et al., 2007; Arensburger et al., 2010; Kirkness et al., 2010). Among the GPCR superfamily, the biogenic amine receptors are of particular interest because of their crucial roles in insect physiology and behavior (Hauser et al., 2006; Fuchs et al., 2014). For example, the biogenic amine dopamine and its receptors are implicated in a variety of arthropod behaviors, including arousal (Kume et al., 2005), locomotion (Yellman et al., 1997; Draper et al., 2007; Mustard et al., 2010), and olfactory learning (Kim et al., 2007; Riemensperger et al., 2011). It is also notable that dopamine is associated with the salivary function of vectors (Ali, 1997; Sauer et al., 2000; Šimo et al., 2011, 2014), suggesting potential roles for the mediation of pathogen acquisition and transmission during blood feeding. In Aedes aegypti, dopamine is also implicated in sclerotization and ovarian/egg development, as increased dopamine levels were observed in newly emerged adults and following a blood meal (Andersen et al., 2006). The central roles of dopamine systems in fundamental biologic processes offer the dopamine receptors as potential insecticide targets.
A recent study from our invertebrate receptor group supports the pursuit of D1-like dopamine receptors (AaDOP1 and AaDOP2) from the yellow fever mosquito, A. aegypti, as targets for novel mode-of-action insecticides (Meyer et al., 2012). Specifically, AaDOP2 was used as a prototypical target for a "genome-to-lead" approach for the discovery of target-based insecticides, where genomic sequence data were used to drive in vitro functional characterization of recombinant AaDOP receptors in human embryonic kidney (HEK) 293 cells (Meyer et al., 2012). Following pharmacologic characterization, high-throughput screening (HTS)–amenable evaluation of pharmacologically active compounds identified AaDOP2 antagonists that display significant in vivo toxicity to mosquito larvae (Meyer et al., 2012), supporting the validity of targeting AaDOP2 for A. aegypti control.
The present study entailed a robust follow-up pharmacologic analysis of AaDOP2 antagonists identified in a small-molecule screen of the LOPAC1280 library (Meyer et al., 2012). To accomplish this, we developed an HTS-amenable cell-based assay that enabled an in-depth study of AaDOP2 antagonism by tricyclic antidepressants and structurally related compounds. Several of these compounds demonstrated enhanced potency for in vitro AaDOP2 antagonism and greater efficacy for larval death in mosquito bioassays. Importantly, we provided evidence that several AaDOP2 antagonists caused toxicity in adult A. aegypti. Furthermore, we improved upon our previously described genome-to-lead pipeline via implementation of efficiency-enhancing in vivo assay technologies.
Materials and Methods
Cis-(Z)-flupenthixol, clozapine, mianserin, nortriptyline, imipramine, protriptyline, norclomipramine, pirenperone, desipramine, haloperidol, trazodone, fluoxetine, fluvoxamine, buspirone, (+)-butaclamol, amoxapine, amitriptyline, chlorpromazine, doxepin, loratadine, ketotifen, chlorprothixene, loxapine, cyproheptadine, asenapine, diphenhydramine, ritanserin, ketanserin, risperidone, 3-isobutyl-1-methylxanthine, G418, and Dulbecco's modified Eagle's medium were purchased from Sigma-Aldrich (St. Louis, MO). Amperozide, methiothepin, clomipramine, SCH-23390 [(R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine], LY-310,762 (1-[2-[4-(4-fluorobenzoyl)-1-piperidinyl]ethyl]-1,3-dihydro-3,3-dimethyl-2H-indol-2-one), R59-022 (6-[2-[4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl]ethyl]-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one), and tomoxetine were purchased from Tocris Bioscience (Ellisville, MO). Benztropine was purchased from Enzo Life Sciences (Farmingdale, NY). The antibiotic-antimycotic 100× solution was purchased from Life Technologies (Grand Island, NY). FetalClone I serum, bovine calf serum, HEPES, and Hanks' balanced salt solution were purchased from Hyclone (Logan, UT). The homogenous time-resolved fluorescence (HTRF) cAMP kit was purchased from Cisbio Bioassays (Bedford, MA).
Cisbio HTRF cAMP Dynamic 2 Cell-Based Assay
HEK293 cells stably expressing AaDOP2 (HEK-AaDOP2) or the human D1 dopamine receptor (HEK-hD1) were maintained and cryogenically frozen as previously described (Meyer et al., 2012). To prepare for pharmacologic analysis, cells were thawed and resuspended in assay buffer (Hanks' balanced salt solution, 20 mM HEPES, 0.1% fatty acid–free bovine serum albumin). To remove cryogenic freezing media, cell suspensions were centrifuged at 500g for 5 minutes, followed by aspiration of the supernatant. Cell pellets were resuspended in assay buffer and seeded into 384-well plates (CulturPlate-384; PerkinElmer, Waltham, MA) at 2000–2500 cells per well and incubated at 37°C and 5% CO2 for 1 hour. Test compounds were added using a 384-well pin tool (V&P Scientific, San Diego, CA). A MultiFlo (BioTek, Winooski, VT) low-volume bulk reagent dispenser was used to dispense 3 μM dopamine (in assay buffer containing 500 μM 3-isobutyl-1-methylxanthine and 0.02% ascorbic acid) to activate AaDOP2. Drug stimulation was carried out at room temperature for 1 hour. Cells were lysed by sequential addition of cAMP-d2 and anti-cAMP cryptate conjugate, both diluted 1:39 in lysis buffer, and were incubated at room temperature for 1 hour. Time-resolved fluorescence resonance energy transfer was measured with a lag time of 100 microseconds and integration time of 300 microseconds using a Synergy4 (BioTek) fluorescence plate reader with a 330/80-nm excitation filter and emission filters of 620/10 and 665/8 nm. Sensitivity parameters were set by reading the cAMP standard curve using the autosensitivity setting. All experimental conditions were read using sensitivity settings obtained for the cAMP standard curve. Cellular cAMP concentrations were estimated in GraphPad Prism (GraphPad Software, La Jolla, CA) by applying the 620/665-nm fluorescence ratio values to a standard curve of known cAMP concentrations.
Cyclic AMP measurements in HEK293 cells stably expressing the human D1 dopamine receptor were performed as described earlier, but 500 nM dopamine was used to stimulate cAMP accumulation.
In Vivo A. aegypti Larval Screen.
Test compounds were evaluated for in vivo toxicity in bioassays against L3 stage A. aegypti larvae in a double-blind manner. In brief, compounds were resuspended in water and added to wells of a 24-well plate (BD Bioscience, San Jose, CA) in duplicate, with each well containing five A. aegypti larvae in 1-ml total volume to achieve a final concentration of 400 μM per well (see Supplemental Fig. 1 for illustrations of the assay format). Plates were incubated at 22°C, and the assay was scored for larval mortality at 24, 48, and 72 hours. Larvae unresponsive to gentle tapping of the plate or touch with a sterile probe were scored as dead.
A. aegypti Adult Concentration-Response Curves.
Test compounds were dissolved in deionized water to a 200 mM stock concentration and serially diluted in Aedes saline (Hayes, 1953) to achieve a dose range of 0.25–20 mM. Four-day-old A. aegypti adult females [average wing length of 3.4 mm, measured as described by Briegel (1990)] were anesthetized on ice, and groups of 20 females were injected with the indicated amounts of test compounds (0.5 μl per mosquito) or Aedes saline alone (control) using a pulled glass capillary needle. Additional uninjected mosquito controls were also included. Mosquitoes were housed in 10-cm diameter × 20-cm height paper coffee cup cages with lace screens (secured with rubber bands) and maintained at 75% humidity with 10% sucrose provided ad libitum via a cotton wick (see Supplemental Fig. 2 for illustrations of injections and mosquito housing). Observations of mortality were made daily for up to 4 days post-treatment. Mosquitoes were scored as "dead" if no movement was observed and confirmed by no response to a gentle touch of the legs with a metal probe. When observed at any time point, moribund adult mosquitoes (i.e., insects incapable of standing, walking, or flying) were scored as dead. At the 24-hour time point, and to a lesser extent at the 48-hour time point, we observed a percentage of the adult mosquito population that was moribund. These mosquitoes did not recover and died by assay endpoint. The moribund phenotype was negligible at 96 hours (less than 1% of the adult population for any replicate dose). LD50 values for test compounds injected into adult mosquitoes were calculated by nonlinear regression using the sigmoidal dose-response equation in the GraphPad Prism software.
In Vitro Evaluation of AaDOP2 Antagonism.
Our previous studies indicated potential value in pursuing AaDOP2 in a target-first approach for developing new insecticides against A. aegypti (Meyer et al., 2012). We also demonstrated the success of utilizing a heterologous cell model, where recombinant AaDOP2 receptors are expressed in HEK293 cells (HEK-AaDOP2) for identification and pharmacologic evaluation of novel AaDOP2 ligands (Meyer et al., 2012). To improve upon our genome-to-lead pipeline for novel insecticide discovery, HEK-AaDOP2 cells were used to develop a cell-based assay that enabled rapid and efficient study of receptor antagonists. The Cisbio HTRF cAMP Dynamic 2 detection methodology was chosen as the assay platform, allowing for the direct detection of cAMP in a 384-well format, and initial experiments were focused on validating cAMP responses to dopamine stimulation using this assay format. As AaDOP2 is a Gαs-coupled D1-like dopamine receptor, stimulation with dopamine results in an enhanced level of cAMP (Meyer et al., 2012). As expected, dopamine treatment displayed a concentration-dependent enhancement of cAMP accumulation with an EC50 of 950 ± 190 nM (n = 5). The EC50 of dopamine was similar to that determined in the previous [3H]cAMP-based quantification method (Meyer et al., 2012). Furthermore, the potency of amitriptyline (the prototypical mosquito-toxic AaDOP2 antagonist) for inhibition of dopamine-stimulated cAMP in the HEK-AaDOP2 cells was similar to that previously reported (Tables 1 and 2) (Meyer et al., 2012; Hill et al., 2013), demonstrating suitability of the HTRF cAMP detection technology for high-throughput cell-based pharmacologic studies of AaDOP2.
Our previous screen of the LOPAC1280 library identified 51 active compounds as AaDOP2 antagonists, including several tricyclic antidepressants (Meyer et al., 2012). Importantly, two tricyclic antidepressant compounds, amitriptyline and doxepin, cause significant mortality of mosquito larvae in whole-organism bioassays, suggesting the potential of tricyclic antidepressants as insecticide lead compounds (Meyer et al., 2012). To gain an understanding of the chemical features that are important for AaDOP2 antagonist activity, pharmacologic evaluation of additional small molecules that are structurally related to the tricyclic leads was carried out in the cell-based assay described earlier. Specifically, nine tricyclic antidepressants and five antidepressant compounds lacking a tricyclic core were studied for their ability to antagonize the cAMP accumulation in response to dopamine treatment (3 μM) in HEK-AaDOP2 cells. All nine tricyclic compounds displayed concentration-dependent antagonist activity against AaDOP2, with IC50 values less than 3 μM, whereas compounds representing other classes of antidepressants displayed less than 10% inhibition at 3 μM (Fig. 1; Table 1).
To identify novel AaDOP2 antagonists with chemical structures distinct from the tricyclic antidepressant ring scaffold, we evaluated concentration-dependent effects of a suite of additional active compounds identified in our previous small-molecule screen (Meyer et al., 2012) together with structurally related compounds, enabling an initial in vitro structure-activity relationship (SAR) analysis. As performed earlier, test compounds were studied for their ability to modulate dopamine-stimulated (3 µM) cAMP accumulation in HEK-AaDOP2 cells (Table 2). Interestingly, six compounds were more potent antagonists than the prototypical AaDOP2 antagonist amitriptyline (Table 2). Furthermore, asenapine, methiothepin, and cis-(Z)-flupenthixol displayed subnanomolar IC50 values for inhibition of dopamine-stimulated cAMP in HEK-AaDOP2 cells (Table 2).
Pharmacologic selectivity for the targeted insect over humans and other animals is a critical attribute of potential insecticides. To address this concern, several of the most potent AaDOP2 antagonists were evaluated for antagonist activity in HEK293 cells stably expressing the human D1 dopamine receptor (HEK-hD1 cells) and compared with the hD1 antagonist, SCH-23390. Each compound inhibited 500 nM dopamine-stimulated cAMP in the HEK-hD1 cells and displayed IC50 values between 19 and 13,000 nM (Table 3). However, in contrast to the hD1-selective antagonist SCH-23390, all of these compounds were more potent antagonists of AaDOP2 than hD1, suggesting potential species-selective pharmacologic profiles for these compounds.
In Vivo Toxicity of AaDOP2 Antagonists: Effects on A. aegypti Larvae.
An important second step in our insecticide discovery effort was the evaluation of the in vivo activity of compounds identified and characterized in the cell-based in vitro studies. We developed an A. aegypti larval screen that can be performed in a 24-well plate format, allowing rapid assessment of in vivo toxicity for compounds identified as potent antagonists in the in vitro studies. This assay was designed to also enable evaluation of speed-to-kill and support prioritization of compounds for further study. Twenty-five compounds were tested using this approach (Table 4), and 10 compounds [asenapine, chlorpromazine, benztropine, methiothepin, cis-(Z)-flupenthixol, chlorprothixene, loxapine, mianserin, amperozide, and clomipramine) caused 70–100% larval mortality within 24 hours post-treatment. These compounds were faster-acting and caused greater mortality of mosquito larvae at the 24-hour treatment time point than our previously identified lead compound for insecticide development, amitriptyline. Notably, asenapine, chlorpromazine, and amperozide caused greater than 70% mortality of the mosquito population within 30 minutes, and cis-(Z)-flupenthixol, chlorprothixene, mianserin, loxapine, and methiothepin caused greater than 70% mortality within 3 hours (data not shown). We also identified five compounds with moderate mosquito toxicity (i.e., 40–70% mortality at 24 hours postexposure) and nine compounds with limited or no toxicity to mosquito larvae (i.e., 0–40% mortality at 24 hours) (Table 4). The in vivo larval mortality data show a significant correlation with in vitro potency values for antagonism of dopamine-stimulated cAMP in HEK-AaDOP2 cells (r = −0.770, n = 25, P > 0.0001; Fig. 2), providing an important line of evidence that AaDOP2 antagonism is linked to larval toxicity.
In Vivo Toxicity of AaDOP2 Antagonists: Effects on Adult A. aegypti.
Toxicity to adult female A. aegypti is considered an important property of any lead molecule because only adult female mosquitoes are responsible for the transmission of disease-causing agents. Therefore, we developed an adult A. aegypti assay to evaluate the effects of AaDOP2 antagonists following introduction to the insect hemocoel via microinjection. Four of the most potent in vitro and/or most efficacious compounds in the larval bioassay were assessed for toxicity (LD50) and speed-to-kill in adult mosquito bioassays (Fig. 3; Table 5). All compounds tested caused dose-dependent toxicity to adult A. aegypti and were capable of providing 100% mortality at all time points, whereas <6% mortality was observed for the saline-injected and uninjected controls throughout the 96-hour experiments. Cis-(Z)-flupenthixol was the most potent compound, having an LD50 of 1.26 nmol/mosquito following 24-hour exposure (Fig. 3). Chlorpromazine and cis-(Z)-flupenthixol became more potent over the course of the 4-day assay, as LD50 values decreased by ∼2- to 3-fold from the 24- to 96-hour time points for these compounds. In contrast, the LD50 values for amitriptyline and amperozide remained relatively stable over the same treatment duration, suggesting that these compounds reach their maximum potency earlier than chlorpromazine and cis-(Z)-flupenthixol (Table 5).
The active ingredients of the major existing neurotoxic classes of insecticides target acetylcholinesterases (organophosphates and carbamates), GABA receptors (organochlorines), and sodium channels (pyrethroids) within insect nervous systems (Hemingway and Ranson, 2000). Continued efficacy of modern commercial insecticides is threatened by the development of insect populations that are resistant to these chemicals (Hemingway and Ranson, 2000), emphasizing the urgency of developing insecticides with new modes of action. Our recent study identified the dopamine receptors of A. aegypti as potential targets for yellow fever mosquito control (Meyer et al., 2012). Specifically, larval toxicity was observed for two tricyclic antidepressant compounds (amitriptyline and doxepin) that display AaDOP2 antagonism (Meyer et al., 2012).
To better understand the chemical basis of A. aegypti toxicity observed in vivo, compounds with activity profiles similar to amitriptyline and doxepin at human targets (i.e., GPCRs and/or biogenic amine transporters) were evaluated for in vitro AaDOP2 modulation and in vivo efficacy in larval bioassays. Several known GPCR-targeting ligands, including tricyclic antidepressants and antipsychotics, demonstrated potent AaDOP2 antagonism and insecticide activity. However, compounds from other antidepressant classes (e.g., selective serotonin reuptake inhibitors and selective norepinephrine reuptake inhibitors) were largely inactive, suggesting GPCR modulation, rather than biogenic amine transporters, as a contributing mechanism for the observed larval toxicity. Further validating AaDOP2 as a viable insecticide target, our data revealed a significant correlation between the in vitro potency of AaDOP2 antagonists and the toxicity of these compounds to mosquito larvae in vivo (Fig. 2). However, it should be noted that benztropine and amperozide, which caused rapid and high larval mortality (Table 4), had somewhat moderate in vitro potency at AaDOP2 (IC50 values of 340 ± 41 and 570 ± 110 nM, respectively). Amperozide and benztropine interact with several different mammalian GPCR families (U'Prichard et al., 1977; Kanba and Richelson, 1984; Bolden et al., 1992; Arnt and Skarsfeldt, 1998), suggesting the possibility that modulation of additional A. aegypti GPCRs could contribute to the in vivo toxicity of these compounds. Alternatively, such differences between the in vitro potency and the magnitude of in vivo toxicity for a given compound may reflect complex factors that impact in vivo insecticidal activity, including differences in the physicochemical properties of compounds that affect absorption through the insect cuticle, dissemination through insect tissues to the target site, and detoxification by insect gut and hemolymph enzymes. Nonetheless, the correlation between the in vitro potencies for AaDOP2 antagonism and larval toxicities suggests that optimizing compounds for potency and selectivity in vitro may be an efficient way to identify and prioritize new lead compounds.
The in vitro evaluation of the chosen compounds for modulation of AaDOP2 provided preliminary insight into the relationship between chemical structure and the potency of AaDOP2 antagonism. One SAR trend suggests that conformational rigidity contributes to the potency of AaDOP2 antagonists. For example, compounds with 6- or 7-membered central rings were generally the most potent AaDOP2 antagonists (Supplemental Fig. 3; Tables 1 and 2). However, the moderate potency of R59-022, risperidone, benztropine, and amperozide (IC50 values ranging from 53 to 570 nM) indicates that the central ring is not essential for antagonist activity. Benztropine was ∼22-fold more potent than diphenhydramine, suggesting that conformational control of the amine moiety also contributes to the potency of compounds with no central ring (Fig. 4A). Another SAR trend suggested that greater lipophilicity may enhance AaDOP2 antagonist potency, as was observed by the ∼3-fold greater potency of amitriptyline over doxepin (Fig. 4B). Furthermore, ligands with tertiary amines (clomipramine, imipramine, amitriptyline, and loxapine) were ∼5- to 100-fold more potent than the secondary amine analogs of these compounds (norclomipramine, desipramine, nortiptyline, and amoxapine, respectively), demonstrating the influence of amine state on AaDOP2 antagonist potency (Fig. 4C; Supplemental Fig. 3). Also, clomipramine and norclomipramine displayed ∼5- to 6-fold more potent IC50 values than imipramine and desipramine, respectively, suggesting that halide substituents of aromatic rings within the tricyclic core can increase the potency of the identified antagonists (Fig. 4C). Enhancements in AaDOP2 antagonist potency were also apparent when considering combinations of chemical properties, such as lipophilicity and halide substitution (Fig. 4C, clomipramine versus desipramine) or lipophilicity and conformational rigidity (Fig. 4D). The chemical scaffolds identified earlier and their key structural features that contribute to AaDOP2 antagonism may be used to guide further lead optimization studies.
The in vitro and in vivo data presented here support the hypothesis that targeting GPCR-mediated processes is a viable strategy for identifying insecticidal compounds. However, a major challenge associated with this approach is the development of ligands that are selective for disruption of biologic activity in A. aegypti but not in humans or other higher eukaryotes. To date, all reports indicate that compounds that display both AaDOP2 antagonism and in vivo efficacy are also known to have biologic activity in humans. Our studies identified compounds that are highly selective for targeting AaDOP2 receptors over the human D1 dopamine receptor (Table 3), but antipsychotics and tricyclic antidepressants potently interact with other families of mammalian GPCRs, including histamine, serotonin, adrenergic, and muscarinic receptors (Cusack et al., 1994; von Coburg et al., 2009). The development of ligands that selectively target biologic activity in A. aegypti over humans and other animals could potentially be addressed by using cell-based in vitro assays to screen against panels of human GPCRs. Also, virtual or in silico screening methods are emerging as promising approaches for the study of GPCR modulators (Shoichet and Kobilka, 2012). Such computational methodologies for lead optimization of antipsychotics and tricyclic antidepressants are strengthened by recently reported human GPCR crystal structures from histamine (Shimamura et al., 2011), serotonin (Wang et al., 2013), dopamine (Chien et al., 2010), adrenergic (Rasmussen et al., 2007; Warne et al., 2008), and muscarinic (Haga et al., 2012; Kruse et al., 2012) receptor families. The combination of these in vitro and in silico approaches is expected to provide insight into the molecular determinants of selectivity for AaDOP2 versus human GPCRs, and may ultimately produce mosquito GPCR-selective small molecules.
Pharmacologic selectivity considerations are multifold, as ligand selectivity for AaDOP2 receptors over nontarget insects (e.g., honeybees), in addition to selectivity over human GPCRs, is also paramount. Pharmacologic screening panels can be assembled for invertebrate targets to better understand ligand pharmacology at these receptors. For example, cross-species comparative pharmacologic studies of invertebrate dopamine receptor modulation can be expanded to include GPCRs from nontarget insects. Furthermore, upon genome mining and cloning of additional biogenic amine receptors (in addition to AaDOP1 and AaDOP2), AaDOP2 antagonists can be screened for modulation of other A. aegypti GPCRs, including muscarinic acetylcholine, serotonin, and octopamine/tyramine receptors (Nene et al., 2007). These pharmacologic efforts are expected to provide a deeper understanding of small-molecule modulation of invertebrate GPCRs, and may ultimately allow for target-based pesticide discovery efforts related to other pest arthropods.
Here we report significant advancements and modifications to our genome-to-lead insecticide discovery pipeline (Meyer et al., 2012). Incorporation of an in vitro HTRF assay enabled efficient in vitro pharmacologic evaluation and SAR profiling, while offering several advantages over the previously used CRE-mediated luciferase reporter assay for HTS. Direct measurement of cAMP eliminates false positives associated with CRE-luciferase reporter assays that include cAMP/protein kinase A–independent modulation of CRE-mediated transcription (George et al., 1997; Hill et al., 2001) or direct modulation of luciferase (Thorne et al., 2010). Furthermore, the HTRF screening platform was robust enough to support future HTS of small molecules for AaDOP2 antagonist activity in a 384-well format in singlet (i.e., Z′ > 0.5; J. Conley, C. Hill, and V. Watts, unpublished observations), enabling sufficient throughput to carry out the in vitro pharmacologic profiling proposed earlier. The HTRF screening platform also provides flexibility, as it can be used to detect modulation of additional downstream GPCR signaling pathways, including extracellular signal-regulated kinase 1/2 and Ca2+/inositol trisphosphate (Degorce et al., 2009). Other improvements to our established insecticide discovery pipeline for small-molecule modulators of AaDOP2 (Meyer et al., 2012) include the enhanced-throughput larval mosquito bioassay (Table 4) to rapidly assess larval toxicity and the utilization of an injection assay to evaluate toxicity in adult A. aegypti.
In addition to the antipsychotic and tricyclic antidepressant lead optimization and GPCR profiling studies suggested earlier, HTS of diverse small-molecule libraries for the identification of AaDOP2 modulators with novel chemical scaffolds may also be a fruitful endeavor. Especially enticing is the possibility of screening for allosteric modulators of AaDOP2 receptors, as drug discovery campaigns targeting multiple human GPCRs have identified allosteric modulators with unmatched specificity and selectivity (Conn et al., 2009; Wootten et al., 2013). Allosteric modulators are attractive because the orthosteric sites (i.e., the sites of endogenous ligand binding) are largely conserved between the human D1 and AaDOP2, but our published studies suggest that there are opportunities to exploit allosteric sites in the intracellular and extracellular loops where sequence similarity between species is reduced (Meyer et al., 2012; Hill et al., 2013). Our emerging understanding of the chemical basis of AaDOP2 receptor antagonism, together with advancements in assay throughput, suggest that the diverse molecular approaches described earlier can be combined to expedite the discovery of novel ligands that selectively modulate GPCRs of target insects.
The present study describes the in vitro pharmacologic characterization and in vivo efficacy of several AaDOP2 antagonists and demonstrates improvements upon our "genome-to-lead" pipeline (Meyer et al., 2012). Specifically, we report the characterization of compounds with unparalleled in vitro potency for AaDOP2 inhibition and improved efficacy for A. aegypti larval toxicity, and demonstrate toxicity of these compounds to adult mosquitoes. Collectively, our findings provided a major advancement in the development of invertebrate GPCR-targeting technology for novel mode-of-action insecticides.
The authors thank Dr. Eric Barker (Purdue University) and Dr. David Nichols (Purdue University) for generously providing venlafaxine and aripiprazole, respectively. The authors also thank Isabelle Verona Brust for assistance with figure preparation.
Authorship Contributions
Participated in research design: Conley, Hill, Meyer, Nuss, Watts.
Conducted experiments: Conley, Doyle, Meyer, Nuss.
Contributed new reagents or analytic tools: Hill, Watts.
Performed data analysis: Conley, Doyle, Hill, Meyer, Nuss, Savinov, Watts.
Wrote or contributed to the writing of the manuscript: Conley, Hill, Meyer, Nuss, Savinov, Watts.
- Received September 4, 2014.
- Accepted October 17, 2014.
This work was supported by a US Department of Defense, Deployed War Fighter Project award [Grant W911QY]; and a Purdue Research Foundation Trask Innovation award (to C.A.H. and V.J.W.). Additional support was supplied by the Indiana Clinical and Translational Sciences Institute funded, in part, by the National Institutes of Health National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award [Grant UL1-TR001108].
↵This article has supplemental material available at jpet.aspetjournals.org.
- G protein–coupled receptor
- human embryonic kidney
- HEK-AaDOP2
- HEK293 cells stably expressing AaDOP2
- HEK-hD1
- HEK293 cells stably expressing the human D1 dopamine receptor
- homogenous time-resolved fluorescence
- high-throughput screening
- 1-[2-[4-(4-fluorobenzoyl)-1-piperidinyl]ethyl]-1,3-dihydro-3,3-dimethyl-2H-indol-2-one
- 6-[2-[4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl]ethyl]-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one
- structure-activity relationship
- (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics


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Do you ever just want to get up go? Well, we've put together some great ideas for you weekend warriors who just need to get away. Here are a few quick roadtrips that every San Diegan should experience at least once in their life.
There's a Bear Grylls in us all and Joshua Tree is the place to unleash your rustic outdoorsman side. With miles and miles of Joshua trees and desert chaparral, the scene is like that of a fairy tale. The surrounding area makes it impossible not to explore what's around you. The trails can be pretty unforgiving during the hotter months; we are in the desert. However, no desert is complete without thriving oases. Spend your day hiking to one of five of these desert oases and you'll finish your day feeling completely accomplished.
Need more of a challenge? Joshua Tree welcomes rock climbers and boulderers from around the world! There are thousands of climbing routes available no matter your climbing experience. However, be sure to come accompanied with an experienced climber to avoid any possible injuries during your endeavour.
If driving non-stop you're looking at a two and a half hour drive from El Cajon. This route takes you through the vineyards of temecula as well as just outside of Palm Springs. So, if you're looking to make this a long weekend, there are plenty of fun destinations along the way!
Sometimes you just gotta float. For the most part this is not exactly a relaxing experience, but an all out party. Salt River Tubing is an experience like no other and it'll cost you next to nothing (once you get there). Here's the gist: park, grab a tube, take a shuttle to the top of the river, float back down to your car. If you're a River Rat, you know that beer goes hand-in-hand with floating. So, you will probably need to rent a second tube to squeeze your cooler into and be prepared for a little bit of public drunkenness.
Here are a few essentials for your trip to the Salt River: sunscreen, waterproof bags, and rope to tie you and your friends' tubes together.
This venture is a bit of a trek so you might want to save this trip for a three day weekend. It'll take a little over five hours to drive out there and another day of recovery time. So, you'll probably want to make a reservation at a nearby hotel or stay with a friend. Remember, to always drink responsibly. Bring a sober driver if you plan to partake in the Salt River's drinking past time.
Apple picking, wine tasting, and devouring a tasty pie is what Julian is all about. If you've never given Julian a visit you are missing out on this small town, country experience. There are horse and carriage rides available to those who prefer to have more of a guided tour, but the town is small enough for you to just stroll up and down the streets. If you're a wine lover there are several little spots to give your tastebuds a tannen treat – Witch Creek and Blue Door are probably the most popular stops in town. Not a wine drinker? Check out Nickel Beer Co. and enjoy a hoppy refreshment!
If you prefer to avoid adult beverages altogether, there is still plenty to do in this historical little town. The Julian Pioneer Cemetery is enough to give you chills and plenty of photo opportunities with its rustic designs and historical grave sites. You can also get the group together for an Old Time photo. Who doesn't love dress up?
Don't forget, your trip to Julian is never complete without a visit to Julian Pie Company where you can buy by the slice or take a whole pie home. My personal recommendation – the dutch apple. You can't go wrong with an all American apple pie with a sweet crumb top!
The trip out is only about an hour drive giving you the option of making this road trip just a short day adventure or, if you'd prefer, book a room at the Julian Gold Rush Hotel and make a weekend out of it!


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How to Get Your Smile Ready for Valentines Day
Valentine's Day is on its way and couples all over the country are seeking strategies to help them prepare their smile and breath for the big night. As everyone wants to be a desirable companion for a romantic evening out with their partner, they will need to walk a fine line between a savory dinner and savoring the evening. Whether you'll be dining in a 5-star restaurant or enjoying a quiet night in, keeping your breath fresh can play a big role in your enjoyment of the rest of the evening.
Keep Yourself Kissable on Valentine's Day
A lot of pressure is put on the Valentines dinner and dessert, but while some foods are more exciting to eat, they can leave your breath a little pungent. Try to avoid foods and beverages with strong flavors during your dinner, because that will be the foundation of keeping yourself appealing. Anything flavored with garlic or onions should be off the table for the evening because they contain sulfur compounds that not only linger in the mouth even after brushing, but they also create body odor by getting into the bloodstream. You should also avoid fatty fish such as tuna, spicy foods of any type, and coffee.
On the other hand, herbs such as rosemary, parsley, cardamom, and cilantro can help keep breath fresh, so consider ordering an entree that's prepared with one or more of these herbs. Entre's like Rosemary Chicken or Pasta Primavera and drinks like Green Tea instead of coffee can help to start your meal off on the right foot. You should also consider "dining continental" style by enjoying your salad after the main course. Nibbling a light salad and things like almonds after dinner can help clean stronger flavors and debris out of your mouth.
Other Ways to Get Your Smile Ready for Valentines Day
After dinner is over, try to slip away for a quick brush-and-floss. If you aren't sure about killing stronger flavors, a mouth wash will have a better long term effect than taking a mint. Most mints are primarily sugar, and will eventually leave your mouth sour again.
We hope you have an enjoyable Valentines celebration and we hope these tips will help.


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by Hannah Keppler
A few weeks ago, I watched the compelling documentary "Fire in the Blood," an account of how Western pharmaceutical companies thwarted access of potentially life-saving AIDS drugs in Africa and the opposing grassroots effort to bypass these obstacles. I will summarize the key issues addressed in this controversial story, which also apply to the larger context of medical access in the developing world, and the roles of Western nations in global health. This article will serve as a jumping-off point for future articles, where I will explore further some of the issues that are raised. With this article, I will try to demonstrate the problem, but you will have to stay tuned to hear about possible solutions.
View original post 1,453 more words


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The 'Craving Citrus' orange kitchen towel offers a sweet design and adds cheerful color to any space. The original orange fruit artwork was created by artist Keri Keene, and product designs created by Katy Keene; twin sisters and native Floridians inspired by the fruit of Florida.
Features: Waffle fabric texture, design printed on one side.
Material: 100% polyester
Made in USA from imported materials
Care Instructions: Machine wash separately in cold water, use delicate cycle and tumble dry on low. Do not bleach. Do not iron.
*Due to the printing process, towels may shrink - up to 1" width may be lost
Production and ship time : 4 business days for production, then your selected shipping time selected at checkout.


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Here you can create a custom frame to suit an artwork you already own.
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Many thanks to those of you who love PIE and enjoy supporting WCHA! On Wednesday, August 24th, there was a "Dine-In Night" at Polly's Pies on Atlantic Avenue, between the hours of 3:00pm and 8:30pm. On that night, Polly's Pies donated 20% of their sales to our organization. Thank you for bringing your friends, family, and enjoying home-style cooking, without the home-style cleanup — and of course, a delicious pie! Pictures from the event can be viewed here: http://tiny.cc/WCHAheartsPIE.
If you haven't renewed your membership or would like to sign up to be a member of Willmore City Heritage Association you are in luck. Visit our membership / donations page for additional information. https://willmorecity.org/?page_id=567
There are several events happening in September. We hope you and yours will attend.
We Love Long Beach Breakfast in the First 9/17/11
Happy to announce that We Love Long Beach is hosting a pancake breakfast in our neighborhood on Saturday, September 17th from 9am – 11am. The 1st District Council Office, Long Beach Heritage – Bembridge House, Community Partners Council (CPC) and Willmore City Heritage Association are a few of the groups attending from the 1st District. These breakfasts are wonderful gatherings to meet your fellow neighbors and celebrate all that is great about Long Beach + 1st District + Downtown + Willmore City Historic District!!!
Code Enforcement Meeting
This meeting gives neighbors a platform to report code violations they observe occurring in the Willmore City / Drake Park Historic District and the West End neighborhoods with a City of Long Beach Code Enforcement representative.
PLACE: Willmore Community Police Center, 910 Daisy Ave.
DATE: Wednesday, September 21, 2011
TIME: 6:00 pm – 7:00 pm
Monthly Meeting: Last one at the Willmore Community Police Center
Come say farewell to the Community Police Center during this months meeting, share story, and celebrate a place that has been great to the improvement of our neighborhood. Details will be given on the future monthly meeting location.
PLACE: Willmore Community Police Center, 910 Daisy Ave.
DATE: Wednesday, September 21, 2011
TIME: 7:00 pm – 8:30 pm
Bembridge House Docent Training: Available Every Tuesday!
Come learn about Long Beach history, the Bembridge House, and be a docent at the Bembridge House Historic Site to share the home's story. It takes approximately 2 hours. Docent training available most Tuesday's between 1-4pm. Contact Jill Black at email@example.com or 562-495-3377 to schedule your class.
953 N Park Circle Drive, Long Beach CA
In the Drake Park, Willmore City Historic District of Long Beach California
Saturday, September 10th Latin American Parade & Festival
The 3rd Annual Latin American Parade & Festival returns to the streets of Downtown Long Beach on September 10th. This year, we anticipate 1,000 parade participants, more than 15 live performances and 40 vendor booths. Don't miss the fun!
The parade steps off at Pine and 6th at 3PM and will make its way to the new Festival location at 4th Street and The Promenade. The Festival will run from 3-9PM.
For more info visit: http://downtownlongbeach.org/event/lapf/
Rose Park Restoration Trade Fair: Sunday, September 25, 2011
WCHA is proud to be participating in this years Rose Park Restoration Trade Fair! We will have a booth showcasing all that is great about our neighborhood and look forward to gathering with our fellow Long Beach historic districts! This is a wonderful event celebrating all that is great about living in some of Long Beach's best communities and support the Rose Park neighborhood. See you there!
Show your Willmore City PRIDE by Sport'n a WCHA Market Tote around town!
"Beginning today – August 1, 2011, large retailers in Long Beach will no longer distribute plastic bags. Customers are being encouraged to bring their own reusable bags to help reduce litter on our streets and preserve landfill space. Small retailers and farmer's markets must comply with this new ordinance beginning January 1, 2012." – Litterfreelb.org
Share your pride for your neighborhood and get your Willmore Market Tote today for a $3 donation. Send an email to email@example.com if you would like one. They can also be purchased at Willmore Five and Dime near the corner of 10th & Daisy Avenue.
Love Coffee + Love Willmore = Order Newhall Coffee
Whenever you enjoy your coffee passion, Newhall Coffee for a Cause offers an opportunity to sample some of California's finest blends from the Newhall Coffee Roasting Company. And best of all, fifty percent of your payment is donated directly to Willmore City Heritage Association. Visit the link below to participate.


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In a continuation of a previous post, I've found 2 more sites that reflect emergency department overcrowding and diversions in real or near-real time. The first is MIEMSS, the Maryland Institute for Emergency Medical Services Systems. The far left column on the bottom half of their site has a section titled CHATS (for County Hospital Alerts Tracking System). Below CHATS are links to three different regions of the state, listing all the hospitals in that region and color coded status for various degrees of overcrowding and bypass.
This Friday shows 13 out of 58 hospitals showing some sort of divert status (that's 22 percent). The MIEMSS Annual Report notes (page 15) that emergency room overcrowding has gotten more acute in the current reporting period (2003-2004) than in previous years, with record peaks during the flu and respiratory season. While not as cool as the Med Media website that the Emergency Health Services Federation in Pennsylvania has, the MIEMSS site is informative and updated regularly.
Our second status site covers 17 Montreal area hospitals. This site is updated at 8 am daily. For each hospital the site shows "stretcher capacity", i.e., beds, occupied stretchers and stretchers occupied for more than 48 hours (yikes!). As of 8am this morning (Friday, March 18, 2005), capacity was at 120 percent, and 79 patients had been boarded more than 48 hours waiting for a hospital room.


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Vault Press is the premium wordpress backup plugins that automatically backup you wordpress blog to its server. VaultPress usually cost 15$ a month for its standard backup service. The cost might seem higher and unaffordable for newbie blogger. There are some free vaultpress alternative which are almost similar to Vaultpress backup service and provides free automatic backup, all these alternatives are listed below.
1. IDrive Plugin for wordpress
IDrive Plugin will backup your database, as well as files, and will store them in your online IDrive account. IDrive account comes with a 2GB of free space. You can store multiple copies of your backup for free without migrating to paid account. You can refer to your friends and can another 10GB space for free instantly. You can download this plugin from here.
2. Back WP UP
This is not a exact alternative to VaultPress, but some of its feature are similar. This plugin backups your blog to FTP Server
,Amazon S3, Google Storage, Microsoft Azure (Blob), RackSpaceCloud
,Dropbox and SugarSync according to the settings you have selected. You can also choose the files and settings you want to backup. Download plugin from here.
3. XCloner – Backup and Restore
XCloner is a Backup and Restore component designed for PHP/Mysql websites, it can work as a native plugin for WordPress and Joomla!.
XCloner Generate, Move and Restore process:
- Generate and Store the backups
- Move the backup and restore script to the new location
- Restore the backup by launching the XCloner.php restore script
- Backup and Restore any PHP/Mysql application
- Create custom backups
- Generate automatic backups based on cronjobs
- Restore your backups anywhere
- Share your custom backups with your clients
Download Plugin from here.
4. Codeguard
Codeguard can be used in any type of site. They can be used for wordpress backup without plugin but they have developed a separate plugin for wordpress as well which simplifies the task of WordPress backup process. CodeGuard is a secure, automatic daily backup service and notifies you when something changes and lets you undo it with ease. Besides backup it also helps you monitor Google Blacklisting and scans for malware. It has free and paid plan, free plan offers 1gb of free storage. Enough for a decent blog.
Download the Codeguard plugin from here.
If you have found an other free vaultpress alternative, list it in the comment below and let us know about it. You can find W3 Total Cache Alternative here.
Also Read : List of other automatic wordpress backup plugin
Speed up wordpress speed


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Team Rock In The Dominican Republic
Team Rock (TR) ministers to people all over the world and this spring TR was in the Dominican Republic. Seen in this photo, Dewayne flips over audience members during an outreach in a Haitian Village during the Dominican Republic tour. We exist to proclaim Christ...read more
Lena Ford Gives Us A Shout Out
Lena Ford Writes- Team Rock Ministry Thank you guys (and girls) for coming out to New Hope Christian Church today and sharing a part of yourselves and your ministry. We enjoyed it and thank you for what you guys do all over the world sharing Christ to others. Thanks...read more


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But will it work?
Electroneum aims to be the most "user friendly cryptocurrency" and comes with both a wallet, and a miner built into a mobile app.
Which leads to another huge plus, instant peer to peer transactions with other users of the currency (as long as they have the app too).
So far, in while other ICO's have struggled amid a few recent flops, Electroneum seems to have managed to gain momentum. At the time of writing this article has raised 25821 ether and 2528 bitcoin.
Now back to the "will it work?" question I started with. Well, looking into the development team, I do feel confident in saying - this one isn't a scam.
However "not a scam" doesn't equal "this will be a success".
The success of Electroneum will depend on people using the app, meaning it's going to take active users, not just people throwing money at it to make it work. Does anyone really want their phone mining all day? Hard to say when there's nothing similar to do a case study on.
That is the part you'll be rolling the dice on.
The ICO is now live here.
Author: Justin Derbek
New York News Desk


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Pros and Cons of an Operating Room Technician Career
Operating room technicians, also called surgical technologists, assist doctors and nurses during surgical procedures. The following pros and cons of being an operating room technician can help you decide if this is the right career for you.
|Pros of Being an Operating Room Technician
|Much-faster-than-average employment growth (expected 30% between 2012 and 2022)*
|Decent wage potential (average wage of about $45,000 as of May 2014)*
|Technical training or associate's degree programs sufficient for entry-level positions**
|May work in various healthcare settings**
|Cons of Being an Operating Room Technician
|May be required to spend long periods standing*
|Work may be stressful*
|Work hours may include evening, weekends and holidays*
|May be exposed to potential hazards, including communicable diseases*
Source: *U.S. Bureau of Labor Statistics, **American Medical Association.
Essential Career Info
Job Description
Operating room technicians are medical professionals who assist surgeons and nurses during surgical procedures. They have a variety of duties that take place pre-surgery, during surgery and post-surgery. Prior to the surgery, they prepare the operating room by making sure it's correctly stocked with sterilized equipment and tools. Operating room technicians help prepare the patient for the procedure by washing, shaving and disinfecting the incision sites. They may also answer questions patients may have.
The techs help transport patients to the operating room and help position them on the table. During the surgery, they assist the surgeons and nurses by handing them the supplies and instruments they need. They also assist the surgical staff with putting on gloves and gowns. They may also help apply dressings to the patient. After the surgery, they transport patients back to their rooms and make sure the operating room is cleaned and restocked with supplies.
Job Growth and Salary
According to the U.S. Bureau of Labor Statistics (BLS), surgical technologists were predicted to see an employment growth of 30% between 2012 and 2022. The aging population and the increase in surgical procedures being performed both contribute to the favorable employment projections. The BLS reported that surgical technologists earned a mean annual wage of around $45,000 as of May 2014. Surgical technologists who complete accredited training programs and keep up with certifications should have the best employment opportunities, according to the BLS.
Education and Training Requirements
To become an operating room technician, you usually need to complete a training program. To meet admission requirements, an applicant may need to provide a high school diploma, completed health examination and health insurance. In addition to submitting to a criminal background check, applicants may need to provide proof of current immunizations, CPR certification and a completed shadow experience at a local clinic.
Training programs, which may be diploma, certificate or associate's degree programs, are found at vocational/technical schools and community colleges. They range in length from nine months to two years. The curriculum includes lectures, laboratory studies and clinical education to obtain supervised hands-on training. Course topics may include anatomy and physiology, patient care skills, medical terminology, microbiology, surgical wound care, surgical technology and pharmacology. As an OR tech, some additional skills are important to the job, such as:
- Ability to work under stress
- Good stamina
- Excellent communication skills
- Detail-oriented
- Dexterity
Job Postings from Real Employers
Education, job duties and work requirements may vary by employer. However, in addition to the necessary education requirements, most employers are seeking applicants with the above-mentioned skills as well as those who are willing to gain additional certifications, such as the Association of Surgical Technologists (AST) certification. Although this isn't a picture of the entire job market for OR technicians, here are a couple of real job postings from April 2012:
- A Connecticut hospital is in need of an operating room technician to assist the surgical team with direct patient care activities. The OR tech will assist with departmental work as needed, participate in staff or patient/family education, evaluate and plan patient care and be aware of the legal aspects regarding surgical technologists. Applicants should be graduates of approved SCT programs with at least six months of experience as surgical techs or have documented on-the-job training. Association of Surgical Technologists (AST) certification is desired but not required.
- An ambulatory surgical center in Wisconsin is seeking an operating room technician to assist with surgical procedures and patient care, while working under the supervision of a charge nurse and/or a registered nurse. Applicant must have completed an accredited operating room technician program and must have experience working in a multispecialty operating room, along with technical skills needed for this position.
- A healthcare service provider in Michigan is seeking a surgical technician to assist in surgical procedures. Candidate requirements include completion of a surgical technician program, certification as a surgical technician and certification in automated external defibrillators (AED) and CPR. Job duties include preparing instruments and surgical medication, cleaning and sterilizing surgical equipment, performing administrative duties and providing patients with post-surgical care.
How to Maximize Your Skills
Once you earn your degree and obtain some work experience, you'll want to make yourself as competitive as possible. One way you can maximize your skills is through certification. Certification, although not required, demonstrates knowledge and skill and is looked at favorably by potential employers. Several organizations offer certification programs for surgical technologists. Eligible candidates can obtain the Certified Surgical Technologist (CST) certification from the National Board of Surgical Technology and Surgical Assisting (NBSTSA). The Tech in Surgery - Certified (TS-C) certification is available through the National Center for Competency Testing. The National Healthcare Association (NHA) offers the Certified Operating Room and Surgical Technician (CORST) certification. To be eligible for certifications, candidates must meet education and/or work experience requirements and pass written examinations. Continuing education is usually required to maintain certification.
Other Fields to Consider
Choosing a career can be a big deal, and you're going to want to be sure you're making the right choice. If you're still uncertain about becoming an operating room technician, here are a couple of other related careers you may wish to consider.
Licensed Practical or Licensed Vocational Nurse
Licensed practical nurses (LPNs) or licensed vocational nurses (LVNs) provide medical care to sick or injured patients while under the supervision of registered nurses and doctors. Becoming an LPN or LVN requires completion of an associate's degree program in nursing. The nursing curriculum includes coursework, lab studies and clinical education. Before you can work as a nurse, you're required to obtain licensure by passing an examination. According to the BLS, these workers can expect an employment growth of 22% between 2010 and 2020. As of May 2011, the mean annual wage for licensed practical nurses and licensed vocational nurses was around $42,000.
Medical and Clinical Laboratory Technologist or Technician
If you know you want to be part of the medical profession but want less or no hands-on patient contact, you may find a career as a medical and clinical laboratory technologist or technician rewarding and challenging. These workers spend most of their time in the lab collecting samples and performing tests used to help with the diagnoses and treatment of diseases. As similar as some of their duties may be, the technicians actually work under the technologists. Becoming a medical and clinical lab technologist requires completion of a bachelor's degree program, while the technicians only need an associate's degree. Both may be required to obtain licensure. According to the BLS, medical and clinical laboratory technologists and technicians were predicted to experience an employment growth of 13% between 2010 and 2020. The BLS also reported that technologists earned a mean annual wage around $58,000, while technicians earned near $39,000 as of May 2011.


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A TWELVE POWER MEDITATION EXERCISE by CHARLES ROTH. Long after Unity's co-founder Charles Filmore wrote about the twelve powers, people continue to study his exploration of our spiritual faculties. In this book Roth provides an easy to understand introduction to this areas of Filmore's work.
£11.75 inc. Postage & Packing.


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Home > Accommodations
Coastal Luxury in Our Cabo San Lucas Suites
Relax in our luxury suites, designed with warm coastal decor that reflects the tranquility and beauty of Los Cabos itself. Get swept away by the sounds of th beach, as you relax in a plush king bed. Order room service and enjoy drinks on your private terrace as you lounge in the cool breeze. Enhance your vacation with satellite TV, wet bars, kitchens, dining rooms and Jacuzzi bathtubs. With a vibrant location in that celebrates the best of Cabo San Lucas, our suites are your paradise at beautiful Land's End.
Deluxe Junior Suite
Comfort comes naturally in this gorgeous, well-appointed suite, featuring a king bed, queen-size sofa sleeper, full tiled bath with tub, air conditioning, kitchenette and balcony. 633 SqFt.
Luxury Suite
Embrace uncommon luxury in this expansive suite, featuring a bedroom with king bed, 2 full tiled baths, living room with a queen-size sofa sleeper, a modern kitchen and balcony. 850 SqFt.
Two Bedroom Presidential Suite
Find your coastal refuge in this 1858-square-foot paradise. Enjoy two spacious bedrooms, a kitchen, dining and living rooms, 3 bathrooms and cool breezes from your private balcony.
3 Bedroom Penthouse Suite
Spectacular views of the Pacific Ocean and Sea of Cortez. It features a separate parlor with an elegant dining room, guest bathroom, spacious living area, queen murphy bed and fully equipped kitchen. Private terrace with jacuzzi, grill and deck chairs in this 2,994 square feet Suite.
4 Bedroom Penthouse Suite
This 5700 square feet units offer unparalleled views of the pacific ocean and feature a separate parlor with elegant dining room, five bathrooms, walk-in closet and large terrace with jacuzzi.
4 Bedroom Penthouse 2551
This 5555 square feet unit offers unparalleled views of the pacific ocean and feature a separate parlor with elegant dining room, four bathrooms, walk-in closet and large terrace with private jacuzzi. With room for up to 10 people, guests can choose from two king beds, 3 double beds and a sofa bed.


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One of the responsibilities of the HTCdev team includes providing a method to unlock the bootloader on several HTC devices. They now allow users to unlock the bootloader on over 30 devices worldwide. Last month we saw them unlock six more handsets on devices near 2 years old. This time around, they continue to open more devices around the same age and some even older.
Recently added to list are the:
- HTC Hero
- HTC Legend
- Droid Eris
- MyTouch 3G
- MyTouch 4G
As far as i know, there are already methods of obtaining S-OFF (unlocked bootloader) on all these devices without having to jump through the hoops at HTCdev.com. I would recommend methods like unrEVOked for devices like the Hero or Eris.
From my personal experience working with an HTC EVO 3D, the provided method from HTCdev doesn't even give you S-OFF. This means the bootloader is still locked down, and its forcing people to use Fastboot methods when flashing custom ROMs and kernels.
While it's nice to see these OEM's make an effort to appease the dev community, they still aren't letting us have full control of the device. Someday they might learn what we really expect from them, but until then we are stuck with this.
Visit HTCdev.com to learn more
Let us know your experiences with the HTCdev bootloader unlocking methods.


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Our very own Taarak Mehta Ka Ooltah Chashmah is the second longest running Indian television series by episodes. Over the time we laughed, cried and celebrated with these actors. These actors have a special place in our hearts and also in our drawing rooms. They have left such indelible imprint on our hearts that they are a part of our family now. It is impossible for us to imagine our lives without their daily arrival on small screens at 8.30 every evening. As audience we are curious to know every small detail regarding their personal lives. Here is a list of 23 actors with their real regions.
1. Dilip Joshi aka Jethala Gada
Dilip Joshi's character Jethalal who calls himself a Kacchi Manus, is a hardworking businessman and is always stuck in some sort of trouble which proves to be a really comical for the audience to watch. He is also originally from Gujarat.
2. Disha Vakani aka Dayaben Gada
Disha Vakani plays extremely honest, caring, kind, sensitive yet witty and humorous wife of Jethalal Gada who is a hardcore Amdavadi (Gujarati) woman who is even originally from Gujarat.
3. Mayur Vakani aka Sundar
Mayur Vakani plays role of Sundar, brother of Disha Vakani aka Dayaben and other female characters of Tarak Mehta ka Ooltah Chashmah. He plays a role of a trouble maker as he always creates some trouble for his brother-in-law Jethalal. He is elder brother of Disha in real life and is from Gujarat.
4. Amit Bhatt aka Champaklal Jayantilal Gada
Amit plays a man of older age in serial, which is shown as giving helpful advices to everyone. He is originally from Gujarat.
5. Bhavya Gandhi aka Tipendra Gada (Tapu)
Bhavya Gandhi plays role of Tipendra Gada, son of Daya and Jethalal, and is popularly known as Tapu in serial. Like both his onscreen parents he is also from Gujarat. He is the most important character of Taarak Mehta Ka Ooltah Chashmah.
Few months back Raj Anadkat replaced Bhavya Gandhi as Tapu, and is now a new leader of Tapu-sena. Raj is originally from Maharashtra.
6. Shailesh Lodha aka Tarak Mehta
Tarak Mehta is the fire brigade friend of Jethalal Gada. Shailesh Lodha plays this character, he is originally from Rajasthan.
7. Neha Mehta aka Anjali Mehta
Neha Mehta plays the character of Anjali Mehta who is an extremely strict dietician and caring wife. She is originally from Gujarat.
8. Mandar Chandwadkar aka Atmaram Tukaram Bhide
Mandar Chandwadkar plays a role of tuition teacher Atmaram Tukaram Bhide who is a Marathi in serial and in real life too, as he is originally from Maharashtra.
9. Sonalika Joshi aka Madhavi Atmaram Bhide
Sonalika Joshi plays Madhavi Atmaram Bhide who makes delicious achaar (pickle) and papad. She is originally from Maharashtra.
10. Jheel Mehta aka Sonalika Bhide (Sonu)
Studious daughter of Madhavi and Atmaram Bhide, Sonu Bhide is played by Jheel Mehta who is originally from Gujarat.
Nidhi Bhanushali replaced Jheel in year 2012 and plays a role of Sonu. She is also from Gujarat.
11. Shyam Pathak aka Patrakar Popatlal Pandey
Shyam Pathak plays the character of Patrakar Popatlal Pandey who is a forever bachelor staying alone. He is originally from Gujarat. Taarak Mehta Ka Ooltah Chashmah cannot be complete with this man.
12. Tanuj Mahashabde aka Krishnan Iyer
Geeky scientist guy Krishnan Iyer is a South Indian in the serial. This character is played by Tanuj Mahashabde who is originally from Maharashtra.
13. Munmun Datta aka Babita Iyer
Munmum plays role of an extremely gorgeous Babita Iyer who is shown to be a Bengali in serial, whereas even in real life she is from Bengal.
14. Gurucharan Singh aka Roshan Sodhi
Gurucharan Singh plays character of ever happy Roshan Sodhi. He plays a role of a Punjabi Sikh and in real life also he is from Punjab.
15. Jennifer Mistry Bansiwal aka Mrs. Roshan Sodhi
Mrs. Roshan Sodhi, wife of Mr. Roshan Sodhi is played by Jennifer Mistry Bansiwal is originally from Madhya Pradesh.
16. Samay Shah aka Gurucharan Singh Sodhi (Gogi)
Samay Shah plays a role of punjabi boy Gogi in Taarak Mehta Ka Ooltah Chashmah. Samay and Bhavya are cousins in real life. He is originally from Gujarat.
17. Kavi Kumar Azad aka Dr. Hansraj Hathi
Kavi Kumar plays the character of food loving Dr. Hansraj Hathi. He is originally from Bihar.
18. Ambika Ranjankar aka Komal Hathi
Ambika Ranjankar aka Komal Hathi is originally from Maharashtra.
19. Kush Shah aka Gulabkumar Hathi (Goli)
Goli Hathi is the naughty son of Komal and Hansraj Hathi. Kush Shah plays this character, he is originally from Gujarat.
20. Ghanshyam Nayak aka Natu Kaka
Faithful employee at Gada Electronics, character of Natu Kaka is played by Ghanshayam Nayak in Taarak Mehta Ka Ooltah Chashmah. He is originally from Gujarat.
21. Tanmay Vekaria aka Bagha
Another employee at Gada Electronics, Bagha with a slightly crooked back is a fun character to watch. Tanmay Vekaria plays this character, who is originally from Gujarat.
22. Priya Ahuja Rajda aka Rita Reporter
Priya plays role of Rita Reporter and works for Kal Tak news channel. She is originally from Maharashtra.
23. Daya Shankar Pandey aka Inspector Chalu Pandey
Daya Shankar Pandey plays a role of an honest and couregous inspector Chalu Pandey who is not afraid of anyone other than his wife Basundi. He is originally from Uttar Pradesh.
Referred From: www.tmkoc.com