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Since I am so relaxed and chilled out I also would prefer my home to be such. This means no hyper screaming little people. Well mannered, quiet behaved children are cool by me.
I am very quiet unless I think it is taking you too long to feed me. I am also terrified of Storms…but all you have to do is keep the bathroom door open so I can hide in the bath tub and we are all good.
So, get busy and fill out the Adoption Application so you can add me to your home.
I am Fully vetted to include, spay, micro chip, disease and parasite free on monthly preventatives. I also just had a dental
I am being fostered in Greenville, SC and my Adoption Donation is $250 which as you know is the cost of an overage yearly exam!
If you meet the above qualifications and you think you can be my perfect home, please continue reading----Do not send an e-mail unless you have read all the way to the bottom of this Page
1. Go to our web site and fill out an application. Nothing will move forward until you do. http://carolina-loving-hound-rescue.com/Adoption_Application.php
2. We will give you a call within 7 Days after receiving a complete application, please do not serial e-mail us, we will work your application in the order it was received. We also work these applications between our paying jobs, taking care of our animals and families. Patience is the key!
3. Once we speak to you and feel you are a good fit, we will set up a meet and greet
4. Once we meet and do a home visit we can then and only then finalize an adoption. Every member of your household must be present for this meeting
5. We do adopt out of state and will do a home visit out of state as well
6. We will not put our dogs on mass transport or hand off transport to get to your home. .it is your responsibility to pick her up. We will make every effort to meet you part of the way but that is not always possible.
7. All animals in your house MUST be spayed/neutered unless you have medical documentation preventing you from doing so. NO EXCEPTIONS.
8. Unless you have outside working dogs we will not adopt to a home with outside dogs
9. Fence is required for me.
10. Must be 25 or above to adopt (exceptions may be made case by case)
If you have any additional questions please e-mail email@example.com
Upon approval you and your new family member will enjoy a long and lasting relationship. Adoption fee's vary based on breed/age of animal!
Thank You!
Your message has been sent to Carolina Loving Hound Rescue.
You'll receive a copy, too, at to help you keep track of which pets you've inquired about, and which shelters and rescues you've emailed.
NOTE: Some shelters have physical locations you can visit; some of these shelters may only have pets for a limited time, so please do not wait for a reply—just go visit the shelter! Other organizations are rescue groups run by busy volunteers who may take a while to reply. You can find information about the shelter or rescue group caring for this pet, and their adoption procedures, on the pet's details page on Adopt-a-Pet.com.


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Dighton Uses Molasses To Treat Snowy RoadsThe town is using molasses to help keep the roads clear of snow.
Snow Smashes Windshield On I-95 In AttleboroSnow and ice flew off a car and smashed a windshield in Attleboro.
Nashua Cleans Up After 'Nuisance' StormThe city of Nashua woke up to ten fresh inches of snow on Monday.
Avalanche Warning Issued For Mount WashingtonThe Mount Washington Avalanche Center has issued an extreme avalanche danger warning on two trails after more than 16 inches of snow has fallen on the highest peak in the Northeast.
Gov. Baker: MassDOT Used Lessons From 2015 Blizzards To Fight Latest StormsMassachusetts Gov. Charlie Baker praised the work of MassDOT Monday morning in clearing the roads and railways of the Commonwealth after the latest storm dumped several more inches of snow across the area.
Delayed Start For State Workers Monday, Gov. Baker SaysThe governor said this move was to help crews continue cleaning roads around the Commonwealth, as well as keeping commuter rail and MBTA tracks clear.
Boston & Dozens Of Other School Districts Close Monday For StormAs New Englanders continued to dig out from a Thursday's Nor'easter and additional accumulation on Saturday, yet another storm was bringing several more inches of snow to the area from Sunday to Monday.
Storm Moves Into New EnglandThe "backlash" of the storm coming on Monday is looking a bit less dire for our coastline.
Winter Storm That Hit Boston Gets Official Blizzard DesignationThe large Nor'easter that hit Massachusetts with up to 18 inches of snow officially became a blizzard as of 4:30 p.m. Thursday.
'Exceptional Amount' Of Thundersnow Marks BlizzardThe first official blizzard in Boston in more than two years brought "an exceptional amount of thundersnow."
Snow Totals - February 9 StormA major nor'easter is hitting southern New England with blizzard-like conditions.
Beyond The Forecast: More Storms In The PipelineThursday's storm may be just the beginning leading to several storm threats over the next 7-10 days.

subset: commoncrawl


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Likely to sell out
Porto Wine and Tapas Tour
Duration: 1 day
This exciting tour will help you to understand the importance of wine to Porto, and to taste some paired with delicious food.
PortoSee all 229 tickets & tours
Want to discover all there is to do in Museu do Vinho do Porto? Click here for a full list.


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- Services
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Patients seeking treatment at Quintessential Chiropractic with Drs. Nick Quinn & Tanya Quinn are assured of receiving only the finest quality care through the use of modern chiropractic equipment and technology. Drs. Nick & Tanya Quinn and staff have a genuine concern for your well-being! Located in Portsmouth, New Hampshire and serving the communities of Portsmouth, Rye, Kittery, Newington, Stratham and Greenland, as well as the seacoast area, the husband and wife team at Quintessential Chiropractic are dedicated to helping you achieve your wellness objectives -- combining skill and expertise that spans the entire chiropractic wellness spectrum. Dr. Nick Quinn, D.C. & Dr. Tanya Quinn, D.C. are committed to bringing you better health and a better way of life by teaching and practicing the true principles of chiropractic wellness care. If your mission is simply to get out of pain, we understand, and will tailor a care plan to suit your needs.
If you are new to our website, please feel free to discover and learn about chiropractic wellness. If you are interested in starting your journey towards wellness please subscribe to our award winning newsletter. If you are already a newsletter subscriber, please explore the member wellness section of our website for wellness articles, resources, and health facts---specifically targeted by Drs. Nick & Tanya Quinn to your wellness needs and interests.
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Are you looking for Stamped Concrete in OHIO? Hotfrog showcases more than 100 popular businesses related to Stamped Concrete in OHIO! Find a specific region or suburb using the left navigation.
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Orrville, OH
Deco-Crete Supply is proud provider of quality decorative concrete products for stamped concrete, stenciled concrete, decorative epoxies, colored concrete, acid staining, concrete overlays, and much more. Serving Northea…
Colored ConcreteDecorative Concrete SuppliesDecorative ConcreteBrick Block Pavers
Northfield, OH
Contact our concrete contractor at (330) 310-4926 in Northfield, OH, to request driveway removal, excavating services, or stamped concrete.
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Jamestown, OH
concrete construction , driveways,color and stamped concrete , framing, decks , pavers
Concrete Construction
Lewis Center, OH
We are pioneers in the stamped concrete process, developing techniques in the art of texturing and mixes, since 1983
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North Royalton, OH
Commercial, Residential, Northeast Ohio, Landscapes, Stamped Concrete, Tree Service
Newark, OH
Citizen Construction Group provides carpentry work and stamped concrete for remodels, kitchens, bathrooms, decks, sunrooms and fences throughout greater Newark OH
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Hartville, OH
Our services consist of decorative concrete, stamped concrete, concrete staining, antiquing concrete, overlays, stencils, countertops and much more
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Mentor, OH
Alba Contractors, Inc. is a recognized industry leader in concrete, waterproofing and a luxury residential remodeler. We are one of Cleveland, Ohio's premier and diversified construction companies. Founded in 1995 by the…
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Goshen, OH
Walkers Concrete specializes in Stamped Concrete Patios, Driveways and Sidewalks in the Cincinnati, Mason, Lovealnd Ohio Area. We also custom build outdoor Fire Places, Fire Pits and seating walls.
Stamped Concrete PatioDecorative Concrete PatiosConcrete Driveways
Dublin, OH
We offer a wide range of services from emergency repairs to fine custom home additions. We are proud to serve everyone from upscale to the average working man. We provide excellent customer service and back up our work…
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Cleveland, OH
We are a concrete contractor supply store with a wide selection of decorative concrete solutions to choose from. We can help you achieve the look you want from stamped concrete, stenciled concrete, colored concrete and m…
VERTICAL CONCRETEDecorative ConcreteConcrete CountertopsConcrete Design
Our decks around our custom built pools are not limited to stamped concrete Each swimming pool and spa we build is crafted and custom created to fit you and your families specific needs
Batavia, OH
Created in 2005 as a wholly owned subsidiary of Towne Properties, TowneScapes exists as Cincinnati's most honored Stamped Concrete, Commercial and General Site Services Contractor
Concrete PatternsStained ConcreteColored ConcreteConcrete Stamping
Columbus, OH
Atlantis Concrete & Construction LLC is a stamped concrete, standard concrete and paver installation contractor in Columbus, Ohio. Specialty areas include: patio - driveway - sidewalk - wall - fire pit - all types of con…
Decorative Stamped ConcreteAcid Stainingplain concreteResidential Concrete
Miamitown, OH
Bill Drout Concrete is a decorative concrete contractor specializing in the installation of stamped concrete, vibrant stained concrete and polymer cement overlays. We pour patios, driveways, sidewalks, porches, garage fl…
Decorative Concrete ContractorConcrete Pool DecksStained ConcretePolymer Cement
Xenia, OH
We are locally owned and operated landscapers here to help you with any of your outdoor needs. We offer premier landscaping design and installation services, so we are your go to company for any of your landscape needs! …
Concrete ContractorHardscapesRetaining Walls
Broadview Heights, OH
1st Impressions Concrete has been a recognized name in Northeast Ohio for over 14 years. Since 1996, 1st Impressions Concrete has been installing quality concrete and color stamped concrete driveways, patios and walkw…
Stained ConcreteColored ConcreteDecorative ConcreteConcrete Driveways
Elyria, OH
Roig's Edging & Design Inc. is a family owned and operated concrete installing company, serving Northeast Ohio and specializing in Stamped Concrete, Concrete Overlays, Acid Staining and Decorative and Commercial Concrete…
Acid StainingConcrete OverlaysCommercial Concrete CurbingConcrete Product
Toledo, OH
Whether you are investing a little or investing a lot, you are
investing into your most important asset, your home. Once you make the
decision to have Dustin Collins Hardscaping design and create your very
own custom…
Concrete Counter TopsPool DecksFlagstone PatioPaver Contractor
Bradford, OH
Carl Ellis Enterprises, LLC has been a recognized name in
the Miami Valley of Ohio for over 10 years. Carl Ellis Enterprises, LLC is a full-range Property Maintenance Services Company offering a range of commercial, in…
C and W Concrete Services provides the best decorative, colorized, and stamped concrete for both residential and commercial projects in Columbus, OH, and surrounding areas
Colored ConcreteDecorative ConcreteConcrete DrivewaysRenovation & Restoration
Amelia, OH
We are a Distributor, Installer and Certified Trainer for Granicrete International Systems.Our product line includes Countertop Overlays, Epoxy Flooring, 3D (boulders and waterfalls, etc.), Wall Systems (including authen…
Stamped Concrete PatternsStamped Concrete DrivewaysStamped Concrete PatioStained Concrete
Middletown, OH
LANDSCAPING SERVICES: Mowing, Pruning, Clean-up, Mulching, Irrigation, Paver Walkways, Retaining Walls, Sod Installation, Concrete Stamping, Decorative Lighting, Fertilizer, Weed Control, Landscape Design, Landscape Inst…
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Hamilton, OH
We specialize in any kind of concrete work (driveways, patios, slabs, sidewalks, repair damaged parking lots & more) for the best price in the Hamilton, OH,Furmandale, OH , Fairfield, OH. Sam and Mike Neal Contractors ha…
Concrete installationConcrete ContractorConcrete CompanyConcrete Work
Brecksville, OH
Sal's Landscaping Co., serving Cleveland, OH specializes in a wide variety of services including lawn installations, trees, shrubs, patios, retaining walls and much more. 440-746-9788
PatiosRetaining Walls
Concrete contractor, driveways,patio's, waterproofing and parking lots.
Boardman, OH
To find out if hiring concrete contractors is something that you may benefit from, consider the following information: Choosing Good Concrete Contractors
Concrete DrivewayConcrete FlooringConcrete ConstructionConcrete Pavers
Loveland, OH
We have a solution for all concrete polishing needs Cincinnati area residents. Polishing Concrete Floors is the greatest flooring solution. (513)-265-9162
Concrete OverlaysConcrete FloorsConcrete FlooringConcrete Counters
We are a general construction company, focusing on concrete (driveways, patios, sidewalks, etc). We also do small carpentry work (decks, fences, small buildings), erosion control, and bush hogging.
Concrete DrivewaysNew DrivewaysSidewalksCement Work
Oregoinia, OH
Concrete Driveways Foundations Concrete barn floors Complete Excavation Drainage solutions Equestrian Riding Arenas Underground Utilities Pond Construction Snow Removal Grading Seed and straw Sod
Concrete DrivewaysNew DrivewaysGravel DrivewaysCement Work
Elite Crete of the Ohio Valley is your supplier of Elite Crete Systems Products and installation tools in the Ohio, West Virginia and Pittsburgh area. Please contact us about your decorative concrete needs and see for …
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Springboro, OH
We solely specialize in creating tailor made outdoor living spaces by utilizing distinctive hardscape design strategies and unparalleled craftsmanship to fully satisfy sophisticated tastes in a customer driven environmen…
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Campbell, OH
Painting is a painting contractor serving the Northern VA area, wholesale buying account numbers are provided so people can buy there own paint for do it yourself jobs
Westerville, OH
Website of hardscape contractor Curt's Complete Landscape LLC, Ohio Patio Builders. Building brick paver patios, driveways, steps and walls at low prices. Design and installation services. Westerville, Ohio Curt Livingst…
Wauseon, OH
Cutting Edge Masonry Serving Northwest Ohio Cutting Edge Masonry, LLC was formed in July of 2004 Formed in 2004, Cutting Edge Masonry is a small business located in Alvada, Ohio, providing many Residential…
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E&N Concrete, LLC is your source for everthing concrete in the greater Columbus, Ohio area. E&N Concrete puts the highest emphasis on workmenship, quality, and most of all customer satisfaction
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We have been in the concrete business for the past 12 years, working in the Central Ohio area We are a fully licensed and insured contractor in the state of Ohio as well as a member of the Buckeye Valley Buildi…
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Middlefield, OH
Local supplier of a full line of Stucco, EIFS, Concrete, and Waterproof Contractor Materials
Colored ConcreteConcrete SealantsConcreteCenturion Stone
Lima, OH
concrete contractorsConcrete
Chillicothe, OH
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I have written a small python script which converts kml file (as it is exported from google my maps) to upoi file, so that this output can be used as IGO Primo favourites (the output file name should be renamed to "user.upoi" and put under the save directory).
Fix - Please notice, if you have downloaded the attachement before I have made this update to the post, reload it. I had a minor error in it (latitude and longitude replaced).
Fix2 - May 4th - Improved version


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On these frigid winter mornings, your tender little tootsies take a real beating, especially when you forget to wear socks to bed and you wince in anticipation as you lower your feet to the cold, bare floor. Thankfully, this is an easy problem to remedy. Make your own rug, and you can even customize it to suit your bedroom's dimensions and colors.
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Review: My Maker's Keeper by Tiffany Current
My Maker's Keeper Author: Tiffany Current Genre: Young Adult, Paranormal Links: Amazon, Reading Alley Review An Absolute 5 Stars!! A Definite Keeper!! Rating: 5 Stars While I really like YA themed titles, I am usually a bit hesitant when vampire themes are involved. Perhaps it's because the last YA vampire book I read — Twilight — raised…… Continue reading Review: My Maker's Keeper by Tiffany Current


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Arrhythmogenic right ventricular cardiomyopathy (ARVC) termed a 'disease of the desmosome' is an inherited cardiomyopathy that recently underwent reclassification owing to the identification of left-dominant and biventricular disease forms. Homozygous loss-of-function mutations in the desmosomal component, desmoplakin, are found in patients exhibiting a biventricular form of ARVC; however, no models recapitulate the postnatal hallmarks of the disease as seen in these patients. To gain insights into the homozygous loss-of-function effects of desmoplakin in the heart, we generated cardiomyocyte-specific desmoplakin-deficient mice (DSP-cKO) using ventricular myosin light chain-2-Cre mice. Homozygous DSP-cKO mice are viable but display early ultrastructural defects in desmosomal integrity leading to a cardiomyopathy reminiscent of a biventricular form of ARVC, which includes cell death and fibro-fatty replacement within the ventricle leading to biventricular dysfunction, failure and premature death. DSP-cKO mice also exhibited ventricular arrhythmias that are exacerbated with exercise and catecholamine stimulation. Furthermore, DSP-cKO hearts exhibited right ventricular conduction defects associated with loss of connexin 40 expression and electrical wavefront propagation defects associated with loss of connexin 43 expression. Dose-dependent assessment of the effects of loss of desmoplakin in neonatal ventricular cardiomyocytes revealed primary loss of connexin 43 levels, phosphorylation and function independent of the molecular dissociation of the mechanical junction complex and fibro-fatty manifestation associated with ARVC, suggesting a role for desmoplakin as a primary stabilizer of connexin integrity. In summary, we provide evidence for a novel mouse model, which is reminiscent of the postnatal onset of ARVC while highlighting mechanisms underlying a biventricular form of human ARVC.
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac muscle disease that is associated with a high frequency of ventricular arrhythmias leading to sudden cardiac death in young children and adults including athletes (1). Compelling evidence from human genetic studies and genetic mouse models has linked ARVC to genetic deficiencies in components of the desmosomes, which are specialized cell–cell junctions within cardiac muscle (2–11). Desmosomes are composed of transmembrane desmosomal cadherins (desmocollin 2 and desmoglein 2), armadillo proteins [plakoglobin (γ-catenin) and plakophilin 2] and the centralized plakin protein, desmoplakin, which links the junctional complex to the intermediate filament, desmin, to maintain mechanical integrity of cardiac muscle (12). Desmoplakin was the first desmosomal gene linked to autosomal dominant ARVC following the identification of a missense mutation in desmoplakin in a family of Italian descent harboring ARVC (3). These patients exhibited right ventricular dilatation and arrhythmias, causing sudden cardiac death in some of the family members (3). Both dominant and recessive ARVC-associated desmoplakin mutations have been identified in humans (3,13–16), one of which is a homozygous loss of function mutation that causes truncation and loss of the major cardiac isoform of desmoplakin (13). However, current mouse models targeting loss or mutations in desmoplakin (7,10,17) do not recapitulate the postnatal severity and pathogenic hallmarks of the disease as seen in patients with homozygous loss of function mutations in desmoplakin (13).
Although genetic mouse models harboring heterozygous loss or mutations in desmosomal genes (7–11,17,18), including desmoplakin, have paved the way to better understanding the pathophysiology of ARVC, there remain a number of open questions regarding the manifestation of the disease features and mechanisms ascribed to the postnatal onset of ARVC. For instance, fibro-fatty replacement of the ventricle is considered a hallmark of ARVC and has been widely postulated to be a key pathological factor that elicits the fatal arrhythmias; however, it is yet to be determined whether there is a direct and early mechanistic consequence of desmosomal (e.g. desmoplakin) deficiency on gap junction components and cell–cell coupling in a system independent of the fibro-fatty manifestation. This is important to determine because a recent study suggested that electrophysiological abnormalities were thought to appear prior to fibro-fatty replacement of the myocardium in human ARVC patients carrying desmoplakin mutations and heterozygous desmoplakin-deficient mice (19). Adding to the complexity of identifying common mechanisms underlying the pathogenesis of ARVC is the recent discovery of complex and heterogeneous disease patterns for ARVC, which not only include the classic right ventricle-dominant form but also left ventricle-dominant and biventricular (parallel involvement of both ventricles) forms, which have led to including the adopted term, arrhythmogenic cardiomyopathy to reference this class of diseases (20). The discovery of left ventricle-dominant and biventricular forms of ARVC arose partly from the reevaluation of previously diagnosed patients exhibiting dilated cardiomyopathy, which were now shown to exhibit classic features of ARVC, such as tachyarrhythmias (20). These observations help to not only explain the identification of desmosomal mutations in dilated cardiomyopathy patients (21) but also offer insights into the complex disease presentation of ARVC among different patients (22). Recent studies have linked mutations in the desmosomal gene, desmoplakin, to patients primarily exhibiting left ventricular-dominant and biventricular forms of ARVC (22–24), highlighting the need to reevaluate molecular mechanisms (loss of junctional plakoglobin) and pathological (fatty deposition) hallmarks in these new settings. This would be important to determine because a current theory suggests that loss of junctional plakoglobin and its nuclear translocation following desmosomal disruption is thought to suppress canonical Wnt/β-catenin signaling to drive adipogenesis specifically in secondary (right) heart field progenitor cells (7,25,26). Furthermore, there is clinical and histological evidence of fibro-fatty replacement in the left ventricle in 47–76% of ARVC cases (27,28). Loss of plakoglobin from the cardiac muscle cell–cell junction has also been proposed as a potential marker for ARVC (29); however, recent studies suggest that not all patients display loss of junctional plakoglobin (30), highlighting that there may be human ARVC subpopulations that exhibit distinct molecular hallmarks associated with ARVC disease pathogenesis. Thus, developing genetic mouse models, which distinguish the postnatal onset of these forms of ARVC, could serve as important tools to help uncover mechanisms that commonly and specifically underlie these disease subtypes as well as a platform to uncover specific treatment modalities.
In the current study, we present a novel mouse model of ARVC that is reminiscent of the postnatal onset of a biventricular form of human ARVC at the histological, physiological and electrophysiological levels. We identify the cellular origin of a specific region of the fatty deposition in DSP-cKO hearts by showing that subepicardial fat deposition originates from a cardiac muscle lineage and that the mechanisms are independent of junctional loss of plakoglobin as we show retained plakoglobin at cell–cell junctions in DSP-cKO hearts. We also provide evidence for desmoplakin as a direct stabilizer of connexin integrity, which includes the demonstration that loss of desmoplakin has direct mechanistic consequences on connexin 43 levels and phosphorylation, leading to conduction abnormalities prior to the molecular dissociation of the mechanical junction complex within a system that is independent of the fibro-fatty manifestation observed in ARVC.
Characterization of cardiomyocyte-specific desmoplakin knockout mice
To determine the homozygous loss-of-function effects of desmoplakin in the heart, desmoplakin-floxed mice (DSPflox/flox) were crossbred with the well-established cardiomyocyte-specific ventricular myosin light chain-2-Cre recombinase (MLC2v-Cre) knock-in mice (31–34). A breeding strategy was utilized to generate ventricular cardiomyocyte-specific homozygous DSP-cKO [DSPflox/flox:MLC2vCre(+)], heterozygous DSP-cKO [DSPflox/+: MLC2vCre(+)] as well as littermate controls [DSPflox/flox:MLC2vCre(−)] (Fig. 1A). Homozygous DSP-cKO were viable and born at expected Mendelian ratios (data not shown); however, they displayed a striking susceptibility to postnatal lethality from the age of 1 month onwards (Fig. 1B). Kaplan–Meier survival analyses of litters revealed that 50% of homozygous DSP-cKO mice die within 2 months of age, eventually resulting in 100% mortality by the age of 5 months compared with littermate controls (Fig. 1B) as well as heterozygous DSP-cKO mice, which display no mortality at this stage (data not shown). As a result, all subsequent studies on DSP-cKO mice were performed on homozygous DSP-cKO mice, which were designated as DSP-cKO.
To determine the efficiency of desmoplakin knock-down in DSP-cKO hearts in vivo, we assessed desmoplakin protein expression in purified ventricular cardiomyocytes from DSP-cKO and littermate control hearts at 3 weeks of age. A significant 83 ± 5.6% (n = 3) and 87 ± 4.3% (n = 3) reduction in desmoplakin protein levels was observed in both total and insoluble (intercalated disc-enriched) fractions, respectively, in DSP-cKO cardiomyocytes when compared with littermate controls (Fig. 1C). Immunofluorescence staining analyses of cardiac sections highlighted the dramatic loss of desmoplakin within intact intercalated discs (white arrows), which is delineated by the retained presence of N-cadherin, in DSP-cKO hearts, when compared with littermate hearts (Fig. 1D). The retained desmoplakin expression in DSP-cKO cardiac sections could either reflect retained desmoplakin expression in cardiomyocytes not targeted by MLC2v-Cre (as it is not 100% efficient). It could also reflect desmoplakin expression in non-muscle cells, because desmoplakin expression is also reported in non-muscle cell types (e.g. endothelial cells) (35).
DSP-cKO hearts display early ultrastructural defects in desmosomal integrity
As desmoplakin is localized to the desmosomes within the intercalated disc, transmission electron microscopy was performed on DSP-cKO mouse hearts to examine the integrity of these structures at the ultrastructural level. Representative micrographs from DSP-cKO hearts (Fig. 2C and D) revealed specific defects in intercalated disc integrity as early as 2.5 weeks, which included rupture of desmosomes and loss of myocyte–myocyte adhesion between neighboring cells, as evidenced by the intercellular gaps, when compared with littermate control hearts (Fig. 2A and B). Similar ultrastructural findings have been observed in human skin of patients with DSP mutations and human hearts from ARVC patients (36,37). It should be noted that in very severely affected regions, DSP-cKO hearts also displayed some loss of muscle architecture coupled with the observed adhesion defects as evidenced by the intercellular gaps being associated with loss or thickening of Z-lines (Fig. 2C and D). These results altogether suggest that defects in desmosomal structural integrity is one of the primary causes of dysfunction in this model.
DSP-cKO mice display a severe form of ventricular cardiomyopathy similar to a biventricular form of arrhythmogenic right ventricular cardiomyopathy
To evaluate whether DSP-cKO mice recapitulate key clinical features of human ARVC, we performed morphological, immunohistochemical and functional analyses of DSP-cKO versus littermate control hearts. At 4 weeks of age, morphological analyses of DSP-cKO hearts revealed that they were grossly enlarged in size, when compared with littermate controls (Fig. 3A). Further histological analyses at this age showed that DSP-cKO mice displayed significant right and left ventricular chamber dilation when compared with controls; however, it was evident that the extent of dilation and wall thinning was more pronounced in the right ventricle (Fig. 3B). DSP-cKO hearts also displayed extensive fibrosis, which could be detected in the right and left ventricles as well as septum (Fig. 3C). However, the effects on fibrosis were exclusive to the ventricular chambers as no fibrosis could be detected in the enlarged atria of DSP-cKO hearts, similar to controls (Fig. 3C). DSP-cKO hearts also displayed an increase in TUNEL-positive cells, which was found in the cardiac mid-wall but more pronounced in the subepicardial region of the heart (Supplementary Material, Fig. S1), suggesting that apoptotic cell death is a contributing factor to the massive increase in collagen deposition. Oil Red O staining of cardiac sections from DSP-cKO right hearts at 6 weeks of age further revealed pronounced pockets of fat deposition within the subepicardium (Fig. 3D). Punctate staining of Oil Red O could also be observed in the mid-wall region of DSP-cKO hearts (Fig. 3D), suggesting that there is a gradient of fat deposition decreasing toward the endocardium. Similar patterns of fibro-fatty deposition have been reported in human hearts from ARVC patients (27). These features of fatty deposition were also observed in the left ventricle of DSP-cKO hearts (data not shown). Fat deposition could not be detected in control hearts (Fig. 3D). To determine the cellular origin of the fat deposition in DSP-cKO hearts at postnatal stages, homozygous DSP-cKO mice were cross-bred with Cre-inducible R26RtdTomato reporter mice (38), to generate DSPflox/flox:MLC2vCre(+):R26RtdTomato mice (Fig. 3E). R26RtdTomato reporter mice specifically express Tomato Red (TomRed) when exposed to Cre recombinase. Thus, expression of TomRed in the background of DSPflox/flox: MLC2vCre(+) mice genetically marks all MLC2v-positive (+) cells in this model, from the earliest stage when MLC2vCre is active (embryonic day 7.75) (34), thus marking cells of a cardiomyocyte muscle lineage (Fig. 3E). Similar to Oil Red O staining results, we show that DSP-cKO hearts contain two pools of lipid-positive cells. We show strong co-localization of perilipin, a marker of mature lipid droplets, with TomRed, specifically within the subepicardial layer of postnatal DSP-cKO hearts (Fig. 3F), suggesting that the subepicardial fat deposition in DSP-cKO hearts originates from a cardiac muscle lineage. However, our studies also show that punctate perilipin staining could also be observed in cells within the mid-wall that are TomRed negative (Fig. 3F), suggesting that non-muscle cells may also contribute to the lipid deposition observed in DSP-cKO hearts.
To determine how the morphological and histological defects in DSP-cKO hearts impact cardiac function, we utilized a noninvasive magnetic resonance imaging (MRI) technique to simultaneously measure right and left ventricular dimensions, wall thickness and function (39). This is important because the crescentic shape and size of the right ventricle has provided challenges in its imaging as well as reliable functional assessment in the mouse using echocardiography (40). To determine the feasibility of MRI as a method to simultaneously assess anatomical changes in the left and especially right ventricle in a mouse model of heart disease, we obtained a series of MRI (short axis) images at end-diastole and end-systole and, from the segmented endocardial borders, created three-dimensional (3D) biventricular models of each heart (Fig. 4A). Despite the unique shape and size of the right ventricle, we provide evidence of the feasibility of using 3D reconstruction methods to accurately quantify the extensive remodeling in DSP-cKO hearts, as evidenced by the enlargement and extension of the right ventricle over the left ventricle (Fig. 4A). These anatomical changes were reflective of the gross morphological changes observed in DSP-cKO versus control hearts via histology (Fig. 3B). Assessment of right and left ventricular end-diastolic and end-systolic volumes, as well as ejection fraction was subsequently performed on DSP-cKO and littermate control mice. Representative long-axis MRI images from DSP-cKO versus littermate control hearts at end-systole further highlight the abnormalities in right and left ventricular dimensions in DSP-cKO mice (Fig. 4B). No significant changes in heart rate and body weight were observed in DSP-cKO mice when compared with littermate controls at this stage. However, DSP-cKO hearts displayed significantly (i) increased end-diastolic and end-systolic volumes, (ii) reduced ejection fraction as well as (iii) reduced wall thickness in both the right and left ventricle when compared with littermate controls (Fig. 4C). The significant changes in cardiac dimensions, wall thickness and function in DSP-cKO hearts were indicative of biventricular dilation and depressed systolic function. These results show that the DSP-cKO model recapitulates cardiac features similar to a form of ARVC where both right and left ventricles are affected. These results are consistent with the recent discovery of left ventricle-dominant and biventricular forms of human ARVC that are primarily associated with desmoplakin mutations (23,24,41). Furthermore, findings from a human patient harboring a recessive desmoplakin mutation that results in loss of desmoplakin, which mimics the loss of desmoplakin in our DSP-cKO model, was shown to lead to a form of ARVC impacting both right and left ventricles (13).
DSP-cKO hearts exhibit ventricular arrhythmias that can be exacerbated with exercise and catecholamine stimulation
As ventricular arrhythmias are the leading cause of death in ARVC patients, we examined whether conduction defects might account for the sudden death exhibited by DSP-cKO mice. Surface ECG analysis performed on anesthetized DSP-cKO mice showed that they displayed no significant differences in heart rates (control: 446 ± 23 (n = 4) versus DSP-cKO: 478 ± 50 (n = 3) bpm) and PR intervals (Fig. 5A) when compared with littermate control mice. However, significantly increased QRS intervals were observed in DSP-cKO mice when compared with littermate controls, suggestive of ventricular depolarization delay (Fig. 5A). ECG tracings from DSP-cKO mice also revealed spontaneous ectopic premature ventricular contractions (PVC) that were not present in littermate control mice (Fig. 5B). To more closely examine the origin of the spontaneous PVCs, we performed optical mapping on DSP-cKO and littermate control hearts. Langendorff-perfused hearts were mapped while spontaneously depolarizing. Optical maps from control hearts revealed an action potential propagation pattern consistent with fast conduction through the Purkinje network [Fig. 5C (left panel)]. However, DSP-cKO mice revealed spontaneous ectopic multifocal premature beats originating from either the left (Fig. 5C [middle panel)] or right [Fig. 5C (right panel)] ventricles, consistent with the observed PVCs in DSP-cKO hearts in vivo. Exercise-induced catecholamine release is a major trigger that has been linked to the onset of ventricular arrhythmias and sudden death in athletes harboring ARVC (42,43). To determine whether exercise and catecholamine stimulation influence the number of ventricular arrhythmias observed in DSP-cKO mice, we subjected mice to treadmill exercise in the absence and presence of low and high doses of epinephrine. We show that the numbers of PVC per second were significantly increased in DSP-cKO mice when exposed to exercise and epinephrine stimulation (Fig. 5D). Interestingly, high doses of epinephrine in combination with exercise could also induce sudden cardiac death in DSP-cKO mice that was not observed in littermate controls (Fig. 5D).
DSP-cKO hearts exhibit right ventricular conduction defects associated with loss of connexin 40 expression
Growing evidence points to a key role for anchoring cell–cell junction proteins, such as desmosomes, in cells of the cardiac conduction system (44). Given that loss of desmoplakin leads to ventricular arrhythmias, we sought to examine whether desmoplakin plays a specific role in the ventricular conduction system. Co-staining of cardiac sections with desmoplakin and hyperpolarization-activated cyclic nucleotide-gated cation channel-4 (HCN4), a marker for the cardiac conduction system, in control mice revealed their strong co-localization within the His bundle (HB) and Bundle Branches (RBB and LBB) (Fig. 6A), suggesting that desmoplakin is expressed in cardiomyocytes of the ventricular conduction system. To determine whether loss of desmoplakin has specific functional effects on the ventricular cardiac conduction system, we optically mapped DSP-cKO and littermate control hearts subsequent to atrial pacing. We show that littermate control hearts display rapid action potential depolarization throughout the left and right ventricles following atrial pacing (Fig. 6B). In stark contrast, we show evidence of delayed conduction to the right ventricle in DSP-cKO hearts following atrial pacing, consistent with right bundle branch block (Fig. 6B). Quantitative analyses of action potential propagation in DSP-cKO versus littermate control hearts revealed significantly prolonged activation time, action potential durations (APD20, APD50 and APD80) as well as action potential dispersion (APD80disp) in DSP-cKO hearts when compared with littermate controls (Fig. 6C). These results are consistent with observations in human ARVC patients, who display right ventricular conduction defects, including incomplete or complete right bundle branch block (45). Studies have associated molecular loss of the gap junction protein, connexin 40, with right bundle branch blocks in genetic mouse models (46,47). We show, through co-staining of cardiac sections with connexin 40 and HCN4, that connexin 40 expression is dramatically downregulated in cardiomyocytes of the ventricular conduction system in DSP-cKO hearts, when compared with littermate controls (Fig. 6D). No significant differences in connexin 40 transcript levels could be detected in DSP-cKO versus control hearts, suggesting that the downregulation of Cxn40 is at the post-transcriptional level (Supplementary Material, Fig. S2). These results highlight a direct and important molecular link between desmoplakin and gap junction proteins in the heart and suggest that loss of this connection underlies the right ventricular conduction defects (right bundle branch block) observed in our model.
DSP-cKO hearts exhibit electrical wavefront propagation defects associated with loss of connexin 43 expression
Alterations in geometric patterns of action potential propagation have been associated with arrhythmogenic diseases (48). Thus, to further determine the underlying cause of the ventricular arrhythmias in DSP-cKO hearts, we sought to assess the epicardial pattern of action potential propagation in DSP-cKO versus littermate control hearts, following ventricular epicardial pacing. Optical mapping studies revealed that littermate controls displayed smooth wavefront propagation patterns across the entire epicardium, whereas DSP-cKO hearts displayed pronounced conduction breaks in wavefront propagation within the epicardium (Fig. 7A). Although no significant differences in overall activation times were observed between mice, we show significant changes in negative wavefront curvature in DSP-cKO versus littermate control hearts (Fig. 7A). These results highlight localized regions of conduction slowing in DSP-cKO hearts that is consistent with a highly arrhythmogenic substrate.
To determine how primary loss of desmoplakin alters the molecular components within the intercalated disc in DSP-cKO hearts, we assessed the expression of desmosomal, fascia adherens and gap junction proteins in whole ventricles from DSP-cKO and littermate control mice at 6 weeks of age (Fig. 7B). A significant 91.2 ± 7.3% (n = 3) reduction in desmoplakin protein expression was observed in DSP-cKO versus control hearts (Fig. 7B). In addition, specific changes in the levels of desmosomal components were observed, which included a significant reduction in desmoglein-2 [70.4 ± 8.2% (n = 3)], desmocollin-2 [56.3 ± 6.4% (n = 3)] and plakophilin-2 [69.7 ± 4.2% (n = 3)] in DSP-cKO whole ventricles compared with controls (Fig. 7B). Interestingly, no significant differences in the levels of fascia adherens proteins, plakoglobin, β-catenin and N-cadherin were observed in DSP-cKO ventricles (Fig. 7B). Reduced levels of plakoglobin at the intercalated disc have been suggested as a common feature in ARVC patients and a potential marker for ARVC (29). To establish whether plakoglobin is lost from the intercalated disc in response to loss of desmoplakin, we specifically assessed the expression level of plakoglobin in the insoluble (intercalated disc-enriched) fractions of DSP-cKO hearts (Fig. 7C) using two independent plakoglobin antibodies. No significant differences in plakoglobin levels were observed in the insoluble protein fractions of DSP-cKO hearts compared with controls (Fig. 7C), similar to what was observed in total protein levels (Fig. 7B). Immunofluorescence staining analyses of cardiac sections further demonstrated that expression of plakoglobin was retained at the intercalated disc with the absence of desmoplakin in DSP-cKO hearts comparable with littermate controls (Fig. 7C), suggesting that the loss of plakoglobin may not be a striking molecular feature of biventricular forms of ARVC resulting from homozygous loss of desmoplakin. Most striking were the effects on gap junctions as significant reductions in levels of connexin 43 [87.2 ± 4.8% (n = 3)] and phosphorylated connexin 43 [S368; 94.3 ± 5.7% (n = 3)] were observed in DSP-cKO ventricles compared with controls (Fig. 7B). Immunofluorescence staining analyses of cardiac sections further affirmed that expression of connexin 43 is downregulated in cardiomyocytes of DSP-cKO hearts when compared with littermate controls (Supplementary Material, Fig. S3). Taken together these results further highlight crosstalk between the desmosomes (desmoplakin) and gap junctions at the intercalated disc in ventricular cardiac muscle that is lost with the loss of desmoplakin in DSP-cKO hearts.
Loss of desmoplakin in neonatal ventricular cardiomyocytes results in primary loss of connexin 43 levels, phosphorylation and function
To establish whether the loss of connexin 43 in DSP-cKO hearts is a primary or secondary consequence of loss of desmoplakin, we evaluated the dose-dependent effects of loss of desmoplakin (AdCre) versus controls (AdlacZ) on key gap junction (connexin 43), desmosomal (plakophilin-2) and fascia adherens (N-cadherin) proteins in neonatal cardiomyocyte cultures generated from DSP-floxed mice (Fig. 8A). We show that the AdCre virus is efficient in causing a dose-dependent reduction in desmoplakin protein expression in neonatal cardiomyocytes, when compared with AdLacZ controls (Fig. 8B). Western blot analysis further demonstrated that a dramatic downregulation of total and phosphorylated (S368) connexin 43 protein levels is observed with the loss of desmoplakin in cardiomyocytes in vitro (Fig. 8B), consistent with the reduction found in whole hearts from DSP-cKO mice (Fig. 7B). We further show that the level of downregulation in total and phosphorylated connexin 43 (i) follows the dose-dependent knock-down of desmoplakin in cardiomyocytes and (ii) is found independent of any molecular dissociation of the desmosomal and fascia adherens junction complex, as evidenced by the robust levels of plakophilin-2 and N-cadherin in neonatal cardiomyocytes following desmoplakin knock-down (Fig. 8B). These results suggest that loss of desmoplakin has primary consequences on connexin 43 levels and the gap junction complex.
To determine the functional consequences of primary loss of desmoplakin levels on cardiomyocyte conduction, we optically mapped cardiac action potential propagation in aligned neonatal control (AdlacZ) and desmoplakin-deficient (AdCre) mouse ventricular myocyte cultures (Fig. 8C) (49–51). Activation maps from cardiomyocyte cultures were evaluated for area and distance of action potential propagation along different paths (longitudinal, transverse and arbitrary angle) from the pacing electrode (Fig. 8D). Representative optical maps from AdCre- and AdLacZ-infected cardiomyocytes indicate that both the total area and distance of linear action potential propagation prior to conduction block are markedly less in desmoplakin-deficient versus control cardiomyocytes (Fig. 8E). Quantitative analyses of the distance of continuous propagation in both groups revealed that desmoplakin-deficient cardiomyocytes display significantly reduced distances of continuous propagation across longitudinal, transverse and arbitrary angles when compared with control cardiomyocytes (Fig. 8F). Similarly, desmoplakin-deficient cardiomyocytes displayed significantly reduced areas of continuous propagation compared with control cells, indicative of an increased incidence of conduction block in confluent monolayers (Fig. 8G), suggesting that loss of desmoplakin has a direct impact on cardiac conduction, consistent with loss of connexin 43 function in a setting independent of fibro-fatty manifestation associated with ARVC (Fig. 9).
The classic view of ARVC is that it is a right ventricular cardiomyopathy with left ventricular involvement being more secondary with disease progression (28,52). However, more recent long-term follow-up studies of ARVC patients and family members via comprehensive ECG and CMR imaging have identified complex and heterogeneous disease patterns for ARVC, which include left ventricle-dominant and biventricular forms of the disease (20). In addition, recent studies have linked mutations in the desmosomal protein, desmoplakin, to patients primarily exhibiting left ventricular-dominant and biventricular forms of ARVC (22–24), further suggesting that the mechanisms underlying the pathogenesis of specific ARVC subtypes may be dependent on the desmosomal mutation carried by the patient. We provide evidence of a novel mouse model of ARVC (DSP-cKO) resulting from homozygous loss of desmoplakin in cardiomyocytes, which recapitulates the postnatal disease features of a biventricular form of ARVC. By exploiting MRI and 3D reconstruction methods, we have been able to accurately depict the extensive morphological remodeling that occurs in DSP-cKO hearts and show that loss of desmoplakin leads to dramatic changes in both right and left ventricular volumes and function resulting in heart failure and premature death. Most importantly, our mouse model recapitulates findings in a pediatric patient harboring a homozygous loss-of-function mutation in desmoplakin, which was shown to lead to a complete loss of the major isoform of desmoplakin in the heart, resulting in the postnatal onset of a severe biventricular form of ARVC, also resulting in heart failure and premature death (13), thus mimicking the postnatal severity and pathogenic hallmarks as seen in these patients.
The homozygous DSP-cKO mouse model further displays features that broadly fit major criteria proposed by the Taskforce for Diagnosis of Classic Right Dominant Human ARVC (53). Specific features related to these criteria include the appearance of fibro-fatty replacement within the right ventricular free-wall and QRS prolongation. However, we also show increased susceptibility to exercise and catecholamine-induced arrhythmias in DSP-cKO mice, suggesting that exercise can exacerbate the ARVC phenotype, closely mimicking the fact that ARVC is a common cause of sudden death associated with exertion in young athletes (54). Through the development of this novel model of biventricular ARVC, we now have the ability to uncover postnatal mechanisms that are common and unique to the classic RV dominant form of ARVC but also highlight the need to broaden task force criteria to encompass features of left-dominant and biventricular forms of ARVC.
Recent efforts have focused on understanding the molecular and cellular mechanisms underlying the development of fibro-fatty tissue replacement in ARVC. We demonstrate using MLC2v-Cre and the Tomato Red reporter line that DSP-cKO hearts harbor subepicardial fat deposition that originates from a cardiomyocyte lineage, highlighting the propensity of cardiac progenitor cells to transdifferentiate into adipocytes within our ARVC model. These results support previous studies suggesting differentiation of cardiac progenitors into adipocytes in ARVC (25,26) but are likely independent of a mechanism that involves a switch to adipogenesis because of suppressed canonical Wnt/β-catenin signaling as a result of junctional loss and nuclear translocation of plakoglobin (7,25), because no junctional loss or differences in levels of plakoglobin were observed in our DSP-cKO hearts versus littermate control hearts. The predominant and unique fatty deposition at the subepicardium in our ARVC model, which is similar to what is observed in ARVC patients (27), suggests that signals evoked to the subepicardium may be important to consider when understanding mechanisms regulating myocardial fat accumulation. Recent studies suggest that the epicardium has the capacity to signal to adjacent cardiac muscle during development and in the setting of injury that these cells have the capacity to replenish lost cardiomyocytes by contributing to multiple non-cardiac muscle cell types repopulating the infarcted heart (55,56). Thus, future studies directed at understanding whether these signals are affected in ARVC may help propel the field in understanding the mechanisms underlying the fatty deposition associated with ARVC.
The discovery that homozygous loss of desmoplakin leads to a biventricular form of ARVC encompassing ventricular arrhythmias and conduction defects in the absence of loss of junctional plakoglobin, at stages similar to and after the appearance of fibro-fatty replacement further suggests that these mechanisms do not play a pivotal causal role in the pathogenesis of ARVC in our model. Independent studies suggest that suppressed canonical Wnt/β-catenin signaling is also not a feature in mice deficient in plakoglobin that develop a biventricular form of ARVC with spontaneous ventricular arrhythmias and slow conductivity occurring with disease progression (9), suggesting that signaling pathways associated with loss of junctional plakoglobin may not be a predominant feature of left and biventricular forms of ARVC. In support of this, recent studies in humans suggest that not all ARVC patients display loss of junctional plakoglobin (30); thus, future studies determining whether there are distinct molecular hallmarks for biventricular and left-dominant forms of ARVC in humans may expand our diagnostic criteria and therapies for human ARVC.
Aberrant expression and distribution of connexin 43 referred to as gap junction remodeling has been identified as a common feature of ARVC (29) and may adversely affect cardiomyocyte electrical coupling and contribute to conduction defects and slowing observed in ARVC. However, the molecular mechanisms underlying electrical uncoupling and the development of conduction abnormalities remain poorly understood. Our studies highlight a direct link between desmosomal protein deficiency and connexin remodeling, which may explain the varying clinical presentation of ARVC. We show that the downregulation of connexin 43 is not because of the massive loss of cardiomyocytes as we show that there is specific downregulation of connexin 43 in cardiomyocytes of DSP-cKO hearts. These studies are reinforced as we show that a dose-dependent loss of desmoplakin has direct mechanistic consequences on connexin 43 levels and phosphorylation, leading to conduction abnormalities prior to the molecular dissociation of the mechanical junction complex in a system independent of the fibro-fatty manifestation observed in ARVC. These findings suggest that the histological (fibro-fatty replacement) changes are not necessary to generate an arrhythmogenic substrate but highlight a molecular trigger that may be important for arrhythmias observed in the concealed phase of ARVC. We further show that apoptotic cell death is a contributing factor to the massive increase in collagen deposition observed in DSP-cKO hearts and that these combined histological defects could potentially contribute to worsening of arrhythmias in ARVC, as evidenced by the pronounced conduction breaks in wavefront propagation in DSP-cKO hearts harboring fibro-fatty deposition. Furthermore, we highlight a novel link between desmoplakin and gap junction remodeling at the post-transcriptional level in the ventricular cardiac conduction system, as we show that the loss of connexin 40 protein may underlie the ventricular conduction defects (right bundle branch block) observed in our ARVC model. These studies support recent findings of connexin defects in ARVC patients (57), while extending previous findings that suggest that electrophysiological abnormalities associated with reduced connexin 43 may appear prior to fibro-fatty replacement in the myocardium of human ARVC patients carrying desmoplakin mutations and heterozygous desmoplakin-deficient mice (19). This notion of molecular crosstalk between desmosomal and gap junction proteins has been supported by previous studies that demonstrate that connexin 43 can coexist in macromolecular complexes with desmosomal proteins, such as plakophilin-2 and desmocollin-2a (58,59). Furthermore, desmosomal deficiencies via ARVC causing mutations or loss-of-function studies were shown to abolish this crosstalk (58,59). Our studies also suggest that posttranslational modifications in connexin 43 at S368 are dramatically affected with desmoplakin deficiency. Although the biological significance of this phosphorylation site remains to be determined, previous studies have linked the loss of connexin 43 (S368) phosphorylation and signaling to reduced conduction velocity and resulting ventricular arrhythmias in isolated rat hearts (60), which suggests the possibility that it could be an important contributor to the arrhythmias observed in our ARVC model. Thus, future studies aimed at identifying domains and phosphorylation sites within connexin 43 that mediate this crosstalk could allow for early detection of ARVC prior to the loss of connexins and the occurrence of structural remodeling at the desmosome.
In summary, we present a novel mouse model that is reminiscent of the postnatal onset of a biventricular form of human ARVC at the histological, physiological and electrophysiological levels, as well as highlight underlying molecular changes that directly contribute to the observed electrophysiological abnormalities. The DSP-cKO model has the potential to serve as an invaluable tool for understanding the molecular mechanisms underlying ARVC subclasses, and identifying new diagnostic markers and therapeutic targets for ARVC.
Generation of cardiomyocyte-specific desmoplakin knockout mouse lines
Desmoplakin-floxed (Dspflox/flox) mice (kind gift from Dr. Elaine Fuchs, Rockefeller University) and heterozygous ventricular myosin light chain-2 Cre [MLC2v(cre+)] mice (kind gift from Dr. Ju Chen, University of California-San Diego) have been previously characterized (34,61) and were used to generate cardiomyocyte-specific desmoplakin knockout (DSP-cKO) mice, as shown in Figure 1A. DSP-cKO mice and their control littermates were kept in a congenic C56Bl/6 background. Survival of DSP-cKO mice (n = 15) and their control littermates (n = 15) was calculated using a Kaplan–Meier survival curve. For fat lineage tracing studies, DSPflox/flox:MLC2v(cre+) mice were crossed with a well-established Ai14 heterozygous reporter (strain: B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato/Hze/) mouse line (Jackson Laboratory, USA) (38), as illustrated in Figure 3E. Reporter mice were kept in a mixed genetic background of C56Bl/6 and Black Swiss. All animal procedures were in full compliance with the guidelines approved by the University of California–San Diego Animal Care and Use Committee.
Mouse cardiac myocyte isolation
Ventricular cardiac myocytes were isolated from adult mouse hearts and neonatal (1–2 days old) DSP-floxed mouse hearts as previously described (62,63).
Protein analysis
Total protein was isolated from cardiomyocytes and ventricles as previously described (31). Insoluble (intercalated disc-enriched) protein extracts were isolated from cardiomyocytes and ventricles using the Triton X-100 method as previously described (10). Immunodetection of desmoplakin (mouse, 1:1000, AbDSerotec), desmoglein-2 (mouse, 1:100, Fitzgerald), desmocollin-2 (rabbit, 1:1000, Fitzgerald), plakophilin-2 (mouse, 1:1000, Fitzgerald), plakoglobin (mouse, 1:1000, Sigma; goat, 1:1000, Santa Cruz Biotechnology), β-catenin (mouse, 1:1000, BIOMOL International), N-cadherin (rabbit, 1:1000, Abcam), connexin 43 (rabbit, 1:1000, Invitrogen), S368 connexin 43 (rabbit, 1:1000, Invitrogen), glyceraldehyde 3-phosphate dehydrogenase (mouse, 1:2000, Santa Cruz Biotechnology) and α-tubulin (mouse, 1:2000, Sigma) was performed as previously described (31).
Immunofluorescence microscopy
Adult heart cryosections and neonatal ventricular cardiomyocytes were fixed in 100% acetone at −20°C and stained with primary antibodies against desmoplakin (mouse, 1:1000, AbDSerotec), N-cadherin (rabbit, 1:100, Abcam), perilipin (rabbit, 1:100, Cell Signaling), hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (rat, 1:100, Abcam), connexin 40 (rabbit, 1:100, Invitrogen), Cypher (rabbit, 1:100, kind gift from Ju Chen, UCSD), plakoglobin (mouse, 1:100, Sigma; goat, 1:100, Santa Cruz Biotechnology), connexin 43 (rabbit, 1:100, Invitrogen) and sarcomeric α-actinin (mouse, 1:200, Sigma). Sections and cells were subsequently stained with fluorescently labeled secondary antibodies (1:100, Jackson ImmunoResearch Inc.) and Hoescht nuclear stain in cases where indicated, followed by imaging using confocal microscopy (Olympus FV1000).
Electron microscopy
Cardiac ventricles were processed for electron microscopy, and images were captured with a Zeiss 10 electron microscope as previously described (62).
Histological analysis
Hearts from 3-week-old mice were perfused in relaxation buffer consisting of 100 mm KCl in phosphate-buffered saline followed by perfusion fixation with 4% paraformaldehyde solution and cryopreservation. Whole-heart (8 μM) cryosections were obtained and stained with Masson's trichrome (Sigma–Aldrich, St. Louis, MO, USA) to detect collagen deposition and Oil Red O (Sigma–Aldrich) to detect lipid deposition according to standard procedures.
Magnetic resonance imaging
In vivo MRI cardiac imaging was performed on a 7T horizontal-bore MR scanner. A 1.9-mm custom-built dual quadrature-driven transverse electromagnetic mode coil was used for transmission and reception of the RF signal. High-resolution bright blood MRI experiments were conducted using an ECG-triggered Fast Low Angle SHot (FLASH) Gradient Echo (GE) pulse sequence tailored for murine imaging as previously described (64), and time series averaging was employed with a FOV of 25 mm2 and data matrix of 1282 to yield a sub-millimeter spatial resolution. Equatorial frames containing the largest and smallest chamber diameters were selected to define the end-diastolic and end-systolic times, respectively, with a temporal resolution of 5 ms. For MRI image analyses, four two-dimensional (2D) contours were manually planimetered for each heart at end-diastole and end-systole (LV and RV epi and endo) using image analysis software (ImageJ, NIH). Contour areas were calculated, multiplied by the slice thickness and summed together to approximate calibrated volumes. LV mass was obtained by subtracting endocardial from epicardial volumes and scaling by the specific gravity of myocardial tissue (1.055 g/ml). Stroke volume was defined as LVEDV–LVESV whereas ejection fraction was calculated as 100 × SV/LVEDV. Wall thickness was measured directly from the 2D short-axis images. For 3D reconstruction, endocardial and epicardial contours were fitted to prolate spheroidal meshes with 64 volume elements using linear least-squares minimization (39). Wall and chamber volumes were determined from the mesh, and wall volume multiplied by myocardial density gave the wall mass.
Surface ECG
Adult (4 weeks old) mice were anesthetized with 1% isoflurane. Needle electrodes (30 gauge) were inserted subcutaneously into right forearm and left leg. ECG signals were amplified using Warner Instruments DP-304 Differential Amplifier bandpass filtered between 0.1 and 100 Hz. Signal was further filtered through a Quest Scientific HumBug 50/60 Hz Noise Eliminator and digitized at 3000 Hz.
Whole-heart optical mapping
Optical mapping studies were conducted as previously described (65). Briefly, hearts were isolated from 4-week-old mice and Langendorff-perfused with a heated, oxygenated modified Krebs–Henseleit solution. The heart was submersed in heated perfusate in an optical bath chamber, and a volume-conducted ECG was recorded throughout the experiment. The voltage-sensitive dye di-4-ANEPPS (1 mL bolus, 26 µM) was injected into the perfusion line. Two 470-nm wavelength LED lamps illuminated the ventricular epicardium, and fluorescence was passed through a 610-nm long-pass filter, focused with a fast video lens (F/0.95; Navitar) and recorded by a 12-bit charge-coupled device camera (64 × 64 pixels, 950 frames/s, CA-D1–0128T; DALSA). During recording of paced beats, the atrium or ventricle was stimulated epicardially with a unipolar platinum electrode at a constant current (×1.5 threshold) using a digital stimulator (DS8000; WPI). To eliminate motion artifacts, 15 mm of the electromechanical uncoupler 2,3-butanedione monoxime was perfused. Data were analyzed with custom software in MATLAB. Activation time was identified as at the maximum slope of the action potential upstroke, and action potential duration was calculated at the 20, 50 and 80% levels of repolarization (APD20, APD50 and APD80, respectively). Dispersion of repolarization was defined as the standard deviation of action potential duration (APD) values over the entire anterior epicardium. Disorganization of the wavefront during ventricular epicardial pacing was quantified by calculating the curvature of the activation map as previously described (66). A greater negative curvature indicates increased heterogeneous areas of local conduction block.
Telemetry ECG
Conscious telemetry using an implantable wireless transmitter/receiver system (Datasciences/Ponemah) was performed on 4-week-old mice as previously described (65). For all implantation procedures, the mice were anesthetized with ketamine (100 mg/kg)–xylazine (5 mg/kg) and monitored continuously. A radio transmitter (ETA- F20; Datasciences) was inserted subcutaneously at the subscapular thoracic region and secured via sutures to the skin. Two leads were tunneled through the subcutaneous space cranially and caudally. Transmitters were detected by receiver platforms placed directly underneath the animal's cage. The receiver platforms were linked to an immediately adjacent computer. Dataquest ART 2.0 data acquisition software (Datasciences) was used for digitization of the signal (at a sampling rate of 1000 Hz) and for on-line display of the electrocardiogram as well as for data storage onto a hard disk. Studies were recorded continuously beginning 96 h following recovery from the implantation. Exercise testing was performed on mice as previously described (67). Following treadmill acclimatization, mice were exercised for at least 45 min on a horizontal treadmill at incrementally faster running speeds (from 12 to 40 m/min), until exhaustion defined by the inability to sustain exercise at 34 m/min or greater. At this point, mice received a high (2 mg/kg) or low (0.5 mg/kg) epinephrine dose via intraperitoneal injection.
Adenoviral infection of cardiomyocyte cultures
Adenovirus vectors containing the lacZ (AdLac-Z) and Cre recombinase (AdCre) cDNAs were prepared (UCSD Viral Vector Core) and used at multiplicity of infections 1.6–12.5 pfu/cell. Neonatal ventricular cardiac myocytes isolated from DSP-floxed mice were plated on laminin and infected with AdLacZ and AdCre, 2 days following isolation, as previously described (68), and as illustrated in Figure 8A. Viral particle carrying medium was changed from cells after 24 h, and cells were subsequently maintained in a media consisting of DMEM, M199, 5% fetal bovine serum, 10% horse serum and 1% penicillin/streptomycin/glutamine for the duration of the study. Cardiomyocytes were analyzed 4 days post-infection.
Cardiomyocyte plating on micropatterned substrates for optical mapping
For optical mapping and immunofluorescence microscopy of aligned cells, a microstructured cell culture environment was created to mimic the myocardium, drawing from earlier works in soft photolithography (50,51,69). Briefly, master molds were patterned on silicon wafers using SU-8 2005-negative photoresist (MicroChem Corp., Newton, MA, USA) and a custom designed photomask (Advance Reproductions Corp., North Andover, MA, USA). These molds were treated with (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane (United Chemical Technologies, Inc., Bristol, PA, USA) before the first use to ensure a non-adhesive surface. Polydimethylsiloxane (PDMS) cell culture substrates were molded from the masters by spin-coating Sylgard 186 silicone elastomer (Dow Corning Corp., Midland, MI, USA) prepared at a 10 parts base-to-1 part curing agent ratio, degassed and thermoset 30 min at 70 degrees. After continuing thermosetting at room temperature overnight, the microstructured PDMS was removed from the molds and mounted into custom cell culture chambers described previously (49–51). The resulting PDMS cell culture substrates present 10-µm wide troughs separated by 5-µm-high and 10-µm-wide walls. Laminin derived from murine sarcoma basement membrane was adsorbed onto the entire microstructured cell culture surface using 350-nm wavelength radiation for 10 min (Sigma–Aldrich). Neonatal ventricular cardiac myocytes isolated from DSP-floxed mice were plated on these micropatterned substrates and infected with adenoviruses, as described earlier.
Electrical activation was detected in cardiomyocytes using the transmembrane voltage-sensitive fluorescent probe di-8-ANEPPS (Molecular Probes, Life Technologies, Grand Island, NY, USA). Dye was loaded in a solution of 0.1% Puloronic F-127 and 30 µM di-8-ANEPPS in antibiotic-free media for 25 min, before switching to antibiotic- and serum-free media for imaging. A 470-nm wavelength LED was used to excite the di-8-ANEPPS, directed to the cell culture with a dichroic mirror reflecting <500 nm (Omega Optical, Brattleboro, VT, USA). Emitted light was passed through the dichroic and a 610-nm long-pass filter before collection by a MiCAM Ultima-L CMOS camera at 500 frames per second, 100 × 100 pixels (SciMedia, Costa Mesa, CA, USA). This system adapts methods used in several earlier optical mapping configurations for a novel application (65,70,71). For an observed culture area of 1 cm2, activation time was mapped according to the time of maximum upstroke rate at each pixel and conduction of the activation front was measured. Cultures were stimulated with a point electrode at either 100- or 300-ms intervals, though spontaneous activation events were not excluded. Cultures in which activation was conducted over <75% of the area observed were considered to exhibit conduction block. In several cases, the test was repeated on a cell culture paced at several unique locations to differentiate between conduction block and altered excitability. Maximum distances and areas of continuous activation front propagation without functional conduction block were measured in ImageJ (U. S. National Institutes of Health, Bethesda, MD, USA), with attention given to maximum distances with respect to the longitudinal and transverse axes of cell culture and at an arbitrary angle, and maximum conducting area surrounding any initiation site. Of the experiments considered (n = 8), one experiment included two replicates each of control (AdLacZ) and desmoplakin knock-down (AdCre) cultures, and two experiments included two AdCre replicates.
Statistical analysis
Data presented in the text and figures are expressed as mean values ± SEM. Significance was evaluated by the two-tailed Student's t-test or repeated-measures ANOVA. For Kaplan–Meier survival analysis, significance was evaluated by the log-rank test. P < 0.05 was considered statistically significant.
Supplementary Material is available at HMG online.
Microscopy work was performed at the University of California–San Diego Neuroscience Microscopy Shared Facility and was supported by a National Institutes of Health grant (P30 NS047101). Optical mapping and MRI studies were supported in part by National Institutes of Health grants (1R01HL105242, P50GM094503, 5P01HL46345) to A.D.M. and J.H.O. R.C.L. was a recipient of an American Heart Association Postdoctoral Fellowship (11POST7170006). A.C. was a recipient of an American Heart Association Summer Undergraduate Research Fellowship (11UFEL7870036). This work was supported by a National Scientist Development grant from the American Heart Association (0730281N) and National Institute of Health grant (1R01HL095780-01) from the National Heart, Lung and Blood Institute to F.S.
We thank the Farquhar Laboratory Transmission Electron Microscopy Core Facility (UCSD, La Jolla, CA, USA) for technical assistance with electron microscopy.
Conflict of Interest statement. None declared.


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Barbara Fassbinder, 40, Nurse With AIDS Traced to Her Job
By TIM HILCHEY
Published: September 22, 1994
Barbara Fassbinder, one of the first health care professionals to be infected with the AIDS virus while on the job, died on Tuesday at the University of Iowa Hospitals and Clinics in Iowa City. She was 40 and lived in Monona, Iowa.
The cause was complications from AIDS, the hospital said.
Mrs. Fassbinder was infected with H.I.V., the virus that causes AIDS, in 1986 while helping treat a patient in the emergency room of Memorial Hospital in Prairie du Chien, Wis.
While she was pressing gauze on a needle puncture, the patient's blood apparently mingled with her blood through small cuts on her hand from gardening, she said in 1990. The young man died, and an autopsy showed he had AIDS. But it was not until January 1987, when she tried to make a blood donation, that she discovered she had been infected.
She and her family kept the infection a secret until she decided to speak out in 1990. "My biggest fear was how the community would react to me and my kids and my husband," she said at a news conference in Iowa City in which she told her story in the hope that it would warn other health-care workers. The 1,500 people of Monona, a farming community in northeastern Iowa, gave her family "nothing but support," she said at the time.
Dr. Michael Osterholm, an AIDS expert and epidemiologist with the Minnesota Department of Health who became a friend of her family, said she "helped bridge the gap between the worlds of the health care provider and the AIDS patient in need of competent and compassionate care like no one else could."
Mrs. Fassbinder traveled extensively, talking to people about AIDS and how to prevent infection by H.I.V., Dr. Osterholm said. She testified about AIDS before Congress, and in 1992 she was recognized by the Surgeon General and the Department of Health and Human Services for her work.
A native of Marion, Ohio, she also served on the National Health Care Reform Committee set up by Hillary Rodham Clinton and was a member of the Iowa State Commission on AIDS, Dr. Osterholm said.
She is survived by her husband, David; two daughters, Eva and Emily, and a son, Joel, all of Monona; her parents, James and Ethel Herring, of Marion, Iowa; three sisters, Kathryn Fassbinder, of Elgin, Iowa, Mary Walston of Kewaunee, Wis., and Susan Valleire of Rock Island, Ill.; four brothers, J. Stephen Herring of Idaho Falls, Idaho, Edward Herring, of Rockford, Ill., Mark Herring, of Manitowoc, Wis., and John Herring of Cedar Rapids, Iowa, and a grandmother, Martha McElroy of Creston, Iowa.


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|Kids & Family
PregTASTIC is the weekly, online radio show by pregnant women, for pregnant women about the fantastic journey to motherhood. Each episode features of expert information and friendly advice, opinions and different perspectives for soon-to-be moms.
October 29th, 2009
Episode 157 of 256 episodes
Natalie's 1 of 4% to give birth on her due date, with a worked-like-it-should epidural at the hospital! Did everything else go so smoothly? How is breastfeeding coming? Also, how hypnobabies, yoga, a birthing ball, water, and mostly the toilet helped Jean's unmedicated birth at the birth center. Why her husband didn't tell her the baby's gender, and what to do with those nipple shields?
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Welcome to the daily audio workout for your head.


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For this purpose we created a web site http://sibza.org, which is built as a Wiki-site and everyone not only can use it for education, but also to contribute to its content after registration.
For quick access to our resource it has been created a mobile application that allows you to access it easily, to search letters, words, phrases, folk songs and stories, and listen to them.
We invite everyone to participate in the development of the project.
version 2.5.4
* Hot fix: link to a new server api (old will be available until the end of the month)
version 2.5.3
version 2.5.2
* Configuration with a choice of language and audio cache cleaning
* An opportunity to add articles to favorites
* Fixed bugs
version 2.0.0
* Improved player application
* Added caching of data
* Display of data, depending on the location
* Improved application interface
version 1.6.0
* The problem with the audio-player
* Improved application interface
* Added English and Turkish languages
version 1.5.0
* Minor improvements in the interface
* Modified built-in player for audio data
version 1.0.6
* The first version of the application


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I'm still at the bank - 5 years next February. I'm still running the Fraud Department, and with my training and tracking we have prevented over $250,000 in losses to fraud in two years. They like me.
I was recently moved to a different, newer branch. There were a lot of issues at the location I had been at for some time, so the move came at a perfect time. Not only does it get me away from a facility that is imploding on itself, but it also allows me to expand my training programs and spend more time working in the fraud arena. I've found my niche. Scary.
In February of this year, I had my third surgery on my finger; the one that I cut with a butter knife in November 2007. For a LONG time it had been arched up where I couldn't straighten it, couldn't move it, nothing. So they went in again, tried and so far, it has not been that successful. Even with splints and a pin in the finger for a couple of weeks after surgery, it is still curling up. At least I can do a little more with it now than I could. Let's just hope it stays that way. The really fun part will be coming soon - the doctor will completely release me next month, then my attorney will want to settle the workers comp claim. This could get ugly.... but I'm optimistic. I'm far too valuable to the bank for them to get rid of me, hopefully...
Other issues have arisen since then, but I won't get into it now. Not important.
Still single. Big fucking surprise there.
Found out yesterday that my Grandmother had a spell on Saturday at the beauty shop where she said she couldn't speak. Why she waited until Sunday night to tell anyone is beyond me, but I digress.
So she got in first thing this morning to see the doctor. The explaination to him was that she was able to speak, but it was a jumbled mess of random words, no coherent thought. Which explaination is more accurate, I don't know. But after talking to her, he said beyond the shadow of a doubt that she had suffered from a mini stroke. He ordered a boat load of different tests and put her on a daily asprin regimine, but the hospital can't get her in for the tests until NEXT WEDNESDAY.... I dunno, I don't like it one bit but what can I do.
So, that's my life. Still insane and boring. How is everyone still around in LJ land?


text: Select an international website Zoover.at Zoover.be Zoover.ch Zoover.com Zoover.com.br Zoover.com.tr Zoover.com.ua Zoover.cz Zoover.de Zoover.dk Zoover.es Zoover.fi Zoover.fr Zoover.gr Zoover.hu Zoover.it Zoover.lt Zoover.nl Zoover.no Zoover.pl Zoover.pt Zoover.ro Zoover.ru Zoover.se Home Destinations Accommodations Awards Add review Add review Add photos Add videos Add new accommodation Add attraction Home Add review England London & South East Chertsey Accommodations Chertsey Reviews (0) Photos & videos (0) Accommodations (4) Attractions (89) Weather forecast Map Tips (0) More Question and Answer (0) There are currently no reviews for Chertsey Weather 3° / 9° 28 Feb 2017 Weather forecast and climate... Unfortunately no photos available Add the first photo - Find Hotel Deals From £ 44 Find the best deal Find the best deals Most visitors found the best deal for Chertsey with: 1 Booking.com Check price TOP More suppliers Collapse Sort by Zoover recommends Rating Reviews Distance from centre 4 Accommodations England, London & South East, Chertsey Location Radius: Select 2 km 10 km 20 km 50 km Country England Region London & South East Destination Chertsey Accommodation Accommodation Campsites in Chertsey Hotels in Chertsey Bed and Breakfasts in Chertsey Facilities Room service Elevator WiFi More facilities User Rating Number of reviews 1 5 Review 7-8.4 Good1 Contributions 3 Search within results: 7.3 click Add review Campsite: Camping & Caravanning Club Chertsey, 0 km from center England London & South East Chertsey - click Add review Hotel: Crown Chertsey, 0.6 km from center England London & South East Chertsey - click Add review Hotel: The Bridge Chertsey, 1.3 km from center England London & South East Chertsey - click Add review Bed and Breakfast: The Orchard Barn Chertsey, 0.8 km from center England London & South East Chertsey Hotels Hotels in Brighton / Hove Hotels in Canterbury Hotels in Oxford Hotels in Southampton Hotels in Reading Hotels in Dover Hotels in Maidstone Information about Chertsey Weather Chertsey Map Chertsey Photos Chertsey Videos Chertsey Question and Answer Chertsey Destinations nearby Laleham Weybridge Ottershaw Shepperton Staines Virginia Water Addlestone Are you looking for a well-reviewed accommodation for your holiday in Chertsey? Below you can find all accommodations in Chertsey. read more Are you looking for a good reviewed accommodation for your holiday in Chertsey? Below you can find all accommodations in Chertsey. You can find comments regarding 1 Campsite, 2 Hotels and 1 Bed and Breakfast in Chertsey. Please use the filters on the left to find your ideal accommodation. - - Less text


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Qi S.Z.,Guangdong Power Grid Corporation |
Zhou S.B.,Guangdong Institute of Automation
Applied Mechanics and Materials | Year: 2014
Aiming at the performance evaluation of mixed-mode sensing interface of the integrated-sensor RFID system, an integrated-sensor RFID system was constructed. Then the mechanism of performance evaluation of mixed-mode interface was analyzed. According to the validity and reliability principle, a mixed-mode interface performance testing model was established. Moreover, a Plug-and-Play performance testing method was presented. Finally, the physical platform was set up to test the reading speed and the recognition rate indicator in Plug-and-Play process. Results showed the reading speeds of two STIM maintained at around 656.56ms and 645.24ms steadily during Plug-and-Play process whose Bit Error Rate was 10-9 Experiments indicate that effective transverse comparison of performance of similar products could be implemented by the proposed method in static environment as well as dynamic environment. © (2014) Trans Tech Publications, Switzerland. Source
Zhou X.,Guangdong Institute of Automation |
Guan Y.,Guangdong University of Technology |
Zhu H.,Guangdong University of Technology |
Wu W.,Guangdong University of Technology |
And 3 more authors.
International Journal of Humanoid Robotics | Year: 2014
Most of current biped robots are active walking platforms. Though they have strong locomotion ability and good adaptability to environments, they have a lot of degrees of freedom (DoFs) and hence result in complex control and high energy consumption. On the other hand, passive or semi-passive walking robots require less DoFs and energy, but their walking capability and robustness are poor. To overcome these shortcomings, we have developed a novel active biped walking robot with only six DoFs. The robot is built with six 1-DoF joint modules and two wheels as the feet. It achieves locomotion in special gaits different from those of traditional biped robots. In this paper, this novel biped robot is introduced, four walking gaits are proposed, the criterion of stable walking is addressed and analyzed, and walking patterns and motion planning are presented. Experiments are carried out to verify the locomotion function, the effectiveness of the presented gaits and to illustrate the features of this novel biped robot. It has been shown that biped active walking may be achieved with only a few DoFs and simple kinematic configuration. © 2014 World Scientific Publishing Company. Source
Wu W.,South China University of Technology |
Guan Y.,Guangdong University of Technology |
Zhu H.,South China University of Technology |
Su M.,South China University of Technology |
And 2 more authors.
Harbin Gongye Daxue Xuebao/Journal of Harbin Institute of Technology | Year: 2014
For solving the problem of finding suitable robot configurations to meet the specific tasks in the application of reconfigurable modular robot, a task-oriented multi-objective configuration optimization method is discussed. At beginning, the basic structure of the modular robot system is introduced, including the main modules and components of the control system. Then, based on the description of the task, an optimization model of modular robot configuration design is represented. Finally, for the specific tasks of climbing and manipulating, the configuration optimal design of modular robot is processed. The feasibility and effectiveness of the method have been well verified by the optimization simulation. The method is task-oriented and multi-objective, and it includes the performance optimization of degrees-of-freedom, reach-ability, energy consumption, and so on. Source
Wang Z.,South China University of Technology |
Wang Z.,Guangdong Institute of Automation |
Wang Z.,Chinese Academy of Sciences |
Xie Y.,South China University of Technology |
And 2 more authors.
Dianli Zidonghua Shebei/Electric Power Automation Equipment | Year: 2014
The input current SVM (Space Vector Modulation) algorithm of matrix rectifier in normal conditions is analyzed and its affect on the output DC voltage waveform in abnormal conditions is studied, aiming at which, an input current SVM algorithm with feed forward fuzzy compensation control is proposed. The modulus of voltage vector is measured, calculated and compared with the normal value in real time, according to which, the modulation coefficient of present control cycle is dynamically adjusted by the fuzzy control to keep the output voltage stable. The operating principle of the proposed compensation strategy is analyzed in detail, and the simulation model and experimental prototype of matrix rectifier are established. Simulative and experimental results verify the feasibility and effectiveness of the proposed compensation control strategy. Source
Ye T.,Guangdong Institute of Automation |
Ye T.,Guangdong Industry Technical College |
Cheng T.,Guangdong Institute of Automation |
Zhou S.,Guangdong Institute of Automation
WIT Transactions on Information and Communication Technologies | Year: 2014
Aiming for the space trajectory planning problem of robot teaching, the paper proposed a trajectory planning method based on multi-knots spline interpolation. The method obtains key points data of space trajectory by robot teaching, and it uses cubic multi-knots spline interpolation method to realize the fitting of robot space trajectory by reversely solving control coefficients of multi-knots spline space curve; and then, it smoothes the robot teaching action by an interpolation algorithm of space trajectory under the control of arc length error. The simulation research shows the trajectory planning method can realize accurate planning of space curve under a given error. © 2014 WIT Press. Source


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The normally discreet world of men's tailoring has been shaken by a dispute over who has the best tailors: Britain or Italy?
Pitti Uomo, Italy's annual showcase for menswear, opens this week in Florence, but for the first time in the its 70-year history, the show will feature clothing by Savile Row tailors, together with an exhibition entitled The London Cut: Savile Row Bespoke Tailoring.
The inclusion has incensed Italian designers. Gianluca Isaia, deputy head of the Italian menswear consortium Classico Italia, said that the Pitti show was "intended to represent menswear made in Italy. I would expect it to


text:
The Art of the 'Shark Tank' Pitch; Investment Flops and Successes.
Learn how to produce highly profitable 3-dimensional motorcycle tanks and fenders using Dennis Mathewson's innovative techniques. In this presentation you'll learn step-by-step how to apply and paint a skull on a tank and a Tiki design on a motorcycle fender.


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Also we paid for it all. So we could do what we wanted.
Also we paid for it all. So we could do what we wanted.
TheGooch (13-11-2015)
I wish I had chosen a dress for me and not my mum. I wish I hadn't gotten married in a church for my grandmother. That's all. The rest was awesome.
I wish we had spent the money on a videographer or at least had someone film the speeches and ceremony. We had such unexpectedly beautiful speeches but I can't remember what was said! And my DH cried through the whole day but you can't really tell through just the photos. Hope your day is wonderful!
I wish I had a Videographer, we had a friend film what he could, but you can so tell when you watch it, especially in the evening!
The only other thing I would have changed, some of the guests which we invited out of pressure and obligation.
Apart from that, it was a magical time, I loved it.
All the best for your wedding day OP, hope it all goes to plan for you. x
I would've reduced the guest list and stopped trying to please others. We invited DHs cousins cause it would've upset his parents if we hadn't. We wished we hadn't after.
Eat the food and have some cake! I was so stressed I didn't touch anything and so I never got to try our wedding cake! :-0
I wish we'd gone with a professional photographer rather than the person who was cheap and 'ok'. I also wish we'd had video of some moments.
I also wish I'd spoken up when the hairdresser did my hair differently from the agreed hairstyle. I ended up with awful hair because they did something that was the opposite of what I'd wanted and I didn't speak up! Must. be. more. assertive.
And I wish we'd paid for an extra hour at the reception. The time just disappeared and we didn't get to speak to everyone.
But - even with all that, we still loved our wedding. So the moral I guess is that even if things go wrong, don't stress, it can still all be okay and you can still have a great time.
Word of mouth is definitely worth listening to! I was recommended a particular mobile hair and make up service by multiple people, but chose a different group as they were slightly cheaper. While my hair and makeup on the day were lovely, all the stress the business owner gave me wasn't worth it. I.e. I asked for airbrushed make up yet my trial was booked with a makeup artist who doesn't do it. She also did my hair and then wasn't the one who turned up on the actual wedding day (luckily the one who came was 100x better)! Also I contacted the owner a few days before the wedding to ask them to arrive an hour earlier at the request of the photographers (so they weren't just sitting around waiting for us to finish hair and makeup) which she assured me was no trouble. Shame she didn't communicate that to the 2 girls who came to do our hair and makeup!
Also don't pick an obscure song for your wedding dance. I did (still stand by my choice though) and the DJ f-ed it up. I told home he wouldn't find it on iTunes and that I could provide him with the file either email/burnt to cd etc and he declined saying it "was better for him to find it". Found out the day before the wedding he couldn't find it (which I had already told him), so provided him with my iPhone on the day with the song lined up ready to go. He still played the wrong song. It's the only thing that came close to ruining my day. Lucky I was able to let it go at the time. I'm still annoyed by it though!
Don't believe it when people tell you that "all brides lose weight before the wedding". I went to have my final fitting and pick up my dress on the Wednesday before the wedding. It didn't fit. They'd made it too small and then the dressmaker had the hide to complain because there wasn't much of the seams that she could let out. Like seriously lady. It's a $3000 dress and I've been having regular fittings for the last 3 months. I had not put on weight, but I hadn't lost any either. Annoyed because it was from a very well known wedding dress designer and the lady doing my fittings was amazing. Shame she wasn't the one sewing it!
Actually, on the weight thing, don't lose heaps of weight. You read and hear of women all the time wanting to weigh what they did on their wedding day, wanting to fit back into their wedding dress etc etc. I stayed the weight I was and now, 8 years later and after 2 kids, I'm actually 5 kilos lighter. I still looked good in my photos and I have the bonus of being able to say yeah, I fit into my wedding dress with some room to spare. Eat the food 😁
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