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Legislative Assembly for the ACT: 2013 Week 6 Hansard (7 May) . . Page.. 1646..
The final instrument facilitates the transfer of $160,000 in expenses on behalf of the territory, territorial appropriation, from the Office of the Legislative Assembly to the ACT executive associated with a transfer of staffing costs.
Section 16B of the FMA allows for appropriations to be preserved from one financial year to the next. This package includes one such instrument signed under section 16B. This instrument authorises a total of $8.458 million in capital injection controlled rollovers for the Economic Development Directorate. The appropriation being rolled over was not disbursed during the 2011-12 fiscal year and is required in 2012-13 for the completion of projects identified in the instrument.
Section 18 of the act provides for the Treasurer to authorise expenditure from the Treasurer's advance. This package includes one instrument signed under section 18. This instrument provides an increase of $354,743 in net cost of outputs controlled appropriation for the Legal Aid Commission of the Australian Capital Territory to meet expenses related to the board of inquiry into the conviction of Mr David Eastman.
Additional details regarding all instruments are provided in the statement of reasons accompanying each of the instruments I have tabled this afternoon. I commend these instruments to the Assembly.
Public Accounts—Standing Committee
Paper and statement by minister
MR RATTENBURY (Molonglo—Minister for Territory and Municipal Services, Minister for Corrections, Minister for Housing, Minister for Aboriginal and Torres Strait Islander Affairs and Minister for Ageing): For the information of members, I present the following paper:
Public Accounts—Standing Committee—Inquiry—Auditor-General's Report 5/2012—Management of Recycling Estates and E-waste—Government submission.
I ask leave to make a brief statement in relation to the paper.
Leave granted.
MR RATTENBURY: Thank you, members. For the information of members, I have tabled the government's submission to the Standing Committee on Public Accounts on Auditor-General's report No 5 of 2012 entitled Management of recycling estates and e-waste. That report's objective was to provide an independent opinion on whether the ACT recycling estates at Parkwood Road and Hume are effectively planned, regulated and managed to optimise recycling activities to meet the ACT's sustainability agenda and that the management of computer and television e-waste is consistent with government legislative and policy requirements.
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Hello everyone. Just wanted to show you some cards I have just made. We will be making three of these in our May Card Classes. I'm not totally sure which three yet. When I start making cards I just can't stop!!! I'm sure my fellow cardmakers understand what I mean. We will definately be making the clock one & the little bike one & I haven't decided which one will be the third one, you will have to wait & see!!! As you can see this months theme I have gone with chipboard card accessories from 2 Crafty & lots of yummy flowers, ribbons & lace from Green Tara of course.
At our shop at The Entrance we have lots of really sweet little 2 Crafty chipboard pieces made especially for cards, including a good selection of little chipboard "card greetings" like the ones I have used here on these cards. They really do make cardmaking so easy.
Card Classes
Friday 11th May 10am to 2pm
Friday 25th May 10am to 2pm
Thursday 26th May 7pm to 10.30pm
In our card class you will make 3 cards.
Cost is $20 class fee plus $10 kit fee & learn a hole lot of new skills, but best of all, have fun while you are learning.
Ring the shop on 43344451 or pop in to the shop to book in.
See you in class. Cheers Di xoPin It Now!

subset: commoncrawl


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tag:blogger.com,1999:blog-8958853673360052269.post3461877220143980117..comments2019-01-22T18:31:22.038+01:00Comments on Complaint Centre Lyoness: FlexKom: the new email@example.com:blogger.com,1999:blog-8958853673360052269.post-64677400878942242842013-11-26T14:32:00.007+01:002013-11-26T14:32:00.007+01:00Do you have any information about Beepxtra? This f...Do you have any information about Beepxtra? This firm is now opening up in the UK. It is run by a former Flexkom member. The scheme is roughly the same as for Lyoness and Flexkom. Membership however is firstname.lastname@example.com:blogger.com,1999:blog-8958853673360052269.post-49615553434823896112013-11-20T17:42:35.069+01:002013-11-20T17:42:35.069+01:00The FTC (Federal Trade Commission) Business Opport...The FTC (Federal Trade Commission) Business Opportunity Rule...

If you sell business opportunities, the FTC's Business Opportunity Rule requires that you give potential buyers a one-page disclosure document

http://www.business.ftc.gov/multimedia/videos/business-opportunity-rule

http://www.ftc.gov/opa/2012/11/lostopp.shtm

http://email@example.com:blogger.com,1999:blog-8958853673360052269.post-27018863221309723152013-11-07T23:56:31.374+01:002013-11-07T23:56:31.374+01:00You're absolutely right. Their math is not (ne...You're absolutely right. Their math is not (never) correct. Flexkom is just an ordinary scam conceived by scammer Cengiz Ehliz. For naieve people: don't believe a word of what this fake company is promoting!

email@example.com:blogger.com,1999:blog-8958853673360052269.post-48633789729950687102013-11-07T22:35:39.912+01:002013-11-07T22:35:39.912+01:00Flexkom will never ever launche, not in 2013, not ...Flexkom will never ever launche, not in 2013, not in 2014, not in Belgium, not in Netherlands, not in France, not in Swiss, not in USA, not in Russia, not in Italy, not in ......... Only small 'pilots' in certain places to keep the sheeps on board. email@example.com:blogger.com,1999:blog-8958853673360052269.post-60316054199465464772013-10-26T08:08:45.393+02:002013-10-26T08:08:45.393+02:00We soon will be in November and, as far as I know,...We soon will be in November and, as far as I know, Flexkom should be launched in Belgium at least end november, begin december. No much time to wait anymore...firstname.lastname@example.com:blogger.com,1999:blog-8958853673360052269.post-8121969430926592672013-10-19T19:37:58.764+02:002013-10-19T19:37:58.764+02:00Hello I write from Italy, my friends have borrowed...Hello I write from Italy, my friends have borrowed money to subscribe to Flexkom, now the license costs € 2200 for only one idea of back-office.Le their meetings more and more similar to the meetings of fanatics know how religiosi.Non do to tell my friends that they are email@example.com:blogger.com,1999:blog-8958853673360052269.post-39740478787545112052013-10-17T04:25:19.446+02:002013-10-17T04:25:19.446+02:00Flexkom America claims that when you pay your $2,2...Flexkom America claims that when you pay your $2,200 at the time of sign up, $2,000 of that money will be distributed as a $2 credit on 1,000 apps you will receive at the time of launch. The problem with is this, once you begin signing people up during pre launch you can earn your $2,200 back by signing up 5 people in the first thirty days. How do you get $2,200 back when $2,000 of each sign up firstname.lastname@example.com:blogger.com,1999:blog-8958853673360052269.post-71970386299900634952013-10-08T06:41:41.329+02:002013-10-08T06:41:41.329+02:00FlexKom America is currently charging $2,200 to si...FlexKom America is currently charging $2,200 to sign up as a Global team member. Unfortunately, they break the law the minute they sign someone up. How? There is no actual product sold at sign up. They claim the sign up fee is paying for a back end virtual office to manage the team member's group and sales. According to the FTC, tools and training to grow the network are NOT considered firstname.lastname@example.com:blogger.com,1999:blog-8958853673360052269.post-6501306642100689132013-09-13T10:09:03.505+02:002013-09-13T10:09:03.505+02:00Just another example to illustrate the complete ma...Just another example to illustrate the complete madness of this SCAM company.

On the Flexkom FB page you can read that they have won an astonishing famous award, this time from Trinidad & Tobago ….

Please try not to laugh because this is a very serious matter for FK and their blind followers.

https://www.facebook.com/Flexkom.global?bookmark_t=page
<br firstname.lastname@example.com:blogger.com,1999:blog-8958853673360052269.post-29000071104380608162013-09-12T09:48:31.257+02:002013-09-12T09:48:31.257+02:00As stated above by someone- thank god for the inte...As stated above by someone- thank god for the internet! I live on Maui Hawaii, and someone is using Craigslist to try to start his "team". Funny thing is, he lives in Las Vegas Nevada in the USA- where their "USA Headquarters" are supposed to be! I can't wait to call him up in the morning, and blow him away with all of this awesome information! Thanks to all who email@example.com:blogger.com,1999:blog-8958853673360052269.post-46592649978480103752013-09-11T22:09:37.185+02:002013-09-11T22:09:37.185+02:00Thanks!Thanks!Anti Lyonesshttps://email@example.com:blogger.com,1999:blog-8958853673360052269.post-34446756456174762372013-09-11T21:44:43.634+02:002013-09-11T21:44:43.634+02:00We have in Holland 4 blogs on www.wouterhol.nl. Al...We have in Holland 4 blogs on www.wouterhol.nl. Almost 1.500 reactions in 2 months!

1. The pyramid game of Flexkom?
2. Waiting for a reaction of Flexkom
3. In Turkey game over for Flexkom
4. The ICT leak at Flexkom

This company with all teir CEO's and COO's is ONE BIG BIG JOKE - everything is fake. You realy can not take them serious with all FAKE firstname.lastname@example.com:blogger.com,1999:blog-8958853673360052269.post-43407959247017839782013-09-11T20:19:41.576+02:002013-09-11T20:19:41.576+02:00In other countries they are just beginning, compla...In other countries they are just beginning, complaints will come soon enough.Anti Lyonesshttps://email@example.com:blogger.com,1999:blog-8958853673360052269.post-78313822013528256242013-09-11T20:18:55.788+02:002013-09-11T20:18:55.788+02:00In Turkey there is a large amount of people and sh...In Turkey there is a large amount of people and shops extremely unhappy with FlexKom. If you speak Turkish, have a look at the website of the Turkish newspaper Zaman, and search for FlexKom there.

If not, you can still visit the website and use Google Translate or so.Anti Lyonesshttps://email@example.com:blogger.com,1999:blog-8958853673360052269.post-77672067344221342242013-09-11T19:44:32.141+02:002013-09-11T19:44:32.141+02:00Thank you for the info.Question: Has anyone heard ...Thank you for the info.Question: Has anyone heard or know if any of the Shops in Turkey or any other country have been scammed?Would love to firstname.lastname@example.com:blogger.com,1999:blog-8958853673360052269.post-51021634629441429262013-09-05T02:42:20.262+02:002013-09-05T02:42:20.262+02:00People believe what they want and need to believe....People believe what they want and need to believe. The problem is that the names of these 'companies' are witheld until an upline is present, or the prospect attends a big recruitment meeting, where the levels of exercised psychological pressure and manipulation are amazingly high. So when these people first hear of Lyoness, FlexKom, etc., they have already been influenced by the Anti Lyonesshttps://firstname.lastname@example.com:blogger.com,1999:blog-8958853673360052269.post-6517105738568289062013-09-05T00:47:11.665+02:002013-09-05T00:47:11.665+02:00What a complete bogus letter, how do people get fo...What a complete bogus letter, how do people get fooled to believe this nonsense despite having all the resources available to you at your fingertips... STAY firstname.lastname@example.com:blogger.com,1999:blog-8958853673360052269.post-44416321504731166642013-09-04T16:54:54.223+02:002013-09-04T16:54:54.223+02:00Thank God for the internet! We just spent an hour...Thank God for the internet! We just spent an hour with two very plausible gents here in the UK who were introduced via an attendee at an FSB event and another business networking event.

On the face of it the proposition seemed reasonable, and not unlike a variety of other 'Save the UK High Street' loyalty card schemes being spoken about on TV news programmes (so good for UK email@example.com:blogger.com,1999:blog-8958853673360052269.post-25475956136688907282013-09-04T02:19:03.459+02:002013-09-04T02:19:03.459+02:00And the mentioned footer: ---- footer: Kind re...And the mentioned footer:

----

footer:

Kind regards,

Erwin Fally,
you can cheque me out to if you want for i have no secrets, here is one of many of my cash payed for and morgage free property's, but here i am most often at the moment.
Address:****, the most expensive road in the most rich area of the whole of Belgium and the best area Anti Lyonesshttps://firstname.lastname@example.com:blogger.com,1999:blog-8958853673360052269.post-45995588602239946362013-09-04T02:18:33.377+02:002013-09-04T02:18:33.377+02:00Thanks, Anonymous, for sending us the email. Here ...Thanks, Anonymous, for sending us the email. Here it is (we removed the contact details from it).

"Hi guys as promised, please see below email from flexkom employee sent to me recently, check his footer!!! awesome!

-----

From: Erwin Fally
Sent: Sat 8/17/13 2:35 AM
To: *********************

Dear Sir

I have recieved your email inAnti Lyonesshttps://email@example.com:blogger.com,1999:blog-8958853673360052269.post-14199385210467007472013-09-01T21:00:28.896+02:002013-09-01T21:00:28.896+02:00For those who have been scammed, I highly encourag...For those who have been scammed, I highly encourage each and every one of you to report your experiences here: http://www.econsumer.gov/

A concerted effort to report the scam will most expeditiously stop the scammers in their tracks, and hopefully bring with it a jail sentence!

We need to start caring more about people than how to earn a fast dollar from international firstname.lastname@example.com:blogger.com,1999:blog-8958853673360052269.post-53132503067734387952013-08-30T13:44:13.851+02:002013-08-30T13:44:13.851+02:00Glad to be of help, and congratulations on your wi...Glad to be of help, and congratulations on your wise decision not to invest any money into this!

We indeed saw that the Kenyan press is picking up on the whole FlexKom scam too (albeit by copy pasting various fragments from our Blog):

http://www.nairobiexposed.com/2013/08/10/flexikom-now-in-kenya-scam-or-legitimate/Anti Lyonesshttps://email@example.com:blogger.com,1999:blog-8958853673360052269.post-66292584203291948202013-08-30T10:57:23.822+02:002013-08-30T10:57:23.822+02:00and they have now entered the Kenyan market. Thank...and they have now entered the Kenyan market.
Thanks God I have this handy information. they will not get a single of my firstname.lastname@example.com:blogger.com,1999:blog-8958853673360052269.post-85962060661348547672013-08-29T11:50:09.959+02:002013-08-29T11:50:09.959+02:00Obviously, we would love to see that email (as the...Obviously, we would love to see that email (as the billions they claim to have may very well be coming from Lyoness, Galvagin, GTS, etc.). We would immensely appreciate it if you could post a copy of the email.

Also, if you would want to communicate with us in private, please leave your email address in a separate comment - it will not be published.Anti Lyonesshttps://email@example.com:blogger.com,1999:blog-8958853673360052269.post-36742277062627247382013-08-25T02:58:50.757+02:002013-08-25T02:58:50.757+02:00This is so hilarious, this company is a complete f...This is so hilarious, this company is a complete fake, absolutely nothing to independently verify their claims apart from ebcon!! which even if it exists wont mean anything in real terms, any company can have registrations, patents, web directory listings etc etc, but it never means that because you have a little buzz about yourself/company which in this case is completely self created that you email@example.com


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My gladii. On top is a pompeii with a reinforced tip and below that is a earlier Mainz style
My pugio, the legionnaire's side-arm. These were typically highly decorative. This one is done with silver inlay.
Pila, the roman javelin, consisting of a mild steel shank attached to ash wood shaft. Designed to bend upon
impact, they could be easily straightened out after the battle. These are styled after those from Oberaden.


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The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV) gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9). Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.
Citation: Grose WE, Clark KR, Griffin D, Malik V, Shontz KM, Montgomery CL, et al. (2012) Homologous Recombination Mediates Functional Recovery of Dysferlin Deficiency following AAV5 Gene Transfer. PLoS ONE 7(6): e39233. https://doi.org/10.1371/journal.pone.0039233
Editor: Paul McNeil, Medical College of Georgia, United States of America
Received: February 10, 2012; Accepted: May 17, 2012; Published: June 15, 2012
Copyright: © 2012 Grose et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Day Foundation, MDA, and Jesse's Journey Foundation for Gene and Cell Therapy. The muscle physiology core is supported by National Institutes of Health (NIH) P30 NS045758. The project described was also supported by Award Number UL1RR025755 from the National Center For Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center For Research Resources or the National Institutes of Health. Dr. Rodino-Klapac was supported by an NIH sponsored NRSA Fellowship (1F32AR055008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Mutations in the dysferlin gene cause allelic autosomal recessive disorders including limb girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy [1], [2] and distal anterior compartment myopathy [3], [4], [5], collectively known as the dysferlinopathies. A less common phenotype of dysferlin deficiency presents with rigid spine syndrome [1], [2], [3], [4], [6]. Typically patients present in their early twenties with slowly progressive weakness and high serum creatine kinase (CK) [7]. Approximately one-third of patients become wheelchair-dependent within 15 years of onset. Clinically the heart is only mildly affected in one third of cases [8] and cognitive function is spared. The phenotypic variants with a relatively restricted distribution of muscle weakness set the stage for potential regional vascular gene replacement therapy that could impact quality of life for this disorder [9], [10]. Single nucleotide changes [11], [12], the typical DYSF gene mutation, also favors success in gene transfer serving to protect the transgene product from immunorejection.
The dysferlin gene is large, with 55 exons so far identified spanning at least 150 kb of genomic DNA. These exons predict a cDNA of approximately 6.5 kb and a protein of 2,088 amino acids [1], [2], [11], [13]. Dysferlin is a 237 kDa protein composed of a C-terminal hydrophobic transmembrane domain and a longer cytoplasmic oriented hydrophilic region with multiple C2 domains with implications for calcium and phospholipid binding [14]. Recent work has shown that loss of dysferlin compromises Ca2+-dependent membrane repair in skeletal muscle [15], [16]. Dysferlin-null muscle fibers fail to exclude dye entry even in the presence of Ca2+ strongly suggesting that Ca2+-dependent membrane repair requires dysferlin [17]. There is also evidence from LGMD2B patients that ultrastructural membrane defects are a present and contributing factor to disease pathology [18], [19]. The importance of this system is emphasized when considering that skeletal muscle is mechanically active and predisposed to injury; thus, a robust membrane resealing mechanism must be present. Absent or mutant dysferlin leads to impaired membrane repair and a cascade of events starting with muscle fiber necrosis resulting in muscle fiber loss and progressive limb weakness [16], [17].
Packaging limitations of AAV, estimated at ∼5 kb, present obstacles for gene replacement strategies requiring cDNA cassettes exceeding this size constraint [20], [21], [22]. Alternative methods to bypass packaging limits include miniaturizing genes and trans-splicing approaches. These tactics have often compromised function and often result in reduced levels of gene expression at standard dosing levels [23], [24], [25], [26]. A recent report suggests that the dysferlin gene can be delivered to muscle using a dual trans-splicing vector strategy with functional improvement of the defect in membrane repair [27]. A second study using AAV to deliver a naturally occurring minidysferlin protein also showed some improvement in the membrane repair defect [28]. A sentinel report indicating that AAV5 can package large transcripts up to 8.9 kb in size encouraged translational investigators to pursue gene replacement requiring inserts as large as the dysferlin gene [29], [30]. Subsequent studies showed the mechanism for AAV5 mediated delivery of genes >5 kb was homologous recombination of 5′ and 3′ products of partially packaged virions rather than ability to package intact full size genomes [31], [32]. In our own studies, we attempted to take advantage of the potential for AAV5 to deliver a dysferlin expression cassette of 7.7 kb in a single vector, including an optimized cDNA and muscle specific promoter to avoid off target effects. Our findings demonstrate highly favorable results with full restoration of dysferlin without compromise in function. In the diaphragm muscle of a mouse model of dysferlin deficiency, tetanic force was restored to normal and there was full resistance to fatigue. Importantly, the newly restored dysferlin fully repaired membrane defects in dysferlin deficient mice. In addition, rAAV5.DYSF was successfully delivered through isolated limb perfusion to the limb muscles preferentially affected in entities such as Miyoshi myopathy and distal anterior compartment myopathy. Even more proximal sites can potentially be transduced by vascular delivery as we have demonstrated in the non-human primate [10]. Of particular note, consistent with recent observations, the full-length dysferlin product that restored these favorable results was mediated by recombination of homologous region of ∼1 kb present in 5′ and 3′ packaged vector genomes, [29], [31], [32]. Herein we present our findings that AAV5 delivery is a viable treatment modality for dysferlin gene replacement with far reaching implications for other monogenic disorders caused by mutations in large genes.
rAAV5.dysferlin gene transfer vector
We constructed a human dysferlin cassette driven by the muscle specific MHCK7 promoter [33] (Fig. 1A). A chimeric intron was added to augment RNA processing. The cassette (7.7 kb total) was packaged into an AAV2/5 vector using standard triple transfection and purified using iodixanol gradients and ion exchange chromatography. To test whether the full-length transgene was packaged or if homologous recombination was occurring, we performed alkaline electrophoresis and southern blot analysis on vector genomes purified from the vector preparation as previously described [32]. Two probes were designed, one within the 5′ MHCK7 promoter and one located in the 3′ end of dysferlin. The findings unequivocally demonstrated that packaging did not exceed ∼5.2 kb with either probe consistent with recent reports [29], [31], [32] (Fig. 1B). AAV is known to package single strand genomes with 3′ to 5′ polarity into the pre-formed particle in an ATP dependent process starting at the 3′ ITR terminus. This is consistent with a vector "breakpoint" within this region based on the Southern blot data demonstrating packaging of up to ∼5.2 kb in length starting at a 3′ ITR termini. Despite these packaging constraints, full-length dysferlin protein was readily demonstrated in skeletal muscle consistent with a process of homologous recombination occurring within transduced myocytes to generate the full-length intact dysferlin gene (Fig. 2C) [29], [31], [32]. Using electron microscopy, AAV5 virions had normal morphology, providing further evidence that the genomes packaged did not exceed capacity (Fig. 1C).
DNA was isolated from rAAV5.DYSF vector preparation and used for Southern blot and PCR analysis. (A) Schematic of rAAV5.DYSF cassette. Strand specific hybridization probes used for Southern blot analysis are indicated by red bars. (B) Southern blot analysis of rAAV5.DYSF genomic DNA with 5′ MHCK7 probe (lane D, left side) and 3′ dysferlin probe (Lane D, right side). A 4.2 kb control vector genome was used as a standard for packaging (C in each blot). "M" denotes marker lane. (C) Electron microscopy of rAAV5 vector prep revealed virions with normal morphology.
4–6 week old dysferlin deficient mice were injected into the tibialis anterior muscle with 1011 vg of rAAV5.DYSF (n = 4 per group). Endpoint analysis occurred at 4 weeks post gene transfer and analyzed by histology, immunofluorescence and western blot analysis. (A) Hematoxylin and eosin staining demonstrates very mild pathology in Dysf−/− control mice which was not exacerbated by rAAV5.Dysf delivery. (B) Animals treated with rAAV5.DYSF demonstrated positive dysferlin expression by immunostaining compared to controls. (C) Western blot analysis confirmed a 237 kd full-length dysferlin band which was absent in control tissue except in SJL/J controls which have ∼15% residual dysferlin protein (PBS). Scale bar = 100 µm.
Intramuscular rAAV5.DYSF gene transfer to limb muscle
To test whether our AAV5.dysferlin vector could efficiently transduce muscle and express full-length intact protein, we performed intramuscular injections into the tibialis anterior (TA) muscle of 4–6 week old dysferlin deficient mice 129-Dysftm1Kcam/J (hereafter referred to as the 129Dysf−/−) [17], [34], [35] with1011 vg of rAAV5.DYSF. Animals were sacrificed after 4 weeks and muscle sections were immunostained with anti-human dysferlin antibody and assessed for histopathological changes. As shown by previous studies, Dysf−/− mice demonstrate very mild pathology in young mice which is limited to a small number of centrally nucleated fibers and isolated necrotic fibers [15], [35]. There was no evidence of toxicity in the muscle following AAV5.Dysf gene transfer (Fig. 2A). In control muscles, 3.8±0.8% of fibers had central nuclei versus 2.9±1.6% in treated muscles and no evidence of necrosis. Widespread dysferlin expression was achieved which correctly localized to the sarcolemma, whereas there was no dysferlin expression in contralateral control muscles (Fig. 2B). Cytoplasm dysferlin was also encountered as noted in newly regenerating fibers in dystrophic patients [36] and is consistent with other studies demonstrating exogenously expressed dysferlin [27], [28]. The number of muscle fibers transduced was quantified with 67.3±14.4% expressing dysferlin. Western blot analysis confirmed the immunostaining results. Treated mice had a well-defined 237 kDa band that was absent in PBS control-treated animals (Fig. 2C). Efficient gene transfer was confirmed in two other models of dysferlin deficiency, SJL-Dysf and A/J for potential use in pre-clinical outcomes analyses (Fig. 2C). As expected, SJL-Dysf mice express residual dysferlin protein which increased following gene transfer (Fig. 2C).
To fully address whether the recombination events that are leading to dysferlin expression are specific in generating only full-length transcript and protein, we performed RT-PCR and western blot analysis on samples isolated from injected tissue. We extracted RNA from treated and control injected TA muscles and following cDNA conversion, three overlapping PCR products specific for human dysferlin were amplified in AAV5 injected tissue and not in control tissue (Fig. 3A). The products were sequenced and found to be 100% identical to the human dysferlin transcript encoded by the transgene cassette (Fig. S1). Moreover, only full-length dysferlin was present on western blot using both N and C-terminal dysferlin antibodies (Fig. 3B).
(A) RNA was extracted from injected tissue, converted to cDNA, and analyzed by PCR using 3 overlapping primer sets (Top to Bottom, 5′ to 3′ end) which showed that the entire Dysferlin transcript was amplified in injected muscle. Lane 1 – no template, lane 2 – injected muscle, lane 3 – injected muscle with no reverse transcriptase enzyme, lane 4 – uninjected muscle, lane 5 – human muscle control RNA, lane 6 – pAAV.MHCK7.DYSF control DNA. (B) Protein was extracted from injected tissue and analyzed by western blot. 4 mice were analyzed following injection with rAAV5.Dysf. A wild-type mouse muscle sample was used as a control. Both an N-terminal antibody and a C-terminal antibody were used for protein analysis in these muscles. Only the full-length Dysferlin band is unique to injected muscles.
Functional outcomes of dysferlin deficiency in skeletal muscle
Development of a therapeutic transgene intended for clinical application requires preclinical efficacy with a functional outcome measure. Prior to the initiation of our study, a physiological outcome measure for tetanic force or resistance to contraction induced injury had not been defined in dysferlin deficient mice. We first assessed skeletal muscle function using the extensor digitorum longus muscle (EDL) in 6 month old animals in all three dysferlin-deficient mouse strains and corresponding strain controls (8 per group). Animals were euthanized and the EDL was dissected for in vitro force measurements. Dysferlin deficient muscles showed no deficits in maximum isometric force compared to strain controls when normalizing for the cross-sectional area of the muscle (ANOVA, P>0.05) (Fig. S2A). After assessment of specific force, the muscles were then subjected to mechanical damage by repetitive eccentric contractions. Dysferlin deficient muscles showed no significant reduction in force generation by repetitive eccentric contractions compared to their corresponding strain control muscles (Fig. S2B–D, 2-way analysis of variance, P>0.05).
Functional deficits were further examined in the diaphragm muscle that exhibits progressive signs of dystrophy [35]. Diaphragm strips from 24 week old 129-Dysf−/−, SJL-Dysf, and A/J animals along with corresponding strain control animals (8 per group) were dissected with rib attachments and central tendon intact. A 1–2 mm wide section (from rib to tendon) of diaphragm was isolated, and attached to a force transducer. The muscle was stretched to the length where twitch contractions were optimal, allowed to rest for 10 minutes, and subjected to a protocol consisting of a series of eight tetanic contractions occurring at 2 minute intervals, each with duration of 500 ms. Following a 5 minute rest, the muscle underwent a fatigue protocol which measured the force exerted by the muscle when stimulated every second for 90 seconds (500 ms tetanus at 100 Hz). All measurements were normalized to cross sectional area. Dysferlin deficient diaphragms demonstrated significant deficits in maximum isometric force compared to strain controls (ANOVA, P<0.05) (Fig. 4A). Dysferlin deficient diaphragms were also significantly more affected (larger loss of force) by muscle fatigue compared to their corresponding strain control muscles (Fig. 4B, 2-way analysis of variance, P<0.001).
Diaphragms from SJL-Dysf, A/J, and 129-Dysf−/−animals along with control animals C57/BL10, A/HeJ, and 129S1/SvImJ (8 per group) were isolated. (A) All three models demonstrated reduced specific force compared to their corresponding strain control (t-test P<0.05). (B) Fatigue induced by stimulation every second revealed significantly lower resistance to fatigue in all three Dysferlin deficient models compared to their corresponding strain controls (2-way analysis of variance, P<0.001). Force retention following ten contractions is shown.
Functional deficiency in the diaphragm provided a substrate to test AAV5.DYSF gene transfer in dysferlin deficient mice. To deliver the vector to the diaphragm, a single incision was made from the base of the sternum to just above the pelvis in 10 week old 129-Dysf−/− mice (n = 6 per group). The diaphragm was identified and 30 µl of the vector preparation (2×1011 vg) was delivered using a 32 gauge needle prior to closing the abdominal wall. Animals were sacrificed 10 weeks post treatment and the diaphragm was isolated and subjected to maximum tetanic contractions and a muscle fatigue protocol. Treated diaphragms demonstrated a significant improvement in tetanic force (Fig. 5A, P<0.05, ANOVA) which was not different from WT force (129S1/SvImJ). Treated diaphragms also demonstrated a significant improvement in resistance to fatigue compared to saline treated controls (Fig. 5B, 2-way analysis of variance, P<0.001), and were not significantly different than WT strain controls (129S1/SvImJ).
The diaphragm of 10 week old dysferlin deficient mice (129-Dysf−/−) (n = 6 per group) was treated with 1011 vg of rAAV5.DYSF via a peritoneal incision. Ten weeks post gene transfer, diaphragm muscle strips were harvested and subjected to a protocol to assess tetanic force and resistance to fatigue. (A) rAAV5.DYSF treated diaphragms demonstrated significant improvement in tetanic force (P>0.05, ANOVA) which was not different from wild-type force (129S1/SvImJ). (B) rAAV5.DYSF treated diaphragms demonstrated significant resistance to fatigue compared to untreated Dysf−/− controls (2-way analysis of variance, P<0.001) and were not different than SVJIM wild-type controls. Force retention following ten contractions is shown.
Restoration of membrane repair following intramuscular delivery of AAV5.DYSF
We next evaluated the ability of AAV5.DYSF treatment to restore membrane repair capability in dysferlin deficient muscle. To test this we performed a membrane wounding/resealing assay using a multi-photon laser scanning microscope on fibers isolated from the flexor digitorum brevis (FDB) muscle. We injected 2 month old 129-Dysf−/− (5 per group) with 3×1010 vg AAV5.DYSF in the (FDB) muscle. WT (129S1/SvImJ), 129-Dysf−/− and AAV5.DYSF treated 129-Dysf−/− mice were sacrificed 8 weeks post-treatment, the FDB muscle was isolated, and individual fibers were isolated following collagenase treatment. Sarcolemmal damage was induced in isolated fibers using the multiphoton laser (20% power for 5s) in the presence of FM 1–43 dye. A small area of fluorescence was detected in all fibers immediately after laser injury at the site of damage. In WT muscle fibers the sarcolemma is repaired and the amount of dye that integrates into the membrane stabilizes (Fig. 6). In contrast, dye continued to integrate into the sarcolemma of the fibers from 129-DYSF−/− muscle which resulted in significantly higher levels of fluorescence following a 3 min time course (Fig. 6). Expression of dysferlin from AAV5.DYSF-transduced fibers resulted in membrane repair that was equivalent to WT fibers further indicating that the exogenously expressed protein is fully functional (Fig. 6). Taken together, these data demonstrate that a large, potentially therapeutic cDNA can be delivered to muscle and efficiently express full-length functional dysferlin protein in muscle using AAV5.
(A) Individual flexor digitorum brevis fibers were isolated from WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF mice and the sarcolemma was damaged in the context of a solution containing FM 1–43. Images were taken before injury (−5 s) and every 5 s for a total of 195 s. Representative initial and final images for WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF fibers are shown. (B) Fluorescence intensity from injured fibers was measured using ImageJ software, converted to change in fluorescence intensity over time, and then graphed. For clarity, only values corresponding to 15 s intervals are shown following injury. (C) The total change in fluorescence intensity over time is shown for WT, 129-DYSF−/−, and 129-DYSFrAAV.DYSF fibers at 195 s post-injury. (2-way analysis of variance, P<0.05).
Regional vascular delivery of rAAV5.DYSF
Translational goals of dysferlin gene replacement require vascular delivery to reach multiple muscle groups. Therefore, we addressed whether rAAV5.DYSF could effectively cross the vascular barrier and transduce the lower hindlimb muscles of dysferlin deficient mice. rAAV5.DYSF (1012 vg) was delivered via the femoral artery to the hindlimb of Dysf−/− mice using a methodology previously described for other AAV serotypes and transgenes [9]. Four weeks post transfer; animals were analyzed for dysferlin expression. Efficient transduction of the tibialis anterior muscle was observed (Fig. 7A) with 75.0±16.2% muscle fibers expressing dysferlin. Western blot analysis again confirmed protein expression as evidenced by a clear 237 kDa band corresponding to full-length dysferlin (Fig. 7B).
rAAV5.DYSF (1012 vg) was delivered via the femoral artery to the hindlimb of 3–4 week old Dysf−/− mice. (A) Four weeks post transfer, immunostaining demonstrated dysferlin expression in treated animals (right). (B) Western blot confirmed 237 kd dysferlin protein in treated muscle which is absent in PBS controls. Scale bar = 100 µm.
AAV-mediated gene delivery remains a potential treatment option for some patients with muscular dystrophy. Challenges, however, for DYSF gene replacement relate to AAV packaging limits for genomes significantly larger than wild-type length (4.8 kb). Fortunately, the sentinel report of Alloca et al. brought enhanced packaging by AAV5 to light and gave new hope for expanding the capacity of AAV gene transfer for monogenic disorders of large genes with the potential to be carried to the bedside [30]. In their studies, efficient packaging, transduction, and expression of an 8.9 kb cassette of a large inherited gene causing blindness implied the ability to overcome the hurdle of a 5 kb size limitation for AAV for replacement therapy. Our current study along with three others [29], [31], [32] have demonstrated that homologous recombination of partially packaged genomes is the mechanism responsible for the generation of full-length transcripts rather than oversized packaging of the whole genome. Regardless, AAV5 does exhibit enhanced plasticity regarding packaging constraints which is likely contributing to its ability to mediate production of full-length transcripts in muscle following homologous recombination [30]. With regard to neuromuscular diseases, the findings provided a new perspective for conditions caused by mutations of large genes. DMD is the most common severe childhood muscular dystrophy and would seem to benefit from expression of the larger transcripts than mini- and micro-dystrophins that only partially restore physiologic function in the mdx mouse [9], [23]. Less common disorders, such as titin deficiency causing LGMD2J, and variants of congenital muscular dystrophy such as phenotypes caused by LARGE gene mutations would also benefit from expression of the full length protein [37], [38], [39]. Because of the success of Alloca et al., it was our intent to take advantage of the transduction capabilities of AAV5 for skeletal muscle [30]. We targeted the dysferlin gene because of its relative frequency amongst the LGMDs [5], [40]. Our findings are supportive of the unique properties of AAV5 to express a 6.5 kb cDNA producing full length protein but the mechanism for expression of the dysferlin gene pointed in a different direction than reported for the series of retinal genes protecting against blindness [30]. Our studies confirmed that AAV5 followed traditional packaging limits for DYSF because analysis of packaged virions was consistent with a packaging limitation of ∼5 kb. Thus, AAV5 virions transducing muscle fibers contained partial dysferlin vector genomes that mapped to both the 5′ and 3′ ends of the expression cassette and produced an intact expression cassette likely through homologous recombination upon reaching the nucleus [29], [31], [32]. This enabled translation of the full length 237 kDa protein demonstrated by western blots following gene transfer. The findings were equally robust following intramuscular and intravascular delivery, and the validity of the newly expressed protein was confirmed by its full restoration of physiologic function in the diaphragm and membrane repair capabilities in skeletal muscle.
Of particular importance, the potential for DYSF gene replacement has also been demonstrated by Lostal et al. with their comparable findings demonstrating expression of full-length dysferlin [27]. These researchers used a dual vector strategy permitting the dysferlin cDNA to be split at the exon 28/29 junction and cloned it into two independent AAV vectors carrying the appropriate splicing sequences. Their work tested the efficiency of dual packaging into AAV2/1 by intramuscular injection of both vectors into a dysferlin-deficient mouse and a novel strategy using systemic delivery of dual vector administration of both rAAV2/1 and rAAV2/9. Overall, both IM and systemic vascular delivery via the tail vein of dysferlin-deficient mice showed improvement of muscle pathology. Their study also noted an improvement in membrane repair in the FDB that did not reach WT control levels. By systemic delivery they found low levels of transduction (1–4%) which is a current limitation of the dual vector approach. In a clinical setting, higher levels of gene expression may be required to achieve a clinically meaningful outcome. Lostal et al suggested that 30% levels of dysferlin may be needed (26).
An additional report by Krahn et al. described a human minidysferlin protein that was identified in a patient with late-onset moderate dysferlinopathy [28]. The 73 kDa protein lacks the consensus dysferlin N-terminus but maintains the wild-type C-terminus including the last two C2 domains and transmembrane domain. The authors packaged this minidysferlin cDNA in a cassette containing a C5.C12 promoter and delivered it to A/J dysferlin deficient mice using AAV2/1 and AAV2/9. Expression of the protein was confirmed in the TA and found to localize primarily to the cytoplasm of muscle fibers and to a lesser extent in the sarcolemma. An examination of function revealed that the minidysferlin led to partial improvement in the membrane repair deficits accompanying the dysferlin-null FDB muscle fibers [28].
Our studies demonstrate clinical applicability for AAV5.DYSF gene transfer for dysferlin deficiency. There are several advantages to using AAV5 to deliver the full-length dysferlin cDNA. One particular advantage is the ability to deliver the entire cassette with one vector, significantly reducing viral load compared to the dual vector strategy. This is especially important with regards to translation in terms of feasibility of vector production and limiting capsid exposure for patients. A second advantage relates to full-length protein versus a miniature version. Although mini-genes are desirable for practicality of standard AAV delivery, there is a compromise in protein function [28] and the potential for novel epitopes that may be immunogenic when delivered to patients [41]. One potential concern with AAV5.dysferlin delivery is the presence of non-recombined vector genome fragments; however we showed only one mRNA and protein species was present in transduced muscle. As with any other naturally occurring truncated transcript that could be produced; elimination would occur by nonsense mediated decay. An informal discussion with the FDA (Rodino-Klapac and Mendell personal communication) defined a potential path for a dysferlin clinical gene therapy trial assuming no problems were encountered in the toxicology/biodistribution studies done with the same rigor as other approved AAV vectors [41], [42], [43].
In conclusion, we have shown that AAV5.dysferlin delivery is a very promising therapeutic approach that could restore functional deficits in dysferlinopathy patients. Specific muscle groups could be targeted by intramuscular delivery for dysferlin phenotypes that include Miyoshi myopathy and distal anterior compartment myopathy. In addition, based on our experience using fluoroscopy guided vascular delivery studies in the non-human primate we can thread the intravascular catheter to the take off point of specific vessels 9. This opens up clinical pathways for gene delivery to particular muscle groups relevant to either LGMD2B or Miyoshi myopathy including quadriceps and hamstring muscles, and the anterior or the posterior compartments of the lower limb.
Materials and Methods
Dysferlin gene construction
The full-length human dysferlin cDNA was used for all gene transfer studies. The MHCK7 promoter is derived from the MCK promoter with an additional 5′ enhancer from the myosin heavy chain (gift of S. Hauschka) [33]. The cassette includes a consensus Kozak sequence, an SV40 chimeric intron, and a synthetic polyadenylation site (53 bp). The dysferlin expression cassettes was cloned between AAV2 ITRs using flanking Xba I restriction enzyme sites in a plasmid derived from pCMVβ (Clontech). Msc I/Sma I restriction enzyme digestions were used to confirm ITR integrity.
rAAV Vector production
rAAV2/5 vectors were produced by a modified cross-packaging approach whereby the AAV type 2 vector genome can be packaged into multiple AAV capsid serotypes [21]. Production was accomplished using a standard 3 plasmid DNA/CaPO4 precipitation method using HEK293 cells. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin. The production plasmids were: (i) pAAV.MHCK7.dysferlin, (ii) rep2-cap5 helper plasmid encoding serotype 5 capsid proteins, and (iii) an adenovirus type 5 helper plasmid (pAdhelper) expressing adenovirus E2A, E4 ORF6, and VA I/II RNA genes. Virus was purified from 1% DOC detergent lysed clarified cell pellets using iodixanol gradients and anion-exchange column chromatography as previously described [44]. A quantitative PCR-based titration method was used to determine an encapsidated vector genome (vg) titer utilizing a Prism 7500 Taqman detector system (PE Applied Biosystems) [45].
Vector genome analysis
DNA was isolated from the rAAV5.DYSF vector preparation as previously described [32] with some modifications. Briefly, 5×1011 vg was subjected to DNase (Invitrogen) treatment (225 U at 37°C 30 min, 95°C 10 min, 4°C) followed by Proteinase K (Invitrogen) treatment (20 µg at 50°C 60 min, 95°C 20 min, 4°C). Vector DNA was purified using a Qiagen PCR purification kit according to the manufacturer's instructions (Qiagen Inc, Cat No. 28104). Southern blot analysis was performed as previously described [32]. Fifteen ng of probe (MHCK7 and Dysferlin) was used for each Southern blot hybridization.
RNA analysis
RNA was isolated from TA muscles using an RNeasy® Mini Kit (QIAGEN). cDNA was synthesized from 100 ng RNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems™) and PCR was performed using PFU Ultra II (Strategene) with the following primers: Dysf 1F (5′ATGCTGAGGGTCTTCATCCTCTA3′) – Dysf 1976R (5′ACCACAGGTTTCACGTTACC3′), Dysf 1957F (5′GGTAACGTGAAACCTGTGGT3′) – Dysf 5005R (5′TGTTCTCCAGGTCGACGAC3′), Dysf 3820F (5′TTGAGCTCATCCAGAGAGAGAAGC3′) – Dysf 6243R (5′TCAGCTGAAGGGCTTCACCAG3′). Human skeletal muscle total RNA (Biochain) and the pAAV.MHCK7.Dysf plasmid (50 ng) were used as positive controls. PCR products were gel purified using the Qiaquick Gel Extraction Kit (QIAGEN) and sequenced using the following primers: Dysf 1F, Dysf 499R (5′ CAGTGAGTCCCTGGTCCTCT3′), Dysf 501F (5′ AGATGAGGCGGAGCCATTCC3′), Dysf 900F (5′GTTCCGGATGGACGTGGGCA3′), Dysf 1976R (5′ACCACAGGTTTCACGTTACC3′), Dysf 1957F, Dysf 2400R (5′TCCCTGCAGCATCCAGATGA3′), Dysf 3339R (5′CCTCAAGGGCAAACACAGCTGC3′), Dysf 3377F (5′TTCCATGTCCGTCTCCACCTTG3′), Dysf 3820F, Dysf 4750F (5′ ATTGTCCGAGCATTTGGCCT3′), Dysf 5005R (5′TGTTCTCCAGGTCGACGAC3′), Dysf 5750F (5′ CCTTTGATGATTTTCTGGGC3′), and Dysf 6243R. The sequenced cDNA from injected tissue was then compared to the reference Dysferlin sequence (Genbank# NM_003494.3) using the ClustalW online alignment tool.
Dysferlin deficient mouse strains
Stocks of A/J, A/HeJ, SJL/J, C57BL/10, 129S1/SvImJ, and 129-Dysf−/− mice were bred and maintained as homozygous animals in standardized conditions in the Vivarium at the Research Institute at Nationwide Children's Hospital. They were maintained on Teklad Global Rodent Diet (3.8% Fiber, 18.8% Protein, 5% fat chow) with a 12∶12 h dark∶light cycle. Procedures used in the experiments were approved by the Institutional Animal Care and Use Committee at Nationwide Childrens Hospital (AR08-00017).
rAAV5.DYSF intramuscular gene transfer
The anterior compartment (containing tibialis anterior muscle and EDL) of 4–6 week old dysferlin deficient mice was injected with 1011 vg of rAAV5.DYSF diluted in normal saline buffer (50 µl volume). Control mice were sham injected with normal saline. The TA and EDL muscles from both limbs were harvested at 4 weeks post administration to assess efficiency of gene transfer. For histological analysis, muscles were embedded in 7% gum tragacanth and flash frozen in isopentane cooled in liquid nitrogen. Cryostat sections (12 µm) were collected for immunohistochemistry.
Intramuscular injections into the diaphragm
Ten week old dysferlin deficient mice (n = 6 per group) were treated with 2×1011 vg of rAAV5.DYSF via a peritoneal incision. Anesthetized mice (Ketamine/Xylazine 100 mg/kg and 10 mg/kg, respectively) were secured to a warm surgical table and the abdomen prepped and draped in a sterile fashion. A single incision was made from the base of the sternum to just above the pelvis (approximately 1 cm incision). The diaphragm was identified and 30 µl of the vector preparation in sterile saline was delivered using a 32 gauge needle. The abdominal wall was closed with 4.0 Vicryl Plus continuous sutures and skin wound closed with sterile surgical staples. Mice were treated with a post-op dose of Buprenorphine 0.01 mg/kg subcutaneously for pain. Ten weeks post gene transfer; diaphragm muscle strips were harvested and subjected to a protocol to assess resistance to fatigue.
Membrane Repair Assay
The ability of the sarcolemma to repair following injury was assessed on at least 5 129-SVLMJ, 129-Dysf−/−, and 129-DysfrAAV5.DYSF mice at 4.5 months of age. Individual fibers were isolated from the flexor digitorum brevis muscle following treatment with a 2% collagenase solution. The fibers were washed in PBS and placed in glass bottom dishes in the presence of 2.5 µM FM® 1–43 (Invitrogen®) with or without 1.5 mM Ca2+. Membrane damage was induced with a FluoView® FV1000 two-photon confocal laser-scanning microscope (Olympus). A circular area (diameter, 5 µm) on the edge of the sarcolemma was irradiated at 20% power for 5 s. Images were captured 5 s prior to injury and every 5 s after injury until 3 min post irradiation. For every image, the fluorescence intensity surrounding the site of damage was analyzed (ImageJ). At least 25 fibers were evaluated for each condition (2-way analysis of variance, P<0.05).
Vascular delivery
Eight adult dysferlin deficient mice (4–6 weeks of age) were perfused with 1012 vg rAAV5.DYSF in 100 µL of normal saline as described [9]. Briefly, mice were anesthetized and the femoral bundle was visualized via a small incision proximal to the mid-thigh. Blood flow through the femoral artery was controlled by catheter placement using a customized heat pulled polypropylene 10 (PE-10) catheter placed into the femoral artery. Prior to vector administration, the arterial catheter was flushed (pre-flush) with 100 µl sterile normal saline. Immediately prior to vector administration all blood flow to the extremity was impeded (isolated limb perfusion – ILP) by tightening the ligature at the mid-thigh. rAAV5.DYSF was perfused through the femoral artery in 100 µl of sterile Tris buffered saline administered at a rate of approximately 2 µl per second (over 60–80 seconds). After 10 minutes of maintained vascular occlusion, 100 µl of normal saline was administered to the arterial catheter (again at about 2 µl per second) as a post-flush and the tourniquet was then released. The wound was flushed with sterile normal saline and closed with a 6-0 proline suture.
Immunostaining for dysferlin was performed on all transduced tissue to assess efficacy of gene transfer. Tissue sections were incubated with dysferlin rabbit anti-human monoclonal (Lifespan Biosciences, Inc., Cat #LS-C138735) antibody to detect dysferlin at a dilution of 1∶100 in block (20% goat serum, 0.1% triton X-100 in PBS) applied for 1 hour at 25°C, in a wet chamber. Sections were washed with PBS 3×20 minutes, reblocked, and then incubated for 30 minutes at 25°C with an Alexa 568 goat anti rabbit antibody at a dilution of 1∶300 (Molecular Probes). Sections were washed in PBS for 3×20 minutes and mounted with Vectashield mounting medium (Vector Laboratories). Images were captured with a Zeiss Axioskop2 Plus Microscope and AxioCam MRC5 camera. Four random 20× images (each field with an average of 150 muscle fibers) were captured per muscle and the number of fibers with dysferlin staining was counted and expressed as percent of total number of fibers.
Centralized nuclei counts were performed on sections of TA muscles stained with hematoxylin and eosin (H&E) from AAV5.DYSF treated Dysf−/− animals. TAs from the contralateral extremity served as controls. Four random 20× fields of 12 µm sections for each muscle were captured and the number of fibers with central nuclei counted and expressed as a percentage of the total number of fibers.
Western Blot Analysis
The TA was isolated from both the treated and contralateral limbs. From each muscle, fresh frozen serial tissue sections were taken for both immunohistochemistry and for western blot analysis. Muscle samples harvested from rAAV5.DYSF and control groups were compared with wild-type mice for levels of vector-mediated dysferlin compared to endogenous dysferlin. Protein (25 µg) extracted from treated and control samples was separated by SDS-PAGE (3–8% Novex NuPAGE gradient gels, Invitrogen), blotted on PVDF membrane and probed with dysferlin rabbit anti-human monoclonal (Lifespan Biosciences, Inc.) primary antibody at a dilution of 1∶1000, NCL-Hamlet (Novocastra) primary antibody at a dilution of 1∶1,000 or actin antibody (NCL-MSA) at a dilution of 1∶6,000 followed by horseradish-peroxidase labeled goat anti-mouse IgG or hoseradish-peroxidase labeled goat anti-rabbit IgG (1∶5,000, Millipore). Immunoreactive bands were detected with ECL Plus detection system (GE Healthcare) and signal captured on Hyperfilm ECL (Amersham).
Force generation and protection from eccentric contractions in EDL
SJL-Dysf, A/J, and 129-Dysf−/− animals along with control animals C57/BL10, A/HeJ, and 129S1/SvImJ (8 per group) were assessed for physiological deficits in the extensor digitorum longus (EDL) muscle at 24 weeks (when histopathological features are present) as previously described [9], [46]. Mice were euthanized and the EDL was removed, the tendons sutured, and bathed in oxygenated circulating Krebs-Henseleit solution at 30°C in an organ bath. For the procedure, one end of the muscle was tied to a force transducer and the other to a high-speed linear servo-controlled motor. The muscle was mounted in the set-up at slack length with a resting tension of 1 g for 10 minutes without electrical stimulation. Stimulation was delivered via two parallel platinum-iridium electrodes on either side of the muscle. Muscles were adjusted to optimum length (L0), defined as the length for maximal twitch and subjected to an isometric tetanus of 150 Hz. Following a 5 minute rest period, muscles were subjected to an eccentric contraction protocol consisting of a series of 10 isometric 700 ms tetani, at 2 minute intervals, with a 5% lengthening of the muscles (0.5 fiber length per second for duration of 200 ms) when maximal force had developed at 500 ms. After the tetanus ended (at t = 700 ms), the muscle was brought back to initial length (at the same speed as the stretch), allowing for full relaxation to the initial length. For comparative purposes, all force measurements are expressed per unit cross-sectional area (normalized isometric force or tension: mN/mm2). Cross-sectional area (CSA) is calculated using the following equation, CSA = (muscle mass in g)/[(optimal fiber length in cm)×(muscle density in g/cm3)], where muscle density is 1.06 g/cm3.
Diaphragm Tetanic Contraction and Muscle Fatigue Methods
As a second approach, the diaphragm will be tested as a target for a therapeutic outcome measure. SJL-Dysf, A/J, and 129-Dysf−/− animals along with control animals C57/BL10, A/HeJ, and 129S1/SvImJ (8 per group) were assessed at 24 weeks. Mice were euthanized and the diaphragm was dissected with rib attachments and central tendon intact, and placed in K-H buffer at 37 C as previously described [47], [48]. A 1–2 mm wide section (with length from rib to tendon) of diaphragm was isolated, and attached to a force transducer. The diaphragm strip was looped around a basket assembly attached to the transducer (the rib cartilage serves as the anchor), and the tendon was pierced by a pin. The muscle was stretched to optimal length for measurement of twitch contractions, and then allowed to rest for 10 minutes before initiation of the tetanic protocol. The protocol consisted of a series of eight tetanic contractions occurring at 2 minute intervals, each with duration of 500 ms. The force was recorded for each stimulus, and normalized to account for muscle width and length. The muscle was rested for 5 minutes before starting the muscle fatigue protocol. This protocol measures the force exerted by the muscle when stimulated every second for 90 seconds (500 ms tetanus at 100 Hz). Following the muscle fatigue protocol, the muscle strip was removed from the apparatus, the rib cartilage removed and weighed.
Supporting Information
Figure S1.
RNA analysis from injected tissue. Sequence analysis of cDNA from rAAV.DYSF-injected muscle. cDNA from injected tissues was sequenced completely and then aligned to the reference Dysferlin sequence containing UTRs. The sequenced cDNA aligns exactly with the reference.
Figure S2.
Functional assessment of the EDL muscle in Dysf−/− mice. SJL-Dysf, A/J, and 129-Dysf−/− animals along with control animals C57/BL10, A/HeJ, and 129S1/SvImJ (8 per group) were assessed for physiological deficits in the EDL. (A) Dysferlin deficient muscles showed no deficits in maximum isometric force compared to strain controls when normalizing for the cross-sectional area of the muscle (ANOVA, P>0.05). (B–D) Muscles were subjected to mechanical damage by 10 repetitive eccentric contractions. Dysferlin deficient muscles were not significantly more affected (larger loss of force) by repetitive eccentric contractions compared to their corresponding strain control muscles (2-way analysis of variance, P>0.05)
We thank Nationwide Children's Viral Vector Core for vector production and Stephen D. Hauschka for the MHCK7 promoter. We also thank Nancy Davis and Jianchao Zhang for technical assistance.
Author Contributions
Conceived and designed the experiments: WEG JRM LRK. Performed the experiments: WEG KRC DG VM KMS CLM SL PMLJ LRK. Analyzed the data: WEG JRM LRK. Contributed reagents/materials/analysis tools: RHB. Wrote the paper: WEG JRM LRK.
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- 13. Liu J, Wu C, Bossie K, Bejaoui K, Hosler BA, et al. (1998) Generation of a 3-Mb PAC contig spanning the Miyoshi myopathy/limb-girdle muscular dystrophy (MM/LGMD2B) locus on chromosome 2p13. Genomics 49: 23–29.
- 14. Therrien C, Dodig D, Karpati G, Sinnreich M (2006) Mutation impact on dysferlin inferred from database analysis and computer-based structural predictions. J Neurol Sci 250: 71–78.
- 15. Bansal D, Miyake K, Vogel SS, Groh S, Chen CC, et al. (2003) Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature 423: 168–172.
- 16. Lennon NJ, Kho A, Bacskai BJ, Perlmutter SL, Hyman BT, et al. (2003) Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing. J Biol Chem 278: 50466–50473.
- 17. Han R, Campbell KP (2007) Dysferlin and muscle membrane repair. Curr Opin Cell Biol 19: 409–416.
- 18. Piccolo F, Moore SA, Ford GC, Campbell KP (2000) Intracellular accumulation and reduced sarcolemmal expression of dysferlin in limb–girdle muscular dystrophies. Ann Neurol 48: 902–912.
- 19. Cenacchi G, Fanin M, De Giorgi LB, Angelini C (2005) Ultrastructural changes in dysferlinopathy support defective membrane repair mechanism. J Clin Pathol 58: 190–195.
- 20. Dong JY, Fan PD, Frizzell RA (1996) Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther 7: 2101–2112.
- 21. Gao G, Vandenberghe LH, Wilson JM (2005) New recombinant serotypes of AAV vectors. Curr Gene Ther 5: 285–297.
- 22. Hermonat PL, Quirk JG, Bishop BM, Han L (1997) The packaging capacity of adeno-associated virus (AAV) and the potential for wild-type-plus AAV gene therapy vectors. FEBS Lett 407: 78–84.
- 23. Harper SQ, Hauser MA, DelloRusso C, Duan D, Crawford RW, et al. (2002) Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Nat Med 8: 253–261.
- 24. Wells DJ, Wells KE, Asante EA, Turner G, Sunada Y, et al. (1995) Expression of human full-length and minidystrophin in transgenic mdx mice: implications for gene therapy of Duchenne muscular dystrophy. Hum Mol Genet 4: 1245–1250.
- 25. Duan D, Yue Y, Yan Z, Engelhardt JF (2000) A new dual-vector approach to enhance recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation. Nat Med 6: 595–598.
- 26. Ghosh A, Yue Y, Long C, Bostick B, Duan D (2007) Efficient whole-body transduction with trans-splicing adeno-associated viral vectors. Mol Ther 15: 750–755.
- 27. Lostal W, Bartoli M, Bourg N, Roudaut C, Bentaib A, et al. (2010) Efficient recovery of dysferlin deficiency by dual adeno-associated vector-mediated gene transfer. Hum Mol Genet 19: 1897–1907.
- 28. Krahn M, Wein N, Bartoli M, Lostal W, Courrier S, et al. (2010) A naturally occurring human minidysferlin protein repairs sarcolemmal lesions in a mouse model of dysferlinopathy. Sci Transl Med 2: 50ra69.
- 29. Lai Y, Yue Y, Duan D (2010) Evidence for the failure of adeno-associated virus serotype 5 to package a viral genome > or = 8.2 kb. Mol Ther 18: 75–79.
- 30. Allocca M, Doria M, Petrillo M, Colella P, Garcia-Hoyos M, et al. (2008) Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice. J Clin Invest 118: 1955–1964.
- 31. Dong B, Nakai H, Xiao W (2010) Characterization of genome integrity for oversized recombinant AAV vector. Mol Ther 18: 87–92.
- 32. Wu Z, Yang H, Colosi P (2010) Effect of genome size on AAV vector packaging. Mol Ther 18: 80–86.
- 33. Salva MZ, Himeda CL, Tai PW, Nishiuchi E, Gregorevic P, et al. (2007) Design of tissue-specific regulatory cassettes for high-level rAAV-mediated expression in skeletal and cardiac muscle. Mol Ther 15: 320–329.
- 34. Bittner RE, Anderson LV, Burkhardt E, Bashir R, Vafiadaki E, et al. (1999) Dysferlin deletion in SJL mice (SJL-Dysf) defines a natural model for limb girdle muscular dystrophy 2B. Nat Genet 23: 141–142.
- 35. Ho M, Post CM, Donahue LR, Lidov HG, Bronson RT, et al. (2004) Disruption of muscle membrane and phenotype divergence in two novel mouse models of dysferlin deficiency. Hum Mol Genet 13: 1999–2010.
- 36. Roche JA, Ru LW, O'Neill AM, Resneck WG, Lovering RM, et al. (2011) Unmasking potential intracellular roles for dysferlin through improved immunolabeling methods. J Histochem Cytochem 59: 964–975.
- 37. Barresi R, Michele DE, Kanagawa M, Harper HA, Dovico SA, et al. (2004) LARGE can functionally bypass alpha-dystroglycan glycosylation defects in distinct congenital muscular dystrophies. Nat Med 10: 696–703.
- 38. Bushby KM, Beckmann JS (2003) The 105th ENMC sponsored workshop: pathogenesis in the non-sarcoglycan limb-girdle muscular dystrophies, Naarden, April 12–14, 2002. Neuromuscul Disord 13: 80–90.
- 39. Longman C, Brockington M, Torelli S, Jimenez-Mallebrera C, Kennedy C, et al. (2003) Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of alpha-dystroglycan. Hum Mol Genet 12: 2853–2861.
- 40. Moore SA, Shilling CJ, Westra S, Wall C, Wicklund MP, et al. (2006) Limb-girdle muscular dystrophy in the United States. J Neuropathol Exp Neurol 65: 995–1003.
- 41. Mendell JR, Campbell K, Rodino-Klapac L, Sahenk Z, Shilling C, et al. (2010) Dystrophin immunity in Duchenne's muscular dystrophy. N Engl J Med 363: 1429–1437.
- 42. Mendell JR, Rodino-Klapac LR, Rosales XQ, Coley BD, Galloway G, et al. (2010) Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdle muscular dystrophy, type 2D. Ann Neurol 68: 629–638.
- 43. Mendell JR, Rodino-Klapac LR, Rosales-Quintero X, Kota J, Coley BD, et al. (2009) Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins. Ann Neurol 66: 290–297.
- 44. Clark KR, Liu X, McGrath JP, Johnson PR (1999) Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses. Hum Gene Ther 10: 1031–1039.
- 45. Schnepp BC, Jensen RL, Chen CL, Johnson PR, Clark KR (2005) Characterization of adeno-associated virus genomes isolated from human tissues. J Virol 79: 14793–14803.
- 46. Martin PT, Xu R, Rodino-Klapac LR, Oglesbay E, Camboni M, et al. (2009) Overexpression of Galgt2 in skeletal muscle prevents injury resulting from eccentric contractions in both mdx and wild-type mice. Am J Physiol Cell Physiol 296: C476–488.
- 47. Rafael-Fortney JA, Chimanji NS, Schill KE, Martin CD, Murray JD, et al. (2011) Early treatment with lisinopril and spironolactone preserves cardiac and skeletal muscle in Duchenne muscular dystrophy mice. Circulation 124: 582–588.
- 48. Beastrom N, Lu H, Macke A, Canan BD, Johnson EK, et al. (2011) mdx(cv) mice manifest more severe muscle dysfunction and diaphragm force deficits than do mdx Mice. Am J Pathol 179: 2464–2474.


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Devotionals for 2019
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Animals are God's gift to humans. They are instruments of His Grace blessed with a special gift for comforting us when we are down, filling us with joy at just the right moment, and encouraging us on our walk of faith. That's why Guideposts created All God's Creatures.
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This beautiful new devotional begins in May to capture that joyous feeling that fills each one of us when spring arrives and warmer weather pushes out the dreary days of winter. Spring officially arrives in March, but May is when all of God's Creatures burst into life with the sweet vibrations of spring. It's the perfect time for a new devotional that brings you a refreshing new way to spend time in His Word. PLUS you'll receive four beautiful, custom-designed Note Cards with envelopes FREE - our gift to you!*
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When writing dialogue, how can you keep your characters from all sounding the same? If you're good at this task you can use regional dialects as a distinguishing characteristic, like my friend Wayne Zurl [http://www.waynezurlbooks.net] does so well in his Sam Jenkins series. If you do it well enough, and Wayne does, before you know it you have books that will sell well as audiobooks where differences in speech usage and patterns are the essential clue in individuating the characters as you listen to the story. At least for me, this is hard to do as you need a very nuanced ear and a very good memory for what they said and how they said it to make people sound different based only on word choice. According to The Guinness Book of Records [http://www.guinnessworldrecords.com/records-2000/most-character-voices-for-an-audio-book-individual] the greatest number of characters given distinct and distinguishable voices by an actor in an audiobook is 224. This amazing record was accomplished in 2004.
What can the aspiring author do to make their characters different from one another? If the educational level of the characters varies (college educated P.I. versus high school dropout thug), you have another way to make their voices quite different. The thug can use a lot of slang and slur his word endings, maybe even say a few bad words here and there. You can make a character stutter or misuse certain words, or even have an accent. One of my characters slips a Spanish word into his English every now and then. The perfect word for Vincent to be overusing for this particular idiosyncrasy is the classic parsley word used promiscuously in conversational Spanish, "claro". Vincent could make a chimichurri with all of the claro parsley he sprinkles into his conversation [check out a previous recent post on this blog on "Salsas" if this doesn't make sense to you, or go directly to the recipe at http://www.simplyrecipes.com/recipes/chimichurri].
My current WIP, "The Deadly Dog Show", poses a challenge in this area because there are a lot of characters in the cast. Y'all will see one of my solutions to this problem in the chapters set in Texas, where regional dialect is easy to transfer to print. One of my characters, a librarian named Saundra, actually sounds a whole lot different than Vincent does in a long conversation between the two of them in a key scene in the book. I found the details involved in making this work difficult to create and even more difficult to write consistently from day to day. I wonder if this comes more easily to authors with musical talent and trained ears?
I haven't figured out as yet how to give the various dogs at several shows distinctive barks in their voices, but you can't win them all. Mostly, I encourage Juliet [the canine heroine of "The Deadly Dog Show] to keep her mouth shut unless she has something really important to say. As a German Shorthaired Pointer, she would have a pretty deep bass "Woof". Maybe I'll just skip the audiobook market for now.


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These are vintage creamy pink glass cabochons that were made in Germany. They have a faceted top. The back is a button back with a thin rim for gluing. They measure 22mm. You will receive 4 cabs.
1 business day
I'll do my best to meet these shipping estimates, but can't guarantee them. Actual delivery time will depend on the shipping method you choose.
Buyers are responsible for any customs and import taxes that may apply. I'm not responsible for delays due to customs.
Just contact me within: 14 days of delivery
Ship items back to me within: 30 days of delivery
But please contact me if you have any problems with your order.
Because of the nature of these items, unless they arrive damaged or defective, I can't accept returns for:
Buyers are responsible for return shipping costs. If the item is not returned in its original condition, the buyer is responsible for any loss in value.
Feb 20, 2013
Very nice, and they came so fast!
Dec 22, 2012
Love them, Thank you :o) Merry Christmas!
May 23, 2012
Jan 5, 2012
★Nice Item and Fast Ship...Thank You ★
Oct 18, 2011


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Place your order by 5PM EST Wed, April 24 and we will ship it by Fri, May 03.
If you need this item sooner please contact us at firstname.lastname@example.com or call us at 800-554-3509 and let us know. We will work our magic to get it to you by your desired date.
The center stone one the engagement ring is a cushion cut diamond, nearly colorless, weighing 1.50 carats. It is surrounded by a halo of 58 round diamonds weighing 0.44 carats. There are 20 round diamond stones, nearly colorless, around the wedding band that weigh 0.20 carats. The center stone is hand selected by our GIA certified gemologist.
Sure to last a lifetime this cushion cut diamond halo bridal set is a representation of your love for one another.
Our Award winning customer service want to make sure you're completely satisfied. which is why we offer ironclad guarantee.
Did you know that after a house and a car, a ring the 3rd biggest purchase a person makes? Our expert jewelers put together this guide to help you navigate such an important purchase.
Cushion Cut Diamond Halo Bridal Set 14k Rose Gold (2.20ct)


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January, 2013 Combating the Flu and Other Helpful Tips
July, 2012 The Doctors Hospitals of the Future:
April, 2012 Robotic Surgery: It's not your grandmother's GYN Sugergy.
March, 2012 Cardiovascular Surgery has Evolved. Patients can be Treated in Hours and be Back to Their Lives in Days not Months. Innovations from the Fogarty Institute
February, 2012 Heart Attack Symptoms in Women Differ from Men
January, 2012 Losing Weight Takes Baby Steps
December, 2011 Nurse Barb Talks About a Great New Health App for Every Woman Who Manages Their Families Health
December, 2011 Family Health: Genetics and Family History Makes a Big Difference
November, 2011 You Don't Have to Live With a Leaky Bladder
October, 2011 Nurse Barb Finds the Most up to Date Ways of Detecting and Treating Breast Cancer
September, 2011 Joint Pain Doesn't Have to Rule Your Life
April, 2011 Nurse Barb Investigates: Commitment to Families at Packard Hospital's PICU – Pediatric ICU Nurses
March, 2011 Preventing Falls in the Home
Are Your Kids Playing 'Choking Games'?
Good Morning America Health
Sally Field and Barb talk about Bone Density Testing
Other appearances:
January, 2012 Nurse Barb and Peggy Fleming Discuss Cold and Flu Season
June, 2011 Nurse Barb Dehn Talk on Food Allergies Are All About Exposure
December, 2010 Whether or Not to Share Breast Milk
September, 2010 Help Raise Awareness on Bone Health
March, 2010 Women's Weight Lose Battles
April, 2010 Swine Flu
December, 2008 Holiday Emergencies
November, 2008 Sharing Your Breast Milk?
July, 2008 How to Talk to Your Teens About Sex.


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Combination Kayak with Snorkeling
All kayak tours are led by expert guides, and last about 2.5 hours. Snorkel gear is also included in the tour price. Kayak Maui's famed Olowalu coast … and then jump into the Pacific Ocean to snorkel and see an abundance of sea life. Many visitors say our tours were the best part of their Maui experience… We have excellent conditions year round for viewing exotic reef wild life… and people visit from all over the world to see whales migrate through our Hawaiian waters.
In addition to the world-famous reef and whale watching location, the Olowalu area has a rich Hawaiian history that dates back to ancient Hawaii, and we operate the only kayak tour on Maui with a private beachfront location.
Tour will begin with a 15-20 min. orientation to familiarize the customer with the use of the kayak and snorkel equipment. Depending on the assessed skill level of the customer, the guide may practice a few skills in the shallow calm waters prior to taking the group out on the tour.
As soon as everyone is comfortable with their kayak the guide will lead the group through the coral gardens to do some snorkeling for 30-45 minutes. Then, we take the tour north to see a few historic sites along the coast line where we take some time to pay tribute to Olowalu's rich history and have some snacks. After about 20-25 min. we're ready to head back out again to do another short snorkeling adventure and head back to base.
(tours vary depending on weather and surf conditions as well as skill level of customers)
Facts and Requirements
- Operates: Daily 8:00 am, 11:00 am and 2:00 pm
- Departure Point: Please check in 15 minutes prior at Olowalu Beach
- Duration: 2.5 hours
- Equipment: Kayak and snorkeling equipment, snack and water
- Inclusions: Light snack and bottled water
- Restrictions: minimum 5 years old
- Cancellation: Associated with cancellation fee (terms and conditions), rescheduling 48 hrs. for all reservations, and 72 hrs. for groups of 6 or more.


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Welcome to my OCEAN of TERROR!!!
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Welcome to my OCEAN of TERROR!!!
The picture above was made by Emeral
Well, well Stormer, I must say you've done amazingly well to get this far..... Well your happiness ends HERE because in this Ocean all you little and puny Stormers will have to fight with all your might against the MIGHTY HURRICANE!! Let me tell you this for free: It isn't going to be easy!!
Now let's get down to the rules to be followed in this OCEAN OF TERROR
1.No Hacks allowed(If i suspect any hacks you lose)
2.Please follow the clauses you've been following all throughout your challenge over here as well(They're to long to write so please follow them)
3.Ruleset (BW OU, UU etc....) will be set upon challenging me
4.If it is a Draw you don't go through(0-0 score doesn't get you through)
5.No crying when you lose(Come on stormers it's already an OCEAN out here!!)
6.The most important rule in this BIG BLUE LAND is to always have fun(If i think tht you're not having fun you're outta here!!)
Those are the rules in this Ocean
Good Luck to all the stormers trying to challenge the MIGHTY HURRICANE!!(You'll need more than tht to get past me!!)
-The great Hurricane himself, Arcena
Hurricane Arcena
- Admin
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Join date : 2012-05-07
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E198 -- Sur le principe de la moindre action
(On the principle of least action)
According to C. G. J. Jacobi, a treatise with this title was presented to the Berlin Academy on February 22, 1753.
Originally published in
Memoires de l'academie des sciences de Berlin
7, 1753, pp. 199-218
Opera Omnia
: Series 2, Volume 5, pp. 179 - 193
Reprinted in
Dissertatio de principio minimae actionis
), 1753, pp. 3, 5, 7, … , 93, 95
Reprinted in
Dissertation sur le principe de la moindre action
(see E1862), 1753, pp. 1-36
Documents Available:
Original publication:
can be viewed or downloaded from
Digitalisierte Akademieschriften und Schriften zur Geschichte
der Königlich Preußischen Akademie der Wissenschaften, which includes serial publications of the Prussian Academy of Science in the 18th and 19th Centuries.
Michael Saclolo and Peter Wake of St. Edward's University have completed a
translation of E198
, which they have made available to the Euler Archive.
Return to the Euler Archive


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One of my many desires for this theme is to have the member login buttons to appear in the "right-panel" on the home page for non-logged in users. I came across the following CSS settings while looking through the FAQ's for the WP-Facebook plugin:
#wp-submit{ height:23px; width:46px; padding:0; margin:0 0 0 7px; }
#user_pass{ height:18px; width:86px; padding: 3px; }
#user_login{ height:18px; width:86px; padding: 3px; }
#forgotText{ display:inline-block; font-size:small; margin-left:9px; height:26px; }
#fbLoginButton{ display:block; height:18px; margin-top:7px; text-align:center; }
#loginform label{ display:inline-block; width:2.75em; }
What I can't seem to do is "properly" insert this code in the "base.css" stylesheet to have the buttons appear above the "Join our Community Now..." button and in the "Network intro post text" area.
Can anyone direct me in this area?
Any responses appreciated.


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External VET (EVET) Courses
While many students enrol full-time at Southern Cross Catholic Vocational College, some courses are also offered on one block session per week (usually in the afternoon) so that students from other schools can attend part-time as external students. The timetable of classes being held in 2019 is shown here.
Calendars for 2019 EVET classes can be found here:
The following EVET courses are being offered in 2019. Most courses are 240 hour courses and run over 2 years.
- Automotive (240 hours over 2 years)
- Beauty Services (Make-Up) (360 hours over 2 years)
- Construction (Pathways) (240 hours over 2 years)
- Early Childhood Education and Care (360 hours over 2 years)*
- Events (Tourism, Travel and Events) (240 hours over 2 years)
- Furniture Making (180 hours in 1 year)
- Hairdressing (180 hours in 1 year)
- Human Services (Health Services Assistance) (300 hours over 2 years)**
- Information and Digital Technology (240 hours over 2 years)
- Music Industry (240 hours over 2 years)
- Property Services (Agency) (300 hours over 2 years)
- Sport Coaching (240 hours over 2 years)*
Most courses allow Year 12 students to complete a part qualification if they join the class for one year only. The exceptions are Early Childhood Education and Care* and Sport Coaching*. The Human Services** course can only be undertaken by Year 12 students if they are enrolled in the 240 hours course in one year.
In some cases students can also undertake specialisation courses related to courses at their own school or in addition to an existing EVET course, enabling them to either extend their competency or even complete the full Certificate III qualification in that course. These courses are an additional 120 hours, over one year.
- Construction (120 hours)
- Hospitality (Catering Operations) (120 hours)
Interested students should visit the Sydney Catholic Schools Vocational Education website to learn more about specific courses, you can also view more information about the VET Courses available in 2019 here
Students who wish to attend these courses in 2019 will be required to complete an EVET Expression of Interest form and discuss with their VET Coordinator at their home school. Once you receive permission from your school, you can apply for a course here.
More information was provided to interested parents and students at an Information Evening held on 31 October 2018 at the College, here is a link to the presentation from the event.
All students are inducted into the College during the first week of classes. A summary of the 2019 presentation can be viewed here.
As well as the VET Courses offered here at the college to students from other schools, SCCVC students have the option of undertaking EVET study in courses offered by other Trade Training/Skills Centres and TAFE NSW and other providers.
The following links provide further information on this option of study:
- Trade Training/Skills Centres
- TAFE NSW TVET information
External VET Student Attendance (School's Use Only)
Schools should follow this link to review NESA enrolment processes


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|Andean Bear Cub
The reason I loved this camera was because it had a view finder. It is difficult to find a point and shoot camera with a view finder. I like to have the choice to either use the view screen or the view finder. Often, the view screen is difficult to see on a sunny day, and I have to just guess and hope that the picture I have taken is what I intended.
I was lucky to have my big DSLR camera for the rest of the cruise, and also my iPhone...but I missed having that small camera that would fit in a pocket and take quick shots and movies. Luck was with me! Ray found my camera in the pocket of my robe. I must have been taking a shot from the balcony in the morning while in my robe waiting for breakfast. I forgot to mention in the other posts that Ray had found my camera.
After our cruise, we travelled to Washington, D.C. to visit Ray's daughter, Valerie. While there we went to the National Zoo. I will write about that later. The sad thing....I have decided that I no longer love my point and shoot camera. I sound like the woman on the insurance commercial who talks about loving her car and that she had even given her car a name...and then got into an accident and the poor car would have to be replaced. Once she hears from the insurance company she is all happy. Well, for me, I loved this camera...until our zoo visit.
Here is a nine second clip of the video I made of these two playful bear cubs.
Here is a 30 second clip of the same bear cubs. If you have a tendency to get sea sick, please don't watch this. If you are ready for an adventure, take a look.
So what happened!!! I realized later, when we had left the zoo, that when I thought I was recording a video I was actually on standby. And when I was on standby, I was really recording. So all of my videos of these two bear cubs were ruined. I have four other clips of them...well, of what I thought was them. Now I do not like my camera. If I had used the view screen instead of the view finder, I would have realized that I was not recording and I would have captured some fun videos.
|Here is a still shot.
All was not lost though. I used Ray's DSLR and recorded two videos of the bears. Here they are. You will understand why I am upset for messing up the other videos. These bears were having lots of fun and putting on quite a show for us.
Here are two more videos of these cute bears.
I think it is time to get a new point and shoot camera.


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Asphalt production:
the secret is in the mixing
According to the WIRTGEN GROUP precepts, CIBER stands out for its investments in technology in order to offer the most modern options when it comes to equipment for building and maintaining roads.
Counter-flow asphalt plant
From the development of its own technology, CIBER's counter-flow asphalt plants with external mixture ensure the quality of the final product while also respecting the environment and bringing to their owners the best cost-benefit ratio. That is why this technology has become a market leader in this segment.
Batch asphalt plant
The batch plant technology was the first to become well known in the paving market and its quality is still unsurpassed with the production of excellent asphalt mixtures that meet the demands of various specifications.
Cold mixture production
Similar to the production of hot asphalt mixtures, CIBER also invests in the research and development of cold technologies in order to offer full-scale solutions for its clients.


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Etteh Aro & Partners is an infrastructural development company that seeks to solve your problems, ensuring that you are satisfied. The firm achieves this by identifying the challenges present, gathering together the required talent and skills to produce your desired solution. We put a smile on your face always. At Etteh Aro & Partners, we are all about PROVIDING SOLUTIONS.
Our services include the following:
Etteh Aro & Partners have a wealth of experience in bridge engineering spanning the full project cycle (planning, design and construction supervision). Our portfolio include landmark bridges in Nigeria. Key examples of our portfolio include:


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Today I want to speak about Sparklines for Excel, "a set of free user defined functions for Excel to create sparklines".
Sparklines ? You know, those little graphics, often cell sized, as seen on a lot of financial / stock exchange / scientific screens.
Well, Frabrice Rimlinger is a smart french guy who coded these sparklines for Excel. VBA code, for all Excel flavors.
Have a look to his blog and download the macros. It is worth a try !
And now, my own personal usage of Sparkline : graphically showing source table sizes, related to my current datawarehouse project in Paris. I'm using the treemap component : each square size is calculated reagarding the table size. Colorization is done with a dynamic color scale.
Nice, isn't it ? And you know what ? Theses sparklines can be used with an OLAP Excel frontend : PALO, TM1 ... your choice...


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A Surefire Investment | Christianity Today | A Magazine of Evangelical Conviction
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My comments:
Philip Yancey has written this article on how to pray during this financial crisis. In this article, he uses the example of Jesus praying in the Garden of Gethsemane and how the prayer line changed from "Take this cup from me" to "May your will be done."
This article explores on the foundations we have been building our lives on, moving on to how we should move beyond our pain and struggles to the greater struggles of our neighbors.


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My host Yair Amsel picked me up in the center of Netanya. He is a great guy and I´m glad to say he is also
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a new friend. We spent the night discussing almost all the big issues and we didn´t really go to bed until very early morning…:-) In the morning we said goodbye and I started a hard day of walking. Hard due to the late night. I had no remorse though, it was so worth it!


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Tuesday, December 1, 2009
Bump...part two
At Xmas time my brother and I received a spectacular gift from my sportsmen uncle in Connecticut. A bow and arrow set, a real one-no rubber suction cups. I mean real arrows that you could hunt big game with and a target. The problem was that it was winter with snow on the ground and we were told that we would have to wait until spring to use it outside. But what eight and ten year old kids could ever wait months to try out such a neat present? The first opportunity was a Saturday morning when the house was empty. Our kitchen was in the basement so we leaned the the straw filled target against the kitchen cabinets and fired away and proceeded to hit everything but the target. Arrow holes were everywhere and when my mother came home she was furious. We, of course felt guilty-lower than whale effluent and it was only when I was an adult looking back did it dawn on me-who in their right minds would send children a deadly weapon and why did my mother allow me and my brother to touch this present? I never figured that one out. That night we went to the seance-the room was dark and I was seated by the window so I could see the slivers of light come and go. Very quickly the deep familiar voice of Shobona(sp.?) and his personality came through Edith"s vocal cords and he was ;laughing calling me and my brother poor hunters, bad marksmen. Apparently we were the laughing stock of the supernatural world. He and the other Indians had been watching us make fools of ourselves earlier in the day. The seance broke up and afterward we ate supper together with everyone contributing a couple of dollars for food. She did not charge for the seance. The meal was a mixture of hamburger, baked beans. onions and ketchup with bread and butter. This is still a favorite meal of mine to this day. Years later my mother was working on a patient trying to convince this person to seek medical treatment for what my mother suspected was a dangerous tumor. Dr. Mary was losing the battle when the phone rang. She said angrily, "I know! I know! but she won't listen to me! Here you tell her." The lady turned white as a proverbial ghost as Edith a thousand miles away told the lady that she had to act immediately-she did and saved her life. My mother had become a Baha'i in Iowa and dutifully shied away from the supernatural in the future as this practice was frowned upon, but she never tired of telling this story. Me, I miss sitting by the window watching the slivers of light come and go.
Posted by Ronnie Tomanio at 6:03 PM
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